Mice are actively immunized after passive monoclonal antibody prophylaxis and ricin toxin challenge. (Reannouncement with new availability information)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lemley, P.V.; Wright, D.C.

1992-12-31

Mice passively immunized by a protective, anti-ricin A-chain monoclonal antibody, then challenged intravenously with ricin, were protected from a subsequent ricin challenge, and were actively immunized. Two significant advantages accrued from this experiment: the monoclonal antibody neutralized the toxicity of the ricin immunogen, and active immunization was achieved with very low antigen load (approx. 0.5 micrograms/mouse). We ruled out the possibility that residual monoclonal antibody provided the protection by using three independent criteria. There was significant (four orders of magnitude) enhancement of the immune response in the presence of the monoclonal antibody; control immunizations of mice with ricin A-chain, ricinmore » B-chain or either chain with the monoclonal antibody did not induce active immunity; and the active immunization could not be replicated when protective goat polyclonal antibody was substituted for the monoclonal antibody. Because high titers were achieved rapidly without any adjuvant, we are currently investigating haptenized ricin to determine if anti-hapten monoclonal antibodies can be produced by this refined procedure.« less

A Randomized Phase 2 Trial of 177Lu Radiolabeled Anti-PSMA Monoclonal Antibody J591 in Patients With High-Risk Castrate Biochemically Relapsed Prostate Cancer

DTIC Science & Technology

2015-09-01

Award Number: W81XWH-09-1-0596 TITLE: A Randomized Phase 2 Trial of 177Lu Radiolabeled Anti- PSMA Monoclonal Antibody J591 in Patients With High...1-0596 A Randomized Phase 2 Trial of 177Lu Radiolabeled Anti- PSMA Monoclonal Antibody J591 in Patients With High-Risk Castrat Biochemically Relapsed...in December 2014 with approval to proceed without modifications. 15. SUBJECT TERMS Prostate cancer, PSA, PSMA , monoclonal antibody

Anti-human neutrophil antigen-1a, -1b, and -2 antibodies in neonates and children with immune neutropenias analyzed by extracted granulocyte antigen immunofluorescence assay.

PubMed

Onodera, Rie; Kurita, Emi; Taniguchi, Kikuyo; Karakawa, Shuhei; Okada, Satoshi; Kihara, Hirotaka; Fujii, Teruhisa; Kobayashi, Masao

2017-11-01

Anti-human neutrophil antigen (HNA) antibodies have been implicated in the development of neonatal alloimmune neutropenia (NAN) and autoimmune neutropenia (AIN). There are many conventional assay methods that detect anti-HNA antibodies. However, a method to measure multiple samples and detect several anti-HNA antibodies simultaneously is needed. We developed a new method, the extracted granulocyte antigen immunofluorescence assay (EGIFA), to analyze anti-HNA-1a, -1b, and -2 antibodies in sera. The results obtained by EGIFA were evaluated in comparison with those from several standard assay methods. Anti-HNA antibodies in serum samples from nine familial cases with suspected NAN (n = 19) and children with suspected AIN (n = 88) were also measured by EGIFA. The evaluation of nine serum samples with anti-HNA antibodies suggested that EGIFA demonstrated equivalent specificity and superior sensitivity to monoclonal antibody-specific immobilization of granulocyte antigens and had comparable sensitivity to the granulocyte indirect immunofluorescence test. EGIFA successfully detected anti-HNA-1a or -1b antibodies in seven of nine familial cases with suspected NAN. EGIFA detected anti-HNA antibodies in 40.9% of children with suspected AIN. Among them, isolated anti-HNA-1a or -1b antibody was detected in 4.5 or 12.5% of children, respectively, and anti-HNA-2 antibody was identified in 3.4% of children. The 30.8% (16 of 52) of children negative for anti-HNA antibody by EGIFA were positive for anti-HLA antibody. EGIFA facilitated the measurement of anti-HNA-1a, -1b, and/or -2 antibodies in sera. The prompt measurement of anti-HNA antibodies will improve the diagnosis and clinical management of patients with suspected NAN or AIN. © 2017 AABB.

[Toxoplasma gondii: the characterization of an anti-P30 monoclonal antibody].

PubMed

Fachado, A; Fernández, N; Hernández, E; Fonseca, L

1996-01-01

A specific monoclonal antibody was characterized to Toxoplasma gondii. The hybridoma produced IgG immunoglobulins. The western blot analysis showed that the monoclonal antibody was specific for the antigen of an apparent molecular mass of 30 kd, which was present on the antigen surface. The monoclonal antibody was purified starting from mouse's ascitic fluid and it was matched with sepharose 4B. This immunoabsorbent was used to purify the specific parasitic antigen. The monoclonal antibody studied may be useful for those techniques contributing to the toxoplasmosis diagnosis.

Suppression of allo-human leucocyte antigen (HLA) antibodies secreted by B memory cells in vitro: intravenous immunoglobulin (IVIg) versus a monoclonal anti-HLA-E IgG that mimics HLA-I reactivities of IVIg.

PubMed

Zhu, D; Ravindranath, M H; Terasaki, P I; Miyazaki, T; Pham, T; Jucaud, V

2014-08-01

B memory cells remain in circulation and secrete alloantibodies without antigen exposure > 20 years after alloimmunization postpartum or by transplantation. These long-lived B cells are resistant to cytostatic drugs. Therapeutically, intravenous immunoglobulin (IVIg) is administered to reduce allo-human leucocyte antigen (HLA) antibodies pre- and post-transplantation, but the mechanism of reduction remains unclear. Recently, we reported that IVIg reacts with several HLA-I alleles and the HLA reactivity of IVIg is lost after its HLA-E reactivity is adsorbed out. Therefore, we have generated an anti-HLA-E monoclonal antibody that mimics the HLA-reactivity of IVIg to investigate whether this antibody suppresses IgG secretion, as does IVIg. B cells were purified from the blood of a woman in whose blood the B memory cells remained without antigen exposure > 20 years after postpartum alloimmunization. The B cells were stimulated with cytokines using a well-defined culture system. The anti-HLA-E monoclonal antibody (mAb) significantly suppressed the allo-HLA class-II IgG produced by the B cells, and that this suppression was far superior to that by IVIg. These findings were confirmed with HLA-I antibody secreted by the immortalized B cell line, developed from the blood of another alloimmunized woman. The binding affinity of the anti-HLA-E mAb for peptide sequences shared (i.e. shared epitopes) between HLA-E and other β2-microglobulin-free HLA heavy chains (open conformers) on the cell surface of B cells may act as a ligand and signal suppression of IgG production of activated B memory cells. We propose that anti-HLA-E monoclonal antibody may also be useful to suppress allo-HLA IgG production in vivo. © 2014 British Society for Immunology.

Long-term effect of rituximab in a case with late-onset Rasmussen´s encephalitis with anti-ganglioside IgGQ1b and anti-GAD antibodies positivity. Case Report.

PubMed

Timarova, Gabriela; Lisa, Iveta; Kukumberg, Peter

2016-07-01

Rasmussen's encephalitis is a rare autoimmune encephalitis usually involving one brain hemisphere, presenting with refractory epileptic seizures, and neurological and cognitive decline. Only 10% of cases start later in adolescence/adulthood. The only effective treatment for refractory seizures in childhood is hemispherectomy. For late-onset cases with mild neurological deficit the hemispherectomy is usually postponed because of its severe consequences. Immunotherapy shows some temporal effect for seizure control and slowing the brain atrophy, mainly in late onset Rasmussen's encephalitis. We report a patient with late onset Rasmussen´s encephalitis with anti-ganglioside IgGQ1b and anti-GAD antibodies positivity, who failed immunotherapy with cytostatics, immunoglobulins and steroids. Anti-ganglioside IgGQ1b antibodies are typically associated with a Miller-Fisher variant of Guillain-Barre syndrome and Bickerstaff's brainstem encephalitis. The association with Rasmussen´s encephalitis was not described before. Patient´s neurological deficit was mild and hemispherectomy was refused. The treatment with rituximab, an anti-CD20+ monoclonal antibody, led to 36-month control of seizures without any signs of progression of neurological deficit and MRI brain atrophy. Although the treatment is associated with long term B-cells depletion, patient doesn´t suffer from any clinically relevant infection. The biological treatment with monoclonal antibodies might be the way to stabilize patients with Rasmussen´s encephalitis, mainly late-onset, to prevent them from harmful and devastating hemispherectomy.

Identification of whole pathogenic cells by monoclonal antibodies generated against a specific peptide from an immunogenic cell wall protein.

PubMed

Kaba, Hani E J; Maier, Natalia; Schliebe-Ohler, Nicole; Mayer, Yvonne; Müller, Peter P; van den Heuvel, Joop; Schuchhardt, Johannes; Hanack, Katja; Bilitewski, Ursula

2015-01-01

We selected the immunogenic cell wall ß-(1,3)-glucosyltransferase Bgl2p from Candida albicans as a target protein for the production of antibodies. We identified a unique peptide sequence in the protein and generated monoclonal anti- C. albicans Bgl2p antibodies, which bound in particular to whole C. albicans cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Production and characterization of monoclonal antibody and its recombinant single chain variable fragment specific for a food-born mycotoxin, fumonisin B1.

PubMed

Min, Won-Ki; Cho, Young-Jin; Park, Jun-Bock; Bae, Yi-Hyun; Kim, Eun-Jeong; Park, Kyungmoon; Park, Yong-Cheol; Seo, Jin-Ho

2010-01-01

Fumonisin B(1) (FMB(1)) is a food-born mycotoxin produced by Fusarium moniliforme. Monoclonal antibody against FMB(1) (anti-FMB(1) mAb) was produced in the hybridoma DV9, which was established from a BALB/c mouse immunized with bovine serum albumin conjugated FMB(1) (FMB(1)-BSA). A competitive direct enzyme-linked immunosorbent assay (ELISA) showed that anti-FMB(1) mAb has about 10 ppb of minimum FMB(1) detection concentration and 220 ppb of 50% inhibition concentration (IC(50)). Much lower cross-reactivity of anti-FMB(1) mAb on ochratoxin A, aflatoxin B(1) and deoxynivalenol provided that anti-FMB(1) mAb was specific for FMB(1). The gene coding single chain variable fragment against FMB(1) (anti-FMB(1) scFv) was cloned from the hybridoma DV9 and was expressed in recombinant Escherichia coli. Insoluble anti-FMB(1) scFv required optimization of its refolding condition, and hence functional scFv was obtained. By using indirect ELISA, about 12-fold lower binding activity of anti-FMB(1) scFv on FMB(1)-BSA was obtained in comparison with that of the parental mAb.

The influence of proteasome inhibitor MG132, external radiation and unlabeled antibody on the tumor uptake and biodistribution of 188Re-labeled anti-E6 C1P5 antibody in cervical cancer in mice

PubMed Central

Phaeton, Rébécca; Wang, Xing Guo; Einstein, Mark H.; Goldberg, Gary L.; Casadevall, Arturo; Dadachova, Ekaterina

2009-01-01

Background Human Papillomavirus (HPV) infection is considered a necessary step for the development of cervical cancer and >95% of all cervical cancers have detectable HPV sequences. We have recently demonstrated the efficacy of radioimmunotherapy (RIT) which targeted viral oncoprotein E6 in treatment of experimental cervical cancer We hypothesized that pre-treatment of tumor cells with various agents which cause cell death and/or elevation of E6 levels would increase the accumulation of radiolabeled antibodies to E6 in cervical tumors. Methods HPV-16 positive CasKi cells were treated in vitro with up to 6 Gy of external radiation, or proteasome inhibitor MG-132 or unlabeled anti-E6 antibody C1P5 and cell death was assessed. Biodistribution of 188Rhenium (188Re)-labeled C1P5 antibody was performed in both control and radiation MG-132 treated CasKi tumor-bearing nude mice. Results . 188Re-C1P5 antibody demonstrated tumor specificity and very low uptake and fast clearance from the major organs. The amount of tumor uptake was enhanced by MG-132 but was unaffected by pre-treatment with radiation. In addition, in vitro studies demonstrated an unanticipated effect of unlabeled antibody on the amount of cell death, a finding that was suggested by our previous in vivo studies in CasKi tumor model. Conclusion We demonstrated that pre-treatment of cervical tumors with proteasome inhibitor MG-132 and with unlabeled antibody to E6 can serve as a means to generate non-viable cancer cells and to elevate the levels of target oncoproteins in the cells for increasing the accumulation of targeted radiolabeled antibodies in tumors. These results favor further development of RIT of cervical cancers targeting viral antigens. PMID:20127955

Generation of monoclonal antibodies to recombinant vascular endothelial growth factor.

PubMed

Shein, S A; Gurina, O I; Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Grinenko, N F; Ivanova, N V; Volgina, N E; Ryabukhin, I A; Chekhonin, V P

2012-05-01

Female BALB/c mice were subcutaneously immunized with recombinant VEGF-164. After 3 immunization cycles, splenic B cells from immunized mouse were fused with immortalized myeloma culture SP2/0-Ag14 cells. Screening of hybrid cells producing anti-VEGF antibodies was performed by ELISA and immunocytochemical analysis on cultured C6 glioma cells. Subsequent cloning yielded hybridoma stably expressing monoclonal anti-VEGF antibodies recognizing recombinant and native VEGF.

BAFF Receptor mAb Treatment Ameliorates Development and Progression of Atherosclerosis in Hyperlipidemic ApoE−/− Mice

PubMed Central

Kyaw, Tin; Cui, Peng; Tay, Christopher; Kanellakis, Peter; Hosseini, Hamid; Liu, Edgar; Rolink, Antonius G.; Tipping, Peter

2013-01-01

Aims Option to attenuate atherosclerosis by depleting B2 cells is currently limited to anti-CD20 antibodies which deplete all B-cell subtypes. In the present study we evaluated the capacity of a monoclonal antibody to B cell activating factor-receptor (BAFFR) to selectively deplete atherogenic B2 cells to prevent both development and progression of atherosclerosis in the ApoE−/− mouse. Methods and Results To determine whether the BAFFR antibody prevents atherosclerosis development, we treated ApoE−/− mice with the antibody while feeding them a high fat diet (HFD) for 8 weeks. Mature CD93− CD19+ B2 cells were reduced by treatment, spleen B-cell zones disrupted and spleen CD20 mRNA expression decreased while B1a cells and non-B cells were spared. Atherosclerosis was ameliorated in the hyperlipidemic mice and CD19+ B cells, CD4+ and CD8+ T cells were reduced in atherosclerotic lesions. Expressions of proinflammatory cytokines, IL1β, TNFα, and IFNγ in the lesions were also reduced, while MCP1, MIF and VCAM-1 expressions were unaffected. Plasma immunoglobulins were reduced, but MDA-oxLDL specific antibodies were unaffected. To determine whether anti-BAFFR antibody ameliorates progression of atherosclerosis, we first fed ApoE−/− mice a HFD for 6 weeks, and then instigated anti-BAFFR antibody treatment for a further 6 week-HFD. CD93− CD19+ B2 cells were selectively decreased and atherosclerotic lesions were reduced by this treatment. Conclusion Anti-BAFFR monoclonal antibody selectively depletes mature B2 cells while sparing B1a cells, disrupts spleen B-cell zones and ameliorates atherosclerosis development and progression in hyperlipidemic ApoE−/− mice. Our findings have potential for clinical translation to manage atherosclerosis-based cardiovascular diseases. PMID:23560095

Enzyme-linked immunosorbent assay for total sennosides using anti-sennside A and anti-sennoside B monoclonal antibodies.

PubMed

Morinaga, Osamu; Uto, Takuhiro; Sakamoto, Seiichi; Tanaka, Hiroyuki; Shoyama, Yukihiro

2009-01-01

Total sennosides concentration is a very important factor when rhubarb and senna will be used as crude drugs. However, one-step analytical technique for total sennosides has not been reported except HPLC. An enzyme-linked immunosorbent assay (ELISA) for total sennosides concentration by using the combination of anti-sennoside A (SA) and anti-sennoside B (SB) monoclonal antibodies (MAbs) in a single assay has been investigated. Total sennosides concentration in rhubarb and senna samples determined by newly developed assay system showed good agreement with those analyzed by ELISA using anti-SA MAb and anti-SB MAb, respectively.

Beta-Catenin Stability in Breast Cancer

DTIC Science & Technology

1999-09-01

monoclonal anti-ß-catenin antibody (Clone#14) and the anti-FLAG™ antibody were purchased from Transduction Labs and Kodak, respectively. Affinity purified...anti-HA (BabCo) and anti-ß-catenin antibodies (Transduction labs ). To test the hypothesis that the tumor suppressor adenomatous polyposis coli (APC...ß-catenin was detected first using 1:50 dilution of a mouse monoclonal antibody (Transduction Labs ) followed by Texas Red-conjugated goat anti

Suppression of in vivo polyclonal IgE responses by monoclonal antibody to the lymphokine B-cell stimulatory factor 1.

PubMed Central

Finkelman, F D; Katona, I M; Urban, J F; Snapper, C M; Ohara, J; Paul, W E

1986-01-01

The lymphokine B-cell stimulatory factor 1 (BSF-1) has been shown to greatly enhance the differentiation of lipopolysaccharide-activated B cells into IgG1- and IgE-secreting cells in vitro. To determine whether in vivo IgG1 and IgE antibody responses are BSF-1 dependent, the ability of a monoclonal rat IgG1 anti-BSF-1 antibody, 11B11, to affect polyclonal IgG1 and IgE production in mice infected with the nematode parasite Nippostrongylus brasiliensis or injected with a purified goat antibody to mouse IgD was studied. 11B11-containing ascites fluid or purified 11B11 strongly inhibited IgE production in both systems but did not affect IgG1 production, while control ascites or normal rat IgG1 had no IgE-inhibitory activity. These results indicate an important physiologic role for BSF-1 in the generation of IgE antibody responses and suggest means for limiting the production of antibodies responsible for allergic reactions without inhibiting protective antibody responses. PMID:3491987

Targeting CD22 in B-cell malignancies: current status and clinical outlook.

PubMed

Sullivan-Chang, Loretta; O'Donnell, Robert T; Tuscano, Joseph M

2013-08-01

CD22 is a B-cell-specific transmembrane glycoprotein found on the surface of most B cells; it modulates B-cell function, survival and apoptosis. CD22 has emerged as an ideal target for monoclonal antibody (mAb)-based therapy of B-cell malignancies including most lymphomas and many leukemias. Epratuzumab, an anti-CD22 mAb, has been developed in various forms, including as an unlabeled (naked) mAb, as a radioimmunotherapeutic, as an antibody drug conjugate (ADC), and as a vehicle for CD22-targeted nanoparticles. While clinical trials with unlabeled epratuzumab have demonstrated modest results, its combination with rituximab in phase II studies has been more encouraging. Based on the potential for CD22 to become internalized, CD22-targeted constructs carrying radioisotopes or toxins have generated promising results. Radioimmunotherapy, utilizing ⁹⁰Y-labeled epratuzumab, was shown to be highly effective in patients with follicular lymphoma, generating a complete response (CR) rate of 92 % and progression-free survival of more than 2 years. ADC therapy is a promising therapeutic approach to B-cell malignancies which includes the direct conjugation of mAbs with cytotoxic agents. Phase II studies of inotuzumab ozogamicin, an ADC which combines anti-CD22 mAb with calicheamicin, an enediyne antibiotic which mediates apoptosis, in patients with acute lymphoblastic leukemia have produced an overall response rate (ORR) of greater than 50 % in treatment-refractory patients. Phase I trials of moxetumomab pasudotox, an ADC which combines anti-CD22 with PE38, a fragment of Pseudomonas exotoxin A, have been completed in hairy cell leukemia with a ORR of 86 %. Finally, a review of CD22-targeted nanoparticles, that include a doxorubicin-containing lipid complex that uses synthetic high-affinity CD22 ligand mimetics as well as anti-CD22 mAb-coated pegylated liposomas doxorubin (PLD), has demonstrated promising results in pre-clinical models of human lymphoma. Moreover, novel anti-CD22 mAb that block CD22 ligand binding as well as second generation ADC that utilize biodegradable linkers and more potent toxins hold great hope for the future of CD22-targeted therapeutics that may translate into better outcomes for patients with CD22-positive malignancies.

Fully human broadly neutralizing monoclonal antibodies against influenza A viruses generated from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Weibin; Chen, Aizhong; Miao, Yi

Whether the 2009 pandemic H1N1 influenza vaccine can induce heterosubtypic cross-protective anti-hemagglutinin (HA) neutralizing antibodies is an important issue. We obtained a panel of fully human monoclonal antibodies from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient. Most of the monoclonal antibodies targeted the HA protein but not the HA1 fragment. Among the analyzed antibodies, seven mAbs exhibited neutralizing activity against several influenza A viruses of different subtypes. The conserved linear epitope targeted by the neutralizing mAbs (FIEGGWTGMVDGWYGYHH) is part of the fusion peptide on HA2. Our work suggests that a heterosubtypic neutralizing antibody response primarilymore » targeting the HA stem region exists in recipients of the 2009 pandemic H1N1 influenza vaccine. The HA stem region contains various conserved neutralizing epitopes with the fusion peptide as an important one. This work may aid in the design of a universal influenza A virus vaccine.« less

Production and characterization of monoclonal antibodies against cathepsin B and cathepsin B-Like proteins of Naegleria fowleri.

PubMed

Seong, Gi-Sang; Sohn, Hae-Jin; Kang, Heekyoung; Seo, Ga-Eun; Kim, Jong-Hyun; Shin, Ho-Joon

2017-12-01

Naegleria fowleri causes fatal primary amoebic meningoencephalitis (PAM) in humans and experimental animals. In previous studies, cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes of N. fowleri were cloned, and it was suggested that refolding rNfCPB and rNfCPB-L proteins could play important roles in host tissue invasion, immune response evasion and nutrient uptake. In this study, we produced anti-NfCPB and anti-NfCPB-L monoclonal antibodies (McAb) using a cell fusion technique, and observed their immunological characteristics. Seven hybridoma cells secreting rNfCPB McAbs and three hybridoma cells secreting rNfCPB-L McAbs were produced. Among these, 2C9 (monoclone for rNfCPB) and 1C8 (monoclone for rNfCPB-L) McAb showed high antibody titres and were finally selected for use. As determined by western blotting, 2C9 McAb bound to N. fowleri lysates, specifically the rNfCPB protein, which had bands of 28 kDa and 38.4 kDa. 1C8 McAb reacted with N. fowleri lysates, specifically the rNfCPB-L protein, which had bands of 24 kDa and 34 kDa. 2C9 and 1C8 monoclonal antibodies did not bind to lysates of other amoebae, such as N. gruberi, Acanthamoeba castellanii and A. polyphaga in western blot analyses. Immuno-cytochemistry analysis detected NfCPB and NfCPB-L proteins in the cytoplasm of N. fowleri trophozoites, particularly in the pseudopodia and food-cup. These results suggest that monoclonal antibodies produced against rNfCPB and rNfCPB-L proteins may be useful for further immunological study of PAM. Copyright © 2017 Elsevier Inc. All rights reserved.

New Dioxaborolane Chemistry Enables [18F]-Positron-Emitting, Fluorescent [18F]-Multimodality Biomolecule Generation from the Solid Phase

PubMed Central

Crisp, Jessica L.; Vera, David R.; Tsien, Roger Y.; Ting, Richard

2016-01-01

New protecting group chemistry is used to greatly simplify imaging probe production. Temperature and organic solvent-sensitive biomolecules are covalently attached to a biotin-bearing dioxaborolane, which facilitates antibody immobilization on a streptavidin-agarose solid-phase support. Treatment with aqueous fluoride triggers fluoride-labeled antibody release from the solid phase, separated from unlabeled antibody, and creates [18F]-trifluoroborate-antibody for positron emission tomography and near-infrared fluorescent (PET/NIRF) multimodality imaging. This dioxaborolane-fluoride reaction is bioorthogonal, does not inhibit antigen binding, and increases [18F]-specific activity relative to solution-based radiosyntheses. Two applications are investigated: an anti-epithelial cell adhesion molecule (EpCAM) monoclonal antibody (mAb) that labels prostate tumors and Cetuximab, an anti-epidermal growth factor receptor (EGFR) mAb (FDA approved) that labels lung adenocarcinoma tumors. Colocalized, tumor-specific NIRF and PET imaging confirm utility of the new technology. The described chemistry should allow labeling of many commercial systems, diabodies, nanoparticles, and small molecules for dual modality imaging of many diseases. PMID:27064381

New Dioxaborolane Chemistry Enables [(18)F]-Positron-Emitting, Fluorescent [(18)F]-Multimodality Biomolecule Generation from the Solid Phase.

PubMed

Rodriguez, Erik A; Wang, Ye; Crisp, Jessica L; Vera, David R; Tsien, Roger Y; Ting, Richard

2016-05-18

New protecting group chemistry is used to greatly simplify imaging probe production. Temperature and organic solvent-sensitive biomolecules are covalently attached to a biotin-bearing dioxaborolane, which facilitates antibody immobilization on a streptavidin-agarose solid-phase support. Treatment with aqueous fluoride triggers fluoride-labeled antibody release from the solid phase, separated from unlabeled antibody, and creates [(18)F]-trifluoroborate-antibody for positron emission tomography and near-infrared fluorescent (PET/NIRF) multimodality imaging. This dioxaborolane-fluoride reaction is bioorthogonal, does not inhibit antigen binding, and increases [(18)F]-specific activity relative to solution-based radiosyntheses. Two applications are investigated: an anti-epithelial cell adhesion molecule (EpCAM) monoclonal antibody (mAb) that labels prostate tumors and Cetuximab, an anti-epidermal growth factor receptor (EGFR) mAb (FDA approved) that labels lung adenocarcinoma tumors. Colocalized, tumor-specific NIRF and PET imaging confirm utility of the new technology. The described chemistry should allow labeling of many commercial systems, diabodies, nanoparticles, and small molecules for dual modality imaging of many diseases.

Isolation of HIV-1-Neutralizing Mucosal Monoclonal Antibodies from Human Colostrum

PubMed Central

Friedman, James; Alam, S. Munir; Shen, Xiaoying; Xia, Shi-Mao; Stewart, Shelley; Anasti, Kara; Pollara, Justin; Fouda, Genevieve G.; Yang, Guang; Kelsoe, Garnett; Ferrari, Guido; Tomaras, Georgia D.; Haynes, Barton F.; Liao, Hua-Xin

2012-01-01

Background Generation of potent anti-HIV antibody responses in mucosal compartments is a potential requirement of a transmission-blocking HIV vaccine. HIV-specific, functional antibody responses are present in breast milk, and these mucosal antibody responses may play a role in protection of the majority of HIV-exposed, breastfeeding infants. Therefore, characterization of HIV-specific antibodies produced by B cells in milk could guide the development of vaccines that elicit protective mucosal antibody responses. Methods We isolated B cells from colostrum of an HIV-infected lactating woman with a detectable neutralization response in milk and recombinantly produced and characterized the resulting HIV-1 Envelope (Env)-specific monoclonal antibodies (mAbs). Results The identified HIV-1 Env-specific colostrum mAbs, CH07 and CH08, represent two of the first mucosally-derived anti-HIV antibodies yet to be reported. Colostrum mAb CH07 is a highly-autoreactive, weakly-neutralizing gp140-specific mAb that binds to linear epitopes in the gp120 C5 region and gp41 fusion domain. In contrast, colostrum mAb CH08 is a nonpolyreactive CD4-inducible (CD4i) gp120-specific mAb with moderate breadth of neutralization. Conclusions These novel HIV-neutralizing mAbs isolated from a mucosal compartment provide insight into the ability of mucosal B cell populations to produce functional anti-HIV antibodies that may contribute to protection against virus acquisition at mucosal surfaces. PMID:22624058

Epitope characterization of pre-existing and developing antibodies to an aglycosylated monoclonal antibody therapeutic of G1m17,1 allotype.

PubMed

Tatarewicz, Suzanna M; Juan, Gloria; Swanson, Steven J; Moxness, Michael S

2012-08-31

Allotypes of IgG1 molecules can influence the immunogenicity of therapeutic monoclonal antibodies and may account for the presence of some pre-existing antibodies. An electrochemiluminescent (ECL) bridging immunoassay was used to characterize the binding epitopes of anti-therapeutic antibodies (ATAs) in a Phase 1 single ascending dose clinical trial of a therapeutic aglycosylated IgG1monoclonal antibody (mAb). There was no evidence for ATAs specific for a possible neo-epitope created due to the lack of glycosylation. ATAs that developed post-treatment were specific for the F(ab')2, whereas, pre-existing ATAs were specific to the Fc region. Further characterization of the pre-existing ATAs identified the specific epitope to be the G1m1 allotype determinant in the Fc of the therapeutic. A novel competitive bridging assay was developed to verify that serum IgG1 from subjects with pre-existing anti-G1m1 antibodies was homozygous for the antithetical allotype (G1m3). The endogenous G1m allotype of all subjects was assessed and correlation to ATA incidence and adverse events was evaluated. Interestingly, the pre-existing anti-allotype antibody in subjects persisted but was not augmented after dosing, indicating the lack of a secondary immune response to this epitope. These studies indicate the relationship of the therapeutic allotype and the corresponding allotype of subjects is an important component to further understand the impact of immunogenicity on the safety and efficacy of therapeutic antibodies. Copyright © 2012 Elsevier B.V. All rights reserved.

Fab fragment labeled with ICG-derivative for detecting digestive tract cancer.

PubMed

Yano, Hiromi; Muguruma, Naoki; Ito, Susumu; Aoyagi, Eriko; Kimura, Tetsuo; Imoto, Yoshitaka; Cao, Jianxin; Inoue, Shohei; Sano, Shigeki; Nagao, Yoshimitsu; Kido, Hiroshi

2006-09-01

In previous studies, we generated infrared ray fluorescence-labeled monoclonal antibodies and developed an infrared ray fluorescence endoscope capable of detecting the monoclonal antibodies to establish a novel diagnostic technique for gastrointestinal cancer. Although the whole IgG molecule has commonly been used for preparation of labeled antibodies, labeled IgG displays insufficient sensitivity and specificity, probably resulting from non-specific binding of the Fc fragment to target cells or interference between fluorochromes on the identical labeled antibody, which might be caused by molecular structure. In this in vitro study, we characterized an Fc-free fluorescence-labeled Fab fragment, which was expected to yield more specific binding to target cells than the whole IgG molecule. An anti-mucin antibody and ICG-ATT, an ICG derivative, were used as the labeled antibody and labeling compound, respectively. Paraffin sections of excised gastric cancer tissues were subjected to staining. The labeled whole IgG molecule (ICG-ATT-labeled IgG) and the labeled Fab fragment (ICG-ATT-labeled Fab) were prepared according to a previous report, and the fluorescence properties, antibody activities, and features of fluorescence microscope images obtained from paraffin sections were compared. Both ICG-ATT-labeled Fab and ICG-ATT-labeled IgG were excited by a near infrared ray of 766nm, and maximum emission occurred at 804nm. Antibody activities of ICG-ATT-labeled Fab were shown to be similar to those of unlabeled anti-MUC1 antibody. The fluorescence intensity obtained from paraffin sections of excised gastric cancer tissues revealed a tendency to be greater with ICG-ATT-labeled Fab than with ICG-ATT-labeled IgG. The infrared ray fluorescence-labeled Fab fragment was likely to be more specific than the conventionally labeled antibodies. Fragmentation of antibodies is considered to contribute to improved sensitivity and specificity of labeled antibodies for detection of micro gastrointestinal cancers.

Development and characterization of monoclonal antibodies against canine PD-1 and PD-L1.

PubMed

Nemoto, Yuki; Shosu, Kazuha; Okuda, Masaru; Noguchi, Shunsuke; Mizuno, Takuya

2018-04-01

Recent research has focused on immunotherapy, particularly with regard to cancer treatment. Programmed death-1 and programmed death ligand 1 (PD-1/PD-L1) pathway blockade is a central topic of the promising immunotherapy field. In veterinary medicine, observations of the PD-1/PD-L1 pathway, including the relationship between immune cells and diseases, have increased. In this study, monoclonal antibodies specific to canine PD-1 and PD-L1 were developed, and the antibodies against PD-1 and PD-L1 bind to PD-1 and PD-L1 overexpressing cells, respectively. Additionally, each antibody interfered with the interaction between PD-1 and PD-L1. The expression of PD-1 and PD-L1 was detected on activated T cells from canine peripheral blood mononuclear cells (PBMC), and, remarkably, was the first recorded instance of PD-L1 expression on canine immature dendritic cells. Production of IFN-γ by activated T cells increased significantly when incubated with anti-PD-1 antibody alone and with both anti-PD-1 and anti-PD-L1 antibodies, revealing the functional effects of the antibodies. The antibodies will be useful for research on immune systems and may be the first passive immunotherapy approach in canine cancer patients. Copyright © 2018 Elsevier B.V. All rights reserved.

Idiotypic Cascades in Cancer Patients Treated with Monoclonal Antibody CO17-1A

NASA Astrophysics Data System (ADS)

Wettendorff, Martine; Iliopoulos, Dimitrios; Tempero, Margaret; Kay, David; Defreitas, Elaine; Koprowski, Hilary; Herlyn, Dorothee

1989-05-01

We have previously shown that gastrointestinal cancer patients treated with monoclonal antibody CO17-1A (Ab1) developed anti-idiotypic antibodies (Ab2) to the Ab1. We now demonstrate that patients produce anti-anti-idiotypic antibodies (Ab3) to their autologous Ab2. Ab3 were demonstrated in culture supernatants of peripheral blood mononuclear cells from five Ab1-treated patients after stimulation of the cells with heterologous Ab2 that functionally mimicked the tumor antigen (Ag) defined by Ab1 and immunologically cross reacted with the patients' Ab2. Ab3 shared idiotopes with Ab1 and were Ab1-like in their binding specificities to tumor cells, Ag, and Ab2. Such antibodies were also elicited by stimulating cells with Ag. However, they were not produced by stimulating posttreatment mononuclear cells with control proteins or by stimulating pretreatment cells with either Ag or Ab2. Our results demonstrate idiotypic cascades in cancer patients treated with monoclonal antibody. Ag-specific Ab3 responses may underlie delayed clinical responses often observed in cancer patients treated with monoclonal antibodies of various specificities.

Production of human monoclonal IgG antibodies against Rhesus (D) antigen.

PubMed Central

Bron, D; Feinberg, M B; Teng, N N; Kaplan, H S

1984-01-01

An Epstein-Barr virus (EBV)-transformed human B-cell line ( LB4r ) producing anti-Rhesus [Rho(D) antigen] antibody was fused with a non-immunoglobulin-producing mouse-human heteromyeloma ( SHM - D33 ) and selected in hypoxanthine/aminopterin/thymidine medium containing 0.5 microM ouabain. Surviving hybrids found to secrete specific anti-Rho(D) antibody were cloned by limiting dilution. Two clones (D4-B2 and E10-C1) producing high levels (12 and 20 micrograms/ml per 10(6) cells per 24 hr, respectively) of monospecific antibody (IgG3, lambda chain) were selected for expansion and further characterization. Compared to the parental cell line ( LB4r ), these hybridoma cell lines presented several advantages: antibody production was increased 10-fold, cloning efficiency was improved, and the EBV genome was not retained. Antibody production has been stable for greater than 8 months. These human monoclonal anti-Rho(D) antibodies have demonstrated utility in routine blood-group typing. They may also prove useful in the biochemical and genetic characterization of the Rh antigen system. Most important, they offer a source of Rh-immune globulin for the prevention of Rh immunization and alloimmune hemolytic disease of the newborn. Images PMID:6427767

Enhanced CDC of B cell chronic lymphocytic leukemia cells mediated by rituximab combined with a novel anti-complement factor H antibody.

PubMed

Winkler, Mark T; Bushey, Ryan T; Gottlin, Elizabeth B; Campa, Michael J; Guadalupe, Eross S; Volkheimer, Alicia D; Weinberg, J Brice; Patz, Edward F

2017-01-01

Rituximab therapy for B cell chronic lymphocytic leukemia (B-CLL) has met with mixed success. Among several factors to which resistance can be attributed is failure to activate complement dependent cytotoxicity (CDC) due to protective complement regulatory proteins, including the soluble regulator complement factor H (CFH). We hypothesized that rituximab killing of non-responsive B-CLL cells could be augmented by a novel human monoclonal antibody against CFH. The B cells from 11 patients with B-CLL were tested ex vivo in CDC assays with combinations of CFH monoclonal antibody, rituximab, and a negative control antibody. CDC of rituximab non-responsive malignant B cells from CLL patients could in some cases be augmented by the CFH monoclonal antibody. Antibody-mediated cytotoxicity of cells was dependent upon functional complement. In one case where B-CLL cells were refractory to CDC by the combination of rituximab plus CFH monoclonal antibody, additionally neutralizing the membrane complement regulatory protein CD59 allowed CDC to occur. Inhibiting CDC regulatory proteins such as CFH holds promise for overcoming resistance to rituximab therapy in B-CLL.

Monoclonal antibodies against Stx1B subunit of Escherichia coli O157:H7 distinguish the bacterium from other bacteria.

PubMed

Li, G; Hong, J; Huo, G; Ren, X

2010-11-01

The Shiga-like toxins (Stx) are critical virulence factors of enterohaemorrhagic Escherichia coli (EHEC). Stx1B subunit plays important roles in EHEC infection. This work aims to generate and characterize monoclonal antibodies (mAbs) against the Stx1B and to investigate their utility in discrimination ELISA. Two newly identified mAbs (designated 2H8 and 1B10, respectively) against the Stx1B protein were prepared via hybridoma techniques. The immunoreactivity of both mAbs to the Stx1B protein was confirmed in ELISA and Western blot. Moreover, they differentiate EHEC from Salmonella enteritis, non-Stx1-producing E. coli, Mycobacterium tuberculosis, Listeria monocytogenes, Streptococcus agalactiae and Staphylococcus aureus. The anti-STx1B mAbs are valuable diagnostic reagents for distinguishing EHEC from other bacteria. This is the first report regarding the usage of anti-STx1B mAbs in discrimination ELISA. The established ELISA may have potential in clinical surveillance of EHEC infection. © 2010 The Authors. © 2010 The Society for Applied Microbiology.

[Identification of Env-specific monoclonal antibodies from Chinese HIV-1 infected person by magnetic beads separating B cells and single cell RT-PCR cloning].

PubMed

Huang, Xiang-Ying; Yu, Shuang-Qing; Cheng, Zhan; Ye, Jing-Rong; Xu, Ke; Feng, Xia; Zeng, Yi

2013-04-01

To establish a simple and practical method for screening of Env-specific monoclonal antibodies from HIV-1 infected individuals. Human B cells were purified by negative sorting from PBMCs and memory B cells were further enriched using anti-CD27 microbeads. Gp120 antigen labbled with biotin was incubated with memory B cells to specifically bind IgG on cells membrane. The memory B cells expressing the Env-specific antibody were harvested by magnetic beads separating, counted and diluted to the level of single cell in each PCR well that loading with catch buffer containing RNase inhibitor to get RNAs. The antibody genes were amplified by single cell RT-PCR and nested PCR, cloned into eukaryotic expression vectors and transfected into 293T cells. The binding activity of recombinant antibodies to Env were tested by ELISA. Three monocolonal Env-specific antibodies were isolated from one HIV-1 infected individual. We can obtain Env-specific antibody by biotin labbled antigen, magnetic beads separating technique coupled with single cell RT-PCR and expression cloning.

Acute inflammatory neuropathy with monoclonal anti-GM2 IgM antibodies, IgM-κ paraprotein and additional autoimmune processes in association with a diffuse large B-cell non-Hodgkin's lymphoma.

PubMed

Milnik, Annette; Roggenbuck, Dirk; Conrad, Karsten; Bartels, Claudius

2013-01-21

Lymphoproliferative disorders are often associated with autoimmune processes preceding or following the occurrence of a lymphoma. Here, we describe a patient with a history of recurrent diffuse large B-cell non-Hodgkin's lymphoma who suffered from an acute inflammatory neuropathy with specific monoclonal anti-GM2 IgM antibodies and associated IgM-κ paraprotein. It was possible in this case to prove that both, anti-GM2 IgM antibodies and IgM-κ paraprotein, share the same binding characteristic. In addition, the patient possibly suffered from an immune thrombocytopenia and an early-stage bullous pemphigoid with anti-BP-230 IgG antibodies. Intravenous immunoglobulin and plasmapheresis alleviated the acute neuropathy and thrombocytopenia, while the bullous pemphigoid has been aggravated. In summary, the simultaneous occurrence of multiple autoimmune processes was a sign of a dysfunctional immune system preceding the relapse of a B-cell non-Hodgkin's lymphoma.

Dog Erythrocyte Antigen 1 (DEA 1): Mode of Inheritance and Initial Characterization

PubMed Central

Polak, Klaudia; Acierno, Michelle; Raj, Karthik; Mizukami, Keijiro; Siegel, Don L.; Giger, Urs

2015-01-01

Background The Dog Erythrocyte Antigen (DEA) 1 blood group system remains poorly defined. Objectives The purpose of the study was to determine the DEA 1 mode of inheritance and to characterize the DEA 1 antigen and alloantibodies. Animals Canine research colony families, clinic canine patients, and DEA 1.2+ blood bank dogs were studied. Methods Canine blood was typed by flow cytometry and immunochromatographic strips using anti-DEA 1 monoclonal antibodies. Gel column experiments with polyclonal and immunoblotting with monoclonal anti-DEA 1 antibodies were performed to analyze select samples. Cross-reactivity of human typing reagents against canine RBCs and one monoclonal anti-DEA 1 antibody against human RBC panels was assessed. Results Typing of 12 families comprising 144 dogs indicated an autosomal dominant inheritance with ≥4 alleles: DEA 1− (0) and DEA 1+ weak (1+), intermediate (2+) and strong (3+ and 4+). Samples from 6 dogs previously typed as DEA 1.2+ were typed as DEA 1+ or DEA 1− using monoclonal antibodies. Human typing reagents produced varied reactions in tube agglutination experiments against DEA 1+ and DEA 1− RBCs. Polypeptide bands were not detected on immunoblots using a monoclonal anti-DEA 1 antibody, therefore the anti-DEA 1 antibody may be specific for conformational epitopes lost during denaturation. Conclusions The autosomal dominant inheritance of DEA 1 with ≥4 alleles indicates a complex blood group system; the antigenicity of each DEA 1+ type will need to be determined. The biochemical nature of the DEA 1 antigen(s) appears different from human blood group systems tested. PMID:26291052

Imaging, biodistribution, and dosimetry of radionuclide-labeled PD-L1 antibody in an immunocompetent mouse model of breast cancer

PubMed Central

Josefsson, Anders; Nedrow, Jessie R.; Park, Sunju; Banerjee, Sangeeta Ray; Rittenbach, Andrew; Jammes, Fabien; Tsui, Benjamin; Sgouros, George

2015-01-01

The programmed cell death ligand 1 (PD-L1) participates in an immune checkpoint system involved in preventing autoimmunity. PD-L1 is expressed on tumor cells, tumor-associated macrophages, and other cells in the tumor microenvironment. Anti-PD-L1 antibodies are active against a variety of cancers, and combined anti-PD-L1 therapy with external beam radiotherapy has been shown to increase therapeutic efficacy. PD-L1 expression status is an important indicator of prognosis and therapy responsiveness, but methods to precisely capture the dynamics of PD-L1 expression in the tumor microenvironment are still limited. In this study, we developed a murine anti-PD-L1 antibody conjugated to the radioactive isotope Indium-111 (111In) for imaging and biodistribution studies in an immune-intact mouse model of breast cancer. The distribution of 111In-DTPA-anti-PD-L1 in tumors as well as the spleen, liver, thymus, heart, and lungs peaked 72 hours after injection. Co-injection of labeled and 100-fold unlabeled antibody significantly reduced spleen uptake at 24 hours, indicating that an excess of unlabeled antibody effectively blocked PD-L1 sites in the spleen, thus shifting the concentration of 111In-DTPA-anti-PD-L1 into the blood stream and potentially increasing tumor uptake. Clearance of 111In-DTPA-anti-PD-L1 from all organs occurred at 144 hours. Moreover, dosimetry calculations revealed that radionuclide-labeled anti-PD-L1 antibody yielded tolerable projected marrow doses, further supporting its use for radiopharmaceutical therapy. Taken together, these studies demonstrate the feasibility of using anti-PD-L1 antibody for radionuclide imaging and radioimmunotherapy, and highlight a new opportunity to optimize and monitor the efficacy of immune checkpoint inhibition therapy. PMID:26554829

Immunotherapy of Alzheimer's disease (AD): from murine models to anti-amyloid beta (Abeta) human monoclonal antibodies.

PubMed

Geylis, Valeria; Steinitz, Michael

2006-01-01

The deposition of amyloid beta (Abeta) protein is a key pathological feature in Alzheimer's disease (AD). In murine models of AD, both active and passive immunization against Abeta induce a marked reduction in amyloid brain burden and an improvement in cognitive functions. Preliminary results of a prematurely terminated clinical trial where AD patients were actively vaccinated with aggregated Abeta bear resemblance to those documented in murine models. Passive immunization of AD patients with anti-Abeta antibodies, in particular human antibodies, is a strategy that provides a more cautious management and control of any undesired side effects. Sera of all healthy adults contain anti-Abeta IgG autoimmune antibodies. Hence antigen-committed human B-cells are easily immortalized by Epstein-Barr virus (EBV) into anti-Abeta secreting cell lines. Two anti-Abeta human monoclonal antibodies which we recently prepared bind to the N-terminus of Abeta peptide and were shown to stain amyloid plaques in non-fixed brain sections from an AD patient. It is anticipated that specifically selected anti-Abeta human monoclonal antibodies could reduce and inhibit deposits of amyloid in brain while avoiding the cognitive decline that characterizes AD. In the future, this type of antibody may prove to be a promising immune therapy for the disease.

Development and characterization of a TAPIR-like mouse monoclonal antibody to amyloid-beta.

PubMed

Wang, Jun; Hara, Hideo; Makifuchi, Takao; Tabira, Takeshi

2008-06-01

Tissue amyloid plaque immuno-reactive (TAPIR) antibody was better related to the effect of immunotherapy in Alzheimer's disease (AD) than ELISA antibody. Here we used a hybridoma technique to develop a TAPIR-like anti-human amyloid-beta (Abeta) mouse monoclonal antibody. The obtained monoclonal antibody, 3.4A10, was an IgG2b isotype and recognized N-terminal portion of Abeta1-42 without binding denatured or native amyloid-beta protein precursor. It had higher affinity to Abeta1-42 than to Abeta1-40 by Biacore affinity analysis and stained preferably the peripheral part of senile plaques and recognized the plaque core less than 4G8. It inhibited the Abeta1-42 fibril formation as well as degraded pre-aggregated Abeta1-42 peptide in a thioflavin T fluorescence spectrophotometry assay. The in vivo studies showed that 3.4A10 treatment decreased amyloid burden compared to the control group and significantly reduced Abeta42 levels rather than Abeta40 levels in brain lysates as well as the Abeta*56 oligomer (12mer) in TBS fraction of the brain lysates. 3.4A10 entered brain and decorated some plaques, which is surrounded by more Iba1-positive microglia. 3.4A10 therapy did not induce lymphocytic infiltration and obvious increase in microhemorrhage. We conclude that 3.4A10 is a TAPIR-like anti-human amyloid monoclonal antibody, and has a potential of therapeutic application for AD.

Anti-respiratory syncytial virus (RSV) G monoclonal antibodies reduce lung inflammation and viral lung titers when delivered therapeutically in a BALB/c mouse model.

PubMed

Caidi, Hayat; Miao, Congrong; Thornburg, Natalie J; Tripp, Ralph A; Anderson, Larry J; Haynes, Lia M

2018-06-01

RSV continues to be a high priority for vaccine and antiviral drug development. Unfortunately, no safe and effective RSV vaccine is available and treatment options are limited. Over the past decade, several studies have focused on the role of RSV G protein on viral entry, viral neutralization, and RSV-mediated pathology. Anti-G murine monoclonal antibody (mAb) 131-2G treatment has been previously shown to reduce weight loss, bronchoalveolar lavage (BAL) cell number, airway reactivity, and Th2-type cytokine production in RSV-infected mice more rapidly than a commercial humanized monoclonal antibody (mAb) against RSV F protein (Palivizumab). In this study, we have tested two human anti-RSV G mAbs, 2B11 and 3D3, by both prophylactic and therapeutic treatment for RSV in the BALB/c mouse model. Both anti-G mAbs reduced viral load, leukocyte infiltration and IFN-γ and IL-4 expression in cell-free BAL supernatants emphasizing the potential of anti-G mAbs as anti-inflammatory and antiviral strategies. Published by Elsevier B.V.

Isolation of high-affinity, neutralizing anti-idiotype antibodies by phage and ribosome display for application in immunogenicity and pharmacokinetic analyses.

PubMed

Chin, Stacey E; Ferraro, Franco; Groves, Maria; Liang, Meina; Vaughan, Tristan J; Dobson, Claire L

2015-01-01

Anti-idiotype antibodies against a therapeutic antibody are key reagents for the development of immunogenicity and pharmacokinetic (PK) assays during pre-clinical and clinical development. Here we have used a combination of phage and ribosome display to isolate a panel of monoclonal anti-idiotype antibodies with sub-nanomolar affinity and high specificity to a human anti-IgE monoclonal antibody. Anti-idiotype antibodies were enriched from scFv libraries using phage display, and a biochemical epitope competition assay was used to identify anti-idiotypes which neutralized IgE binding, which was essential for the intended use of the anti-idiotypes as positive controls in neutralizing anti-drug antibody (Nab) assays. The phage display-derived anti-idiotype antibodies were rapidly affinity-matured using a random point mutagenesis approach in ribosome display. Ten anti-idiotype antibodies with improved neutralizing activity relative to the parent antibodies displayed sub-nanomolar affinity for the anti-IgE antibody, representing up to 20-fold improvements in affinity from just two rounds of affinity-based selection. The optimized anti-idiotype antibodies retained the specificity of the parent antibodies, and importantly, were fit for purpose for use in PK and anti-drug antibody (ADA) assays. The approach we describe here for generation of anti-idiotype antibodies to an anti-IgE antibody is generically applicable for the rapid isolation and affinity maturation of anti-idiotype antibodies to any antibody-based drug candidate. Copyright © 2014 Elsevier B.V. All rights reserved.

Autoantigen-specific B-cell depletion overcomes failed immune tolerance in type 1 diabetes.

PubMed

Henry, Rachel A; Kendall, Peggy L; Thomas, James W

2012-08-01

Eliminating autoantigen-specific B cells is an attractive alternative to global B-cell depletion for autoimmune disease treatment. To identify the potential for targeting a key autoimmune B-cell specificity in type 1 diabetes, insulin-binding B cells were tracked within a polyclonal repertoire using heavy chain B-cell receptor (BCR) transgenic (VH125Tg) mice. Insulin-specific B cells are rare in the periphery of nonautoimmune VH125Tg/C57BL/6 mice and WT/NOD autoimmune mice, whereas they clearly populate 1% of mature B-cell subsets in VH125Tg/NOD mice. Autoantigen upregulates CD86 in anti-insulin B cells, suggesting they are competent to interact with T cells. Endogenous insulin occupies anti-insulin BCR beginning with antigen commitment in bone marrow parenchyma, as identified by a second anti-insulin monoclonal antibody. Administration of this monoclonal antibody selectively eliminates insulin-reactive B cells in vivo and prevents disease in WT/NOD mice. Unexpectedly, developing B cells are less amenable to depletion, despite increased BCR sensitivity. These findings exemplify how a critical type 1 diabetes B-cell specificity escapes immune tolerance checkpoints. Disease liability is corrected by eliminating this B-cell specificity, providing proof of concept for a novel therapeutic approach for autoimmune disease.

The Role of Anti-Drug Antibodies in the Pharmacokinetics, Disposition, Target Engagement, and Efficacy of a GITR Agonist Monoclonal Antibody in Mice.

PubMed

Brunn, Nicholas D; Mauze, Smita; Gu, Danling; Wiswell, Derek; Ueda, Roanna; Hodges, Douglas; Beebe, Amy M; Zhang, Shuli; Escandón, Enrique

2016-03-01

Administration of biologics to enhance T-cell function is part of a rapidly growing field of cancer immunotherapy demonstrated by the unprecedented clinical success of several immunoregulatory receptor targeting antibodies. While these biologic agents confer significant anti-tumor activity through targeted immune response modulation, they can also elicit broad immune responses potentially including the production of anti-drug antibodies (ADAs). DTA-1, an agonist monoclonal antibody against GITR, is a highly effective anti-tumor treatment in preclinical models. We demonstrate that repeated dosing with murinized DTA-1 (mDTA-1) generates ADAs with corresponding reductions in drug exposure and engagement of GITR on circulating CD3(+) CD4(+) T cells, due to rapid hepatic drug uptake and catabolism. Mice implanted with tumors after induction of preexisting mDTA-1 ADA show no anti-tumor efficacy when given 3 mg/kg mDTA-1, an efficacious dose in naive mice. Nonetheless, increasing mDTA-1 treatment to 30 mg/kg in ADA-positive mice restores mDTA-1 exposure and GITR engagement on circulating CD3(+) CD4(+) T cells, thereby partially restoring anti-tumor efficacy. Formation of anti-mDTA-1 antibodies and changes in drug exposure and disposition does not occur in GITR(-/-) mice, consistent with a role for GITR agonism in humoral immunity. Finally, the administration of muDX400, a murinized monoclonal antibody against the checkpoint inhibitor PD-1, dosed alone or combined with mDTA-1 did not result in reduced muDX400 exposure, nor did it change the nature of the anti-mDTA-1 response. This indicates that anti-GITR immunogenicity may not necessarily impact the pharmacology of coadministered monoclonal antibodies, supporting combination immunomodulatory strategies. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

IgM anti-myeloperoxidase antibody-secreting lymphocytes are present in the peripheral repertoire of lupus mice but rarely differentiate into IgG-producing cells

PubMed Central

Vittecoq, O; Brard, F; Jovelin, F; Le Loet, X; Tron, F; Gilbert, D

1999-01-01

Two IgM, κ anti-myeloperoxidase (MPO) monoclonal antibodies, 6D6 and 9B5, bound to MPO in a solid-phase enzyme-linked immunosorbent assay were derived from the splenocytes of (NZB × NZW) F1 and MRL/lpr-lpr mice, respectively. 6D6 gave a characteristic perinuclear immunofluorescence staining pattern on ethanol-fixed human neutrophils, bound to the native form of MPO by immunoblotting and had a high constant affinity for MPO as demonstrated by real-time specific interaction. 9B5 produced a cytoplasmic immunofluorescence staining pattern, reacted with the heavy chain of MPO and had a low constant affinity for MPO. The heavy-and light-chain variable region genes of monoclonal antibodies (mAb) 6D6 and 9B5 were sequenced and found to be highly homologous to germline genes and to contain negatively charged amino acids in the complementarity determining regions. IgM MPO-binding activity was observed in most BW and MRL/lpr-lpr mouse sera, which may correspond to polyclonal activation of B cells, whereas IgG anti-MPO antibodies could be rarely detected. Thus, this study indicates that (i) BW and MRL/lpr-lpr mice do not delete IgM anti-MPO secreting B cells, do not maintain these B cells in a state of anergy, but most individuals are not able to spontaneously induce the class-switching of this autoantibody population; (ii) IgM anti-MPO antibodies can recognize different epitopes on MPO and produce different immunofluorescence staining pattern on ethanol-fixed human neutrophils, as demonstrated by the immunochemical properties of the two lupus-mouse derived mAb. PMID:10540169

B cell-targeted therapy with anti-CD20 monoclonal antibody in a mouse model of Graves' hyperthyroidism

PubMed Central

Ueki, I; Abiru, N; Kobayashi, M; Nakahara, M; Ichikawa, T; Eguchi, K; Nagayama, Y

2011-01-01

Graves' disease is a B cell-mediated and T cell-dependent autoimmune disease of the thyroid which is characterized by overproduction of thyroid hormones and thyroid enlargement by agonistic anti-thyrotrophin receptor (TSHR) autoantibody. In addition to antibody secretion, B cells have recently been recognized to function as antigen-presenting/immune-modulatory cells. The present study was designed to evaluate the efficacy of B cell depletion by anti-mouse (m) CD20 monoclonal antibody (mAb) on Graves' hyperthyroidism in a mouse model involving repeated injection of adenovirus expressing TSHR A-subunit (Ad-TSHR289). We observe that a single injection of 250 µg/mouse anti-mCD20 mAb eliminated B cells efficiently from the periphery and spleen and to a lesser extent from the peritoneum for more than 3 weeks. B cell depletion before immunization suppressed an increase in serum immunoglobulin (Ig)G levels, TSHR-specific splenocyte secretion of interferon (IFN)-γ, anti-TSHR antibody production and development of hyperthyroidism. B cell depletion 2 weeks after the first immunization, a time-point at which T cells were primed but antibody production was not observed, was still effective at inhibiting antibody production and disease development without inhibiting splenocyte secretion of IFN-γ. By contrast, B cell depletion in hyperthyroid mice was therapeutically ineffective. Together, these data demonstrate that B cells are critical not only as antibody-producing cells but also as antigen-presenting/immune-modulatory cells in the early phase of the induction of experimental Graves' hyperthyroidism and, although therapeutically less effective, B cell depletion is highly efficient for preventing disease development. PMID:21235532

B cell-targeted therapy with anti-CD20 monoclonal antibody in a mouse model of Graves' hyperthyroidism.

PubMed

Ueki, I; Abiru, N; Kobayashi, M; Nakahara, M; Ichikawa, T; Eguchi, K; Nagayama, Y

2011-03-01

Graves' disease is a B cell-mediated and T cell-dependent autoimmune disease of the thyroid which is characterized by overproduction of thyroid hormones and thyroid enlargement by agonistic anti-thyrotrophin receptor (TSHR) autoantibody. In addition to antibody secretion, B cells have recently been recognized to function as antigen-presenting/immune-modulatory cells. The present study was designed to evaluate the efficacy of B cell depletion by anti-mouse (m) CD20 monoclonal antibody (mAb) on Graves' hyperthyroidism in a mouse model involving repeated injection of adenovirus expressing TSHR A-subunit (Ad-TSHR289). We observe that a single injection of 250 µg/mouse anti-mCD20 mAb eliminated B cells efficiently from the periphery and spleen and to a lesser extent from the peritoneum for more than 3 weeks. B cell depletion before immunization suppressed an increase in serum immunoglobulin (Ig)G levels, TSHR-specific splenocyte secretion of interferon (IFN)-γ, anti-TSHR antibody production and development of hyperthyroidism. B cell depletion 2 weeks after the first immunization, a time-point at which T cells were primed but antibody production was not observed, was still effective at inhibiting antibody production and disease development without inhibiting splenocyte secretion of IFN-γ. By contrast, B cell depletion in hyperthyroid mice was therapeutically ineffective. Together, these data demonstrate that B cells are critical not only as antibody-producing cells but also as antigen-presenting/immune-modulatory cells in the early phase of the induction of experimental Graves' hyperthyroidism and, although therapeutically less effective, B cell depletion is highly efficient for preventing disease development. © 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.

Generation and testing anti-influenza human monoclonal antibodies in a new humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO. IL-2Rγc KO. NOD).

PubMed

Mendoza, Mirian; Ballesteros, Angela; Qiu, Qi; Pow Sang, Luis; Shashikumar, Soumya; Casares, Sofia; Brumeanu, Teodor-D

2018-02-01

Pandemic outbreaks of influenza type A viruses have resulted in numerous fatalities around the globe. Since the conventional influenza vaccines (CIV) provide less than 20% protection for individuals with weak immune system, it has been considered that broadly cross-neutralizing antibodies may provide a better protection. Herein, we showed that a recently generated humanized mouse (DRAGA mouse; HLA-A2. HLA-DR4. Rag1KO. IL-2Rgc KO. NOD) that lacks the murine immune system and expresses a functional human immune system can be used to generate cross-reactive, human anti-influenza monoclonal antibodies (hu-mAb). DRAGA mouse was also found to be suitable for influenza virus infection, as it can clear a sub-lethal infection and sustain a lethal infection with PR8/A/34 influenza virus. The hu-mAbs were designed for targeting a human B-cell epitope ( 180 WGIHHPPNSKEQ QNLY 195 ) of hemagglutinin (HA) envelope protein of PR8/A/34 (H1N1) virus with high homology among seven influenza type A viruses. A single administration of HA 180-195 specific hu-mAb in PR8-infected DRAGA mice significantly delayed the lethality by reducing the lung damage. The results demonstrated that DRAGA mouse is a suitable tool to (i) generate heterotype cross-reactive, anti-influenza human monoclonal antibodies, (ii) serve as a humanized mouse model for influenza infection, and (iii) assess the efficacy of anti-influenza antibody-based therapeutics for human use.

The role of sulfoglucuronosyl glycosphingolipids in the pathogenesis of monoclonal IgM paraproteinemia and peripheral neuropathy

PubMed Central

ARIGA, Toshio

2011-01-01

In IgM paraproteinemia and peripheral neuropathy, IgM M-protein secretion by B cells leads to a T helper cell response, suggesting that it is antibody-mediated autoimmune disease involving carbohydrate epitopes in myelin sheaths. An immune response against sulfoglucuronosyl glycosphingolipids (SGGLs) is presumed to participate in demyelination or axonal degeneration in the peripheral nervous system (PNS). SGGLs contain a 3-sulfoglucuronic acid residue that interacts with anti-myelin-associated glycoprotein (MAG) and the monoclonal antibody anti-HNK-1. Immunization of animals with sulfoglucuronosyl paragloboside (SGPG) induced anti-SGPG antibodies and sensory neuropathy, which closely resembles the human disease. These animal models might help to understand the disease mechanism and lead to more specific therapeutic strategies. In an in vitro study, destruction or malfunction of the blood-nerve barrier (BNB) was found, resulting in the leakage of circulating antibodies into the PNS parenchyma, which may be considered as the initial key step for development of disease. PMID:21785257

Blockade of epidermal growth factor- or heregulin-dependent ErbB2 activation with the anti-ErbB2 monoclonal antibody 2C4 has divergent downstream signaling and growth effects.

PubMed

Jackson, James G; St Clair, Patricia; Sliwkowski, Mark X; Brattain, Michael G

2004-04-01

Due to heterodimerization and a variety of stimulating ligands, the ErbB receptor system is both diverse and flexible, which proves particularly advantageous to the aberrant signaling of cancer cells. However, specific mechanisms of how a particular receptor contributes to generating the flexibility that leads to aberrant growth regulation have not been well described. We compared the utilization of ErbB2 in response to epidermal growth factor (EGF) and heregulin stimulation in colon carcinoma cells. Anti-ErbB2 monoclonal antibody 2C4 blocked heregulin-stimulated phosphorylation of ErbB2 and ErbB3; activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3'-kinase (PI3K), and Akt; proliferation; and anchorage-independent growth. 2C4 blocked EGF-mediated phosphorylation of ErbB2 and inhibited PI3K/Akt and anchorage-independent growth but did not affect ErbB1 or MAPK. Immunoprecipitations showed that ErbB3 and Grb2-associated binder (Gab) 1 were phosphorylated and associated with PI3K activity after heregulin treatment and that Gab1 and Gab2, but not ErbB3, were phosphorylated and associated with PI3K activity after EGF treatment. These data show that monoclonal antibody 2C4 inhibited all aspects of heregulin signaling as well as anchorage-independent and monolayer growth. Furthermore, we identify ErbB2 as a critical component of EGF signaling to the Gab1/Gab2-PI3K-Akt pathway and anchorage-independent growth, but EGF stimulation of MAPK and monolayer growth can occur efficiently without the contribution of ErbB2.

The combination of anti-KIR monoclonal antibodies with anti-PD-1/PD-L1 monoclonal antibodies could be a critical breakthrough in overcoming tumor immune escape in NSCLC.

PubMed

He, Yayi; Liu, Sangtian; Mattei, Jane; Bunn, Paul A; Zhou, Caicun; Chan, Daniel

2018-01-01

The anti-programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) monoclonal antibody has a good effect in the treatment of non-small cell lung cancer (NSCLC), but not all PD-1/PD-L1 positive patients can get benefit from it. Compensatory expression of other immune checkpoints may be correlated with the poor efficacy of anti-PD-1/PD-L1 monoclonal antibodies. The inhibitory human leukocyte antigen (HLA)/killer cell Ig-like receptor (KIR) can effectively block the killing effect of natural killer (NK) cells on tumors. Our previous studies have confirmed that high expression of KIR was correlated with poor prognosis of NSCLC. Inhibitory KIR expression was positively correlated with the expression of PD-1. The expressions of KIR 2D (L1, L3, L4, S4) (BC032422/ADQ31987/NP_002246/NP_036446, Abcam) and PD-1 (NAT 105, Cell marque) proteins was assessed by immunohistochemistry. The expression of inhibitory KIR in tumor cells or tumor infiltrating lymphocytes (TILs) is associated with PD-1 expression. Among PD-1 positive patients, 76.3% were KIR 2D (L1, L3, L4, S4) positive on tumor cells, and 74.6% were KIR 2D (L1, L3, L4, S4) positive on TILs. We compared the expression of inhibitory KIR before and after treatment with nivolumab in 11 patients with NSCLC. We found that five (45.5%) patients had positive expression of inhibitory KIR in tumor tissue after being treated with anti-PD-1 monoclonal antibodies, two of whom exhibited a significant increase in expression of inhibitory KIR, and three showed no change. PD-1 expression was correlated with KIR 2D (L1, L3, L4, S4) on tumor cells or TILs. The resistance to anti-PD-1 monoclonal antibody treatment might be related to KIR. The inhibitory HLA/KIR could combine with the PD-1/PD-L1 signaling pathway negatively regulating NSCLC tumor immunity.

Fc receptors for mouse IgG1 on human monocytes: polymorphism and role in antibody-induced T cell proliferation.

PubMed

Tax, W J; Hermes, F F; Willems, R W; Capel, P J; Koene, R A

1984-09-01

In previous studies, it was shown that there is polymorphism in the mitogenic effect of mouse IgG1 monoclonal antibodies against the T3 antigen of human T cells. This polymorphism implies that IgG1 anti-T3 antibodies are not mitogenic for T cells from 30% of healthy individuals. The present results demonstrate that this polymorphism is caused by polymorphism of an Fc receptor for mouse IgG1, present on human monocytes. The Fc receptor for murine IgG1 could be detected by a newly developed rosetting assay on monocytes from all individuals responsive to the mitogenic effect of IgG1 anti-T3 antibodies. This Fc receptor was not detectable on monocytes from those individuals exhibiting no mitogenic responses to IgG1 anti-T3 monoclonal antibodies. Cross-linking of T3 antigens appears to be essential for antibody-induced mitosis of T cells, because mononuclear cells that did not proliferate in response to WT 31 (an IgG1 antibody against T3 antigen) showed a proliferative response to Sepharose beads coated with WT 31. The Fc receptor--if functionally present--may be involved in the cross-linking of T3 antigens through anti-T3 antibodies. Further evidence for the involvement of this Fc receptor in antibody-induced T cell proliferation was provided by inhibition studies. Immune complexes containing IgG1 antibodies were able to inhibit the proliferative response to IgG1 anti-T3 antibodies. This inhibition by immune complexes appears to be mediated through the monocyte Fc receptor for mouse IgG1. These findings are important for the interpretation of previously described inhibitory effects of anti-T cell monoclonal antibodies on T cell proliferation, and show that such inhibitory effects may be monocyte-mediated (via immune complexes) rather than caused by a direct involvement of the respective T cell antigens in T cell mitosis. The Fc receptor for mouse IgG1 plays a role in antibody-induced T cell proliferation. Its polymorphism may have important implications for the therapeutic use of IgG1 monoclonal antibodies.

HIV-1 Envelope gp41 Antibodies Can Originate from Terminal Ileum B Cells that Share Cross-Reactivity with Commensal Bacteria

PubMed Central

Trama, Ashley M.; Moody, M. Anthony; Alam, S. Munir; Jaeger, Frederick H.; Lockwood, Bradley; Parks, Robert; Lloyd, Krissey E.; Stolarchuk, Christina; Scearce, Richard; Foulger, Andrew; Marshall, Dawn J.; Whitesides, John F.; Jeffries, Thomas L.; Wiehe, Kevin; Morris, Lynn; Lambson, Bronwen; Soderberg, Kelly; Hwang, Kwan-Ki; Tomaras, Georgia D.; Vandergrift, Nathan; Jackson, Katherine J.L.; Roskin, Krishna M.; Boyd, Scott D.; Kepler, Thomas B.; Liao, Hua-Xin; Haynes, Barton F.

2014-01-01

SUMMARY Monoclonal antibodies derived from blood plasma cells of acute HIV-1-infected individuals are predominantly targeted to the HIV Env gp41 and cross-reactive with commensal bacteria. To understand this phenomenon, we examined anti-HIV responses in ileum B cells using recombinant antibody technology and probed their relationship to commensal bacteria. The dominant ileum B cell response was to Env gp41. Remarkably, a majority (82%) of the ileum anti-gp41 antibodies cross-reacted with commensal bacteria, and of those, 43% showed non-HIV-1 antigen polyreactivity. Pyrosequencing revealed shared HIV-1 antibody clonal lineages between ileum and blood. Mutated immunoglobulin G antibodies cross-reactive with both Env gp41 and microbiota could also be isolated from the ileum of HIV-1 uninfected individuals. Thus, the gp41 commensal bacterial antigen cross-reactive antibodies originate in the intestine, and the gp41 Env response in HIV-1 infection can be derived from a preinfection memory B cell pool triggered by commensal bacteria that cross-react with Env. PMID:25121750

Neuropsychiatric disturbances in SLE are associated with antibodies against NMDA receptors.

PubMed

Omdal, R; Brokstad, K; Waterloo, K; Koldingsnes, W; Jonsson, R; Mellgren, S I

2005-05-01

To determine whether neuropsychiatric manifestations in patients with systemic lupus erythematosus (SLE) are influenced by antibodies against the human N-methyl-D-aspartate (NMDA) receptor types NR2a or NR2b. A decapeptide was synthesized containing a sequence motif present in the extracellular ligand-binding domain of NMDA receptors NR2a and NR2b, bound by the monoclonal murine anti-DNA antibody R4A. In an ELISA with the murine monoclonal R4v as positive control, plasma samples of 57 patients with SLE were examined for the anti-peptide (anti-NR2) antibody after the patients had been subjected to comprehensive psychological and cognitive testing. Poor performance on the Visual Paired Associates test (immediate), the Grooved Pegboard test, as well as high scores on the Beck Depression Inventory, and scales D-2 (depression), Pd-4 (psychopathic deviate), Sc-8 (schizophrenia), and Ma-9 (hypomania) of the MMPI-2 were significantly associated with elevated levels of anti-NR2 antibodies. The findings in several domains indicate an association between anti-NR2 antibodies and depressed mood in addition to decreased short-time memory and learning. Antibodies to NMDA receptors thus may represent one of several mechanisms for cerebral dysfunction in patients with SLE.

An immunogen synthesis strategy for the development of specific anti-deoxynivalenol monoclonal antibodies.

PubMed

Sanders, Melanie; Guo, Yirong; Iyer, Abhishek; García, Yara Ruiz; Galvita, Anastasia; Heyerick, Arne; Deforce, Dieter; Risseeuw, Martijn D P; Van Calenbergh, Serge; Bracke, Marc; Eremin, Sergei; Madder, Annemieke; De Saeger, Sarah

2014-01-01

An immunogen synthesis strategy was designed to develop anti-deoxynivalenol (DON) monoclonal antibodies with low cross-reactivity against structurally similar trichothecenes. A total of eight different DON immunogens were synthesised, differing in the type and position of the linker on the DON molecule. After immunisation, antisera from mice immunised with different DON immunogens were checked for the presence of relevant antibodies. Then, both homologous and heterologous enzyme-linked immunosorbent assays (ELISAs) were performed for hybridoma screening. Finally, three monoclonal antibodies against DON and its analogues were generated. In addition, monoclonal antibody 13H1 could recognise DON and its analogues in the order of HT-2 toxin > 15-acetyldeoxynivalenol (15-ADON) > DON, with IC₅₀ ranging from 1.14 to 2.13 µg ml⁻¹. Another monoclonal antibody 10H10 manifested relatively close sensitivities to DON, 3-acetyldeoxynivalenol (3-ADON) and 15-ADON, with IC₅₀ values of 22, 15 and 34 ng ml⁻¹, respectively. Using an indirect ELISA format decreases the 10H10 sensitivity to 15-ADON with 92%. A third monoclonal antibody 2A9 showed to be very specific and sensitive to 3-ADON, with IC₅₀ of 0.38 ng ml⁻¹. Using both 2A9 and 10H10 monoclonal antibodies allows determining sole DON contamination.

Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to Detect CD19-Specific T Cells in Clinical Trials

PubMed Central

Jena, Bipulendu; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Lee, Dean A.; Champlin, Richard E.; Cooper, Laurence J. N.

2013-01-01

Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19+ tumor targets. This clone can be used to detect CD19-specific CAR+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy. PMID:23469246

Fusion antibody for Alzheimer's disease with bidirectional transport across the blood-brain barrier and abeta fibril disaggregation.

PubMed

Boado, Ruben J; Zhang, Yufeng; Zhang, Yun; Xia, Chun-Fang; Pardridge, William M

2007-01-01

Delivery of monoclonal antibody therapeutics across the blood-brain barrier is an obstacle to the diagnosis or therapy of CNS disease with antibody drugs. The immune therapy of Alzheimer's disease attempts to disaggregate the amyloid plaque of Alzheimer's disease with an anti-Abeta monoclonal antibody. The present work is based on a three-step model of immune therapy of Alzheimer's disease: (1) influx of the anti-Abeta monoclonal antibody across the blood-brain barrier in the blood to brain direction, (2) binding and disaggregation of Abeta fibrils in brain, and (3) efflux of the anti-Abeta monoclonal antibody across the blood-brain barrier in the brain to blood direction. This is accomplished with the genetic engineering of a trifunctional fusion antibody that binds (1) the human insulin receptor, which mediates the influx from blood to brain across the blood-brain barrier, (2) the Abeta fibril to disaggregate amyloid plaque, and (3) the Fc receptor, which mediates the efflux from brain to blood across the blood-brain barrier. This fusion protein is a new antibody-based therapeutic for Alzheimer's disease that is specifically engineered to cross the human blood-brain barrier in both directions.

Rituximab for the treatment of refractory simultaneous anti-glomerular basement membrane (anti-GBM) and membranous nephropathy.

PubMed

Bandak, Ghassan; Jones, Bruce A; Li, Jian; Yee, Jerry; Umanath, Kausik

2014-02-01

Antibody-mediated anti-glomerular basement membrane (anti-GBM) disease occurs rarely in the presence of another B-cell disorder, membranous nephropathy. The coexistence of these two autoimmune disorders would be anticipated to require differing, specific therapies targeted to each disease process. We describe a case of concomitant membranous nephropathy and anti-GBM disease in which conventional therapy, including steroids, plasmapheresis and cyclophosphamide, failed to attenuate the anti-GBM disease, yet responded to an alternative treatment of rituximab. This B-cell directed, monoclonal, chimeric antibody treatment substantially reduced anti-GBM antibody titers and led to discontinuation of plasmapheresis, while maintaining the remission of membranous nephropathy and anti-GBM disease.

Acute neurological worsening after Rituximab treatment in patients with anti-MAG neuropathy.

PubMed

Sala, Emilie; Robert-Varvat, Florence; Paul, Stéphane; Camdessanché, Jean-Philippe; Antoine, Jean-Christophe

2014-10-15

Patients with peripheral neuropathy and anti-MAG monoclonal IgM may respond to Rituximab, a humanized monoclonal anti-CD20 antibody. We report on three patients with peripheral neuropathy and anti-MAG monoclonal IgM who deteriorated under Rituximab and reviewed seven previously published cases. Worsening was acute and severe, and occurred during the treatment period. All the patients improved after deterioration but at final evaluation only one was improved comparatively to baseline, five were worsened and four were stabilized. Deterioration was not clearly associated with an increase of the anti-MAG antibody titer. Two patients received Rituximab prior or after the course which induced worsening without adverse reaction. Although rare, acute worsening of the neuropathy can occur after Rituximab. The deterioration is however reversible within some weeks to several months. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel mAb against a human CD34 peptide reacts with the native protein on CD34+ cells.

PubMed

Shams, Mahmood; Jeddi-Tehrani, Mahmood; Notash Haghighat, Farzaneh; Bayat, Ali Ahmad; Mahmoudian, Jafar; Rezvani, Mohammad Reza

2013-12-01

‎Human CD34 is a transmembrane glycoprotein which is expressed in human hematopoietic stem ‎cells (HSCs) and the small-‎vessel endothelial cells of a variety of tissues. CD34 plays a critical role as a ‎marker for diagnosis ‎and classification of leukemia. Anti CD34 antibodies are used for isolation and ‎purification ‎of HSCs from bone marrow, peripheral blood and cord blood. To characterize a newly produced monoclonal antibody against a human CD34 peptide. Anti CD34 monoclonal antibody (Clone 2C10-D3) was purified from mouse ascitic fluid and hybridoma cell culture supernatants by affinity chromatography and its immune reactivity was examined by ELISA. The purified antibody was further characterized using Western blot and flow cytometry on TF1 (Human Erythroblast) cell line. ‎ELISA experiment revealed that the antibody recognized CD34 peptide. Western ‎blot analysis on TF1 ‎cell lysate confirmed the reactivity of the antibody with a 42 KDa protein. Blocking the antibody with a saturating concentration of specific CD34 peptide resulted in loss of its activity with TF1 lysate in Western blot. The 2C10-D3 antibody reacted with TF1 cells in flow cytometry in a similar manner to a commercial anti CD34 monoclonal antibody.‎ ‎Our data suggest that the anti CD34 monoclonal antibody (Clone 2C10-D3) is an appropriate antibody to study the CD34+ cells by flow cytometry and Western blot.

Mechanisms of Action of Therapeutic Antibodies for Cancer

PubMed Central

Redman, JM; Hill, EM; AlDeghaither, D; Weiner, LM

2015-01-01

The therapeutic utility of antibodies and their derivatives is achieved by various means. The FDA has approved several targeted antibodies that disrupt signaling of various growth factor receptors for the treatment of a number of cancers. Rituximab, and other anti-CD20 monoclonal antibodies are active in B cell malignancies. As more experience has been gained with anti-CD20 monoclonal antibodies, the multifactorial nature of their anti-tumor mechanisms has emerged. Other targeted antibodies function to dampen inhibitory checkpoints. These checkpoint inhibitors have recently achieved dramatic results in several cancers, including melanoma. These and related antibodies continue to be investigated in the clinical and pre-clinical settings. Novel antibody structures that target two or more antigens have also made their way into clinical use. Tumor targeted antibodies can also be conjugated to chemo- or radiotherapeutic agents, or catalytic toxins, as a means to deliver toxic payloads to cancer cells. Here we provide a review of these mechanisms and a discussion of their relevance to current and future clinical applications. PMID:25911943

Mass-Production and Characterization of Anti-CD20 Monoclonal Antibody in Peritoneum of Balb/c Mice

PubMed Central

Sineh sepehr, Koushan; Baradaran, Behzad; Majidi, Jafar; Abdolalizadeh, Jalal; Aghebati, leili; Zare Shahneh, Fatemeh

2013-01-01

Purpose: Monoclonal antibodies are important tools are used in basic research as well as, in diagnosis, imaging and treatment of immunodeficiency diseases, infections and cancers. The purpose of this study was to produce large scale of monoclonal antibody against CD20 in order to diagnostic application in leukemia and lymphomas disorders. Methods: Hybridoma cells that produce monoclonal antibody against human CD20 were administered into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After twelve days, approximately 7 ml ascetic fluid was harvested from the peritoneum of each mouse. Evaluation of mAb titration was assessed by ELISA method. In the present study, we describe a protocol for large scale production of MAbs. Results: We prepared monoclonal antibodies (mAbs) with high specificity and sensitivity against human CD20 by hybridoma method and characterized them by ELISA. The subclass of antibody was IgG2a and its light chain was kappa. Ascetic fluid was purified by Protein-A Sepharose affinity chromatography and the purified monoclonal antibody was conjugated with FITC and Immunofluorescence was done for confirming the specific binding. Conclusion: The conjugated monoclonal antibody could have application in diagnosis B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma cancer stem cells. PMID:24312821

Expression of Surface Protein LapB by a Wide Spectrum of Listeria monocytogenes Serotypes as Demonstrated with Anti-LapB Monoclonal Antibodies

PubMed Central

Boivin, Teela; Elmgren, Cathie; Brooks, Brian W.; Huang, Hongsheng; Pagotto, Franco

2016-01-01

ABSTRACT Protein antigens expressed on the surface of all strains of Listeria monocytogenes and absent from nonpathogenic Listeria spp. are presumably useful targets for pathogen identification, detection, and isolation using specific antibodies (Abs). To seek such surface proteins expressed in various strains of L. monocytogenes for diagnostic applications, we focused on a set of surface proteins known to be involved or putatively involved in L. monocytogenes virulence and identified Listeria adhesion protein B (LapB) as a candidate based on the bioinformatics analysis of whole-genome sequences showing that the gene coding for LapB was present in L. monocytogenes strains and absent from strains of other Listeria spp. Immunofluorescence microscopy (IFM), performed with rabbit polyclonal antibodies against the recombinant LapB protein (rLapB) of L. monocytogenes serotype 4b strain L10521, confirmed expression of LapB on the surface. A panel of 48 mouse monoclonal antibodies (MAbs) to rLaB was generated, and 7 of them bound strongly to the surface of L. monocytogenes cells as demonstrated using IFM. Further characterization of these 7 anti-LapB MAbs, using an enzyme-linked immunosorbent assay (ELISA), revealed that 6 anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) reacted strongly with 46 (86.8%) of 53 strains representing 10 of the 12 serotypes tested (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4ab, 4b, 4d, and 4e). These results indicate that LapB, together with companion anti-LapB MAbs, can be targeted as a biomarker for the detection and isolation of various L. monocytogenes strains from contaminated foods. IMPORTANCE Strains of L. monocytogenes are traditionally grouped into serotypes. Identification of a surface protein expressed in all or the majority of at least 12 serotypes would aid in the development of surface-binding monoclonal antibodies (MAbs) for detection and isolation of L. monocytogenes from foods. Bioinformatics analysis revealed that the gene coding for Listeria adhesion protein B (LapB), a surface protein involved in L. monocytogenes virulence, was present in L. monocytogenes strains and absent from other Listeria spp. Polyclonal antibodies against recombinant LapB (rLapB) detected the exposed epitopes on the surface of L. monocytogenes. Production and extensive assessment of 48 MAbs to rLapB showed that 6 anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) detected the expression of LapB in a wide range of L. monocytogenes isolates representing 10 of 12 serotypes tested, suggesting that LapB, together with specific MAbs, can be targeted as a biomarker for pathogen detection and isolation. PMID:27613687

Expression of Surface Protein LapB by a Wide Spectrum of Listeria monocytogenes Serotypes as Demonstrated with Anti-LapB Monoclonal Antibodies.

PubMed

Boivin, Teela; Elmgren, Cathie; Brooks, Brian W; Huang, Hongsheng; Pagotto, Franco; Lin, Min

2016-11-15

Protein antigens expressed on the surface of all strains of Listeria monocytogenes and absent from nonpathogenic Listeria spp. are presumably useful targets for pathogen identification, detection, and isolation using specific antibodies (Abs). To seek such surface proteins expressed in various strains of L. monocytogenes for diagnostic applications, we focused on a set of surface proteins known to be involved or putatively involved in L. monocytogenes virulence and identified Listeria adhesion protein B (LapB) as a candidate based on the bioinformatics analysis of whole-genome sequences showing that the gene coding for LapB was present in L. monocytogenes strains and absent from strains of other Listeria spp. Immunofluorescence microscopy (IFM), performed with rabbit polyclonal antibodies against the recombinant LapB protein (rLapB) of L. monocytogenes serotype 4b strain L10521, confirmed expression of LapB on the surface. A panel of 48 mouse monoclonal antibodies (MAbs) to rLaB was generated, and 7 of them bound strongly to the surface of L. monocytogenes cells as demonstrated using IFM. Further characterization of these 7 anti-LapB MAbs, using an enzyme-linked immunosorbent assay (ELISA), revealed that 6 anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) reacted strongly with 46 (86.8%) of 53 strains representing 10 of the 12 serotypes tested (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4ab, 4b, 4d, and 4e). These results indicate that LapB, together with companion anti-LapB MAbs, can be targeted as a biomarker for the detection and isolation of various L. monocytogenes strains from contaminated foods. Strains of L. monocytogenes are traditionally grouped into serotypes. Identification of a surface protein expressed in all or the majority of at least 12 serotypes would aid in the development of surface-binding monoclonal antibodies (MAbs) for detection and isolation of L. monocytogenes from foods. Bioinformatics analysis revealed that the gene coding for Listeria adhesion protein B (LapB), a surface protein involved in L. monocytogenes virulence, was present in L. monocytogenes strains and absent from other Listeria spp. Polyclonal antibodies against recombinant LapB (rLapB) detected the exposed epitopes on the surface of L. monocytogenes Production and extensive assessment of 48 MAbs to rLapB showed that 6 anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) detected the expression of LapB in a wide range of L. monocytogenes isolates representing 10 of 12 serotypes tested, suggesting that LapB, together with specific MAbs, can be targeted as a biomarker for pathogen detection and isolation. © Crown copyright 2016.

Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okazaki, Shogo; Nakatani, Fumi; Masuko, Kazue

2016-01-29

The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-basedmore » cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix. - Highlights: • We newly generated four clones of human ErbB4 specific mAb. • ErbB4 mAb clone P6-1 blocks ErbB4 phosphorylation induced by NRG-1. • ErbB4 mAb clone P6-1 suppresses NRG-1-promoted breast cancer cells proliferation on three dimensional culture condition.« less

Development of a novel monoclonal antibody to human inducible co-stimulator ligand (ICOSL): Biological characteristics and application for enzyme-linked immunosorbent assay.

PubMed

Hu, Xiaohan; Wu, Jian; An, Jingnan; Hu, Yumin; Shen, Yu; Liu, Cuiping; Zhang, Xueguang

2016-07-01

ICOSL (B7-H2, CD275), a co-stimulatory molecule of the B7 superfamily, functions as a positive signal in immune response. To investigate whether ICOSL could be released into sera and the possible biological function of soluble ICOS (sICOSL), we generated and characterized a functional anti-human ICOSL monoclonal antibody (mAb), 20B10, and developed a novel enzyme-linked immunosorbent assay (ELISA) based on two anti-human ICOSL antibodies with different epitope specificities. Using the ELISA system, we found that sICOSL in the serum of healthy donors increases in an age-dependent manner and that the matrix metalloproteinase inhibitor (MMPI) could suppress sICOSL production. Together, these data demonstrate that the existence of circulating sICOSL in human serum might play an important role in immunoregulation. Copyright © 2016. Published by Elsevier B.V.

Functional Activity of Antibodies Directed towards Flagellin Proteins of Non-Typhoidal Salmonella

PubMed Central

Boyd, Mary A.; Wang, Jin Y.; Tulapurkar, Mohan E.; Pasetti, Marcela F.; Levine, Myron M.; Simon, Raphael

2016-01-01

Non-typhoidal Salmonella (NTS) serovars Typhimurium and Enteritidis are major causes of invasive bacterial infections in children under 5 years old in sub-Saharan Africa, with case fatality rates of ~20%. There are no licensed NTS vaccines for humans. Vaccines that induce antibodies against a Salmonella Typhi surface antigen, Vi polysaccharide, significantly protect humans against typhoid fever, establishing that immune responses to Salmonella surface antigens can be protective. Flagella proteins, abundant surface antigens in Salmonella serovars that cause human disease, are also powerful immunogens, but the functional capacity of elicited anti-flagellar antibodies and their role in facilitating bacterial clearance has been unclear. We examined the ability of anti-flagellar antibodies to mediate microbial killing by immune system components in-vitro and assessed their role in protecting mice against invasive Salmonella infection. Polyclonal (hyperimmune sera) and monoclonal antibodies raised against phase 1 flagellin proteins of S. Enteritidis and S. Typhimurium facilitated bacterial uptake and killing of the homologous serovar pathogen by phagocytes. Polyclonal anti-flagellar antibodies accompanied by complement also achieved direct bacterial killing. Serum bactericidal activity was restricted to Salmonella serovars expressing the same flagellin used as immunogen. Notably, individual anti-flagellin monoclonal antibodies with complement were not bactericidal, but this biological activity was restored when different monoclonal anti-flagellin antibodies were combined. Passive transfer immunization with a monoclonal IgG antibody specific for phase 1 flagellin from S. Typhimurium protected mice against lethal challenge with a representative African invasive S. Typhimurium strain. These findings have relevance for the use of flagellin proteins in NTS vaccines, and confirm the role of anti-flagellin antibodies as mediators of protective immunity. PMID:26998925

Development of monoclonal antibodies against axenic amastigotes of Leishmania infantum strain in Iran: implication for diagnosis of Kala-azar

PubMed Central

Nourizadeh, Ezzat; Zargar, Seyed Jalal; Alimohammadian, Mohammad Hossein; Ajdary, Soheila; Mahdavi, Mahdi

2018-01-01

Objective(s): Leishmaniasis is endemic in 88 countries. Amastigote forms of Leishmania are experts at exploiting host cell processes to establish infection. Monoclonal antibodies are key reagents used in the diagnosis of infectious and non-infectious diseases. The aim of this study was to produce monoclonal antibodies against axenic amastigotes of the Leishmania infantum strain in Iran. Materials and Methods: First, standard strains were cultured and axenic amastigote antigens of L. infantum were obtained. Since then, BALB/c smice were immunized and antibody titers were determined. For hybridoma cell formation, lymphocytes isolated from spleen of immunized mice and myeloma cells were fused at a ratio of 10 to 1 in the presence of polyethylene glycol, followed by limiting dilution for the isolation of monoclones. Subsequently, antibody isotypes were determined by using the isotyping kit. The best clone was injected intraperitoneally to pristane-primed mice for large scale production of monoclonal antibodies. The specificity of antibody was determined with Western blotting. Results: Approximately 25 positive monoclones were obtained, of which four hybrids producing anti-amastigotes L. infantum monoclonal antibodies with high optical density (OD), selected and designated as 8D2 FVI6, 8D2 FVI3, 6G2 FV4 and 6G2 FV3. Results from isotype determination showed the IgG2b sub-class in 6G2FV2 and 8D2FVI6 monoclones. Conclusion: This study produced monoclonal antibody against amastigotes of Iranian strain of L. infantum for the first time. These antibodies have reactivity against Iranian strain of L. infantum and can be used in the diagnosis of Kala-azar.

Anti-CD22 and anti-CD79b antibody-drug conjugates preferentially target proliferating B cells.

PubMed

Fuh, Franklin K; Looney, Caroline; Li, Dongwei; Poon, Kirsten A; Dere, Randall C; Danilenko, Dimitry M; McBride, Jacqueline; Reed, Chae; Chung, Shan; Zheng, Bing; Mathews, William Rodney; Polson, Andrew; Prabhu, Saileta; Williams, Marna

2017-04-01

CD22 and CD79b are cell-surface receptors expressed on B-cell-derived malignancies such as non-Hodgkin's lymphoma (NHL). An anti-mitotic agent, monomethyl auristatin E, was conjugated to anti-CD22 and anti-CD79b antibodies to develop target-specific therapies for NHL. The mechanism of action (MOA) and pharmacological and pharmacokinetic (PK) profiles of these antibody-drug conjugates (ADCs) were investigated in cynomolgus monkeys. Animals were administered anti-CD22 or anti-CD79b ADCs, respective unconjugated antibodies or vehicle. Pharmacodynamic effects on total and proliferating B cells and serum PK were then assessed. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of the ADCs were evaluated in vitro. Depletion of B cells was observed after administration of either ADC or the respective unconjugated antibodies. An extended duration of depletion was observed in animals administered ADCs. Similarly, preferential depletion of proliferating B cells in blood and germinal centre B cells in spleen were only observed in animals administered ADCs. Serum PK profiles of ADCs and respective unconjugated antibodies were comparable. In vitro, anti-human CD22 and anti-human CD79b antibodies showed no or only moderate ADCC activity, respectively; neither antibody had CDC activity. The findings support the proposed MOA: initial depletion of total B cells by antibody-mediated opsonization, followed by preferential, sustained depletion of proliferating B cells by the auristatin conjugate due to its anti-mitotic action. Delivering potent anti-mitotic agents to B cells via the specificity of monoclonal antibodies provides a means to eliminate pathogenic B cells in NHL with improved risk-benefit profiles over traditional chemotherapeutics. © 2016 Genentech. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.

Antigenic modulation limits the efficacy of anti-CD20 antibodies: implications for antibody selection.

PubMed

Beers, Stephen A; French, Ruth R; Chan, H T Claude; Lim, Sean H; Jarrett, Timothy C; Vidal, Regina Mora; Wijayaweera, Sahan S; Dixon, Sandra V; Kim, Hyungjin; Cox, Kerry L; Kerr, Jonathan P; Johnston, David A; Johnson, Peter W M; Verbeek, J Sjef; Glennie, Martin J; Cragg, Mark S

2010-06-24

Rituximab, a monoclonal antibody that targets CD20 on B cells, is now central to the treatment of a variety of malignant and autoimmune disorders. Despite this success, a substantial proportion of B-cell lymphomas are unresponsive or develop resistance, hence more potent anti-CD20 monoclonal antibodies (mAbs) are continuously being sought. Here we demonstrate that type II (tositumomab-like) anti-CD20 mAbs are 5 times more potent than type I (rituximab-like) reagents in depleting human CD20 Tg B cells, despite both operating exclusively via activatory Fcgamma receptor-expressing macrophages. Much of this disparity in performance is attributable to type I mAb-mediated internalization of CD20 by B cells, leading to reduced macrophage recruitment and the degradation of CD20/mAb complexes, shortening mAb half-life. Importantly, human B cells from healthy donors and most cases of chronic lymphatic leukemia and mantle cell lymphoma, showed rapid CD20 internalization that paralleled that seen in the Tg mouse B cells, whereas most follicular lymphoma and diffuse large B-cell lymphoma cells were far more resistant to CD20 loss. We postulate that differences in CD20 modulation may play a central role in determining the relative efficacy of rituximab in treating these diseases and strengthen the case for focusing on type II anti-CD20 mAb in the clinic.

Comparison of humanized IgG and FvFc anti-CD3 monoclonal antibodies expressed in CHO cells.

PubMed

Serpieri, Flavia; Inocencio, Andre; de Oliveira, Jose Marcelino; Pimenta, Alécio A; Garbuio, Angélica; Kalil, Jorge; Brigido, Marcelo M; Moro, Ana Maria

2010-07-01

Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.

Radioimmunotherapy of nude mice with intraperitoneally growing ovarian cancer xenograft utilizing 211At-labelled monoclonal antibody MOv18.

PubMed

Andersson, H; Lindegren, S; Back, T; Jacobsson, L; Leser, G; Horvath, G

2000-01-01

The aim of this study was to investigate the therapeutic efficacy of 211At-labelled monoclonal antibody given intraperitoneally to nude mice with intraperitoneal growth of a human ovarian cancer cell line. Female nude mice were inoculated intraperitoneally with 1 x 10(7) cells of the human ovarian cancer cell line NIH:OVCAR 3. After about two weeks they were injected with the 211At-labelled specific monoclonal antibody MOv18 intraperitoneally. For comparison, other groups of mice were given the same labelled antibody intravenously, 211At-labelled unspecific antibody C242 intraperitoneally or unalbelled MOv18 intraperitoneally. Six weeks later the animals were sacrificed and the occurrence of tumour and ascites was determined. When the mice were treated with 211At-labelled MOv18 intraperitoneally 9 out of 10 were apparently free of both ascites and tumour compared to none of the mice given unlabelled antibody. 211At-labelled MOv18 given intravenously or 211At-labelled unspecific antibody given intraperitoneally were less effective. Regional radioimmunotherapy with the alpa-emitter 211Astatine seems to be an effective treatment of nude mice with intraperitoneally growing human ovarian cancer. Hopefully this treatment can be given in an adjuvant setting to women with minimal residual ovarian cancer in the future.

Monoclonal antibodies against the rat liver glucocorticoid receptor.

PubMed Central

Okret, S; Wikström, A C; Wrange, O; Andersson, B; Gustafsson, J A

1984-01-01

Splenic cells from one BALB/c mouse and one C57/BL mouse, immunized with purified rat liver glucocorticoid receptor (GR), were fused with the mouse myeloma cell line Sp 2/0-Ag 14. Screening for production of anti-GR-antibodies by the hybridomas was carried out with an enzyme-linked immunosorbent assay, using partially purified rat liver GR as antigen. Further screening was by a second-antibody immunoprecipitation assay using [3H]triamcinolone acetonide-GR complex from rat liver cytosol as tracer. Hybridomas from 10 different microplate wells, positive in both assays, were successfully cloned by the limiting dilution method to monoclonality. The different origins of the monoclonal antibodies were confirmed by their various isoelectric points when analyzed by isoelectric focusing. Four of the monoclonal hybridoma cell lines secreted IgM antibodies; two, IgG1; three, IgG2a; and one, IgG2b. The GR-antibody complex was identified in glycerol density gradients by a shift of the 4S GR to an 8.5S or 19S GR-antibody complex when incubated with monoclonal IgG or IgM antibody, respectively. The 10 monoclonal antibodies recognized different determinants on the GR, all situated on that domain of the receptor that is separate from the ligand and DNA-binding domains. Also, the cross-reactivity to the mouse liver GR varied among the monoclonal antibodies. No cross-reactivity was observed to the human lymphocytic GR. NaDodSO4 electrophoresis of a 0.5% pure GR preparation followed by immunoblotting using one of the monoclonal antibodies identified a single peptide with a molecular weight of 94,000, identical to the purified rat liver GR. Images PMID:6200880

[Anti-PD-1 antibody: basics and clinical application].

PubMed

Tanaka, Yoshimasa; Okamura, Haruki

2013-09-01

Although the treatment of cancer with monoclonal antibodies has long been pursued, T cell-directed immunotherapy has met with limited success. Recently, much attention has been devoted to the blockade of PD-1 signaling to activate an immune response to cancer. PD-1, a protein expressed on T cells, is a member of the CD28 superfamily, and it transmits coinhibitory signals upon engagement with its ligands PD-L1 and PD-L2. Accumulating evidence suggests that the PD-1 system plays pivotal roles in the regulation of autoimmunity, transplantation immunity, infectious immunity, and tumor immunity. Because the interaction of PD-1 with its ligands occurs in the effector phase of killer T cell responses in peripheral blood, anti-PD-1 and anti-PD-L1 monoclonal antibodies are ideal as specific agents to augment T cell responses to tumors with fewer adverse events than with the inhibition of CTLA-4, because the interaction of CTLA-4 with its ligands occurs in the priming phase of T cell responses within lymph nodes. In recent phase I clinical trials, objective responses were observed in patients with melanoma, renal cell carcinoma, and non-small cell lung cancer who underwent immunotherapy with an anti-PD-1 monoclonal antibody. In addition, the antitumor activity of an anti-PD-L1 monoclonal antibody was observed in patients with melanoma, renal cell carcinoma, non-small cell lung cancer, and ovarian cancer. The next frontier of immunotherapy targeting the PD-1 axis is to define patient selection criteria and explore combination therapy with other therapeutic manipulations such as adoptive immunotherapies.

Generation and characterization of a potent fully human monoclonal antibody against the interleukin-23 receptor.

PubMed

Sasaki-Iwaoka, Haruna; Ohori, Makoto; Imasato, Akira; Taguchi, Katsunari; Minoura, Kyoko; Saito, Tetsu; Kushima, Kiyoshi; Imamura, Emiko; Kubo, Satoshi; Furukawa, Shigetada; Morokata, Tatsuaki

2018-06-05

Interleukin (IL)-12 and IL-23 share a common subunit (p40) and function in T-helper (Th) 1 and Th17 immunity, respectively. Anti-IL-12/23p40 and specific anti-IL-23 antibodies are currently in clinical use for psoriasis and undergoing trials for autoimmune diseases. Since expression levels of the IL-23 receptor are likely to be much lower than those of IL-23, an anti-IL-23 receptor antibody might offer greater promise in inhibiting the IL-23-IL-17 pathways involved in inflammatory disorders. To our knowledge, no anti-IL-23 receptor antibody has been trialed in clinical studies to date. This study describes the generation and characterization of AS2762900-00, a fully human monoclonal antibody against the IL-23 receptor. AS2762900-00 bound both human and cynomolgus monkey IL-23 receptors. AS2762900-00 showed potent inhibitory effects on IL-23-induced Kit-225 cell proliferation compared to the existing anti-IL-12/23p40 antibody, ustekinumab. In a single dose administration pharmacodynamics study in cynomolgus monkeys, 1 mg/kg of AS2762900-00 significantly inhibited (> 85%) IL-23-induced STAT3 phosphorylation in blood for up to 84 days. Therefore, AS2762900-00 represents a potent novel IL-23-IL-17 pathway inhibitor with the potential to be developed into a new therapy for the treatment of autoimmune diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

CT-SPECT fusion plus conjugate views for determining dosimetry in iodine-131-monoclonal antibody therapy of lymphoma patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koral, K.F.; Zasadny, K.R.; Kessler, M.L.

A method of performing {sup 131}I quantitative SPECT imaging is described which uses the superimposition of markers placed on the skin to accomplish fusion of computed tomography (CT) and SPECT image sets. To calculate mean absorbed dose after administration of one of two {sup 131}I-labeled monoclonal antibodies (Mabs), the shape of the time-activity curve is measured by daily diagnostic conjugate views, the y-axis of that curve is normalized by a quantitative SPECT measurement (usually intra-therapy), and the tumor mass is deduced from a concurrent CT volume measurement. The method is applied to six B-cell non-Hodgkin`s lymphoma patients. For four tumorsmore » in three patients treated with the MB1 Mab, a correlation appears to be present between resulting mean absorbed dose and disease response. Including all dosimetric estimates for both antibodies, the range for the specific absorbed dose is within that found by others in treating B-cell lymphoma patients. Excluding a retreated anti-B1 patient, the tumor-specific absorbed dose during anti-B1 therapy is from 1.4 to 1.7 mGy/MBq. For the one anti-B1 patient, where quantitative SPECT and conjugate-view imaging was carried out back to back , the quantitative SPECT-measured activity was somewhat less for the spleen and much less for the tumor than that from conjugate views. The quantitative SPECT plus conjugate views method may be of general utility for macro-dosimetry of {sup 131}If therapies. 18 refs., 3 figs., 5 tabs.« less

Development of a highly-sensitive multi-plex assay using monoclonal antibodies for the simultaneous measurement of kappa and lambda immunoglobulin free light chains in serum and urine.

PubMed

Campbell, John P; Cobbold, Mark; Wang, Yanyun; Goodall, Margaret; Bonney, Sarah L; Chamba, Anita; Birtwistle, Jane; Plant, Timothy; Afzal, Zaheer; Jefferis, Roy; Drayson, Mark T

2013-05-31

Monoclonal κ and λ immunoglobulin free light chain (FLC) paraproteins in serum and urine are important markers in the diagnosis and monitoring of B cell dyscrasias. Current nephelometric and turbidimetric methods that use sheep polyclonal antisera to quantify serum FLC have a number of well-observed limitations. In this report, we describe an improved method using specific mouse anti-human FLC monoclonal antibodies (mAbs). Anti-κ and anti-λ FLC mAbs were, separately, covalently coupled to polystyrene Xmap® beads and assayed, simultaneously, in a multi-plex format by Luminex® (mAb assay). The mAbs displayed no cross-reactivity to bound LC, the alternate LC type, or other human proteins and had improved sensitivity and specificity over immunofixation electrophoresis (IFE) and Freelite™. The assay gives good linearity and sensitivity (<1 mg/L), and the competitive inhibition format gave a broad calibration curve up to 437.5 mg/L and prevented anomalous results for samples in antigen excess i.e. high FLC levels. The mAbs displayed good concordance with Freelite™ for the quantitation of normal polyclonal FLC in plasma from healthy donors (n=249). The mAb assay identified all monoclonal FLC in serum from consecutive patient samples (n=1000; 50.1% with monoclonal paraprotein by serum IFE), and all FLC in a large cohort of urine samples tested for Bence Jones proteins (n=13090; 22.8% with monoclonal κ, 9.0% with monoclonal λ, and 0.8% with poly LC detected by urine IFE). Importantly this shows that the mAbs are at least close to the ideal of detecting FLC from all patients and neoplastic plasma cell clones. Given the overall effectiveness of the anti-FLC mAbs, further clinical validation is now warranted on serial samples from a range of patients with B cell disorders. Use of these mAbs on other assay platforms should also be investigated. Copyright © 2013 Elsevier B.V. All rights reserved.

Endotoxin reduces specific pulmonary uptake of radiolabeled monoclonal antibody to angiotensin-converting enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muzykantov, V.R.; Puchnina, E.A.; Atochina, E.N.

The biodistribution of radiolabeled monoclonal antibody (Mab) to angiotensin-converting enzyme (ACE) was examined in normal and endotoxin-treated rats. Endotoxin administration at a dose of 4 mg/kg induced mild or middle pulmonary edema. The ACE activity in lung homogenate remained virtually unchanged, while the activity of serum ACE increased 15 hr after endotoxin infusion. In normal rats, anti-ACE Mab accumulates specifically in the lung after i.v. injection. Endotoxin injection induces reduction of specific pulmonary uptake of this antibody. Even in non-edematous endotoxemia, the accumulation of anti-ACE Mab antibody (Mab 9B9) decreased from 19.02 to 11.91% of ID/g of tissue without anymore » change in accumulation of control nonspecific IgG. The antibody distribution in other organs and its blood level were almost the same as in the control. In a case of endotoxemia accompanied by increased microvascular permeability, the lung accumulation of Mab 9B9 was reduced to 9.17% of ID/g of tissue, while the accumulation of nonspecific IgG increased to 1.44% versus 0.89% in the control.« less

Monitoring of antigen-specific CD8 T cells in patients with type 1 diabetes treated with antiCD3 monoclonal antibodies.

PubMed

Cernea, Simona; Herold, Kevan C

2010-02-01

The way in which anti-CD3 monoclonal antibodies (mAbs) modify human immune responses in type 1 diabetes (T1DM) is not known. We prepared a panel of Class I HLA-A2.1 tetramers with peptides from diabetes-associated antigens and studied the frequency and phenotype of the cells in patients with T1DM and blood donors and in patients treated with anti-CD3 mAb (Teplizumab). More patients with T1DM showed positive staining for at least 1 tetramer using frozen and fresh samples (p<0.05). Three months following treatment with anti-CD3 mAb, the proportion of GAD65- and InsB-peptide reactive CD8+ T cells increased (p<0.05). The phenotype of these cells was modulated from naïve to effector memoryRA+. We concludethat Class I MHC tetramers can identify antigen specific CD8+ T cells in patients with T1DM. The frequency of certain specificities increases after treatment with anti-CD3 mAb. Their modulated phenotype may have functional consequences for their pathogenicity. Copyright 2009 Elsevier Inc. All rights reserved.

A non-toxic enzyme-linked immunosorbent assay for aflatoxin B1 using anti-idiotypic antibodies as substitutes.

PubMed

Hu, Li; Liu, Aiping; Chen, Weifeng; Yang, Hongxiu; Wang, Xiaohong; Chen, Fusheng

2017-03-01

Immunoassays are widely employed techniques to detect aflatoxins since they are rapid, selective and sensitive. One common disadvantage of them is using aflatoxins as standard substances, which may trigger exposure risks to operators and environmental contamination without proper handling. Anti-idiotypic antibodies (anti-Ids or Ab2s), also named as internal-image anti-Ids, are able to mimic and function as antigens, so a non-toxic enzyme-linked immunosorbent assay (ELISA) for aflatoxin B 1 (AFB 1 ) is developed and validated using anti-Ids as substitutes. Mouse monoclonal anti-idiotypic antibody (McAb2) to AFB 1 was generated by the hybridoma technique using Fab fragments of rabbit anti-AFB 1 idiotype antibody (Ab1) as immunogen. As indicated by indirect competitive ELISA, McAb2, represented an internal-image of antigen AFB 1 , was able to bind Fab with competition to AFB 1 . Then, analysis of AFB 1 in spiked samples by non-toxic ELISA using anti-Ids as substitutes was developed, and it showed no significant differences with comparison to AFB 1 as competitive antigens. Our work demonstrated that anti-Ids could be used as internal-image mimicry of AFB 1 , and it had potential applications in immunoassays for antigen substitution to reduce operational risk for operators and environmental contamination. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Vectored Intracerebral Immunization with the Anti-Tau Monoclonal Antibody PHF1 Markedly Reduces Tau Pathology in Mutant Tau Transgenic Mice.

PubMed

Liu, Wencheng; Zhao, Lingzhi; Blackman, Brittany; Parmar, Mayur; Wong, Man Ying; Woo, Thomas; Yu, Fangmin; Chiuchiolo, Maria J; Sondhi, Dolan; Kaminsky, Stephen M; Crystal, Ronald G; Paul, Steven M

2016-12-07

Passive immunization with anti-tau monoclonal antibodies has been shown by several laboratories to reduce age-dependent tau pathology and neurodegeneration in mutant tau transgenic mice. These studies have used repeated high weekly doses of various tau antibodies administered systemically for several months and have reported reduced tau pathology of ∼40-50% in various brain regions. Here we show that direct intrahippocampal administration of the adeno-associated virus (AAV)-vectored anti-phospho-tau antibody PHF1 to P301S tau transgenic mice results in high and durable antibody expression, primarily in neurons. Hippocampal antibody levels achieved after AAV delivery were ∼50-fold more than those reported following repeated systemic administration. In contrast to systemic passive immunization, we observed markedly reduced (≥80-90%) hippocampal insoluble pathological tau species and neurofibrillary tangles following a single dose of AAV-vectored PHF1 compared with mice treated with an AAV-IgG control vector. Moreover, the hippocampal atrophy observed in untreated P301S mice was fully rescued by treatment with the AAV-vectored PHF1 antibody. Vectored passive immunotherapy with an anti-tau monoclonal antibody may represent a viable therapeutic strategy for treating or preventing such tauopathies as frontotemporal dementia, progressive supranuclear palsy, or Alzheimer's disease. We have used an adeno-associated viral (AAV) vector to deliver the genes encoding an anti-phospho-tau monoclonal antibody, PHF1, directly to the brain of mice that develop neurodegeneration due to a tau mutation that causes frontotemporal dementia (FTD). When administered systemically, PHF1 has been shown to modestly reduce tau pathology and neurodegeneration. Since such antibodies do not readily cross the blood-brain barrier, we used an AAV vector to deliver antibody directly to the hippocampus and observed much higher antibody levels and a much greater reduction in tau pathology. Using AAV vectors to deliver antibodies like PHF1 directly to brain may constitute a novel approach to treating various neurodegenerative disorders, such as FTD and Alzheimer's disease. Copyright © 2016 the authors 0270-6474/16/3612425-11$15.00/0.

Varied distribution of RhD epitopes in the Indian population.

PubMed

Kulkarni, S S; Gupte, S C; Vasantha, K; Mohanty, D; Ghosh, K

2007-01-01

Inhabited by more than 4000 caste and tribal groups, India has an extremely heterogenous population. For thousands of years many tribal groups have practised endogamy and are practically genetically isolated. Traditionally, polyclonal anti-D reagent has been used for RhD typing; though monoclonal antibodies are increasingly being used. As a result, blood banks find it difficult to assign the RhD status to an increasing number of people. As monoclonal anti-D typing reagents may not detect all RhD antigen epitopes, we studied the RhD antigen epitope heterogeneity in different population groups in India. Red cells of 5315 RhD-positive individuals belonging to different castes and tribes of India were tested with 30 different epitope-specific monoclonal anti-D antibodies. No single monoclonal antibody could detect all RhD-positive red cells detected by polyclonal antisera. The highest proportion of D antigen was detected by LHM 76/55 and BRAD-8 (98%) monoclonal antibodies. We need to determine the correct mix of monoclonal antibodies that will detect nearly all RhD antigens detected by polyclonal anti-D sera. Similarly, before accepting monoclonal anti-D for therapeutic use, it would be necessary to determine the appropriate ones for use in the Indian population.

Indium-111 labeled anti-melanoma monoclonal antibodies

DOEpatents

Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

1984-04-30

A monoclonal antibody to a high molecular weight melanoma-associated antigen was chelated and radiolabeled with indium-111. This material shows high affinity for melanoma and thus can be used in the detection, localization and imaging of melanoma. 1 figure.

HPA antibodies in Algerian multitransfused patients: Prevalence and involvement in platelet refractoriness.

PubMed

Brouk, Hacene; Bertrand, Gérald; Zitouni, Selma; Djenouni, Amel; Martageix, Corinne; Griffi, Fatiha; Kaplan, Cecile; Ouelaa, Hanifa

2015-06-01

Patients receiving cellular blood components may form HLA or HPA antibodies. The frequency and the specificity of HPA antibodies after a series of blood transfusions have never been reported in the Algerian population which is ethnically diverse and runs a higher risk of platelet alloimmunization due to high b allelic frequencies observed for the HPA systems. 117 polytransfused patients were included in this study; the detection of HPA antibodies was performed by the Monoclonal Antibody-specific Immobilization of Platelet Antigens method (MAIPA). Post-transfusion platelet effectiveness was evaluated by the calculation of corrected count increment (CCI). The antibodies against platelets were detected in 10.26% of the patients. In this study, the platelet systems concerned by the alloimmunizations were specifically HPA-1, -3 and -5 with particular predominance of HPA-1. Twenty two patients were refractory to platelet transfusion, as assessed by a CCI; in which 64% have factors associated with increased platelet consumption. Platelet Immunization was found in 14% of platelet refractoriness (PTR) cases. 03 Anti-platelet antibodies were directed against GPIb-IX (n = 1), anti-HPA-1b (n = 1) and anti HPA-5b (n = 1) associated with anti-HLA antibodies in two cases. HLA and HPA alloimmunization is common among chronically transfused patients. PTR detection, identification of the underlying causes, and selection of the appropriate product for transfusion are fundamental to reduce the risk of major bleedings. Copyright © 2014 Elsevier Ltd. All rights reserved.

Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

PubMed Central

2012-01-01

Background The epidermal growth factor receptor (EGFR) is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib. PMID:23234355

Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry.

PubMed

Papac, D I; Hoyes, J; Tomer, K B

1994-09-01

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.

Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry.

PubMed Central

Papac, D. I.; Hoyes, J.; Tomer, K. B.

1994-01-01

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP. PMID:7530543

Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B.

PubMed

Cheng, Luisa W; Henderson, Thomas D; Lam, Tina I; Stanker, Larry H

2015-11-27

Botulinum neurotoxins (BoNT) are some of nature's most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL) immunoassay for BoNT/B, using monoclonal antibodies (mAbs) MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism.

Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B

PubMed Central

Cheng, Luisa W.; Henderson, Thomas D.; Lam, Tina I.; Stanker, Larry H.

2015-01-01

Botulinum neurotoxins (BoNT) are some of nature’s most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL) immunoassay for BoNT/B, using monoclonal antibodies (mAbs) MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism. PMID:26633496

The main immunogenic region of acetylcholine receptors does not provoke the formation of antibodies of a predominant idiotype.

PubMed

Killen, J A; Hochschwender, S M; Lindstrom, J M

1985-08-01

Anti-idiotype antibodies were induced in rats by immunization with rat monoclonal antibodies to the main immunogenic region of acetylcholine receptors. These anti-idiotype antibodies showed very little crossreaction with other rat monoclonal antibodies which bind to the same region of the receptor. When the rats producing these anti-idiotype antibodies were immunized with receptor, they showed no net decrease in anti-receptor antibody production. These data indicate that, although more than half of the antibodies produced by rats immunized with receptor are directed at a small region, many anti-receptor idiotypes are involved in this response and anti-idiotype therapy is not beneficial.

Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki; Oshita, Masatoshi

2009-09-11

Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizingmore » ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.« less

Neutralising Antibodies against Ricin Toxin

PubMed Central

Prigent, Julie; Panigai, Laetitia; Lamourette, Patricia; Sauvaire, Didier; Devilliers, Karine; Plaisance, Marc; Volland, Hervé; Créminon, Christophe; Simon, Stéphanie

2011-01-01

The Centers for Disease Control and Prevention have listed the potential bioweapon ricin as a Category B Agent. Ricin is a so-called A/B toxin produced by plants and is one of the deadliest molecules known. It is easy to prepare and no curative treatment is available. An immunotherapeutic approach could be of interest to attenuate or neutralise the effects of the toxin. We sought to characterise neutralising monoclonal antibodies against ricin and to develop an effective therapy. For this purpose, mouse monoclonal antibodies (mAbs) were produced against the two chains of ricin toxin (RTA and RTB). Seven mAbs were selected for their capacity to neutralise the cytotoxic effects of ricin in vitro. Three of these, two anti-RTB (RB34 and RB37) and one anti-RTA (RA36), when used in combination improved neutralising capacity in vitro with an IC50 of 31 ng/ml. Passive administration of association of these three mixed mAbs (4.7 µg) protected mice from intranasal challenges with ricin (5 LD50). Among those three antibodies, anti-RTB antibodies protected mice more efficiently than the anti-RTA antibody. The combination of the three antibodies protected mice up to 7.5 hours after ricin challenge. The strong in vivo neutralising capacity of this three mAbs combination makes it potentially useful for immunotherapeutic purposes in the case of ricin poisoning or possibly for prevention. PMID:21633505

Growth inhibition of tumor cells in vitro by using monoclonal antibodies against gonadotropin-releasing hormone receptor.

PubMed

Lee, Gregory; Ge, Bixia

2010-07-01

As the continuation of a previous study, synthetic peptides corresponding to the extracellular domains of human gonadotropin-releasing hormone (GnRH) receptor were used to generate additional monoclonal antibodies which were further characterized biochemically and immunologically. Among those identified to recognize GnRH receptor, monoclonal antibodies designated as GHR-103, GHR-106 and GHR-114 were found to exhibit high affinity (Kd < or = 1 x 10(-8) M) and specificity to GnRH receptor as judged by the whole cell binding immunoassay and Western blot assay. Both anti-GnRH receptor monoclonal antibodies and GnRH were shown to compete for the same binding site of GnRH receptor on the surface of cultured cancer cells. Growth inhibitions of cancer cells cultured in vitro were demonstrated by cellular apoptosis experiments (TUNEL and MTT assays) under different conditions of treatment with GHR-106 monoclonal antibody or GnRH analogs. It was generally observed that both GnRH I and GHR-106 effectively induce the apoptosis of cultured cancer cells as determined by TUNEL and MTT assays. Consistently, suppressions of gene expressions at mRNA levels were demonstrated with several ribosomal proteins (P0, P1, P2 and L37), when cancer cells were incubated with GnRH or GHR-106. The widespread expressions of GnRH receptor in almost all of the studied human cancer cell lines were also demonstrated by RT-PCR and Western blot assay, as well as indirect immunofluorescence assay with either of these monoclonal antibodies as the primary antibody. In view of the longer half life of antibodies as compared to that of GnRH or its analogs, anti-GnRH receptor monoclonal antibodies in humanized forms could function as GnRH analogs and serve as an ideal candidate of anti-cancer drugs for therapeutic treatments of various cancers in humans as well as for fertility regulations.

Rapid High-Level Production of Functional HIV Broadly Neutralizing Monoclonal Antibodies in Transient Plant Expression Systems

PubMed Central

Rosenberg, Yvonne; Sack, Markus; Montefiori, David; Forthal, Donald; Mao, Lingjun; -Abanto, Segundo Hernandez; Urban, Lori; Landucci, Gary; Fischer, Rainer; Jiang, Xiaoming

2013-01-01

Passive immunotherapy using anti-HIV broadly neutralizing monoclonal antibodies (mAbs) has shown promise as an HIV treatment, reducing mother-to-child-transmission (MTCT) of simian/human immunodeficiency virus (SHIV) in non-human primates and decreasing viral rebound in patients who ceased receiving anti-viral drugs. In addition, a cocktail of potent mAbs may be useful as mucosal microbicides and provide an effective therapy for post-exposure prophylaxis. However, even highly neutralizing HIV mAbs used today may lose their effectiveness if resistance occurs, requiring the rapid production of new or engineered mAbs on an ongoing basis in order to counteract the viral resistance or the spread of a certain HIV-1 clade in a particular region or patient. Plant-based expression systems are fast, inexpensive and scalable and are becoming increasingly popular for the production of proteins and monoclonal antibodies. In the present study, Agrobacterium-mediated transient transfection of plants, utilizing two species of Nicotiana, have been tested to rapidly produce high levels of an HIV 89.6PΔ140env and several well-studied anti-HIV neutralizing monoclonal antibodies (b12, 2G12, 2F5, 4E10, m43, VRC01) or a single chain antibody construct (m9), for evaluation in cell-based viral inhibition assays. The protein-A purified plant-derived antibodies were intact, efficiently bound HIV envelope, and were equivalent to, or in one case better than, their counterparts produced in mammalian CHO or HEK-293 cells in both neutralization and antibody dependent viral inhibition assays. These data indicate that transient plant-based transient expression systems are very adaptable and could rapidly generate high levels of newly identified functional recombinant HIV neutralizing antibodies when required. In addition, they warrant detailed cost-benefit analysis of prolonged incubation in plants to further increase mAb production. PMID:23533588

Protection by meningococcal outer membrane protein PorA-specific antibodies and a serogroup B capsular polysaccharide-specific antibody in complement-sufficient and C6-deficient infant rats.

PubMed

Toropainen, Maija; Saarinen, Leena; Vidarsson, Gestur; Käyhty, Helena

2006-05-01

The relative contributions of antibody-induced complement-mediated bacterial lysis and antibody/complement-mediated phagocytosis to host immunity against meningococcal infections are currently unclear. Further, the in vivo effector functions of antibodies may vary depending on their specificity and Fc heavy-chain isotype. In this study, a mouse immunoglobulin G2a (mIgG2a) monoclonal antibody (MN12H2) to meningococcal outer membrane protein PorA (P1.16), its human IgG subclass derivatives (hIgG1 to hIgG4), and an mIgG2a monoclonal antibody (Nmb735) to serogroup B capsular polysaccharide (B-PS) were evaluated for passive protection against meningococcal serogroup B strain 44/76-SL (B:15:P1.7,16) in an infant rat infection model. Complement component C6-deficient (PVG/c-) rats were used to assess the importance of complement-mediated bacterial lysis for protection. The PorA-specific parental mIgG2a and the hIgG1 to hIgG3 derivatives all induced efficient bactericidal activity in vitro in the presence of human or infant rat complement and augmented bacterial clearance in complement-sufficient HsdBrlHan:WIST rats, while the hIgG4 was unable to do so. In C6-deficient PVG/c- rats, lacking complement-mediated bacterial lysis, the augmentation of bacterial clearance by PorA-specific mIgG2a and hIgG1 antibodies was impaired compared to that in the syngeneic complement-sufficient PVG/c+ rat strain. This was in contrast to the case for B-PS-specific mIgG2a, which conferred similar protective activity in both rat strains. These data suggest that while anti-B-PS antibody can provide protection in the infant rats without membrane attack complex formation, the protection afforded by anti-PorA antibody is more dependent on the activation of the whole complement pathway and subsequent bacterial lysis.

Immune checkpoint inhibitors and targeted therapies for metastatic melanoma: A network meta-analysis.

PubMed

Pasquali, Sandro; Chiarion-Sileni, Vanna; Rossi, Carlo Riccardo; Mocellin, Simone

2017-03-01

Immune checkpoint inhibitors and targeted therapies, two new class of drugs for treatment of metastatic melanoma, have not been compared in randomized controlled trials (RCT). We quantitatively summarized the evidence and compared immune and targeted therapies in terms of both efficacy and toxicity. A comprehensive search for RCTs of immune checkpoint inhibitors and targeted therapies was conducted to August 2016. Using a network meta-analysis approach, treatments were compared with each other and ranked based on their effectiveness (as measured by the impact on progression-free survival [PFS]) and acceptability (the inverse of high grade toxicity). Twelve RCTs enrolling 6207 patients were included. Network meta-analysis generated 15 comparisons. Combined BRAF and MEK inhibitors were associated with longer PFS as compared to anti-CTLA4 (HR: 0.22; 95% confidence interval [CI]: 0.12-0.41) and anti-PD1 antibodies alone (HR: 0.38; CI: 0.20-0.72). However, anti-PD1 monoclonal antibodies were less toxic than anti-CTLA4 monoclonal antibodies (RR: 0.65; CI: 0.40-0.78) and their combination significantly increased toxicity compared to either single agent anti-CTLA4 (RR: 2.06; CI: 1.45-2.93) or anti-PD1 monoclonal antibodies (RR: 3.67; CI: 2.27-5.96). Consistently, ranking analysis suggested that the combination of targeted therapies is the most effective strategy, whereas single agent anti-PD1 antibodies have the best acceptability. The GRADE level of evidence quality for these findings was moderate to low. The simultaneous inhibition of BRAF and MEK appears the most effective treatment for melanomas harboring BRAF V600 mutation, although anti-PD1 antibodies appear to be less toxic. Further research is needed to increase the quality of evidence. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cryo-EM structures elucidate neutralizing mechanisms of anti-chikungunya human monoclonal antibodies with therapeutic activity

DOE PAGES

Long, Feng; Fong, Rachel H.; Austin, Stephen K.; ...

2015-10-26

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe acute and chronic disease in humans. Although highly inhibitory murine and human monoclonal antibodies (mAbs) have been generated, the structural basis of their neutralizing activity remains poorly characterized. In this paper, we determined the cryo-EM structures of chikungunya virus-like particles complexed with antibody fragments (Fab) of two highly protective human mAbs, 4J21 and 5M16, that block virus fusion with host membranes. Both mAbs bind primarily to sites within the A and B domains, as well as to the B domain’s β-ribbon connector of the viral glycoprotein E2. The footprints ofmore » these antibodies on the viral surface were consistent with results from loss-of-binding studies using an alanine scanning mutagenesis-based epitope mapping approach. The Fab fragments stabilized the position of the B domain relative to the virus, particularly for the complex with 5M16. Finally, this finding is consistent with a mechanism of neutralization in which anti-CHIKV mAbs that bridge the A and B domains impede movement of the B domain away from the underlying fusion loop on the E1 glycoprotein and therefore block the requisite pH-dependent fusion of viral and host membranes.« less

Anti-ErbB-2 mAb therapy requires type I and II interferons and synergizes with anti-PD-1 or anti-CD137 mAb therapy.

PubMed

Stagg, John; Loi, Sherene; Divisekera, Upulie; Ngiow, Shin Foong; Duret, Helene; Yagita, Hideo; Teng, Michele W; Smyth, Mark J

2011-04-26

Trastuzumab, a monoclonal antibody targeting human epidermal growth factor receptor-2 (HER2/ErbB-2), has become the mainstay of treatment for HER2-positive breast cancer. Nevertheless, its exact mechanism of action has not been fully elucidated. Although several studies suggest that Fc receptor-expressing immune cells are involved in trastuzumab therapy, the relative contribution of lymphocyte-mediated cellular cytotoxicity and antitumor cytokines remains unknown. We report here that anti-ErbB-2 mAb therapy is dependent on the release of type I and type II IFNs but is independent of perforin or FasL. Our study thus challenges the notion that classical antibody-dependent, lymphocyte-mediated cellular cytotoxicity is important for trastuzumab. We demonstrate that anti-ErbB-2 mAb therapy of experimental tumors derived from MMTV-ErbB-2 transgenic mice triggers MyD88-dependent signaling and primes IFN-γ-producing CD8+ T cells. Adoptive cell transfer of purified T cell subsets confirmed the essential role of IFN-γ-producing CD8+ T cells. Notably, anti-ErbB-2 mAb therapy was independent of IL-1R or IL-17Ra signaling. Finally, we investigated whether immunostimulatory approaches with antibodies against programmed death-1 (PD-1) or 41BB (CD137) could be used to capitalize on the immune-mediated effects of trastuzumab. We demonstrate that anti-PD-1 or anti-CD137 mAb can significantly improve the therapeutic activity of anti-ErbB-2 mAb in immunocompetent mice.

3B11-N, a monoclonal antibody against MERS-CoV, reduces lung pathology in rhesus monkeys following intratracheal inoculation of MERS-CoV Jordan-n3/2012

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Reed F., E-mail: johnsonreed@mail.nih.gov; Bagci, Ulas; Center for Research in Computer Vision

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was identified in 2012 as the causative agent of a severe, lethal respiratory disease occurring across several countries in the Middle East. To date there have been over 1600 laboratory confirmed cases of MERS-CoV in 26 countries with a case fatality rate of 36%. Given the endemic region, it is possible that MERS-CoV could spread during the annual Hajj pilgrimage, necessitating countermeasure development. In this report, we describe the clinical and radiographic changes of rhesus monkeys following infection with 5×10{sup 6} PFU MERS-CoV Jordan-n3/2012. Two groups of NHPs were treated with either a humanmore » anti-MERS monoclonal antibody 3B11-N or E410-N, an anti-HIV antibody. MERS-CoV Jordan-n3/2012 infection resulted in quantifiable changes by computed tomography, but limited other clinical signs of disease. 3B11-N treated subjects developed significantly reduced lung pathology when compared to infected, untreated subjects, indicating that this antibody may be a suitable MERS-CoV treatment. - Highlights: • MERS-CoV Jordan-n3/2012 challenge of rhesus monkeys results in a mild disease. • CT can be used to monitor disease progression to aid models of human disease. • Treatment with the human monoclonal antibody 3B11-N resulted in decreased disease.« less

Induction of suppressor cells in vitro by Candida albicans.

PubMed

Cuff, C F; Rogers, C M; Lamb, B J; Rogers, T J

1986-06-01

Normal splenocytes cultured with Formalin-killed Candida albicans were shown to acquire significant suppressor cell activity in a period of 3 days. These cells were found to suppress both the phytohemagglutinin-induced mitogen response as well as the anti-sheep erythrocyte antibody response. Experiments were carried out to determine the nature of the suppressor cell population. Results showed that these cells were not susceptible to treatment with anti-Thy 1 antibody and complement. Panning experiments showed that the suppressor cells were not plastic-adherent or Mac-1 antigen-positive. The suppressor cells were, however, adherent to anti-mouse immunoglobulin (F(ab')2-fragment)-coated dishes. Additional experiments showed that the suppressor cell activity was susceptible to treatment with monoclonal anti-Lyb 2.1 antibody and complement. These results suggest that the suppressor cell induced in vitro by Candida is a member of the B-lymphocyte lineage.

Maternal Brain-Reactive Antibodies and Autism Spectrum Disorder

DTIC Science & Technology

We have determined that one monoclonal antibody cloned from the memory B cell population of the mother of a child with ASD binds Caspr2. This... child with ASD exhibit antibodies to Caspr2; thus this antibody may contribute to approximately 5% of cases of ASD. We have also determined that the pathogenicity of anti-Caspr2 antibodies is determined, in part, by epitope specificity.

In situ demonstration of tissue proliferative activity using anti-bromo-deoxyuridine monoclonal antibody.

PubMed Central

Veronese, S; Gambacorta, M; Falini, B

1989-01-01

Immunohistochemical staining with anti-bromo-deoxyuridine (BrdU) monoclonal antibody was performed on a variety of human tissues following in vitro incubation with BrdU. The effect of different fixatives and DNA denaturation techniques on the reactivity with anti-BrdU was investigated. Optimal preservation of the antigenicity of BrdU incorporated into the DNA of proliferating cells was seen in tissues fixed in Bouin's fluid, while samples which had been fixed with cross-linking reagents, such as formalin, were usually unreactive. Positivity for BrdU was restored in formalin fixed tissues after digestion with pepsin, but this was usually associated with loss of morphological details. Acid and thermal DNA denaturation techniques gave similar results. It is concluded that Bouin fixation followed by acid or thermal denaturation of DNA is the method of choice for the in situ detection of cells in S-phase using anti-BrdU monoclonal antibody. Images Fig 1 Fig 1 PMID:2475528

Synthetic Human Monoclonal Antibodies toward Staphylococcal Enterotoxin B (SEB) Protective against Toxic Shock Syndrome*

PubMed Central

Karauzum, Hatice; Chen, Gang; Abaandou, Laura; Mahmoudieh, Mahta; Boroun, Atefeh R.; Shulenin, Sergey; Devi, V. Sathya; Stavale, Eric; Warfield, Kelly L.; Zeitlin, Larry; Roy, Chad J.; Sidhu, Sachdev S.; Aman, M. Javad

2012-01-01

Staphylococcal enterotoxin B (SEB) is a potent toxin that can cause toxic shock syndrome and act as a lethal and incapacitating agent when used as a bioweapon. There are currently no vaccines or immunotherapeutics available against this toxin. Using phage display technology, human antigen-binding fragments (Fabs) were selected against SEB, and proteins were produced in Escherichia coli cells and characterized for their binding affinity and their toxin neutralizing activity in vitro and in vivo. Highly protective Fabs were converted into full-length IgGs and produced in mammalian cells. Additionally, the production of anti-SEB antibodies was explored in the Nicotiana benthamiana plant expression system. Affinity maturation was performed to produce optimized lead anti-SEB antibody candidates with subnanomolar affinities. IgGs produced in N. benthamiana showed characteristics comparable with those of counterparts produced in mammalian cells. IgGs were tested for their therapeutic efficacy in the mouse toxic shock model using different challenge doses of SEB and a treatment with 200 μg of IgGs 1 h after SEB challenge. The lead candidates displayed full protection from lethal challenge over a wide range of SEB challenge doses. Furthermore, mice that were treated with anti-SEB IgG had significantly lower IFNγ and IL-2 levels in serum compared with mock-treated mice. In summary, these anti-SEB monoclonal antibodies represent excellent therapeutic candidates for further preclinical and clinical development. PMID:22645125

Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies

PubMed Central

Pietsch, E C; Dong, J; Cardoso, R; Zhang, X; Chin, D; Hawkins, R; Dinh, T; Zhou, M; Strake, B; Feng, P-H; Rocca, M; Santos, C Dos; Shan, X; Danet-Desnoyers, G; Shi, F; Kaiser, E; Millar, H J; Fenton, S; Swanson, R; Nemeth, J A; Attar, R M

2017-01-01

CD47, a broadly expressed cell surface protein, inhibits cell phagocytosis via interaction with phagocyte-expressed SIRPα. A variety of hematological malignancies demonstrate elevated CD47 expression, suggesting that CD47 may mediate immune escape. We discovered three unique CD47-SIRPα blocking anti-CD47 monoclonal antibodies (mAbs) with low nano-molar affinity to human and cynomolgus monkey CD47, and no hemagglutination and platelet aggregation activity. To characterize the anti-cancer activity elicited by blocking CD47, the mAbs were cloned into effector function silent and competent Fc backbones. Effector function competent mAbs demonstrated potent activity in vitro and in vivo, while effector function silent mAbs demonstrated minimal activity, indicating that blocking CD47 only leads to a therapeutic effect in the presence of Fc effector function. A non-human primate study revealed that the effector function competent mAb IgG1 C47B222-(CHO) decreased red blood cells (RBC), hematocrit and hemoglobin by >40% at 1 mg/kg, whereas the effector function silent mAb IgG2σ C47B222-(CHO) had minimal impact on RBC indices at 1 and 10 mg/kg. Taken together, our findings suggest that targeting CD47 is an attractive therapeutic anti-cancer approach. However, the anti-cancer activity observed with anti-CD47 mAbs is Fc effector dependent as are the side effects observed on RBC indices. PMID:28234345

Functional Analysis of the Anti-adalimumab Response Using Patient-derived Monoclonal Antibodies♦

PubMed Central

van Schouwenburg, Pauline A.; Kruithof, Simone; Votsmeier, Christian; van Schie, Karin; Hart, Margreet H.; de Jong, Rob N.; van Buren, Esther E. L.; van Ham, Marieke; Aarden, Lucien; Wolbink, Gertjan; Wouters, Diana; Rispens, Theo

2014-01-01

The production of antibodies to adalimumab in autoimmune patients treated with adalimumab is shown to diminish treatment efficacy. We previously showed that these antibodies are almost exclusively neutralizing, indicating a restricted response. Here, we investigated the characteristics of a panel of patient-derived monoclonal antibodies for binding to adalimumab. Single B-cells were isolated from two patients, cultured, and screened for adalimumab specificity. Analysis of variable region sequences of 16 clones suggests that the immune response against adalimumab is broad, involving multiple B-cell clones each using different combinations of V(D)J segments. A strong bias for replacement mutations in the complementarity determining regions was found, indicating an antigen-driven response. We recombinantly expressed 11 different monoclonal antibodies and investigated their affinity and specificity. All clones except one are of high affinity (Kd between 0.6 and 233 pm) and compete with TNF as well as each other for binding to adalimumab. However, binding to a panel of single-point mutants of adalimumab indicates markedly different fine specificities that also result in a differential tendency of each clone to form dimeric and multimeric immune complexes. We conclude that although all anti-adalimumab antibodies compete for binding to TNF, the response is clonally diverse and involves multiple epitopes on adalimumab. These results are important for understanding the relationship between self and non-self or idiotypic determinants on therapeutic antibodies and their potential immunogenicity. PMID:25326381

Development and evaluation of a competitive ELISA using a monoclonal antibody for antibody detection after goose parvovirus virus-like particles (VLPs) and vaccine immunization in goose sera.

PubMed

Wang, Qian; Ju, Huanyu; Li, Yanwei; Jing, Zhiqiang; Guo, Lu; Zhao, Yu; Ma, Bo; Gao, Mingchun; Zhang, Wenlong; Wang, Junwei

2014-12-01

An assay protocol based on a monoclonal antibody-based competitive enzyme-linked immunosorbent assay (MAb-based C-ELISA) for detecting antibodies against goose parvovirus (GPV) and its virus-like particles (VLPs) is described. The assay was developed using baculovirus-expressed recombinant VP2 virus-like particles (rVP2-VLPs) as antigens and a monoclonal antibody against GPV as the competitive antibody. Of the four anti-GPV MAbs that were screened, MAb 1G3 was selected as it was blocked by the GPV positive serum. Based on the distribution of percent inhibition (PI) of the known negative sera (n=225), a cut-off value was set at 36% inhibition. Using this cut-off value, the sensitivity of the assay was 93.3% and the specificity was 95.8%, as compared with the gold standard (virus neutralization assay). The rVP2-VLPs did not react with anti-sera to other goose pathogens, indicating that it is specific for the recognization of goose parvovirus antibodies. The assay was then validated with serum samples from goslings vaccinated with several VLPs (rVP1-VLPs, rVP2-VLPs, rVP3-VLPs, and rCGV-VLPs) and other vaccines (inactivated and attenuated). The C-ELISA described in this study is a sensitive and specific diagnostic test and should have wide applications for the sero-diagnosis and immunologic surveillance of GPV. Copyright © 2014 Elsevier B.V. All rights reserved.

Anti-sphingosine-1-phosphate monoclonal antibodies inhibit angiogenesis and sub-retinal fibrosis in a murine model of laser-induced choroidal neovascularization

PubMed Central

Caballero, Sergio; Swaney, James; Moreno, Kelli; Afzal, Aqeela; Kielczewski, Jennifer; Stoller, Glenn; Cavalli, Amy; Garland, William; Hansen, Geneviève; Sabbadini, Roger; Grant, Maria B.

2013-01-01

The efficacy of novel monoclonal antibodies that neutralize the pro-angiogenic mediator, sphingosine-1-phosphate (S1P), were tested using in vitro and in vivo angiogenesis models, including choroidal neovascularization (CNV) induced by laser disruption of Bruch’s membrane. S1P receptor levels in human brain choroid plexus endothelial cells (CPEC), human lung microvascular endothelial cells, human retinal vascular endothelial cells, and circulating endothelial progenitor cells were examined by semi-quantitative PCR. The ability of murine or humanized anti-S1P monoclonal antibodies (mAbs) to inhibit S1P-mediated microvessel tube formation by CPEC on Matrigel was evaluated and capillary density in subcutaneous growth factor-loaded Matrigel plugs was determined following anti-S1P treatment. S1P promoted in vitro capillary tube formation in CPEC consistent with the presence of cognate S1P1–5 receptor expression by these cells and the S1P antibody induced a dose-dependent reduction in microvessel tube formation. In a murine model of laser-induced rupture of Bruch’s membrane, S1P was detected in posterior cups of mice receiving laser injury, but not in uninjured controls. Intravitreous injection of anti-S1P mAbs dramatically inhibited CNV formation and sub-retinal collagen deposition in all treatment groups (p < 0.05 compared to controls), thereby identifying S1P as a previously unrecognized mediator of angiogenesis and subretinal fibrosis in this model. These findings suggest that neutralizing S1P with anti-S1P mAbs may be a novel method of treating patients with exudative age-related macular degeneration by reducing angiogenesis and sub-retinal fibrosis, which are responsible for visual acuity loss in this disease. PMID:18723015

Preparation and biological activities of anti-HER2 monoclonal antibodies with fully core-fucosylated homogeneous bi-antennary complex-type glycans.

PubMed

Tsukimura, Wataru; Kurogochi, Masaki; Mori, Masako; Osumi, Kenji; Matsuda, Akio; Takegawa, Kaoru; Furukawa, Kiyoshi; Shirai, Takashi

2017-12-01

Recently, the absence of a core-fucose residue in the N-glycan has been implicated to be important for enhancing antibody-dependent cellular cytotoxicity (ADCC) activity of immunoglobulin G monoclonal antibodies (mAbs). Here, we first prepared anti-HER2 mAbs having two core-fucosylated N-glycan chains with the single G2F, G1aF, G1bF, or G0F structure, together with those having two N-glycan chains with a single non-core-fucosylated corresponding structure for comparison, and determined their biological activities. Dissociation constants of mAbs with core-fucosylated N-glycans bound to recombinant Fcγ-receptor type IIIa variant were 10 times higher than those with the non-core-fucosylated N-glycans, regardless of core glycan structures. mAbs with the core-fucosylated N-glycans had markedly reduced ADCC activities, while those with the non-core-fucosylated N-glycans had high activities. These results indicate that the presence of a core-fucose residue in the N-glycan suppresses the binding to the Fc-receptor and the induction of ADCC of anti-HER2 mAbs.

Bacillus anthracis Capsular Conjugates Elicit Chimpanzee Polyclonal Antibodies That Protect Mice from Pulmonary Anthrax.

PubMed

Chen, Zhaochun; Schneerson, Rachel; Lovchik, Julie A; Dai, Zhongdong; Kubler-Kielb, Joanna; Agulto, Liane; Leppla, Stephen H; Purcell, Robert H

2015-08-01

The immunogenicity of Bacillus anthracis capsule (poly-γ-D-glutamic acid [PGA]) conjugated to recombinant B. anthracis protective antigen (rPA) or to tetanus toxoid (TT) was evaluated in two anthrax-naive juvenile chimpanzees. In a previous study of these conjugates, highly protective monoclonal antibodies (MAbs) against PGA were generated. This study examines the polyclonal antibody response of the same animals. Preimmune antibodies to PGA with titers of >10(3) were detected in the chimpanzees. The maximal titer of anti-PGA was induced within 1 to 2 weeks following the 1st immunization, with no booster effects following the 2nd and 3rd immunizations. Thus, the anti-PGA response in the chimpanzees resembled a secondary immune response. Screening of sera from nine unimmunized chimpanzees and six humans revealed antibodies to PGA in all samples, with an average titer of 10(3). An anti-PA response was also observed following immunization with PGA-rPA conjugate, similar to that seen following immunization with rPA alone. However, in contrast to anti-PGA, preimmune anti-PA antibody titers and those following the 1st immunization were ≤300, with the antibodies peaking above 10(4) following the 2nd immunization. The polyclonal anti-PGA shared the MAb 11D epitope and, similar to the MAbs, exerted opsonophagocytic killing of B. anthracis. Most important, the PGA-TT-induced antibodies protected mice from a lethal challenge with virulent B. anthracis spores. Our data support the use of PGA conjugates, especially PGA-rPA targeting both toxin and capsule, as expanded-spectrum anthrax vaccines. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Production of polyclonal and monoclonal antibodies against group A, B, and C capsular polysaccharides of Neisseria meningitidis and preparation of latex reagents.

PubMed Central

Nato, F; Mazie, J C; Fournier, J M; Slizewicz, B; Sagot, N; Guibourdenche, M; Postic, D; Riou, J Y

1991-01-01

Polyclonal and monoclonal antibodies against capsular polysaccharides of Neisseria meningitidis serogroups A, B, and C were produced in order to develop immunological reagents allowing both the detection of soluble antigens during meningococcal meningitis and antigenic serogrouping of N. meningitidis cultures. The performance characteristics of monoclonal and polyclonal antibody latex reagents were compared. For the detection of soluble polysaccharide antigen, polyclonal antibody latex reagent was selected for N. meningitidis A and C. The latex reagent prepared with polyclonal antibodies against N. meningitidis B could not detect capsular polysaccharide even at 1 mg/ml. The monoclonal antibody B latex reagent which detected 100 ng of polysaccharide per ml was therefore chosen. For the serogroup identification of N. meningitidis, the use of a confirmatory test results in an overall specificity of 100% with polyclonal or monoclonal antibody latex reagents. PMID:1909346

An ultra-sensitive monoclonal antibody-based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk

USDA-ARS?s Scientific Manuscript database

A rapid lateral flow fluorescent microspheres immunochromatography test strip (FMs-ICTS) has been developed for the detection of aflatoxin M1 (AFM1) residues in milk. For this purpose, an ultra-sensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC50 value of the MA...

A human anti-dsDNA monoclonal antibody caused hyaline thrombi formation in kidneys of ‘leaky’ SCID mice

PubMed Central

Mason, L J; Ravirajan, C T; Latchman, D S; Isenberg, D A

2001-01-01

There are few studies assessing the pathogenicity of human monoclonal anti-DNA antibodies. The use of SCID mice avoids the problem of rejection of the human hybridoma cells thus allowing in vivo assessment of human immunoglobulins. Using electron microscopy we have shown that the human IgG anti-dsDNA monoclonal antibody, RH14, is nephritogenic in SCID mice, causing morphological changes in the kidney due to immunoglobulin deposition. The problem with using SCID mice is that they have an abnormal immune system; normally they are used at about 2 months of age, at which time they have virtually no functional T or B cells. It is known that older SCID mice become increasingly ‘leaky’, that is they develop some mature lymphocyte clones. Our aim was to assess if implanting anti-DNA antibodies into older ‘leaky’ SCID mice would result in pathology which was observable by light microscopy. Eight-month-old SCID mice were implanted with human hybridoma cells secreting either RH14 an anti-dsDNA IgG, CL24, an antiphospholipid antibody or an irrelevant human IgG control. As previously, RH14 deposited in the kidney and caused proteinuria but unexpectedly we also observed hyaline thrombi in the kidney glomeruli and peritubular capillaries. These thrombi occurred only in the case of RH14 implanted mice and were found to stain positively for human IgG and fibrin. However, apart from the interesting thrombi, we did not observe any greater pathological damage resulting from the anti-dsDNA antibody deposition than we had seen in the younger mice; indeed, the electron microscopic findings were more limited. PMID:11678910

Pathogenesis of systemic inflammatory diseases in childhood: "Lessons from clinical trials of anti-cytokine monoclonal antibodies for Kawasaki disease, systemic onset juvenile idiopathic arthritis, and cryopyrin-associated periodic fever syndrome".

PubMed

Yokota, Shumpei; Kikuchi, Masako; Nozawa, Tomo; Kanetaka, Taichi; Sato, Tomomi; Yamazaki, Kazuko; Sakurai, Nodoka; Hara, Ryoki; Mori, Masaaki

2015-01-01

Inflammation has often been considered to be a nonspecific response and to play a bridging role in the activation of adaptive immunity. However, it is now accepted that inflammation is the product of an independent innate immune system closely linked to the adaptive immune system. The key mediators of inflammation are inflammatory cytokines, as determined by multiple lines of evidence both in vitro and in vivo. Due to the crucial role of inflammatory cytokines in the pathogenesis of autoimmune disorders, anti-cytokine treatment has been developed as a therapy for rheumatoid arthritis, juvenile idiopathic arthritis (JIA), and inflammatory bowel diseases. We recently completed several clinical trials of anti-cytokine treatment for children with systemic inflammatory diseases: anti-IL-6 receptor monoclonal antibody (tocilizumab) for children with two subtypes of JIA (poly-JIA and systemic JIA), anti-TNF-α monoclonal antibody (infliximab) for children with Kawasaki disease, and anti-IL-1-β monoclonal antibody (canakinumab) for children with cryopyrin-associated periodic syndrome. This review summarizes the basis of inflammation in terms of innate immunity and adaptive immunity in these systemic inflammatory diseases, clinical efficacy, and tolerability of these biologic agents, and attempts to determine the roles of individual inflammatory cytokines in disease pathogenesis.

Ecobody technology: rapid monoclonal antibody screening method from single B cells using cell-free protein synthesis for antigen-binding fragment formation.

PubMed

Ojima-Kato, Teruyo; Nagai, Satomi; Nakano, Hideo

2017-10-25

We report a rapid and cost-effective monoclonal antibody screening method from single animal B cells using reverse transcription (RT)-PCR and Escherichia coli cell-free protein synthesis (CFPS), which allows evaluation of antibodies within 2 working days. This process is named "Ecobody technology". The method includes strategies to isolate B cells that specifically bind an antigen from the peripheral blood of immunised animals, and single-cell RT-PCR to generate DNA fragments of the V H and V L genes, followed by CFPS for production of fragments of antigen binding (Fab). In the CFPS step, we employed our techniques: 1) 'Zipbody' as a method for producing Fab, in which the association of heavy and light chains is facilitated by adhesive leucine zipper peptides fused at the C-termini of the Fab; and 2) an N-terminal SKIK peptide tag that can increase protein expression levels. Using Ecobody technology, we obtained highly-specific monoclonal antibodies for the antigens Vibrio parahaemolyticus and E. coli O26. The anti-V. parahaemolyticus Zipbody mAb was further produced in E. coli strain SHuffle T7 Express in inclusion bodies and refolded by a conventional method, resulting in significant antigen-binding activity (K D  = 469 pM) and productivity of 8.5 mg purified antibody/L-culture.

New Agents for Taxol-Resistant Breast Adenocarcinoma

DTIC Science & Technology

2004-07-01

mg/ mi2 (14) using 3 hr infusion schedules failed to demonstrate marked improvement in disease response or survival, despite incurring severe sensory...analyzed immediately by flow cytometry. Immunoblotting The anti-human HER-2/neu mouse monoclonal antibody , clone 3B5, isotype IgGI, was obtained from...carboxyl domain of the human c-neu gene product. An affinity-purified sheep anti-mouse IgG whole antibody preparation conjugated to horseradish peroxidase

Electrophoretically deposited reduced graphene oxide platform for food toxin detection

NASA Astrophysics Data System (ADS)

Srivastava, Saurabh; Kumar, Vinod; Ali, Md Azahar; Solanki, Pratima R.; Srivastava, Anchal; Sumana, Gajjala; Saxena, Preeti Suman; Joshi, Amish G.; Malhotra, B. D.

2013-03-01

Reduced graphene oxide (RGO) due to its excellent electrochemical properties and large surface area, has recently aroused much interest for electrochemical biosensing application. Here, the chemically active RGO has been synthesized and deposited onto an indium tin oxide (ITO) coated glass substrate by the electrophoretic deposition technique. This novel platform has been utilized for covalent attachment of the monoclonal antibodies of aflatoxin B1 (anti-AFB1) for food toxin (AFB1) detection. The electron microscopy, X-ray diffraction, and UV-visible studies reveal successful synthesis of reduced graphene oxide while the XPS and FTIR studies suggest its carboxylic functionalized nature. The electrochemical sensing results of the anti-AFB1/RGO/ITO based immunoelectrode obtained as a function of aflatoxin concentration show high sensitivity (68 μA ng-1 mL cm-2) and improved detection limit (0.12 ng mL-1). The association constant (ka) for antigen-antibody interaction obtained as 5 × 10-4 ng mL-1 indicates high affinity of antibodies toward the antigen (AFB1).Reduced graphene oxide (RGO) due to its excellent electrochemical properties and large surface area, has recently aroused much interest for electrochemical biosensing application. Here, the chemically active RGO has been synthesized and deposited onto an indium tin oxide (ITO) coated glass substrate by the electrophoretic deposition technique. This novel platform has been utilized for covalent attachment of the monoclonal antibodies of aflatoxin B1 (anti-AFB1) for food toxin (AFB1) detection. The electron microscopy, X-ray diffraction, and UV-visible studies reveal successful synthesis of reduced graphene oxide while the XPS and FTIR studies suggest its carboxylic functionalized nature. The electrochemical sensing results of the anti-AFB1/RGO/ITO based immunoelectrode obtained as a function of aflatoxin concentration show high sensitivity (68 μA ng-1 mL cm-2) and improved detection limit (0.12 ng mL-1). The association constant (ka) for antigen-antibody interaction obtained as 5 × 10-4 ng mL-1 indicates high affinity of antibodies toward the antigen (AFB1). Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr32242d

Active immunotherapy with anti-idiotypic antibody for patients with nasopharyngeal carcinoma (NPC).

PubMed

Li, Guancheng; Xie, Lu; Zhou, Guohua; Zhu, Jiangao; Hu, Jinyue; Sun, Qubing

2002-12-01

Two anti-idiotypic monoclonal antibodies (Ab2), designated 2H4 and 5D3, against two antitumor antibodies Ab1 (FC2 and HNL5) that recognize nasopharyngeal carcinoma (NPC) associated antigen were generated. They could substitute NPC antigen to induce humoral and cellular immune response against NPC cells in syngeneic mice. Nineteen patients with NPC at stage IV were chosen for active immunotherapy. They were treated with aluminum hydroxide-precipitated Ab2 2H4 or 5D3 accompanying radiotherapy. None of the immunization of anti-idiotypic monoclonal antibody (mAb) was associated with toxicity or allergies reactions. Nine patients with radiotherapy alone served as control. Both anti-anti-idiotypic antibodies (Ab3) and anti-NPC antibodies (Ab1') were increased and human anti-mouse Ig antibodies (HAMA) occurred in nineteen patients of the experimental group; whereas the levels of Ab1' did not rise in the control group. Serum IL-2, IFN-gamma, and TNF-alpha levels were increased in most patients in the experimental group, while in the control group, there were no differences of Ab1' and cytokine level between pretherapy and posttherapy. In addition, IL-2 mRNA expression in peripheral blood mononuclear cells (PBMC) of NPC patients was closely related to serum IL-2 (r = +0.8829) by in situ hybridization. Therefore, mouse anti-idiotypic antibodies 2H4 and 5D3 are safe for active immunotherapy and might enhance humoral and/or cellular immunity of NPC patients receiving radiotherapy.

Local convection-enhanced delivery of an anti-CD40 agonistic monoclonal antibody induces antitumor effects in mouse glioma models

PubMed Central

Shoji, Takuhiro; Saito, Ryuta; Chonan, Masashi; Shibahara, Ichiyo; Sato, Aya; Kanamori, Masayuki; Sonoda, Yukihiko; Kondo, Toru; Ishii, Naoto; Tominaga, Teiji

2016-01-01

Background Glioblastoma is one of the most malignant brain tumors in adults and has a dismal prognosis. In a previous report, we reported that CD40, a TNF-R-related cell surface receptor, and its ligand CD40L were associated with glioma outcomes. Here we attempted to activate CD40 signaling in the tumor and determine if it exerted therapeutic efficacy. Methods CD40 expression was examined in 3 mouse glioma cell lines (GL261, NSCL61, and bRiTs-G3) and 5 human glioma cell lines (U87, U251, U373, T98, and A172). NSCL61 and bRiTs-G3, as glioma stem cells, also expressed the glioma stem cell markers MELK and CD44. In vitro, we demonstrated direct antitumor effects of an anti-CD40 agonistic monoclonal antibody (FGK45) against the cell lines. The efficacy of FGK45 was examined by local convection-enhanced delivery of the monoclonal antibody against each glioma model. Results CD40 was expressed in all mouse and human cell lines tested and was found at the cell membrane of each of the 3 mouse cell lines. FGK45 administration induced significant, direct antitumor effects in vitro. The local delivery of FGK45 significantly prolonged survival compared with controls in the NSCL61 and bRiTs-G3 models, but the effect was not significant in the GL261 model. Increases in apoptosis and CD4+ and CD8+ T cell infiltration were observed in the bRiTs-G3 model after FGK45 treatment. Conclusions Local delivery of FGK45 significantly prolonged survival in glioma stem cell models. Thus, local delivery of this monoclonal antibody is promising for immunotherapy against gliomas. PMID:26917236

Analysis of the 3D structure og agglutinated erythrocyte using CellScan and confocal microscopy: characterization by FLIM-FRET

NASA Astrophysics Data System (ADS)

Riquelme, Bibiana D.; Dumas, Dominique; Valverde de Rasia, Juana; Rasia, Rodolfo J.; Stoltz, Jean Francois

2003-10-01

We report the adhesion of human erythrocyte membranes mediated by monoclonal antibodies anti-glycophorin. The distribution of the linked antibodies on membrane was identified with selective fluorescence labels. To analyze the antibody distribution on interfacial region between two cells agglutinated and on its surface, three types of fluorescence marked strategy were evaluated. The 3D images were obtained in a CellScan and Confocal Laser Scanning Microscopy CLSM. We considered the FRET signal to characterize the agglutination of Red Blood Cells (RBC) by specific monoclonal antibodies (anti-glycophorin A or B). The fluorescence labeling demonstrated that distribution of antibody on erythrocyte membranes is not homogeneous. The fluorescence intensity on contact region in the agglutinated is bigger than the intensity on exterior surface. Tentatively, we interpreted these intensity differences in terms of the mobility of antibody linked to the glycocalix on cell surface. Such mobility has a large consequence in the morphology of cellular agglutinated.

An antagonistic monoclonal antibody (B-N6) specific for the human neurotensin receptor-1.

PubMed

Ovigne, J M; Vermot-Desroches, C; Lecron, J C; Portier, M; Lupker, J; Pecceu, F; Wijdenes, J

1998-06-01

The neuropeptide neurotensin (NT) interacts with two types of human receptors (hNTR) termed hNTR-1 and hNTR-2. This study describes a monoclonal antibody (MAb) specific for hNTR-1, B-N6. This MAb binds specifically to hNTR-1, but not to hNTR-2 transfected CHO cells. B-N6 and NT display a reciprocal competition and react in a similar way to trypsin, suggesting that the B-N6 epitope is at or close to the NT binding site on the third extracellular loop. Unlike B-N6, NT induces hNTR-1 internalization. Although neither NT-FITC nor B-N6 binding was detected by flow cytometry on different human cells, specific mRNA expression for hNTR-1 was detected in these cells. In CHO cells expressing hNTR-1 and a luciferase gene coupled to the krox24 reporter, B-N6 and the antagonist SR 48692 inhibited NT-induced intracellular activation of krox24 in a dose-dependent manner. From these results it is concluded that B-N6 is an antagonistic anti-hNTR-1 MAb.

Purification of HBsAg produced by the human hepatoma cell line PLC/PRE/5 by affinity chromatography using monoclonal antibodies and application for ELISA diagnostic.

PubMed

Merten, O W; Reiter, S; Scheirer, W; Katinger, H

1983-01-01

The human cell line PLC/PRF/5 (5) was used for the production of hepatitis B surface antigen subtype ad (HBsAg ad) and purified by affinity chromatography (AC) with monoclonal antibodies (mAb). mAb to HBsAg from mouse ascites have been purified by Protein A - AC prior coupling to AH-Sepharose 4B (Pharmacia). The combined procedure of ammonium-sulphate-precipitation of HBsAg from culture supernatants and immunosorbent-AC leads to approx. 700-fold purification. ELISA results using the mAb and the HBsAg for diagnostics of human serum, positive for anti-HBsAg-antibodies correlate with the RIA (AUSAB, Abbott).

Detection of antibodies reacting with the antithetical duffy blood group antigens Fy(a) and Fy(b) using recombinant fusion proteins containing the duffy extracellular domain.

PubMed

Sheffield, William P; Bhakta, Varsha; Branch, Donald R; Denomme, Gregory A

2006-12-01

Detecting blood group-specific antibodies in patient sera is essential to the management of blood transfusions or pregnancies. We produced the antithetical forms of the 65 amino acid extracellular domain (ECD) of the Duffy (Fy) blood group protein fused to glutathione sulfotransferase (GST): GST-Fy(a); and GST-Fy(b), differing only in Gly or Asp at position 44, respectively. The purified recombinant proteins were recognized more effectively by reference polyclonal or monoclonal antibodies than the antithetical Fy specificity by either ELISA or immunoblotting. Combined immunoblot and ELISA tests performed at 1:200 dilutions of sera using the recombinant proteins gave results in agreement with undiluted sera and agglutination for 17/19 alloimmunized patients. At 1:200, agglutination detected anti-Fy(a) or anti-Fy(b) in only three of 12 samples that were positive by ELISA. Recombinant ECD-Fy proteins are suitable and sensitive reagents for the detection of anti-Fy that use technology amenable to automation and/or miniaturization and avoid the need for intact red cells.

AFM, CLSM and EIS characterization of the immobilization of antibodies on indium-tin oxide electrode and their capture of Legionella pneumophila.

PubMed

Souiri, Mina; Blel, Nesrine; Sboui, Dejla; Mhamdi, Lotfi; Epalle, Thibaut; Mzoughi, Ridha; Riffard, Serge; Othmane, Ali

2014-01-01

The microscopic surface molecular structures and properties of monoclonal anti-Legionella pneumophila antibodies on an indium-tin oxide (ITO) electrode surface were studied to elaborate an electrochemical immunosensor for Legionella pneumophila detection. A monoclonal anti-Legionella pneumophila antibody (MAb) has been immobilized onto an ITO electrode via covalent chemical bonds between antibodies amino-group and the ring of (3-Glycidoxypropyl) trimethoxysilane (GPTMS). The functionalization of the immunosensor was characterized by atomic force microscopy (AFM), water contact angle measurement, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN)₆](3-/4-) as a redox probe. Specific binding of Legionella pneumophila sgp 1 cells onto the antibody-modified ITO electrode was shown by confocal laser scanning microscopy (CLSM) imaging and EIS. AFM images evidenced the dense and relatively homogeneous morphology on the ITO surface. The formation of the complex epoxysilane-antibodies acting as barriers for the electron transfer between the electrode surface and the redox species in the solution induced a significant increase in the charge transfer resistance (Rct) compared to all the electric elements. A linear relationship between the change in charge transfer resistance (ΔRct=Rct after immunoreactions - Rct control) and the logarithmic concentration value of L. pneumophila was observed in the range of 5 × 10(1)-5 × 10(4) CFU mL(-1) with a limit of detection 5 × 10(1)CFU mL(-1). The present study has demonstrated the successful deposition of an anti-L. pneumophila antibodies on an indium-tin oxide surface, opening its subsequent use as immuno-captor for the specific detection of L. pneumophila in environmental samples. © 2013 Elsevier B.V. All rights reserved.

Rapid detection of Bacillus anthracis using monoclonal antibody functionalized QCM sensor.

PubMed

Hao, Rongzhang; Wang, Dianbing; Zhang, Xian'en; Zuo, Guomin; Wei, Hongping; Yang, Ruifu; Zhang, Zhiping; Cheng, Zhenxing; Guo, Yongchao; Cui, Zongqiang; Zhou, Yafeng

2009-01-01

Since the anthrax spore bioterrorism attacks in America in 2001, the early detection of Bacillus anthracis spores and vegetative cells has gained significant interest. At present, many polyclonal antibody-based quartz crystal microbalance (QCM) sensors have been developed to detect B. anthracis simulates. To achieve a simultaneous rapid detection of B. anthracis spores and vegetative cells, this paper presents a biosensor that utilizes an anti-B. anthracis monoclonal antibody designated to 8G3 (mAb 8G3, IgG) functionalized QCM sensor. Having compared four kinds of antibody immobilizations on Au surface, an optimized mAb 8G3 was immobilized onto the Au electrode with protein A on a mixed self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (11-MUA) and 6-mercaptohexan-1-ol (6-MHO) as adhesive layer. The detection of B. anthracis was investigated under three conditions: dip-and-dry, static addition and flow through procedure. The results indicated that the sensor yielded a distinct response to B. anthracis spores or vegetative cells but had no significant response to Bacillus thuringiensis species. The functionalized sensor recognized B. anthracis spores and vegetative cells specifically from its homophylic ones, and the limit of detection (LOD) reached 10(3)CFU or spores/ml of B. anthracis in less than 30 min. Cyclic voltammogram (CV) and scanning electronic microscopy (SEM) were performed to characterize the surface of the sensor in variable steps during the modification and after the detection. The mAb functionalized QCM biosensor will be helpful in the fabrication of a similar biosensor that may be available in anti-bioterrorism in the future.

Confirmation of a new conserved linear epitope of Lyssavirus nucleoprotein.

PubMed

Xinjun, Lv; Xuejun, Ma; Lihua, Wang; Hao, Li; Xinxin, Shen; Pengcheng, Yu; Qing, Tang; Guodong, Liang

2012-05-01

Bioinformatics analysis was used to predict potential epitopes of Lyssavirus nucleoprotein and highlighted some distinct differences in the quantity and localization of the epitopes disclosed by epitope analysis of monoclonal antibodies against Lyssavirus nucleoprotein. Bioinformatics analysis showed that the domain containing residues 152-164 of Lyssavirus nucleoprotein was a conserved linear epitope that had not been reported previously. Immunization of two rabbits with the corresponding synthetic peptide conjugated to the Keyhole Limpe hemocyanin (KLH) macromolecule resulted in a titer of anti-peptide antibody above 1:200,000 in rabbit sera as detected by indirect enzyme-linked immunosorbent assay (ELISA). Western blot analysis demonstrated that the anti-peptide antibody recognized denatured Lyssavirus nucleoprotein in sodium dodecylsulfonate-polyacrylate gel electrophoresis (SDS-PAGE). Affinity chromatography purification and FITC-labeling of the anti-peptide antibody in rabbit sera was performed. FITC-labeled anti-peptide antibody could recognize Lyssavirus nucleoprotein in BSR cells and canine brain tissues even at a 1:200 dilution. Residues 152-164 of Lyssavirus nucleoprotein were verified as a conserved linear epitope in Lyssavirus. Copyright © 2012 Elsevier B.V. All rights reserved.

Large Scale Generation and Characterization of Anti-Human CD34 Monoclonal Antibody in Ascetic Fluid of Balb/c Mice

PubMed Central

Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Abdolalizadeh, Jalal; Kazemi, Tohid; Aghebati Maleki, Ali; Sineh sepehr, Koushan

2013-01-01

Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC. Results: Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa. Conclusion: The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells. PMID:24312838

Emerging immunotherapy and strategies directly targeting B cells for the treatment of diffuse large B-cell lymphoma.

PubMed

Witkowska, Magdalena; Smolewski, Piotr

2015-01-01

During the last decade, significant prolonged survival in diffusive large B-cell lymphoma (DLBCL) has been observed. The efficacy of initial treatment improved mostly due to addition of a chimeric anti-CD20 monoclonal antibody (rituximab) to standard chemotherapeutic regimens. Moreover, accurate understanding of DLBCL pathogenesis and remarkable progress in gene expression profiling have led to the development of a variety of tumor-specific regimens. Novel agents target directly the pathways involved in signal transduction, lead to apoptosis and cancer cells differentiation. In this article, we mainly focus on new treatment options, such as monoclonal antibodies, tyrosine kinase inhibitors and immunomodulatory drugs, currently investigated in aggressive B-cell lymphoma with particular attention to DLBCL type.

Characterization of chimpanzee/human monoclonal antibodies to vaccinia virus A33 glycoprotein and its variola virus homolog in vitro and in a vaccinia virus mouse protection model.

PubMed

Chen, Zhaochun; Earl, Patricia; Americo, Jeffrey; Damon, Inger; Smith, Scott K; Yu, Fujuan; Sebrell, Andrew; Emerson, Suzanne; Cohen, Gary; Eisenberg, Roselyn J; Gorshkova, Inna; Schuck, Peter; Satterfield, William; Moss, Bernard; Purcell, Robert

2007-09-01

Three distinct chimpanzee Fabs against the A33 envelope glycoprotein of vaccinia virus were isolated and converted into complete monoclonal antibodies (MAbs) with human gamma 1 heavy-chain constant regions. The three MAbs (6C, 12C, and 12F) displayed high binding affinities to A33 (K(d) of 0.14 nM to 20 nM) and may recognize the same epitope, which was determined to be conformational and located within amino acid residues 99 to 185 at the C terminus of A33. One or more of the MAbs were shown to reduce the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro and to more effectively protect mice when administered before or 2 days after intranasal challenge with virulent vaccinia virus than a previously isolated mouse anti-A33 MAb (1G10) or vaccinia virus immunoglobulin. The protective efficacy afforded by anti-A33 MAb was comparable to that of a previously isolated chimpanzee/human anti-B5 MAb. The combination of anti-A33 MAb and anti-B5 MAb did not synergize the protective efficacy. These chimpanzee/human anti-A33 MAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox and other orthopoxvirus diseases.

Investigation of the immunogenicity of different types of aggregates of a murine monoclonal antibody in mice.

PubMed

Freitag, Angelika J; Shomali, Maliheh; Michalakis, Stylianos; Biel, Martin; Siedler, Michael; Kaymakcalan, Zehra; Carpenter, John F; Randolph, Theodore W; Winter, Gerhard; Engert, Julia

2015-02-01

The potential contribution of protein aggregates to the unwanted immunogenicity of protein pharmaceuticals is a major concern. In the present study a murine monoclonal antibody was utilized to study the immunogenicity of different types of aggregates in mice. Samples containing defined types of aggregates were prepared by processes such as stirring, agitation, exposure to ultraviolet (UV) light and exposure to elevated temperatures. Aggregates were analyzed by size-exclusion chromatography, light obscuration, turbidimetry, infrared (IR) spectroscopy and UV spectroscopy. Samples were separated into fractions based on aggregate size by asymmetrical flow field-flow fractionation or by centrifugation. Samples containing different types and sizes of aggregates were subsequently administered to C57BL/6 J and BALB/c mice, and serum was analyzed for the presence of anti-IgG1, anti-IgG2a, anti-IgG2b and anti-IgG3 antibodies. In addition, the pharmacokinetic profile of the murine antibody was investigated. In this study, samples containing high numbers of different types of aggregates were administered in order to challenge the in vivo system. The magnitude of immune response depends on the nature of the aggregates. The most immunogenic aggregates were of relatively large and insoluble nature, with perturbed, non-native structures. This study shows that not all protein drug aggregates are equally immunogenic.

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

PubMed Central

Batsuli, Glaivy; Deng, Wei; Healey, John F.; Parker, Ernest T.; Baldwin, W. Hunter; Cox, Courtney; Nguyen, Brenda; Kahle, Joerg; Königs, Christoph; Li, Renhao; Lollar, Pete

2016-01-01

Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance. PMID:27381905

3B11-N, a monoclonal antibody against MERS-CoV, reduces lung pathology in rhesus monkeys following intratracheal inoculation of MERS-CoV Jordan-n3/2012.

PubMed

Johnson, Reed F; Bagci, Ulas; Keith, Lauren; Tang, Xianchun; Mollura, Daniel J; Zeitlin, Larry; Qin, Jing; Huzella, Louis; Bartos, Christopher J; Bohorova, Natasha; Bohorov, Ognian; Goodman, Charles; Kim, Do H; Paulty, Michael H; Velasco, Jesus; Whaley, Kevin J; Johnson, Joshua C; Pettitt, James; Ork, Britini L; Solomon, Jeffrey; Oberlander, Nicholas; Zhu, Quan; Sun, Jiusong; Holbrook, Michael R; Olinger, Gene G; Baric, Ralph S; Hensley, Lisa E; Jahrling, Peter B; Marasco, Wayne A

2016-03-01

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was identified in 2012 as the causative agent of a severe, lethal respiratory disease occurring across several countries in the Middle East. To date there have been over 1600 laboratory confirmed cases of MERS-CoV in 26 countries with a case fatality rate of 36%. Given the endemic region, it is possible that MERS-CoV could spread during the annual Hajj pilgrimage, necessitating countermeasure development. In this report, we describe the clinical and radiographic changes of rhesus monkeys following infection with 5×10(6) PFU MERS-CoV Jordan-n3/2012. Two groups of NHPs were treated with either a human anti-MERS monoclonal antibody 3B11-N or E410-N, an anti-HIV antibody. MERS-CoV Jordan-n3/2012 infection resulted in quantifiable changes by computed tomography, but limited other clinical signs of disease. 3B11-N treated subjects developed significantly reduced lung pathology when compared to infected, untreated subjects, indicating that this antibody may be a suitable MERS-CoV treatment. Published by Elsevier Inc.

Systemic treatments for metastatic cutaneous melanoma.

PubMed

Pasquali, Sandro; Hadjinicolaou, Andreas V; Chiarion Sileni, Vanna; Rossi, Carlo Riccardo; Mocellin, Simone

2018-02-06

The prognosis of people with metastatic cutaneous melanoma, a skin cancer, is generally poor. Recently, new classes of drugs (e.g. immune checkpoint inhibitors and small-molecule targeted drugs) have significantly improved patient prognosis, which has drastically changed the landscape of melanoma therapeutic management. This is an update of a Cochrane Review published in 2000. To assess the beneficial and harmful effects of systemic treatments for metastatic cutaneous melanoma. We searched the following databases up to October 2017: the Cochrane Skin Group Specialised Register, CENTRAL, MEDLINE, Embase and LILACS. We also searched five trials registers and the ASCO database in February 2017, and checked the reference lists of included studies for further references to relevant randomised controlled trials (RCTs). We considered RCTs of systemic therapies for people with unresectable lymph node metastasis and distant metastatic cutaneous melanoma compared to any other treatment. We checked the reference lists of selected articles to identify further references to relevant trials. Two review authors extracted data, and a third review author independently verified extracted data. We implemented a network meta-analysis approach to make indirect comparisons and rank treatments according to their effectiveness (as measured by the impact on survival) and harm (as measured by occurrence of high-grade toxicity). The same two review authors independently assessed the risk of bias of eligible studies according to Cochrane standards and assessed evidence quality based on the GRADE criteria. We included 122 RCTs (28,561 participants). Of these, 83 RCTs, encompassing 21 different comparisons, were included in meta-analyses. Included participants were men and women with a mean age of 57.5 years who were recruited from hospital settings. Twenty-nine studies included people whose cancer had spread to their brains. Interventions were categorised into five groups: conventional chemotherapy (including single agent and polychemotherapy), biochemotherapy (combining chemotherapy with cytokines such as interleukin-2 and interferon-alpha), immune checkpoint inhibitors (such as anti-CTLA4 and anti-PD1 monoclonal antibodies), small-molecule targeted drugs used for melanomas with specific gene changes (such as BRAF inhibitors and MEK inhibitors), and other agents (such as anti-angiogenic drugs). Most interventions were compared with chemotherapy. In many cases, trials were sponsored by pharmaceutical companies producing the tested drug: this was especially true for new classes of drugs, such as immune checkpoint inhibitors and small-molecule targeted drugs.When compared to single agent chemotherapy, the combination of multiple chemotherapeutic agents (polychemotherapy) did not translate into significantly better survival (overall survival: HR 0.99, 95% CI 0.85 to 1.16, 6 studies, 594 participants; high-quality evidence; progression-free survival: HR 1.07, 95% CI 0.91 to 1.25, 5 studies, 398 participants; high-quality evidence. Those who received combined treatment are probably burdened by higher toxicity rates (RR 1.97, 95% CI 1.44 to 2.71, 3 studies, 390 participants; moderate-quality evidence). (We defined toxicity as the occurrence of grade 3 (G3) or higher adverse events according to the World Health Organization scale.)Compared to chemotherapy, biochemotherapy (chemotherapy combined with both interferon-alpha and interleukin-2) improved progression-free survival (HR 0.90, 95% CI 0.83 to 0.99, 6 studies, 964 participants; high-quality evidence), but did not significantly improve overall survival (HR 0.94, 95% CI 0.84 to 1.06, 7 studies, 1317 participants; high-quality evidence). Biochemotherapy had higher toxicity rates (RR 1.35, 95% CI 1.14 to 1.61, 2 studies, 631 participants; high-quality evidence).With regard to immune checkpoint inhibitors, anti-CTLA4 monoclonal antibodies plus chemotherapy probably increased the chance of progression-free survival compared to chemotherapy alone (HR 0.76, 95% CI 0.63 to 0.92, 1 study, 502 participants; moderate-quality evidence), but may not significantly improve overall survival (HR 0.81, 95% CI 0.65 to 1.01, 2 studies, 1157 participants; low-quality evidence). Compared to chemotherapy alone, anti-CTLA4 monoclonal antibodies is likely to be associated with higher toxicity rates (RR 1.69, 95% CI 1.19 to 2.42, 2 studies, 1142 participants; moderate-quality evidence).Compared to chemotherapy, anti-PD1 monoclonal antibodies (immune checkpoint inhibitors) improved overall survival (HR 0.42, 95% CI 0.37 to 0.48, 1 study, 418 participants; high-quality evidence) and probably improved progression-free survival (HR 0.49, 95% CI 0.39 to 0.61, 2 studies, 957 participants; moderate-quality evidence). Anti-PD1 monoclonal antibodies may also result in less toxicity than chemotherapy (RR 0.55, 95% CI 0.31 to 0.97, 3 studies, 1360 participants; low-quality evidence).Anti-PD1 monoclonal antibodies performed better than anti-CTLA4 monoclonal antibodies in terms of overall survival (HR 0.63, 95% CI 0.60 to 0.66, 1 study, 764 participants; high-quality evidence) and progression-free survival (HR 0.54, 95% CI 0.50 to 0.60, 2 studies, 1465 participants; high-quality evidence). Anti-PD1 monoclonal antibodies may result in better toxicity outcomes than anti-CTLA4 monoclonal antibodies (RR 0.70, 95% CI 0.54 to 0.91, 2 studies, 1465 participants; low-quality evidence).Compared to anti-CTLA4 monoclonal antibodies alone, the combination of anti-CTLA4 plus anti-PD1 monoclonal antibodies was associated with better progression-free survival (HR 0.40, 95% CI 0.35 to 0.46, 2 studies, 738 participants; high-quality evidence). There may be no significant difference in toxicity outcomes (RR 1.57, 95% CI 0.85 to 2.92, 2 studies, 764 participants; low-quality evidence) (no data for overall survival were available).The class of small-molecule targeted drugs, BRAF inhibitors (which are active exclusively against BRAF-mutated melanoma), performed better than chemotherapy in terms of overall survival (HR 0.40, 95% CI 0.28 to 0.57, 2 studies, 925 participants; high-quality evidence) and progression-free survival (HR 0.27, 95% CI 0.21 to 0.34, 2 studies, 925 participants; high-quality evidence), and there may be no significant difference in toxicity (RR 1.27, 95% CI 0.48 to 3.33, 2 studies, 408 participants; low-quality evidence).Compared to chemotherapy, MEK inhibitors (which are active exclusively against BRAF-mutated melanoma) may not significantly improve overall survival (HR 0.85, 95% CI 0.58 to 1.25, 3 studies, 496 participants; low-quality evidence), but they probably lead to better progression-free survival (HR 0.58, 95% CI 0.42 to 0.80, 3 studies, 496 participants; moderate-quality evidence). However, MEK inhibitors probably have higher toxicity rates (RR 1.61, 95% CI 1.08 to 2.41, 1 study, 91 participants; moderate-quality evidence).Compared to BRAF inhibitors, the combination of BRAF plus MEK inhibitors was associated with better overall survival (HR 0.70, 95% CI 0.59 to 0.82, 4 studies, 1784 participants; high-quality evidence). BRAF plus MEK inhibitors was also probably better in terms of progression-free survival (HR 0.56, 95% CI 0.44 to 0.71, 4 studies, 1784 participants; moderate-quality evidence), and there appears likely to be no significant difference in toxicity (RR 1.01, 95% CI 0.85 to 1.20, 4 studies, 1774 participants; moderate-quality evidence).Compared to chemotherapy, the combination of chemotherapy plus anti-angiogenic drugs was probably associated with better overall survival (HR 0.60, 95% CI 0.45 to 0.81; moderate-quality evidence) and progression-free survival (HR 0.69, 95% CI 0.52 to 0.92; moderate-quality evidence). There may be no difference in terms of toxicity (RR 0.68, 95% CI 0.09 to 5.32; low-quality evidence). All results for this comparison were based on 324 participants from 2 studies.Network meta-analysis focused on chemotherapy as the common comparator and currently approved treatments for which high- to moderate-quality evidence of efficacy (as represented by treatment effect on progression-free survival) was available (based on the above results) for: biochemotherapy (with both interferon-alpha and interleukin-2); anti-CTLA4 monoclonal antibodies; anti-PD1 monoclonal antibodies; anti-CTLA4 plus anti-PD1 monoclonal antibodies; BRAF inhibitors; MEK inhibitors, and BRAF plus MEK inhibitors. Analysis (which included 19 RCTs and 7632 participants) generated 21 indirect comparisons.The best evidence (moderate-quality evidence) for progression-free survival was found for the following indirect comparisons:• both combinations of immune checkpoint inhibitors (HR 0.30, 95% CI 0.17 to 0.51) and small-molecule targeted drugs (HR 0.17, 95% CI 0.11 to 0.26) probably improved progression-free survival compared to chemotherapy;• both BRAF inhibitors (HR 0.40, 95% CI 0.23 to 0.68) and combinations of small-molecule targeted drugs (HR 0.22, 95% CI 0.12 to 0.39) were probably associated with better progression-free survival compared to anti-CTLA4 monoclonal antibodies;• biochemotherapy (HR 2.81, 95% CI 1.76 to 4.51) probably lead to worse progression-free survival compared to BRAF inhibitors;• the combination of small-molecule targeted drugs probably improved progression-free survival (HR 0.38, 95% CI 0.21 to 0.68) compared to anti-PD1 monoclonal antibodies;• both biochemotherapy (HR 5.05, 95% CI 3.01 to 8.45) and MEK inhibitors (HR 3.16, 95% CI 1.77 to 5.65) were probably associated with worse progression-free survival compared to the combination of small-molecule targeted drugs; and• biochemotherapy was probably associated with worse progression-free survival (HR 2.81, 95% CI 1.54 to 5.11) compared to the combination of immune checkpoint inhibitors.The best evidence (moderate-quality evidence) for toxicity was found for the following indirect comparisons:• combination of immune checkpoint inhibitors (RR 3.49, 95% CI 2.12 to 5.77) probably increased toxicity compared to chemotherapy;• combination of immune checkpoint inhibitors probably increased toxicity (RR 2.50, 95% CI 1.20 to 5.20) compared to BRAF inhibitors;• the combination of immune checkpoint inhibitors probably increased toxicity (RR 3.83, 95% CI 2.59 to 5.68) compared to anti-PD1 monoclonal antibodies; and• biochemotherapy was probably associated with lower toxicity (RR 0.41, 95% CI 0.24 to 0.71) compared to the combination of immune checkpoint inhibitors.Network meta-analysis-based ranking suggested that the combination of BRAF plus MEK inhibitors is the most effective strategy in terms of progression-free survival, whereas anti-PD1 monoclonal antibodies are associated with the lowest toxicity.Overall, the risk of bias of the included trials can be considered as limited. When considering the 122 trials included in this review and the seven types of bias we assessed, we performed 854 evaluations only seven of which (< 1%) assigned high risk to six trials. We found high-quality evidence that many treatments offer better efficacy than chemotherapy, especially recently implemented treatments, such as small-molecule targeted drugs, which are used to treat melanoma with specific gene mutations. Compared with chemotherapy, biochemotherapy (in this case, chemotherapy combined with both interferon-alpha and interleukin-2) and BRAF inhibitors improved progression-free survival; BRAF inhibitors (for BRAF-mutated melanoma) and anti-PD1 monoclonal antibodies improved overall survival. However, there was no difference between polychemotherapy and monochemotherapy in terms of achieving progression-free survival and overall survival. Biochemotherapy did not significantly improve overall survival and has higher toxicity rates compared with chemotherapy.There was some evidence that combined treatments worked better than single treatments: anti-PD1 monoclonal antibodies, alone or with anti-CTLA4, improved progression-free survival compared with anti-CTLA4 monoclonal antibodies alone. Anti-PD1 monoclonal antibodies performed better than anti-CTLA4 monoclonal antibodies in terms of overall survival, and a combination of BRAF plus MEK inhibitors was associated with better overall survival for BRAF-mutated melanoma, compared to BRAF inhibitors alone.The combination of BRAF plus MEK inhibitors (which can only be administered to people with BRAF-mutated melanoma) appeared to be the most effective treatment (based on results for progression-free survival), whereas anti-PD1 monoclonal antibodies appeared to be the least toxic, and most acceptable, treatment.Evidence quality was reduced due to imprecision, between-study heterogeneity, and substandard reporting of trials. Future research should ensure that those diminishing influences are addressed. Clinical areas of future investigation should include the longer-term effect of new therapeutic agents (i.e. immune checkpoint inhibitors and targeted therapies) on overall survival, as well as the combination of drugs used in melanoma treatment; research should also investigate the potential influence of biomarkers.

Expression of fusion IL2-B7.1(IgV+C) and effects on T lymphocytes.

PubMed

Kong, Linghong; Li, Yaochen; Yang, Ye; Li, Kangsheng

2007-12-01

The search for an effective immunotherapeutic treatment for tumors is an important area of cancer research. To prepare a more effective form of the bifunctional fusion protein IL2-B7.1(IgV+C) and analyze its effect on the stimulation of T lymphocyte proliferation, we used DNAStar 5.03 software to predict the structural diversity and biochemical character of IL2-B7.1(IgV+C). We then prepared fusion protein IL2-B7.1(IgV+C) by establishing its prokaryotic expression system, and tested its effect on the stimulation of T lymphocytes in vitro. The results indicated that IL2-B7.1(IgV+C) correctly formed a secondary structure in which both IL2 and B7.1(IgV+C) maintained their original hydrophilicity and epitopes. Western blot analysis revealed that IL2-B7.1(IgV+C) was efficiently expressed. Our analysis of CTLL-2 and T-cell proliferation showed that recombinant human (rh) IL2-B7.1(IgV+C) exerted the combined stimulating effects of both rhIL2 and rh B7.1(IgV+C) on cell proliferation, and that these effects could be blocked by adding either anti-IL2 or anti-B7.1 monoclonal antibodies. A >2-fold increase in [3H]TdR incorporation compared with that of cells treated with recombinant protein IL2, or B7.1(IgV+C) alone, revealed that rhIL2-B7.1(IgV+C) had dose-dependent synergetic effects on T-cell activation in the presence of anti-CD3 monoclonal antibody. We concluded that the augmented potency of rhIL2-B7.1(IgV+C) resulted in a stronger stimulation of T-cell proliferation than either rhB7.1(IgV+C) or rhIL2 alone.

Combination of an agonistic anti-CD40 monoclonal antibody and the COX-2 inhibitor celecoxib induces anti-glioma effects by promotion of type-1 immunity in myeloid cells and T-cells

PubMed Central

Kosaka, Akemi; Ohkuri, Takayuki

2014-01-01

Malignant gliomas are heavily infiltrated by immature myeloid cells that mediate immuno-suppression. Agonistic CD40 monoclonal antibody (mAb) has been shown to activate myeloid cells and promote antitumor immunity. Our previous study has also demonstrated blockade of cyclooxygenase-2 (COX-2) reduces immunosuppressive myeloid cells, thereby suppressing glioma development in mice. We therefore hypothesized that a combinatory strategy to modulate myeloid cells via two distinct pathways, i.e., CD40/CD40L stimulation and COX-2 blockade, would enhance anti-glioma immunity. We used three different mouse glioma models to evaluate therapeutic effects and underlying mechanisms of a combination regimen with an agonist CD40 mAb and the COX-2 inhibitor celecoxib. Treatment of glioma-bearing mice with the combination therapy significantly prolonged survival compared with either anti-CD40 mAb or celecoxib alone. The combination regimen promoted maturation of CD11b+ cells in both spleen and brain, and enhanced Cxcl10 while suppressing Arg1 in CD11b+Gr-1+ cells in the brain. Anti-glioma activity of the combination regimen was T-cell dependent because depletion of CD4+ and CD8+ cells in vivo abrogated the anti-glioma effects. Furthermore, the combination therapy significantly increased the frequency of CD8+ T-cells, enhanced IFN-γ-production and reduced CD4+CD25+Foxp3+ T regulatory cells in the brain, and induced tumor-antigen-specific T-cell responses in lymph nodes. Our findings suggest that the combination therapy of anti-CD40 mAb with celecoxib enhances anti-glioma activities via promotion of type-1 immunity both in myeloid cells and T-cells. PMID:24878890

Mapping of cat albumin using monoclonal antibodies: identification of determinants common to cat and dog.

PubMed Central

Boutin, Y; Hébert, J; Vrancken, E R; Mourad, W

1989-01-01

Cat and dog albumins from commercial extracts were used to produce monoclonal antibodies (MoAb). Anti-cat albumin MoAb recognized both cat and dog albumin equally, as did anti-dog albumin MoAb; this confirms cross-reactivity between cat and dog. The MoAb were separated into two groups according to their epitopic specificity; they recognized two overlapping epitopes of cat albumin. Furthermore, by competitive inhibition of radio-allergosorbent test (RAST), it was shown that one MoAb group inhibited significantly the binding of human IgE antibodies (from a pool of 13 patients allergic to both cats and dogs) to insolubilized cat or dog extracts. These observations suggest that murine anti-cat or anti-dog MoAb and human IgE antibodies recognize identical or closely related determinants on cat and dog albumin. Images Fig. 1 Fig. 2 PMID:2478325

Blockade of invariant TCR-CD1d interaction specifically inhibits antibody production against blood group A carbohydrates

PubMed Central

Tazawa, Hirofumi; Irei, Toshimitsu; Tanaka, Yuka; Igarashi, Yuka; Tashiro, Hirotaka

2013-01-01

Previously, we detected B cells expressing receptors for blood group A carbohydrates in the CD11b+CD5+ B-1a subpopulation in mice, similar to that in blood group O or B in humans. In the present study, we demonstrate that CD1d-restricted natural killer T (NKT) cells are required to produce anti-A antibodies (Abs), probably through collaboration with B-1a cells. After immunization of wild-type (WT) mice with human blood group A red blood cells (A-RBCs), interleukin (IL)-5 exclusively and transiently increased and the anti-A Abs were elevated in sera. However, these reactions were not observed in CD1d−/− mice, which lack NKT cells. Administration of anti-mouse CD1d blocking monoclonal Abs (mAb) prior to immunization abolished IL-5 production by NKT cells and anti-A Ab production in WT mice. Administration of anti-IL-5 neutralizing mAb also diminished anti-A Ab production in WT mice, suggesting that IL-5 secreted from NKT cells critically regulates anti-A Ab production by B-1a cells. In nonobese diabetic/severe combined immunodeficient (NOD/SCID/γcnull) mice, into which peripheral blood mononuclear cells from type O human volunteers were engrafted, administration of anti-human CD1d mAb prior to A-RBC immunization completely inhibited anti-A Ab production. Thus, anti-CD1d treatment might constitute a novel approach that could help in evading Ab-mediated rejection in ABO-incompatible transplant recipients. PMID:23943651

Antibody-based bacterial toxin detection

NASA Astrophysics Data System (ADS)

Menking, Darrell E.; Heitz, Jonathon M.; Anis, Nabil A.; Thompson, Roy G.

1994-03-01

Fiber optic evanescent fluorosensors are under investigation in our laboratory for the study of drug-receptor interactions for detection of threat agents and antibody-antigen interactions for detection of biological toxins. In a one step assay, antibodies against Cholera toxin or Staphylococcus Enterotoxin B were noncovalently immobilized on quartz fibers and probed with fluorescein-isothiocyanate (FITC)-labeled toxins. In the two-step assay, Cholera toxin or Botulinum toxoid A was immobilized onto the fiber, followed by incubation in an antiserum or partially purified antitoxin IgG. These were then probed with FITC-anti-IgG antibodies. Unlabeled toxins competed with labeled toxins or antitoxin IgG in a dose-dependent manner and the detection of the toxins was in the nanomolar range.

Anti-NGF monoclonal antibody muMab 911 does not deplete neurons in the superior cervical ganglia of young or old adult rats.

PubMed

Marcek, John; Okerberg, Carlin; Liu, Chang-Ning; Potter, David; Butler, Paul; Boucher, Magalie; Zorbas, Mark; Mouton, Peter; Nyengaard, Jens R; Somps, Chris

2016-10-01

Nerve growth factor (NGF) blocking therapies are an emerging and effective approach to pain management. However, concerns about the potential for adverse effects on the structure and function of the peripheral nervous system have slowed their development. Early studies using NGF antisera in adult rats reported effects on the size and number of neurons in the sympathetic chain ganglia. In the work described here, both young adult (6-8 week) and fully mature (7-8 month) rats were treated with muMab 911, a selective, murine, anti-NGF monoclonal antibody, to determine if systemic exposures to pharmacologically active levels of antibody for 1 month cause loss of neurons in the sympathetic superior cervical ganglia (SCG). State-of-the-art, unbiased stereology performed by two independent laboratories was used to determine the effects of muMab 911 on SCG neuronal number and size, as well as ganglion size. Following muMab 911 treatment, non-statistically significant trends toward smaller ganglia, and smaller and fewer neurons, were seen when routine, nonspecific stains were used in stereologic assessments. However, when noradrenergic neurons were identified using tyrosine hydroxylase (TH) immunoreactivity, trends toward fewer neurons observed with routine stains were not apparent. The only statistically significant effects detected were lower SCG weights in muMab 911-treated rats, and a smaller volume of TH immunoreactivity in neurons from younger rats treated with muMab 911. These results indicate that therapeutically relevant exposures to the anti-NGF monoclonal antibody muMab 911 for 1 month have no effect on neuron numbers within the SCG from young or old adult rats. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of an Enzyme-Linked Immunosorbent Assay and Immunoaffinity Column Chromatography for Saikosaponin d Using an Anti-Saikosaponin d Monoclonal Antibody.

PubMed

Sai, Jiayang; Zhao, Yan; Shan, Wenchao; Qu, Baoping; Zhang, Yue; Cheng, Jinjun; Qu, Huihua; Wang, Qingguo

2016-03-01

This work developed a novel immunochemical approach for the quality control of saikosaponin d using an enzyme-linked immunosorbent assay. Splenocytes from mice immunized with the saikosaponin d-bovine serum albumin conjugate were fused with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma SP2/0 cell line, and a hybridoma secreting monoclonal antibody against saikosaponin d was successfully obtained. The prepared anti-saikosaponin d monoclonal antibody 1E7F3 has a novel characteristic, showing weak reactivity with compounds that are structurally related to saikosaponin d. Using monoclonal antibody 1E7F3, a specific and reliable enzyme-linked immunosorbent assay was developed to detect saikosaponin d. The system shows a full measurement range from 156.25 to 5000.00 ng × mL(-1). Both intra-assay and inter-assay repeatability and precision were achieved, with relative standard deviations lower than 10.00%. The recovery rates ranged from 92.36% to 101.00%, meeting the requirements for biological samples. There was a good correlation between the enzyme-linked immunosorbent assay and high-performance liquid chromatography analyses of saikosaponin d, and the saikosaponin d levels in formulated Chinese medicines were successfully determined. Furthermore, immunoaffinity column chromatography was established using this anti-saikosaponin d monoclonal antibody, and the elution profile of saikosaponin d was detected by a Bio-Rad QuadTec UV/Vis detector at 203 nm. The results demonstrate that we generated a reliable and more efficient assay system for measuring saikosaponin d and provide a potential approach for purifying and separating saikosaponin d. Georg Thieme Verlag KG Stuttgart · New York.

Optical Imaging of Mammaglobin Expression of Breast Cancer

DTIC Science & Technology

2003-05-01

monoclonal anti-MMG antibodies with a near infrared fluorescent probe for optical imaging and 64Cu -DOTA for positron emission tomography (mPET...Preliminary results indicate that the mPET imaging with 64Cu -DOTA-anti MMG monoclonal antibodies showed predominant liver uptake in mice. In contrast, the

Treating inflammation by blocking interleukin-1 in a broad spectrum of diseases.

PubMed

Dinarello, Charles A; Simon, Anna; van der Meer, Jos W M

2012-08-01

Interleukin-1 (IL-1) is a highly active pro-inflammatory cytokine that lowers pain thresholds and damages tissues. Monotherapy blocking IL-1 activity in autoinflammatory syndromes results in a rapid and sustained reduction in disease severity, including reversal of inflammation-mediated loss of sight, hearing and organ function. This approach can therefore be effective in treating common conditions such as post-infarction heart failure, and trials targeting a broad spectrum of new indications are underway. So far, three IL-1-targeted agents have been approved: the IL-1 receptor antagonist anakinra, the soluble decoy receptor rilonacept and the neutralizing monoclonal anti-IL-1β antibody canakinumab. In addition, a monoclonal antibody directed against the IL-1 receptor and a neutralizing anti-IL-1α antibody are in clinical trials.

Production and Characterization of Monoclonal Antibodies Against the Protective Antigen Component of Bacillus anthracis Toxin

DTIC Science & Technology

1988-01-21

concentration. Radial immmurnodiffusion plates were prepared with rabbit anti-mouse IgG ( Miles Scientific, Naperville, I) or rabbit anti-mouse IgM (Kirkegaard...6. Ezzell , J. W., B. E. Ivins, and S. H. Leppla. 1964. Immunoelectrophoretic analysis, toxicity, and kinetics o4 in 16 vitro production of the

A Neutralizing Anti-gH/gL Monoclonal Antibody Is Protective in the Guinea Pig Model of Congenital CMV Infection

PubMed Central

Auerbach, Marcy R.; Yan, Donghong; Vij, Rajesh; Hongo, Jo-Anne; Nakamura, Gerald; Vernes, Jean-Michel; Meng, Y. Gloria; Lein, Samantha; Chan, Pamela; Ross, Jed; Carano, Richard; Deng, Rong; Lewin-Koh, Nicholas; Xu, Min; Feierbach, Becket

2014-01-01

Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2–1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective [1]–[3]. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV) glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta) to fetal infection (fetus and amniotic fluid). Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans. PMID:24722349

Generation and functional analysis of monoclonal antibodies against the second extracellular loop of human M3 muscarinic acetylcholine receptor.

PubMed

Tsuboi, Hiroto; Nakamura, Yumi; Iizuka, Mana; Matsuo, Naomi; Matsumoto, Isao; Sumida, Takayuki

2012-04-01

The M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the activation of salivary and lachrymal glands. The M3R contains four extracellular domains (the N-terminal, and the first, second, and third extracellular loops), and we recently detected antibodies against each of these four domains in a subgroup of patients with Sjögren's syndrome (SS). Functional analysis indicated that the influence of such anti-M3R antibodies on salivary secretion might differ based on the epitopes to which they bind. To clarify the relationship between B-cell epitopes on the M3R and its function, we generated two hybridomas producing anti-M3R monoclonal antibodies against the second extracellular loop of M3R (anti-M3R(2nd) mAbs) and analyzed their function by Ca(2+)-influx assays, using a human salivary gland (HSG) cell line. These two anti-M3R(2nd) mAbs suppressed Ca(2+)-influx in the HSG cells induced by cevimeline stimulation, suggesting that autoantibodies against the second extracellular loop of M3R could be involved in salivary dysfunction in patients with SS.

A novel anti-GPC3 monoclonal antibody (YP7) | Center for Cancer Research

Cancer.gov

Glypican-3 (GPC3) is an emerging therapeutic target in hepatoma. A novel anti-GPC3 monoclonal antibody (YP7) has been generated through a combination of peptide immunization and high-throughput flow cytometry screening. YP7 binds cell-surface-associated GPC3 with high affinity and exhibits significant hepatoma xenograft growth inhibition in nude mice. The new antibody may have

Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frankfurt, O.S.

A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to themore » drug dose. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.« less

Gold nanoparticle based Tuberculosis immunochromatographic assay: the quantitative ESE Quanti analysis of the intensity of test and control lines.

PubMed

Mdluli, Phumlani; Tetyana, Phumlani; Sosibo, Ndabenhle; van der Walt, Hendriëtte; Mlambo, Mbuso; Skepu, Amanda; Tshikhudo, Robert

2014-04-15

A rapid dual channel lateral flow assay for the detection of Mycobacterium Tuberculosis antibodies (MTB 38 kDa monoclonal antibody) in human blood was developed. The MTB 6-14-38 kDa fusion antigen and anti-Protein A were used as the capture proteins for test and control lines respectively. Protein A labeled 40 nm gold nanoparticles were used as the detection conjugate. Whole blood and serum were spiked with MTB 38 kDa monoclonal antibody to make a positive sample model. The developed lateral flow was used to test MTB 38 kDa monoclonal antibody, and a detection limit of 5 ng/ml was used as a cut-off concentration of the analytes. The effect of the analyte concentration on the MTB lateral flow assay was studied using the variation of the intensity obtained from a ESE Quanti reader. There was a direct correlation between the analyte (MTB 38 kDa monoclonal antibody) concentration and the intensity of the test line. The intensity increased with an increase in the concentration of MTB 38 kDa monoclonal antibody, while in contrast, an increase in analyte concentration decreased the intensity of the control line. © 2013 Published by Elsevier B.V.

Endothelial connexin 32 regulates tissue factor expression induced by inflammatory stimulation and direct cell-cell interaction with activated cells.

PubMed

Okamoto, Takayuki; Akita, Nobuyuki; Hayashi, Tatsuya; Shimaoka, Motomu; Suzuki, Koji

2014-10-01

Endothelial cell (EC) interacts with adjacent EC through gap junction, and abnormal expression or function of Cxs is associated with cardiovascular diseases. In patients with endothelial dysfunction, the up-regulation of tissue factor (TF) expression promotes the pathogenic activation of blood coagulation, however the relationship between gap junctions and TF expression in ECs remains uncharacterized. ECs express the gap junction (GJ) proteins connexin32 (Cx32), Cx37, Cx40 and Cx43. We investigated the role of endothelial gap junctions, particularly Cx32, in modulating TF expression during vascular inflammation. Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor-α (TNF-α) and TF activity was assessed in the presence of GJ blockers and an inhibitory anti-Cx32 monoclonal antibody. Treatment with GJ blockers and anti-Cx32 monoclonal antibody enhanced the TNF-α-induced TF activity and mRNA expression in HUVECs. TNF-α-activated effector HUVECs or mouse MS-1 cells were co-cultured with non-stimulated acceptor HUVECs and TF expression in acceptor HUVECs was detected. Effector EC induced TF expression in adjacent acceptor HUVECs through direct cell-cell interaction. Cell-cell interaction induced TF expression was reduced by anti-intercellular adhesion molecule-1 (ICAM1) monoclonal antibody. Soluble ICAM1-Fc fusion protein promotes TF expression. GJ blockers and anti-Cx32 monoclonal antibody enhanced TF expression induced by cell-cell interaction and ICAM1-Fc treatment. Blockade of endothelial Cx32 increased TF expression induced by TNF-α stimulation and cell-cell interaction which was at least partly dependent upon ICAM1. These results suggest that direct Cx32-mediated interaction modulates TF expression in ECs during vascular inflammation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

NASA Astrophysics Data System (ADS)

Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

1992-09-01

The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

Tracking Antibody Distribution with Near-Infrared Fluorescent Dyes: Impact of Dye Structure and Degree of Labeling on Plasma Clearance.

PubMed

Cilliers, Cornelius; Nessler, Ian; Christodolu, Nikolas; Thurber, Greg M

2017-05-01

Monoclonal antibodies labeled with near-infrared (NIR) fluorophores have potential use in disease detection, intraoperative imaging, and pharmacokinetic characterization of therapeutic antibodies in both the preclinical and clinical setting. Recent work has shown conjugation of NIR fluorophores to antibodies can potentially alter antibody disposition at a sufficiently high degree of labeling (DoL); however, other reports show minimal impact after labeling with NIR fluorophores. In this work, we label two clinically approved antibodies, Herceptin (trastuzumab) and Avastin (bevacizumab), with NIR dyes IRDye 800CW (800CW) or Alexa Fluor 680 (AF680), at 1.2 and 0.3 dyes/antibody and examine the impact of fluorophore conjugation on antibody plasma clearance and tissue distribution. At 0.3 DoL, AF680 conjugates exhibited similar clearance to unlabeled antibody over 17 days while 800CW conjugates diverged after 4 days, suggesting AF680 is a more suitable choice for long-term pharmacokinetic studies. At the 1.2 DoL, 800CW conjugates cleared faster than unlabeled antibodies after several hours, in agreement with other published reports. The tissue biodistribution for bevacizumab-800CW and -AF680 conjugates agreed well with literature reported biodistributions using radiolabels. However, the greater tissue autofluorescence at 680 nm resulted in limited detection above background at low (∼2 mg/kg) doses and 0.3 DoL for AF680, indicating that 800CW is more appropriate for short-term biodistribution measurements and intraoperative imaging. Overall, our work shows a DoL of 0.3 or less for non-site-specifically labeled antibodies (with a Poisson distribution) is ideal for limiting the impact of NIR fluorophores on antibody pharmacokinetics.

Mechanism of recovery from acute virus infection: treatment of lymphocytic choriomeningitis virus-infected mice with monoclonal antibodies reveals that Lyt-2+ T lymphocytes mediate clearance of virus and regulate the antiviral antibody response.

PubMed Central

Moskophidis, D; Cobbold, S P; Waldmann, H; Lehmann-Grube, F

1987-01-01

After intravenous infection of mice, lymphocytic choriomeningitis virus multiplied in spleens and livers, attaining highest concentrations on days 4 to 6. The subsequent clearance was as rapid, and 8 to 10 days after inoculation, infectivity was usually below detectability. During the effector phase of virus elimination, both cytotoxic T-cell (CTL) activity and the number of cells producing antiviral antibodies were high. Monoclonal antibodies directed against T lymphocytes and T-lymphocyte subsets were inoculated once intravenously 5, 6, or 7 days after infection of the animals, and the effects on antiviral immune responses, as well as on elimination of virus from the organs, were determined. Treatment with anti-Thy-1 and anti-Lyt-2 antibodies blocked elimination of the virus and profoundly diminished the activity of spleen CTLs but reduced the antibody response partially (anti-Thy-1) or increased it (anti-Lyt-2). In contrast, treatment with the anti-L3T4 antibody had essentially no effect on either virus elimination or CTL response but abolished antibody production. We conclude that Lyt-2+ (cytotoxic-suppressive) T lymphocytes are needed for elimination of the virus and also regulate the humoral response but that antiviral antibodies are not essential for control of the infection. PMID:3494855

Solid-phase receptor binding assay for /sup 125/I-hCG

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bortolussi, M.; Selmin, O.; Colombatti, A.

1987-01-01

A solid-phase radioligand-receptor assay (RRA) to measure the binding of /sup 125/I-labelled human chorionic gonadotropin (/sup 125/I-hCG) to target cell membranes has been developed. The binding of /sup 125/I-hCG to membranes immobilized on the wells of microtitration plates reached a maximum at about 3 hours at 37 degrees C, was saturable, displayed a high affinity (Ka = 2.4 X 10(9) M-1) and was specifically inhibited by unlabelled hCG. In comparison with RRAs carried out with membranes in suspension, the solid-phase RRA is significantly simpler and much faster to perform as it avoids centrifugation or filtration procedures. The solid-phase RRA wasmore » adapted profitably to process large numbers of samples at the same time. It proved particularly useful as a screening assay to detect anti-hCG monoclonal antibodies with high inhibitory activity for binding of /sup 125/I-hCG to its receptors.« less

Reversible Opening of the Blood-Brain Barrier by Anti-Bacterial Antibodies

NASA Astrophysics Data System (ADS)

Tuomanen, Elaine I.; Prasad, Sudha M.; George, Jonathan S.; Hoepelman, Andy I. M.; Ibsen, Per; Heron, Iver; Starzyk, Ruth M.

1993-08-01

The leukocyte adhesion molecule CR3 (CD11b/CD18, Mac-1) promotes leukocyte transmigration into tissues by engaging an unknown cognate ligand on the surface of vascular endothelial cells. Filamentous hemagglutinin (FHA), an adhesin of the bacterium Bordetella pertussis, binds to CR3. We hypothesized that FHA mimics the native ligand for the CR3 integrin on endothelial cells and predicted that anti-FHA antibodies should bind to endothelial cells, interfere with leukocyte recruitment, and induce endothelial permeability. Anti-FHA monoclonal antibodies bound to cerebral microvessels in sections from human brain and upon intravenous injection into rabbits. Antibody binding correlated with the ability to recognize two polypeptides in extracts of human cerebral vessels that were also bound by CD18. In vivo, antibody binding not only interfered with transmigration of leukocytes into cerebrospinal fluid but also induced a dose-dependent reversible increase in blood-brain barrier permeability sufficient to improve delivery of intravenously administered therapeutic agents to brain parenchyma.

Clinical significance of BRAF non-V600E mutations on the therapeutic effects of anti-EGFR monoclonal antibody treatment in patients with pretreated metastatic colorectal cancer: the Biomarker Research for anti-EGFR monoclonal Antibodies by Comprehensive Cancer genomics (BREAC) study.

PubMed

Shinozaki, Eiji; Yoshino, Takayuki; Yamazaki, Kentaro; Muro, Kei; Yamaguchi, Kensei; Nishina, Tomohiro; Yuki, Satoshi; Shitara, Kohei; Bando, Hideaki; Mimaki, Sachiyo; Nakai, Chikako; Matsushima, Koutatsu; Suzuki, Yutaka; Akagi, Kiwamu; Yamanaka, Takeharu; Nomura, Shogo; Fujii, Satoshi; Esumi, Hiroyasu; Sugiyama, Masaya; Nishida, Nao; Mizokami, Masashi; Koh, Yasuhiro; Abe, Yukiko; Ohtsu, Atsushi; Tsuchihara, Katsuya

2017-11-07

Patients with BRAF V600E -mutated metastatic colorectal cancer (mCRC) have a poorer prognosis as well as resistance to anti-EGFR antibodies. However, it is unclear whether BRAF mutations other than BRAF V600E (BRAF non-V600E mutations) contribute to anti-EGFR antibody resistance. This study was composed of exploratory and inference cohorts. Candidate biomarkers identified by whole exome sequencing from super-responders and nonresponders in the exploratory cohort were validated by targeted resequencing for patients who received anti-EGFR antibody in the inference cohort. In the exploratory cohort, 31 candidate biomarkers, including KRAS/NRAS/BRAF mutations, were identified. Targeted resequencing of 150 patients in the inference cohort revealed 40 patients with RAS (26.7%), 9 patients with BRAF V600E (6.0%), and 7 patients with BRAF non-V600E mutations (4.7%), respectively. The response rates in RAS, BRAF V600E , and BRAF non-V600E were lower than those in RAS/BRAF wild-type (2.5%, 0%, and 0% vs 31.9%). The median PFS in BRAF non-V600E mutations was 2.4 months, similar to that in RAS or BRAF V600E mutations (2.1 and 1.6 months) but significantly worse than that in wild-type RAS/BRAF (5.9 months). Although BRAF non-V600E mutations identified were a rare and unestablished molecular subtype, certain BRAF non-V600E mutations might contribute to a lesser benefit of anti-EGFR monoclonal antibody treatment.

SIB-DOTA: a trifunctional prosthetic group potentially amenable for multi-modal labeling that enhances tumor uptake of internalizing monoclonal antibodies.

PubMed

Vaidyanathan, G; White, B J; Affleck, D J; Zhao, X G; Welsh, P C; McDougald, D; Choi, J; Zalutsky, M R

2012-12-15

A major drawback of internalizing monoclonal antibodies (mAbs) radioiodinated with direct electrophilic approaches is that tumor retention of radioactivity is compromised by the rapid washout of iodo-tyrosine, the primary labeled catabolite for mAbs labeled via this strategy. In our continuing efforts to develop more versatile residualizing labels that could overcome this problem, we have designed SIB-DOTA, a prosthetic labeling template that combines the features of the prototypical, dehalogenation-resistant N-succinimidyl 3-iodobenzoate (SIB) with DOTA, a useful macrocyclic chelator for labeling with radiometals. Herein we describe the synthesis of the unlabeled standard of this prosthetic moiety, its protected tin precursor, and radioiodinated SIB-DOTA. An anti-EGFRvIII-reactive mAb, L8A4 was radiolabeled with [(131)I]SIB-DOTA in 27.1±6.2% (n=2) conjugation yields and its targeting properties to the same mAb labeled with [(125)I]SGMIB both in vitro and in vivo using U87MG·ΔEGFR cells and xenografts were compared. In vitro paired-label internalization assays showed that the intracellular radioactivity from [(131)I]SIB-DOTA-L8A4 was 21.4±0.5% and 26.2±1.1% of initially bound radioactivity at 16 and 24h, respectively. In comparison, these values for [(125)I]SGMIB-L8A4 were 16.7±0.5% and 14.9±1.1%. Similarly, the SIB-DOTA prosthetic group provided better tumor targeting in vivo than SGMIB over 8 d period. These results suggest that SIB-DOTA warrants further evaluation as a residualizing agent for labeling internalizing mAbs including those targeted to EGFRvIII. Copyright © 2012 Elsevier Ltd. All rights reserved.

A phase 1/2 trial of ublituximab, a novel anti-CD20 monoclonal antibody, in patients with B-cell non-Hodgkin lymphoma or chronic lymphocytic leukaemia previously exposed to rituximab.

PubMed

Sawas, Ahmed; Farber, Charles M; Schreeder, Marshall T; Khalil, Mazen Y; Mahadevan, Daruka; Deng, Changchun; Amengual, Jennifer E; Nikolinakos, Petros G; Kolesar, Jill M; Kuhn, John G; Sportelli, Peter; Miskin, Hari P; O'Connor, Owen A

2017-04-01

This phase 1/2 study evaluated the safety, pharmacokinetic behavior and anti-tumour activity of ublituximab, a unique type I, chimeric, glycoengineered anti-CD20 monoclonal antibody, in rituximab-relapsed or -refractory patients with B-cell non-Hodgkin lymphoma (B-NHL) or chronic lymphocytic leukaemia (CLL). Induction therapy (doses of 450-1200 mg) consisted of 4 weekly infusions in cycle 1 for NHL and 3 weekly infusions in cycles 1 and 2 for CLL. Patients received ublituximab maintenance monthly during cycles 3-5, then once every 3 months for up to 2 years. Enrolled patients with B-NHL (n = 27) and CLL (n = 8) had a median of 3 prior therapies. No dose-limiting toxicities or unexpected adverse events (AEs) occurred. The most common AEs were infusion-related reactions (40%; grade 3/4, 0%); fatigue (37%; grade 3/4, 3%); pyrexia (29%; grade 3/4, 0%); and diarrhoea (26%; grade 3/4, 0%). Common haematological AEs were neutropenia (14%; grade 3/4, 14%) and anaemia (11%; grade 3/4, 6%). The overall response rate for evaluable patients (n = 31) was 45% (13% complete responses, 32% partial responses). Median duration of response and progression-free survival were 9·2 months and 7·7 months, respectively. Ublituximab was well-tolerated and efficacious in a heterogeneous and highly rituximab-pre-treated patient population. © 2017 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.

Construction of a hepatitis B virus neutralizing chimeric monoclonal antibody recognizing escape mutants of the viral surface antigen (HBsAg).

PubMed

Golsaz-Shirazi, Forough; Amiri, Mohammad Mehdi; Farid, Samira; Bahadori, Motahareh; Bohne, Felix; Altstetter, Sebastian; Wolff, Lisa; Kazemi, Tohid; Khoshnoodi, Jalal; Hojjat-Farsangi, Mohammad; Chudy, Michael; Jeddi-Tehrani, Mahmood; Protzer, Ulrike; Shokri, Fazel

2017-08-01

Hepatitis B virus (HBV) infection is a global burden on the health-care system and is considered as the tenth leading cause of death in the world. Over 248 million patients are currently suffering from chronic HBV infection worldwide and annual mortality rate of this infection is 686000. The "a" determinant is a hydrophilic region present in all antigenic subtypes of hepatitis B surface antigen (HBsAg), and antibodies against this region can neutralize the virus and are protective against all subtypes. We have recently generated a murine anti-HBs monoclonal antibody (4G4), which can neutralize HBV infection in HepaRG cells and recognize most of the escape mutant forms of HBsAg. Here, we describe the production and characterization of the chimeric human-murine antibody 4G4 (c-4G4). Variable region genes of heavy and light chains of the m-4G4 were cloned and fused to constant regions of human kappa and IgG1 by splice overlap extension (SOE) PCR. The chimeric antibody was expressed in Chinese Hamster Ovary (CHO)-K1 cells and purified from culture supernatant. Competition ELISA proved that both antibodies bind the same epitope within HBsAg. Antigen-binding studies using ELISA and Western blot showed that c-4G4 has retained the affinity and specificity of the parental murine antibody, and displayed a similar pattern of reactivity to 13 escape mutant forms of HBsAg. Both, the parental and c-4G4 showed a comparably high HBV neutralization capacity in cell culture even at the lowest concentration (0.6μg/ml). Due to the ability of c-4G4 to recognize most of the sub-genotypes and escape mutants of HBsAg, this antibody either alone or in combination with other anti-HBs antibodies could be considered as a potent alternative for Hepatitis B immune globulin (HBIG) as an HBV infection prophylactic or for passive immunotherapy against HBV infection. Copyright © 2017 Elsevier B.V. All rights reserved.

Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice

PubMed Central

Ezzatifar, Fatemeh; Majidi, Jafar; Baradaran, Behzad; Aghebati Maleki, Leili; Abdolalizadeh, Jalal; Yousefi, Mehdi

2015-01-01

Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane. After ten days, ascitic fluid was harvested from the peritoneum of each mouse. The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit. MAb was purified from the ascitic fluid by ion exchange chromatography. The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP. Results: Monoclonal antibodies with high specificity and sensitivity against Human IgA were prepared by hybridoma technology. The subclass of antibody was IgG1 and its light chain was the kappa type. Conclusion: This conjugated monoclonal antibody could have applications in designing ELISA kits in order to diagnose different infectious diseases such as toxoplasmosis and H. Pylori. PMID:25789225

Palladium-109 labeled anti-melanoma monoclonal antibodies

DOEpatents

Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

1984-04-30

The invention consists of new monoclonal antibodies labelled with Palladium 109, a beta-emitting radionuclide, the method of preparing this material, and its use in the radiotherapy of melanoma. The antibodies are chelate-conjugated and demonstrate a high uptake in melanomas. (ACR)

Generation and characterization of monoclonal antibodies against Giardia muris trophozoites.

PubMed

Heyworth, M F; Ho, K E; Pappo, J

1989-11-01

Mouse monoclonal antibodies (mAb) were produced against Giardia muris trophozoite surface antigens. To generate B-cell hybridomas, P3/NS1/1-Ag4-1 myeloma cells were fused with splenic lymphocytes from BALB/c mice that had been immunized parenterally with G. muris trophozoites. Hybridoma culture supernatants were screened for mAb by flow cytometry of G. muris trophozoites incubated with culture supernatant followed by fluorescein-conjugated anti-mouse IgG and IgM. Flow cytometry showed three types of trophozoite staining by mAb: (i) bright staining of greater than 90% of trophozoites, with aggregation of the organisms; (ii) bright staining of approximately 90% of trophozoites, with little or no aggregation; (iii) dull staining of approximately 20% of trophozoites, without aggregation. Western blotting of mAb on G. muris trophozoite antigens separated by polyacrylamide gel electrophoresis showed that a mAb exhibiting the third of these flow cytometry staining patterns recognized trophozoite antigens of MW approximately 31,000 and 35,000. Immunoprecipitation studies indicated that the same mAb specifically precipitated two 125I-labelled trophozoite surface antigens of MW approximately 30,000. Monoclonal antibodies generated in this study may facilitate the purification and biochemical characterization of trophozoite antigens that are targets for protective intestinal antibody in G. muris-infected mice.

Microglial internalization and degradation of pathological tau is enhanced by an anti-tau monoclonal antibody

PubMed Central

Luo, Wenjie; Liu, Wencheng; Hu, Xiaoyan; Hanna, Mary; Caravaca, April; Paul, Steven M.

2015-01-01

Microglia have been shown to contribute to the clearance of brain amyloid β peptides (Aβ), the major component of amyloid plaques, in Alzheimer’s disease (AD). However, it is not known whether microglia play a similar role in the clearance of tau, the major component of neurofibrillary tangles (NFTs). We now report that murine microglia rapidly internalize and degrade hyperphosphorylated pathological tau isolated from AD brain tissue in a time-dependent manner in vitro. We further demonstrate that microglia readily degrade human tau species released from AD brain sections and eliminate NFTs from brain sections of P301S tauopathy mice. The anti-tau monoclonal antibody MC1 enhances microglia-mediated tau degradation in an Fc-dependent manner. Our data identify a potential role for microglia in the degradation and clearance of pathological tau species in brain and provide a mechanism explaining the potential therapeutic actions of passively administered anti-tau monoclonal antibodies. PMID:26057852

A novel anti-EMMPRIN function-blocking antibody reduces T cell proliferation and neurotoxicity: relevance to multiple sclerosis

PubMed Central

2012-01-01

Background Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147, basigin) is an inducer of the expression of several matrix metalloproteinases (MMPs). We reported previously that blocking EMMPRIN activity reduced neuroinflammation and severity of disease in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). Methods To improve upon EMMPRIN blockade, and to help unravel the biological functions of EMMPRIN in inflammatory disorders, we have developed several anti-EMMPRIN monoclonal antibodies. Results Of these monoclonal antibodies, a particular one, clone 10, was efficient in binding mouse and human cells using several methods of detection. The specificity of clone 10 was demonstrated by its lack of staining of EMMPRIN-null embryos compared to heterozygous and wild-type mouse samples. Functionally, human T cells activated with anti-CD3 and anti-CD28 elevated their expression of EMMPRIN and the treatment of these T cells with clone 10 resulted in decreased proliferation and matrix metalloproteinase- 9 (MMP-9) production. Activated human T cells were toxic to human neurons in culture and clone 10 pretreatment reduced T cell cytotoxicity correspondent with decrease of granzyme B levels within T cells. In vivo, EAE mice treated with clone 10 had a markedly reduced disease score compared to mice treated with IgM isotype control. Conclusions We have produced a novel anti-EMMPRIN monoclonal antibody that blocks several aspects of T cell activity, thus highlighting the multiple roles of EMMPRIN in T cell biology. Moreover, clone 10 reduces EAE scores in mice compared to controls, and has activity on human cells, potentially allowing for the testing of anti-EMMPRIN treatment not only in EAE, but conceivably also in MS. PMID:22480370

A novel anti-EMMPRIN function-blocking antibody reduces T cell proliferation and neurotoxicity: relevance to multiple sclerosis.

PubMed

Agrawal, Smriti M; Silva, Claudia; Wang, Janet; Tong, Jade Pui-Wai; Yong, V Wee

2012-04-05

Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147, basigin) is an inducer of the expression of several matrix metalloproteinases (MMPs). We reported previously that blocking EMMPRIN activity reduced neuroinflammation and severity of disease in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). To improve upon EMMPRIN blockade, and to help unravel the biological functions of EMMPRIN in inflammatory disorders, we have developed several anti-EMMPRIN monoclonal antibodies. Of these monoclonal antibodies, a particular one, clone 10, was efficient in binding mouse and human cells using several methods of detection. The specificity of clone 10 was demonstrated by its lack of staining of EMMPRIN-null embryos compared to heterozygous and wild-type mouse samples. Functionally, human T cells activated with anti-CD3 and anti-CD28 elevated their expression of EMMPRIN and the treatment of these T cells with clone 10 resulted in decreased proliferation and matrix metalloproteinase- 9 (MMP-9) production. Activated human T cells were toxic to human neurons in culture and clone 10 pretreatment reduced T cell cytotoxicity correspondent with decrease of granzyme B levels within T cells. In vivo, EAE mice treated with clone 10 had a markedly reduced disease score compared to mice treated with IgM isotype control. We have produced a novel anti-EMMPRIN monoclonal antibody that blocks several aspects of T cell activity, thus highlighting the multiple roles of EMMPRIN in T cell biology. Moreover, clone 10 reduces EAE scores in mice compared to controls, and has activity on human cells, potentially allowing for the testing of anti-EMMPRIN treatment not only in EAE, but conceivably also in MS.

Immuno-PCR for one step detection of H5N1 avian influenza virus and Newcastle disease virus using magnetic gold particles as carriers.

PubMed

Deng, MingJun; Long, Ling; Xiao, XiZhi; Wu, ZhenXing; Zhang, FengJuan; Zhang, YanMing; Zheng, XiaoLong; Xin, XueQian; Wang, Qun; Wu, DongLai

2011-06-15

Detecting avian influenza virus (AIV) and Newcastle disease virus (NDV) at low concentrations from tracheal and cloacal swabs of avian influenza- and Newcastle disease-infected poultry was carried out using a highly sensitive immunological-polymerase chain reaction (immuno-PCR) method. Magnetic gold particles were pre-coated with a capture antibody, either a monoclonal anti-AIV/H5 or monoclonal anti-NDV/F and viruses serially diluted ten-fold from 10(2) to 10(-5)EID(50)/ml. A biotinylated detection antibody bound to the viral antigen was then linked via a streptavidin bridge to biotinylated reporter DNA. After extensive washing, reporter DNA was released by denaturation, transferred to PCR tubes, amplified, electrophoresed and visualized. An optimized immuno-PCR method was able to detect as little as 10(-4)EID(50)/ml AIV and NDV. To further evaluate the specificity and the clinical application of this IPCR assay for AIV H5N1 and NDV, the tracheal swab specimens, taken from chickens which were infected with H5N1/AIV, H9N2/AIV, H7N2/AIV, NDV, IBDV, IBV/H(120), were detected by IPCR. Our data demonstrated that this monoclonal antibody-based immuno-PCR method provides a platform capable of rapid screening of clinical samples for trace levels of AIV H5 and NDV in one step. Copyright © 2011 Elsevier B.V. All rights reserved.

Rehabilitation or the death penalty: autoimmune B cells in the dock.

PubMed

Dahal, Lekh N; Cragg, Mark S

2015-03-01

CD20-based monoclonal antibodies have become established as treatments for lymphoma, rheumatoid arthritis, systemic lupus erythematosus, vasculitis and dermatomyositis, with the principle therapeutic mechanism relating to B-cell depletion through effector cell engagement. An article by Brühl et al. in this issue of the European Journal of Immunology [Eur. J. Immunol. 2015. 45: 705-715] reveals a fundamentally distinct mechanism of silencing autoimmune B-cell responses. Rather than B-cell depletion, the authors use anti-CD79b antibodies to induce B-cell tolerance and suppress humoral immune responses against collagen to prevent the development of arthritis in mice. Here we highlight the differences in the mechanisms used by anti-CD20 and anti-CD79b Ab therapy and discuss why depletion of B cells may not be required to treat autoimmune arthritis and other B-cell-associated pathologies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

L1-CAM-targeted antibody therapy and (177)Lu-radioimmunotherapy of disseminated ovarian cancer.

PubMed

Fischer, Eliane; Grünberg, Jürgen; Cohrs, Susan; Hohn, Alexander; Waldner-Knogler, Karin; Jeger, Simone; Zimmermann, Kurt; Novak-Hofer, Ilse; Schibli, Roger

2012-06-01

The L1-cell adhesion molecule (L1-CAM) is highly expressed in various cancer types including ovarian carcinoma but is absent from most normal tissue. A chimeric monoclonal antibody, chCE7, specifically binds to human L1-CAM and exhibits anti-proliferative effects on L1-CAM-expressing tumor cells. The goal of this study was to evaluate the efficacy of a novel (177)Lu-chCE7 radioimmunotherapeutic agent and to compare it to a treatment protocol with unlabeled, growth-inhibiting chCE7 in a mouse xenograft model of disseminated ovarian cancer. chCE7agl, an aglycosylated IgG1 variant with improved pharmacokinetics, was conjugated with 1,4,7,10-tetraazacyclododecane-N-N'-N'-N‴-tetraacetic acid (DOTA) and labeled with the low-energy β-emitter (177)Lu. Tumor growth and survival were assessed after a single i.v. dose of 8 MBq (60 μg) radioimmunoconjugate in nude mice bearing either subcutaneous or intraperitoneal SKOV3.ip1 human ovarian cancer tumors. Therapeutic efficacy was compared with three times weekly i.p. administration of 10 mg/kg unconjugated chCE7. In vivo analysis of (177)Lu-chCE7agl biodistribution demonstrated high and specific accumulation of radioactivity at the tumor site with maximal tumor uptake of up to 48.0 ± 8.1% ID/g at 168 h postinjection. A single treatment with (177)Lu-DOTA-chCE7agl caused significant retardation of tumor growth and prolonged median survival from 33 to 71 days, while administration of a nontargeted (177)Lu-immunoconjugate had no beneficial effect. Three times weekly i.p. application of unlabeled chCE7 10 mg/kg similarly increased survival from 44 to 72 days. We conclude that a single dose of (177)Lu-DOTA-chCE7agl is as effective as repeated administration of nonradioactive chCE7 for treatment of small intraperitoneal tumors expressing L1-CAM. Copyright © 2011 UICC.

Frequency of subgroups of the antigen “a” among volunteer donors

PubMed

Parra-Jaramillo, Katherine; Chiriboga-Ponce, Rosa F

2018-01-01

The presence of weak variants of blood type A represents a challenge in the practice of immunohematology for discrepancies in the time of the classification. It is common in blood banks to perform a forward and reverse typing for the purpose of confirming the blood type, but not all the people with a subgroup A2 have developed anti-A1 antibodies. We present a descriptive, observational and transversal study that establishes the proportion of subgroups of A antigen with the analysis of manual tube technique and monoclonal antibodies like anti-A, anti-A1 (Dolichus biflorus lectins extract) and anti-H. The analysis involved a total of 818 samples of voluntary blood donor, selected by random sampling, which were initially classified as 737 of Type A, and 81 as Type AB, with a confidence level of 95% (alpha error of 5% and 3% of precision). The present study evaluated the existence of the subgroups A1, A2, A1B, A2B, A intermediate and A intB. It is recommended the identification of subgroups in different types of blood in the laboratory and blood banks. Copyright: © 2018 SecretarÍa de Salud

Co-receptor Binding Site Antibodies Enable CD4-Mimetics to Expose Conserved Anti-cluster A ADCC Epitopes on HIV-1 Envelope Glycoproteins.

PubMed

Richard, Jonathan; Pacheco, Beatriz; Gohain, Neelakshi; Veillette, Maxime; Ding, Shilei; Alsahafi, Nirmin; Tolbert, William D; Prévost, Jérémie; Chapleau, Jean-Philippe; Coutu, Mathieu; Jia, Manxue; Brassard, Nathalie; Park, Jongwoo; Courter, Joel R; Melillo, Bruno; Martin, Loïc; Tremblay, Cécile; Hahn, Beatrice H; Kaufmann, Daniel E; Wu, Xueling; Smith, Amos B; Sodroski, Joseph; Pazgier, Marzena; Finzi, Andrés

2016-10-01

Human immunodeficiency virus type 1 (HIV-1) has evolved a sophisticated strategy to conceal conserved epitopes of its envelope glycoproteins (Env) recognized by antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. These antibodies, which are present in the sera of most HIV-1-infected individuals, preferentially recognize Env in its CD4-bound conformation. Accordingly, recent studies showed that small CD4-mimetics (CD4mc) able to "push" Env into this conformation sensitize HIV-1-infected cells to ADCC mediated by HIV+ sera. Here we test whether CD4mc also expose epitopes recognized by anti-cluster A monoclonal antibodies such as A32, thought to be responsible for the majority of ADCC activity present in HIV+ sera and linked to decreased HIV-1 transmission in the RV144 trial. We made the surprising observation that CD4mc are unable to enhance recognition of HIV-1-infected cells by this family of antibodies in the absence of antibodies such as 17b, which binds a highly conserved CD4-induced epitope overlapping the co-receptor binding site (CoRBS). Our results indicate that CD4mc initially open the trimeric Env enough to allow the binding of CoRBS antibodies but not anti-cluster A antibodies. CoRBS antibody binding further opens the trimeric Env, allowing anti-cluster A antibody interaction and sensitization of infected cells to ADCC. Therefore, ADCC responses mediated by cluster A antibodies in HIV-positive sera involve a sequential opening of the Env trimer on the surface of HIV-1-infected cells. The understanding of the conformational changes required to expose these vulnerable Env epitopes might be important in the design of new strategies aimed at fighting HIV-1. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Detection of specific antibody responses to vaccination in variable flying foxes (Pteropus hypomelanus).

PubMed

Wellehan, James F X; Green, Linda G; Duke, Diane G; Bootorabi, Shadi; Heard, Darryl J; Klein, Paul A; Jacobson, Elliott R

2009-09-01

Megachiropteran bats are biologically important both as endangered species and reservoirs for emerging human pathogens. Reliable detection of antibodies to specific pathogens in bats is thus epidemiologically critical. Eight variable flying foxes (Pteropus hypomelanus) were immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). Each bat received monthly inoculations for 2 months. Affinity-purified IgG was used for production of polyclonal and monoclonal anti-variable flying fox IgG antibodies. ELISA and western blot analysis were used to monitor immune responses and for assessment of polyclonal and monoclonal antibody species cross-reactivity. Protein G, polyclonal antibodies, and monoclonal antibodies detected specific anti-DNP antibody responses in immunized variable flying foxes, with protein G being the most sensitive, followed by monoclonal antibodies and then polyclonal antibodies. While the polyclonal antibody was found to cross-react well against IgG of all bat species tested, some non-specific background was observed. The monoclonal antibody was found to cross-react well against IgG of six other species in the genus Pteropus and to cross-react less strongly against IgG from Eidolon helvum or Phyllostomus hastatus. Protein G distinguished best between vaccinated and unvaccinated bats, and these results validate the use of protein G for detection of bat IgG. Monoclonal antibodies developed in this study recognized immunoglobulins from other members of the genus Pteropus well, and may be useful in applications where specific detection of Pteropus IgG is needed.

In vitro induction of monoclonal antibody-defined T-cell markers in lymphocytes from immunodeficient children by synthetic serum thymic factor (FTS).

PubMed Central

Bene, M C; Faure, G; Bordigoni, P; Olive, D; Duheille, J

1982-01-01

Lymphocytes from five children suffering from ataxia telangectasia or various unclassified immune deficiencies were tested in vitro for their sensitivity to synthetic serum thymic factor (FTS). The percentages of cells bearing T cell markers were elevated after incubation with FTS at graded concentrations (0.25, 2.5 and 25 ng/ml), by microlymphocytotoxicity or indirect immunofluorescence, using monoclonal anti-Lyt1 antibodies. In four cases, more than 30% of the non-T non-B cells acquired the Lyt1 T cell marker. These four children had low levels of circulating FTS. In the fifth child, who had a normal serum FTS level, and in two age-matched controls, there was no significant increase in the percentage of cells bearing the T marker. PMID:7049454

Local convection-enhanced delivery of an anti-CD40 agonistic monoclonal antibody induces antitumor effects in mouse glioma models.

PubMed

Shoji, Takuhiro; Saito, Ryuta; Chonan, Masashi; Shibahara, Ichiyo; Sato, Aya; Kanamori, Masayuki; Sonoda, Yukihiko; Kondo, Toru; Ishii, Naoto; Tominaga, Teiji

2016-08-01

Glioblastoma is one of the most malignant brain tumors in adults and has a dismal prognosis. In a previous report, we reported that CD40, a TNF-R-related cell surface receptor, and its ligand CD40L were associated with glioma outcomes. Here we attempted to activate CD40 signaling in the tumor and determine if it exerted therapeutic efficacy. CD40 expression was examined in 3 mouse glioma cell lines (GL261, NSCL61, and bRiTs-G3) and 5 human glioma cell lines (U87, U251, U373, T98, and A172). NSCL61 and bRiTs-G3, as glioma stem cells, also expressed the glioma stem cell markers MELK and CD44. In vitro, we demonstrated direct antitumor effects of an anti-CD40 agonistic monoclonal antibody (FGK45) against the cell lines. The efficacy of FGK45 was examined by local convection-enhanced delivery of the monoclonal antibody against each glioma model. CD40 was expressed in all mouse and human cell lines tested and was found at the cell membrane of each of the 3 mouse cell lines. FGK45 administration induced significant, direct antitumor effects in vitro. The local delivery of FGK45 significantly prolonged survival compared with controls in the NSCL61 and bRiTs-G3 models, but the effect was not significant in the GL261 model. Increases in apoptosis and CD4(+) and CD8(+) T cell infiltration were observed in the bRiTs-G3 model after FGK45 treatment. Local delivery of FGK45 significantly prolonged survival in glioma stem cell models. Thus, local delivery of this monoclonal antibody is promising for immunotherapy against gliomas. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Case report: diffuse splenic metastasis of occult breast cancer with incompatible blood group antigenic determinants.

PubMed

Baranyay, Ferenc

2009-01-01

Cancer cells with immunogenic properties having altered protein glycosilation, modified blood group substances have been widely studied [Kannagi R, Miyake M, Zenita KM, Itai S, Hiraiwa N, Shigeta K, et al. Cancer-associated carbohydrate antigens: modified blood group substances and oncodevelopmental antigens on tumor cells. Gann Monogr Cancer Res 1988; 34: p. 15-28; Hakomori S. Antigen structure and genetic basis of histo-blood groups A, B and O their changes associated with human cancer. Biochem Biophys Acta 1999; 1473: p. 247-266; Brooks SA, Carter TM, Royle L, Harvey DJ, Fry SA, Kinch C, et al. Altered glycosilation of proteins in cancer: what is the potential for new anti-tumour strategies. Anticancer Agents Med Chem 2008; 8: p. 2-21]. In the study reported here, a 78-year-old female patient was admitted to the hospital with circulatory failure. At autopsy, the spleen (weight: 420 g) was extremely firm with a diffusely blackberry-colored cut surface. There were no signs of carcinomatous process at autopsy. By histology, the spleen showed diffuse metastatic carcinomatous infiltration. Using immunohistochemistry, an antibody to breast carcinoma antigen (BioGenex) labelled metastatic cells of the spleen and bone marrow. The patient was blood group O. Labelling for binding of lectins with and without blood group antigen specificity and monoclonal antibodies was carried out. The B blood group specific Banderiaea simplicifolia agglutinin I and an anti-B blood group monoclonal antibody labelled all the metastatic cells of spleen and bone marrow intensely. There was no detection of blood group A antigen by either binding of Dolichos biflorus agglutinin or anti-blood group A monoclonal antibodies. These observations raise the possibility that the detected incompatible B blood group antigen determinants on the metastatic cells were immunogenic. The surviving carcinoma cells may have found a place of refuge from immune surveillance in the spleen and in the bone marrow, where the complement-mediated tumor cell lysis immune rejection was not effective.

Motility assays using myosin attached to surfaces through specific binding to monoclonal antibodies.

PubMed Central

Winkelmann, D. A.; Bourdieu, L.; Kinose, F.; Libchaber, A.

1995-01-01

We have analyzed the dependence of actin filament movement on the mode of myosin attachment to surfaces. Monoclonal antibodies that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. These monoclonal antibodies were used to provide increasing flexibility in the mode of attachment. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these monoclonal antibodies and the sliding movement of fluorescently labeled actin filaments analyzed by video microscopy. Each of these antibodies produced stable, myosin-coated surfaces that supported uniform movement of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM monoclonal antibodies yielded a maximum velocity of 10 microns/s at 30 degrees C, whereas attachment through anti-LC2 produced a lower velocity of 4-5 microns/s. Each antibody showed a characteristic minimum myosin density below which sliding movement was no longer supported and an exponential dependence of actin filament velocity on myosin surface density below Vmax. Maximum sliding velocity was achieved over a range of myosin surface densities. Thus, the specific mode of attachment can influence the characteristic velocity of actin filament movement and the surface density needed to support movement. These data are being used to analyze the dynamics of sliding filament assays and evaluate estimates of the average number of motor molecules per unit length of actin required to support movement. PMID:7787107

Effects of tissue fixation conditions and protease pretreatment on immunohistochemical performance of a large series of new anti-keratin monoclonal antibodies: value in oncopathology.

PubMed

Bártková, J; Bártek, J; Lukás, Z; Vojtĕsek, B; Stasková, Z; Bursová, H; Pavlovská, R; Rejthar, A; Kovarík, J

1991-01-01

A comparative study with 21 recently raised monoclonal antibodies (3 of which are reported here for the first time) to human keratin polypeptides was performed on a wide range of paraffin-embedded tissues and tumors, aimed at the examination of effects of four different fixatives and protease pretreatment on the immunohistochemical detection of keratins. Our data demonstrated that: (a) formaldehyde-based fixatives modified by acidification and/or addition of methanol gave results superior to those achieved by routinely used formol saline; (b) relatively rare antibodies (4 out of 21) could be identified which gave reliable immunostaining patterns even on routine formalin-fixed material; (c) a proteolytic digestion step preceding the immunostaining was beneficial for the performance of the majority of antibodies in our panel. Additional options which could potentially lead to further improvement of keratin immunohistochemistry in paraffin embedded specimens are also suggested. This work provides the necessary basis for wider application of the anti-keratin antibodies of the C-series in both routine oncopathology and research-oriented retrospective studies.

Anti-PD1 following ipilimumab for mucosal melanoma: durable tumor response associated with severe hypothyroidism and rhabdomyolysis.

PubMed

Min, Le; Hodi, F Stephen

2014-01-01

Treatment with fully human monoclonal antibodies against programmed death 1 (PD1) receptor has shown great promise for a number of advanced malignancies. Although inflammatory adverse events have been well described with anti-CTL antigen 4 (CTLA4) therapy, experience with the range of adverse effects of anti-PD1 remains comparatively limited. Here, we report on a patient with advanced mucosal melanoma who received four doses of MK-3475, a fully human monoclonal antibody against PD1, and experienced a durable near-complete response but developed severe hypothyroidism, rhabdomyolysis, and acute kidney injury. To our knowledge, this is the first case reported of a patient with advanced mucosal melanoma who responded to anti-PD1 therapy. With the promising antitumor effects of anti-PD1 in a wide array of tumors, we expect an increasing number of patients to be exposed to anti-PD1 therapies. Recognition of infrequent presentations of adverse events such as elevated creatine kinase levels and thyroid disorders in patients who receive anti-PD1 therapy is important. ©2014 AACR.

T regulatory cells participate in the control of germinal centre reactions

PubMed Central

Alexander, Carla-Maria; Tygrett, Lorraine T; Boyden, Alexander W; Wolniak, Kristy L; Legge, Kevin L; Waldschmidt, Thomas J

2011-01-01

Germinal centre (GC) reactions are central features of T-cell-driven B-cell responses, and the site where antibody-producing cells and memory B cells are generated. Within GCs, a range of complex cellular and molecular events occur which are critical for the generation of high affinity antibodies. These processes require exquisite regulation not only to ensure the production of desired antibodies, but to minimize unwanted autoreactive or low affinity antibodies. To assess whether T regulatory (Treg) cells participate in the control of GC responses, immunized mice were treated with an anti-glucocorticoid-induced tumour necrosis factor receptor-related protein (GITR) monoclonal antibody (mAb) to disrupt Treg-cell activity. In anti-GITR-treated mice, the GC B-cell pool was significantly larger compared with control-treated animals, with switched GC B cells composing an abnormally high proportion of the response. Dysregulated GCs were also observed regardless of strain, T helper type 1 or 2 polarizing antigens, and were also seen after anti-CD25 mAb treatment. Within the spleens of immunized mice, CXCR5+ and CCR7− Treg cells were documented by flow cytometry and Foxp3+ cells were found within GCs using immunohistology. Final studies demonstrated administration of either anti-transforming growth factor-β or anti-interleukin-10 receptor blocking mAb to likewise result in dysregulated GCs, suggesting that generation of inducible Treg cells is important in controlling the GC response. Taken together, these findings indicate that Treg cells contribute to the overall size and quality of the humoral response by controlling homeostasis within GCs. PMID:21635248

Development of a one-step immunochromatographic strip test for the detection of sennosides A and B.

PubMed

Putalun, Waraporn; Morinaga, Osamu; Tanaka, Hiroyuki; Shoyama, Yukihiro

2004-01-01

An immunochromatographic strip test was developed to detect sennoside A (1) and sennoside B (2) using anti-1 and anti-2 monoclonal antibodies. The qualitative assay was based on a competitive immunoassay in which the detector reagent consisted of colloidal gold particles coated with the respective sennoside antibodies. The capture reagents were 1- and 2-human serum albumin (HSA) conjugates immobilised on a nitrocellulose membrane on the test strip. The sample containing 1 and 2, together with detector reagent, passed over the zone where the capture reagents had been immobilised. The analytes in the sample competed for binding to the limited amount of antibodies in the detector reagent with the immobilised 1- and 2-HSA conjugates on the membrane and hence positive samples showed no colour in the capture spot zone. Detection limits for the strip test were 125 ng/mL for both sennosides. The assay system is useful as a rapid and simple screening method for the detection of 1 and 2 in plants, drugs and body fluids.

Monoclonal Antibodies Against Fusicoccin with Binding Characteristics Similar to the Putative Fusicoccin Receptor of Higher Plants 1

PubMed Central

Feyerabend, Martin; Weiler, Elmar W.

1987-01-01

Monoclonal antibodies were raised against fusicoccin. The toxin, linked to bovine serum albumin through its t-pentenyl moiety, served as immunogen. Hybridomas secreting anti-fusicoccin antibodies were screened by radioimmunoassay employing a novel radioactive derivative, [3H]-nor-fusicoccin-alcohol of high specific activity (1.5 × 1014Bq/mole). The two monoclonal antibodies reported here are of high apparent affinity for fusicoccin (0.71 × 10−9 molar and 1.85 × 10−9 molar). This is comparable to the apparent affinity of rabbit antiserum raised against the same type of conjugate (9.3 × 10−9 molar). A method for the single step purification of the monoclonal antibodies from ascites fluid is reported. A solid-phase immunoassay, using alkaline phosphatase as enzyme, exhibits a measuring range from 0.1 to 1.5 picomoles (about 70 picograms to 1 nanogram) of fusicoccin. The displacement of [3H]-nor-fusicoccin-alcohol from the antibodies by compounds structurally related to fusicoccin exhibits similar selectivity as a microsomal binding assay with the same tracer as radiolabeled probe. Images Fig. 2 PMID:16665786

Radioimmunotherapy of pancreatic cancer xenografts in nude mice using 90Y-labeled anti-α6β4 integrin antibody

PubMed Central

Aung, Winn; Tsuji, Atsushi B.; Sudo, Hitomi; Sugyo, Aya; Ukai, Yoshinori; Kouda, Katsushi; Kurosawa, Yoshikazu; Furukawa, Takako; Saga, Tsuneo

2016-01-01

The contribution of integrin α6β4 (α6β4) overexpression to the pancreatic cancer invasion and metastasis has been previously shown. We have reported immunotargeting of α6β4 for radionuclide-based and near-infrared fluorescence imaging in a pancreatic cancer model. In this study, we prepared yttrium-90 labeled anti-α6β4 antibody (90Y-ITGA6B4) and evaluated its radioimmunotherapeutic efficacy against pancreatic cancer xenografts in nude mice. Mice bearing xenograft tumors were randomly divided into 5 groups: (1) single administration of 90Y-ITGA6B4 (3.7MBq), (2) double administrations of 90Y-ITGA6B4 with once-weekly schedule (3.7MBq × 2), (3) single administration of unlabeled ITGA6B4, (4) double administrations of unlabeled ITGA6B4 with once-weekly schedule and (5) the untreated control. Biweekly tumor volume measurements and immunohistochemical analyses of tumors at 2 days post-administration were performed to monitor the response to treatments. To assess the toxicity, body weight was measured biweekly. Additionally, at 27 days post-administration, blood samples were collected through cardiac puncture, and hematological parameters, hepatic and renal functions were analyzed. Both 90Y-ITGA6B4 treatment groups showed reduction in tumor volumes (P < 0.04), decreased cell proliferation marker Ki-67-positive cells and increased DNA damage marker p-H2AX-positive cells, compared with the other groups. Mice treated with double administrations of 90Y-ITGA6B4, exhibited myelosuppression. There were no significant differences in hepatic and renal functions between the 2 treatment groups and the other groups. Our results suggest that 90Y-ITGA6B4 is a promising radioimmunotherapeutic agent against α6β4 overexpressing tumors. In the future studies, dose adjustment for fractionated RIT should be considered carefully in order to get the optimal effect while avoiding myelotoxicity. PMID:27246980

Combination of treatment with death receptor 5-specific antibody with therapeutic HPV DNA vaccination generates enhanced therapeutic antitumor effects

PubMed Central

Tseng, Chih-Wen; Trimble, Cornelia; Monie, Archana; Alvarez, Ronald D.; Huh, Warner K.; Buchsbaum, Donald J.; Straughn, J. Michael; Wang, Mei-Cheng; Yagita, Hideo; Hung, Chien-Fu; Wu, T.-C.

2008-01-01

There is currently a vital need for the development of novel therapeutic strategies for the control of advanced stage cancers. Antigen-specific immunotherapy and the employment of antibodies against the death receptor 5 (DR5) have emerged as two potentially promising strategies for cancer treatment. In the current study, we hypothesize that the combination of treatment with the anti-DR5 monoclonal antibody, MD5-1 with a DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 (HPV-16) E7 antigen (CRT/E7(detox)) administered via gene gun would lead to further enhancement of E7-specific immune responses as well as antitumor effects. Our results indicated that mice bearing the E7-expressing tumor, TC-1 treated with MD5-1 monoclonal antibody followed by CRT/E7(detox) DNA vaccination generated the most potent therapeutic anti-tumor effects as well as highest levels of E7-specific CD8+ T cells among all the groups tested. In addition, treatment with MD5-1 monoclonal antibody was capable of rendering the TC-1 tumor cells more susceptible to lysis by E7-specific cytotoxic T lymphocytes. Our findings serve as an important foundation for future clinical translation. PMID:18598733

Ta1, a novel 105 KD human T cell activation antigen defined by a monoclonal antibody.

PubMed

Fox, D A; Hussey, R E; Fitzgerald, K A; Acuto, O; Poole, C; Palley, L; Daley, J F; Schlossman, S F; Reinherz, E L

1984-09-01

By using a murine monoclonal antibody produced against an IL 2-dependent human T cell line, we defined a T lineage-specific molecule, termed Ta1, that is expressed strongly on activated T lymphocytes of both the T4 and T8 subsets, as well as on T cell lines and clones, but only weakly on a fraction of resting T cells. SDS-PAGE analysis of immunoprecipitates from 125I-labeled, activated T cells demonstrates a single major band of apparent m.w. 105 KD under both reducing and nonreducing conditions. Unlike anti-IL 2 receptor antibodies, anti-Ta1 does not inhibit T cell proliferative responses to mitogen, antigen, or IL 2-containing medium. Moreover, anti-Ta1 has no effect on T cell-mediated cytotoxicity. Ta1 appears to be a novel human T cell-specific activation antigen that may serve as a useful marker of T cell activation in human disease.

Photoluminescence detection of 2,4,6-trinitrotoluene (TNT) binding on diatom frustule biosilica functionalized with an anti-TNT monoclonal antibody fragment.

PubMed

Zhen, Le; Ford, Nicole; Gale, Debra K; Roesijadi, Guritno; Rorrer, Gregory L

2016-05-15

A selective and label-free biosensor for detection of the explosive compound 2,4,6-trinitrotoluene (TNT) in aqueous solution was developed based on the principle of photoluminescence quenching of upon immunocomplex formation with antibody-functionalized diatom frustule biosilica. The diatom frustule is an intricately nanostructured, highly porous biogenic silica material derived from the shells of microscopic algae called diatoms. This material emits strong visible blue photoluminescence (PL) upon UV excitation. PL-active frustule biosilica was isolated from cultured cells of the marine diatom Pinnularia sp. and functionalized with a single chain variable fragment (scFv) derived from an anti-TNT monoclonal antibody. When TNT was bound to the anti-TNT scFv-functionalized diatom frustule biosilica, the PL emission from the biosilica was partially quenched due to the electrophilic nature of the nitro (-NO2) groups on the TNT molecule. The dose-response curve for immunocomplex formation of TNT on the scFv-functionalized diatom frustule biosilica had a half-saturation binding constant of 6.4 ± 2.4·10(-8)M and statistically-significant measured detection limit of 3.5·10(-8)M. The binding and detection were selective for TNT and TNB (trinitrobenzene) but not RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) or 2,6-DNT (2,6-dinitrotoluene). Copyright © 2016. Published by Elsevier B.V.

A monoclonal IgM smooth muscle antibody reactive with fibroblast stress fibres produced by immunization with Treponema pallidum.

PubMed Central

Strugnell, R A; Underwood, J R; Clarke, F M; Pedersen, J S; Chalmers, P J; Faine, S; Toh, B H

1983-01-01

A monoclonal IgM smooth muscle antibody secreted by a hybrid (MMI-1) of mouse plasmacytoma NS-1 with spleen cells from mouse immunized with Treponema pallidum was detected by indirect immunofluorescence tests on frozen tissue sections and on acetone fixed monolayers of rat and human fibroblasts. The antibody did not react with acetone fixed smears of T. pallidum but reacted with smooth muscle fibres and with striations of skeletal and cardiac muscle. In non-muscle cells, the antibody stained liver in a 'polygonal' pattern, thymus with accentuated staining of the thymic medulla, renal glomeruli and the brush border and peritubular fibrils of renal tubules. In fibroblast monolayers, the antibody stained stress fibres in an interrupted pattern. Immunoblotting with muscle proteins and the antibody showed labelling of a 100K molecule. The cellular distribution of the mouse monoclonal antibody is similar to that obtained with anti-actin antibody suggesting that the corresponding antigen may be an actin binding protein. Images Fig. 3 PMID:6347470

Cloning of the first human anti-JCPyV/VP1 neutralizing monoclonal antibody: epitope definition and implications in risk stratification of patients under natalizumab therapy.

PubMed

Diotti, Roberta Antonia; Mancini, Nicasio; Clementi, Nicola; Sautto, Giuseppe; Moreno, Guisella Janett; Criscuolo, Elena; Cappelletti, Francesca; Man, Petr; Forest, Eric; Remy, Louise; Giannecchini, Simone; Clementi, Massimo; Burioni, Roberto

2014-08-01

JC virus (JCPyV) has gained novel clinical importance as cause of progressive multifocal leukoencephalopathy (PML), a rare demyelinating disease recently associated to immunomodulatory drugs, such as natalizumab used in multiple sclerosis (MS) cases. Little is known about the mechanisms leading to PML, and this makes the need of PML risk stratification among natalizumab-treated patients very compelling. Clinical and laboratory-based risk-stratification markers have been proposed, one of these is represented by the JCPyV-seropositive status, which includes about 54% of MS patients. We recently proposed to investigate the possible protective role of neutralizing humoral immune response in preventing JCPyV reactivation. In this proof-of-concept study, by cloning the first human monoclonal antibody (GRE1) directed against a neutralizing epitope on JCPyV/VP1, we optimized a robust anti-JCPyV neutralization assay. This allowed us to evaluate the neutralizing activity in JCPyV-positive sera from MS patients, demonstrating the lack of correlation between the level of anti-JCPyV antibody and anti-JCPyV neutralizing activity. Relevant consequences may derive from future clinical studies induced by these findings; indeed the study of the serum anti-JCPyV neutralizing activity could allow not only a better risk stratification of the patients during natalizumab treatment, but also a better understanding of the pathophysiological mechanisms leading to PML, highlighting the contribution of peripheral versus central nervous system JCPyV reactivation. Noteworthy, the availability of GRE1 could allow the design of novel immunoprophylactic strategies during the immunomodulatory treatment. Copyright © 2014 Elsevier B.V. All rights reserved.

[Study on the assay of proximal tubular antigen in urine and serum with an anti-human renal monoclonal antibody].

PubMed

Taniai, K

1991-10-01

Monoclonal antibodies (Mabs) were produced by immunizing mice with human kidney microsomal antigen. Mab-B1 recognized brushborder (B1-Ag) in proximal tubules. Using Mab-B1, B1-Ag was assayed in the urine and serum of renal disease patients by sandwich ELISA. The subjects included normal control (Nor), minimal change nephrotic syndrome (MCNS), IgA nephropathy (IgA), membranous nephropathy (MN), membranoproliferative glomerulonephritis (MPGN), and chronic renal failure (CRF) (s-Cr greater than 2 mg/dl). Urinary B1-Ag demonstrated significant increases in the IgA (p less than 0.001), MN (p less than 0.001), MPGN (p less than 0.001) and CRF (p less than 0.01) groups as compared to the Nor group. There was no significant increase in the MCNS group. In the CRF group, B1-Ag in urine showed a significant increase in the progressive CRF group with delta s-Cr greater than 1.0 mg/dl/month as compared to the stationary CRF group with delta s-Cr less than 1.0 mg/dl/month. No correlation was observed between urinary B1-Ag and proteinuria, hematuria, s-Cr, s-BMG and u-NAG. The above findings suggested that the assay of urinary B1-Ag was useful as a new parameter in detecting the site and degree of proximal tubular damage.

Fc gamma receptor 3a genotype predicts overall survival in follicular lymphoma patients treated on SWOG trials with combined monoclonal antibody plus chemotherapy but not chemotherapy alone

PubMed Central

Persky, Daniel O.; Dornan, David; Goldman, Bryan H.; Braziel, Rita M.; Fisher, Richard I.; LeBlanc, Michael; Maloney, David G.; Press, Oliver W.; Miller, Thomas P.; Rimsza, Lisa M.

2012-01-01

Background Fc gamma receptor polymorphisms were linked to outcome in follicular lymphoma patients treated with single-agent rituximab, an anti-CD20 monoclonal antibody. In particular, 158F/F genotype of Fc gamma receptor 3A and 131R/R genotype of Fc gamma receptor 2A correlated with worse outcome compared to high-affinity 158V/V and 131H/H, respectively. We examined this association in the context of anti-CD20 monoclonal antibody combined with chemotherapy, as compared to chemotherapy alone, in follicular lymphoma patients treated on SWOG clinical trials. Design and Methods Tissue from 142 SWOG patients treated with chemotherapy alone (protocol S8809, n=70) or combined chemotherapy and anti-CD20 monoclonal antibody (rituximab and Iodine I-131 tositumomab on protocols S9800 and S9911, n=30 and 42, respectively) was analyzed. DNA was extracted and assayed for Fc gamma receptor 3A V158F and 2A R131H polymorphisms using a TaqMan SNP assay. Stratified Cox’s regression was used to assess association with overall survival. Results For Fc gamma receptor 3A, there was an association with overall survival in the combination therapy trials but not in the chemotherapy-only trial. Having at least one Fc gamma receptor 3A V allele was associated with improved overall survival versus F/F (HR=0.33, 95% CI, 0.11, 0.96, P=0.042). For overall survival, there was evidence of a statistical interaction between the use of mAb and the number of V alleles (0, 1, or 2) (P=0.006). There was no such association for Fc gamma receptor 2A. Conclusions Fc gamma receptor 3A polymorphism status may be predictive of survival in follicular lymphoma patients receiving treatments containing an anti-CD20 antibody but not treatment with chemotherapy alone. Thus, Fc gamma receptor 3A polymorphisms may be important to consider in designing new follicular lymphoma trials and new anti-CD20 monoclonal antibodies. PMID:22271896

Rat brain xenografts reverse hypogonadism in mice immunosuppressed with anti-CD4 monoclonal antibody.

PubMed

Honey, C R; Charlton, H M; Wood, K J

1991-01-01

This study examines the effect of immunosuppression with monoclonal antibodies (MAb) against the murine CD4 (L3T4), a cell surface glycoprotein expressed primarily on helper T-lymphocytes, on the viability and function of rat neural xenografts placed in the third ventricle of hypogonadal (hpg) mice. The hpg mouse fails to synthesize hypothalamic gonadotrophin releasing hormone (GnRH) and consequently there is a drastic reduction in pituitary gonadotrophic hormone content and a failure of postnatal gonadal development (Cattanach et al. 1977). Three groups of male hpg mice received xenografts of day 1 post natal rat preoptic area (POA) tissue, a source of GnRH neurons, to their third ventricle. Those immunosuppressed with anti-CD4 MAb all showed surviving graft tissue thirty days post-transplant and half of this group had enlarged testes with all stages of spermatogenesis. In those hpg mice which were injected with saline alone, or with an anti-CD8 (Lyt-2) antibody there was no xenograft survival. These results suggest that the injection of monoclonal antibodies against the T-helper subset may provide an alternative means of immunosuppression aimed at the enhancement of survival of tissue grafts in the CNS.

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors.

PubMed

Batsuli, Glaivy; Deng, Wei; Healey, John F; Parker, Ernest T; Baldwin, W Hunter; Cox, Courtney; Nguyen, Brenda; Kahle, Joerg; Königs, Christoph; Li, Renhao; Lollar, Pete; Meeks, Shannon L

2016-10-20

Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance. © 2016 by The American Society of Hematology.

Immunization with a novel chimeric peptide representing B and T cell epitopes from HER2 extracellular domain (HER2 ECD) for breast cancer.

PubMed

Mahdavi, Manijeh; Keyhanfar, Mehrnaz; Jafarian, Abbas; Mohabatkar, Hassan; Rabbani, Mohammad

2014-12-01

Because of direct stimulating immune system against disease, vaccination or active immunotherapy is preferable compared to passive immunotherapy. For this purpose, a newly designed chimeric peptide containing epitopes for both B and T cells from HER2 ECD subdomain III was proposed. To evaluate the effects of the active immunization, a discontinuous B cell epitope peptide was selected based on average antigenicity by bioinformatics analysis. The selected peptide was collinearly synthesized as a chimera with a T helper epitope from the protein sequence of measles virus fusion (208-302) using the GPSL linker. Three mice were immunized with the chimeric peptide. Reactive antibodies with HER2 protein in ELISA and immunofluorescence assays with no cross-reactivity were generated. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay indicated that the anti-peptide sera had inhibitory effects on proliferation of SK-BR-3 cells. Hence, the newly designed, discontinuous chimeric peptide representing B and T cell epitopes from subdomain III of HER2-ECD can form the basis for future vaccines design, where these data can be applied for monoclonal antibody production targeting the distinct epitope of HER2 receptor compared to the two broadly used anti-HER2 monoclonal antibodies, Herceptin and pertuzumab.

Monitoring multiple myeloma patients treated with daratumumab: teasing out monoclonal antibody interference.

PubMed

McCudden, Christopher; Axel, Amy E; Slaets, Dominique; Dejoie, Thomas; Clemens, Pamela L; Frans, Sandy; Bald, Jaime; Plesner, Torben; Jacobs, Joannes F M; van de Donk, Niels W C J; Moreau, Philippe; Schecter, Jordan M; Ahmadi, Tahamtan; Sasser, A Kate

2016-06-01

Monoclonal antibodies are promising anti-myeloma treatments. As immunoglobulins, monoclonal antibodies have the potential to be identified by serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE). Therapeutic antibody interference with standard clinical SPE and IFE can confound the use of these tests for response assessment in clinical trials and disease monitoring. To discriminate between endogenous myeloma protein and daratumumab, a daratumumab-specific immunofixation electrophoresis reflex assay (DIRA) was developed using a mouse anti-daratumumab antibody. To evaluate whether anti-daratumumab bound to and shifted the migration pattern of daratumumab, it was spiked into daratumumab-containing serum and resolved by IFE/SPE. The presence (DIRA positive) or absence (DIRA negative) of residual M-protein in daratumumab-treated patient samples was evaluated using predetermined assessment criteria. DIRA was evaluated for specificity, limit of sensitivity, and reproducibility. In all of the tested samples, DIRA distinguished between daratumumab and residual M-protein in commercial serum samples spiked with daratumumab and in daratumumab-treated patient samples. The DIRA limit of sensitivity was 0.2 g/L daratumumab, using spiking experiments. Results from DIRA were reproducible over multiple days, operators, and assays. The anti-daratumumab antibody was highly specific for daratumumab and did not shift endogenous M-protein. As the treatment of myeloma evolves to incorporate novel monoclonal antibodies, additional solutions will be needed for clinical monitoring of patient responses to therapeutic regimens. In the interim, assays such as DIRA can inform clinical outcomes by distinguishing daratumumab from endogenous M-protein by IFE.

A Randomized Phase 2 Trial of 177Lu Radiolabeled Anti-PSMA Monoclonal Antibody J591in Patients with High-Risk Castrate, Biochemically Relapsed Prostate Cancer

DTIC Science & Technology

2012-09-01

micrometastases that may be targeted with radioimmunotherapy. Prostate specific membrane antigen (PSMA) is the single, most well-established, highly restricted...Radiolabeled anti- prostate specific membrane antigen (PSMA) monoclonal antibody J591 (177Lu-J591) for nonmetastatic castration-resistant prostate cancer...rationale for systemic salvage targeted anti- prostate specific membrane antigen radioimmunotherapy. Adv Urol 2012, Article ID 921674, doi:10.1155

Role of Reactive Stroma in Prostate Cancer Progression

DTIC Science & Technology

2007-02-01

buffer with 5% nonfat milk at 4jC overnight. Mouse anti-V5 monoclonal antibody (Invitrogen), diluted at 1:5,000, was used as primary antibody to...detect the presence of CTGF-V5-His fusion protein, and incubated for 2 hours at room temperature. Secondary antibody was biotin-conjugated sheep anti-mouse...genes is associated with reactive stroma that forms at sites of wound repair, microbial invasion, or carcinoma as we have reported previously (1, 32, 35

HLA Class Ia and Ib Polyreactive Anti-HLA-E IgG2a Monoclonal Antibodies (TFL-006 and TFL-007) Suppress Anti-HLA IgG Production by CD19+ B Cells and Proliferation of CD4+ T Cells While Upregulating Tregs

PubMed Central

2017-01-01

The anti-HLA-E IgG2a mAbs, TFL-006 and TFL-007, reacted with all HLA-I antigens, similar to the therapeutic preparations of IVIg. Indeed, IVIg lost its HLA reactivity, when its HLA-E reactivity was adsorbed out. US-FDA approved IVIg to reduce antibodies in autoimmune diseases. But the mechanism underlying IVIg-mediated antibody reduction could not be ascertained due to the presence of other polyclonal antibodies. In spite of it, the cost prohibitive high or low IVIg is administered to patients waiting for donor organ and for allograft recipients for lowering antiallograft antibodies. A mAb that could mimic IVIg in lowering Abs, with defined mechanism of action, would be highly beneficial for patients. Demonstrably, the anti-HLA-E mAbs mimicked several functions of IVIg relevant to suppressing the antiallograft Abs. The mAbs suppressed activated T cells and anti-HLA antibody production by activated B cells, which were dose-wise superior to IVIg. The anti-HLA-E mAb expanded CD4+, CD25+, and Foxp3+ Tregs, which are known to suppress T and B cells involved in antibody production. These defined functions of the anti-HLA-E IgG2a mAbs at a level superior to IVIg encourage developing their humanized version to lower antibodies in allograft recipients, to promote graft survival, and to control autoimmune diseases. PMID:28634589

Development of an immunoaffinity column method using broad-specificity monoclonal antibodies for simultaneous extraction and cleanup of quinolone and sulfonamide antibiotics in animal muscle tissues.

PubMed

Li, Cun; Wang, Zhanhui; Cao, Xingyuan; Beier, Ross C; Zhang, Suxia; Ding, Shuangyang; Li, Xiaowei; Shen, Jianzhong

2008-10-31

This paper describes a novel mixed-bed immunoaffinity column (IAC) method. The IAC was produced by coupling anti-quinolone and anti-sulfonamide broad-specificity monoclonal antibodies to Sepharose 4B for simultaneously isolating 13 quinolones (QNs) and 6 sulfonamides (SAs) from swine and chicken muscle tissues, followed by antibiotic determination using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A new broad-specificity Mab (B1A4E8) toward sulfonamides was produced using sulfamethoxazole as hapten that demonstrated cross-reactivities to 6 SAs in the range of 31-112%. IAC optimized conditions were found that allowed the IAC to be reused for selective binding of both SAs and QNs. Recoveries of all 19 antibiotics from animal muscle ranged from 72.6 to 107.6%, with RSDs below 11.3% and 15.4% for intra-day and inter-day experiments, respectively. The limit of quantification ranged from 0.5 to 3.0ng/g. The strategy used here for a mixed-bed IAC may be used to study other compounds and more than two classes of analytes simultaneously.

Hepatic microsomal cytochromes P450 in mink fed Saginaw Bay carp (SBC)

USGS Publications Warehouse

Melancon, M.J.; LeCaptain, L.; Rattner, B.A.; Heaton, S.; Aulerich, R.; Tillitt, D.; Stegeman, John J.; Woodin, B.

1992-01-01

Livers from mink fed diets containing 0% (n = 12), 10% (n = 11), 20% (n = 12) and 40% (n = 10) SBC for 6 months contained 0.1, 2.2, 3.6, and 6.3 ug/g total PCBs, respectively. Hepatic microsomes were prepared and assayed for protein, arylhydrocarbon hydroxylase (AHH), benzyloxyresorufin-O-dealkylase (BROD), ethoxy-ROD (ER0D), pentoxy-ROD (PROD), and ethoxycoumarin-OD (ECOD). Mink fed SBC had increased AHH, EROD, and ECOD (group means 2.2-3.4 X control means), decreased BROD and unchanged PROD (the latter 2 assays indicators for phenobarbital-type induction in mammals). Three samples from each group were examined by western blot using a polyclonal anti-P450llB antibody and a monoclonal anti-P450lA antibody (MAb 1-12-3). Mink fed SBC showed induction of a protein recognized by anti-P450lA (8 X control), but had little protein recognized by anti-P450IlB. The monooxygenase activities and western blot data give a consistent picture of MC-type but not PB-type induction in mink fed SBC.

Immunization against an IL-6 peptide induces anti-IL-6 antibodies and modulates the Delayed-Type Hypersensitivity reaction in cynomolgus monkeys.

PubMed

Desallais, Lucille; Bouchez, Caroline; Mouhsine, Hadley; Moreau, Gabriel; Ratsimandresy, Rojo; Montes, Matthieu; Do, Hervé; Quintin-Colonna, Françoise; Zagury, Jean-François

2016-01-19

Interleukin-6 (IL-6) overproduction has been involved in the pathogenesis of several chronic inflammatory diseases and the administration of an anti-IL-6 receptor monoclonal antibody has been proven clinically efficient to treat them. However, the drawbacks of monoclonal antibodies have led our group to develop an innovative anti-IL-6 strategy using a peptide-based active immunization. This approach has previously shown its efficacy in a mouse model of systemic sclerosis. Here the safety, immunogenicity, and efficacy of this strategy was assessed in non human primates. No unscheduled death and clinical signs of toxicity was observed during the study. Furthermore, the cynomolgus monkeys immunized against the IL-6 peptide produced high levels of anti-IL-6 antibodies as well as neutralizing antibodies compared to control groups. They also showed an important decrease of the cumulative inflammatory score following a delayed-type hypersensitivity reaction induced by the Tetanus vaccine compared to control groups (minus 57,9%, P = 0.014). These findings are highly significant because the immunizing IL-6 peptide used in this study is identical in humans and in monkeys and this novel anti-IL-6 strategy could thus represent a promising alternative to monoclonal antibodies.

A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Yanlan; Chen, Yicheng; Ding, Guoqing

The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacymore » and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ristow, Sandra S.; Arnzen, Jeanene M.; Leong, JoAnn Ching

Seventeen strains of infectious hematopoietic necrosis virus (IHNV) from different geographical regions and from different fish stocks were typed by polyacrylamide gel electrophoresis, indirect fluorescence with 27 monoclonal antibodies against both the G and N proteins of the virus, and by serum neutralization with six monoclonal anti-glycoprotein antibodies. In addition, many other IHNV isolates have been examined. Studying the isolates with the antibodies has shown that a greater amount of variation exists between isolates than was first predicted by the application of the polyacrylamide technique. Isolates within electrophoretic types I-V may be further classified according to their reactions with themore » monoclonal antibodies in indirect fluorescence. Serum neutralization with selected anti-glycoprotein antibodies in conjunction with fluorescence analysis confirms one of the original findings of Hsu et al. (1986) that two different species in a single facility can be infected with the same isolate. Variation among isolates as measured by reactivity with the monoclonal library appears to be greater within the G protein than within the N protein sequence. 9 refs., 7 figs., 6 tabs.« less

Monoclonal Idiotope Vaccine against Streptococcus pneumoniae Infection

NASA Astrophysics Data System (ADS)

McNamara, Mary K.; Ward, Ronald E.; Kohler, Heinz

1984-12-01

A monoclonal anti-idiotope antibody coupled to a carrier protein was used to immunize BALB/c mice against a lethal Streptococcus pneumoniae infection. Vaccinated mice developed a high titer of antibody to phosphorylcholine, which is known to protect against infection with Streptococcus pneumoniae. Measurement of the median lethal dose of the bacteria indicated that anti-idiotope immunization significantly increased the resistance of BALB/c mice to the bacterial challenge. Antibody to an idiotope can thus be used as an antigen substitute for the induction of protective immunity.

Technetium-99m-labeled rituximab for use as a specific tracer of sentinel lymph node biopsy: a translational research study.

PubMed

Wang, Xuejuan; Yang, Zhi; Lin, Baohe; Zhang, Yan; Zhai, Shizhen; Zhao, Qichao; Xie, Qing; Liu, Fei; Han, Xuedi; Li, Jinfeng; Ouyang, Tao

2016-06-21

We aimed to develop and translate a CD20-antigen-targeted radiopharmaceutical, Technetium-99 m-labeled (99mTc) rituximab, for sentinel lymph node (SLN) detection. 99mTc-rituximab was synthesized and tested for stability in human serum. The binding affinity to CD20 was evaluated in Raji cells by flow cytometric analysis. Biodistribution and sentinel node mapping were carried out in bal b/c mice. Eighty-five patients with breast cancer participated in this study. Dynamic sentinel lymphoscintigraphy was first assessed in 12 patients before planar lymphoscintigraphy was assessed in a larger cohort. All patients underwent sentinel lymph node biopsy (SLNB), followed by axillary lymph node dissection. The cell-binding study showed that 99mTc-rituximab possessed compatible affinity to human CD20. In the mechanism study, 99mTc-labeled anti-mouse CD20 monoclonal antibodies could bind to mouse CD20 and accumulate in the SLN with 2.62±1.25 % of the percentage of injected activity, which could be blocked by excessive unlabeled antibody. Low uptake of non-sentinel nodes and fast clearance from the injection site were observed in the mice. Sentinel nodes were identified in 82 of 85 breast cancer patients (96.5%) by lymphoscintigraphy and SLNB. The sensitivity, specificity, and accuracy were 96.8% (30/31), 100% (51/51), and 98.8% (81/82), respectively. 99mTc-rituximab, specifically binding to CD20, met most of the requirements of an ideal sentinel mapping agent for use in clinical settings.

Optimization of multi-epitopic HIV-1 recombinant protein expression in prokaryote system and conjugation to mouse DEC-205 monoclonal antibody: implication for in-vivo targeted delivery of dendritic cells

PubMed Central

Rahimi, Roghayeh; Ebtekar, Massoumeh; Moazzeni, Seyed Mohammad; Mostafaie, Ali; Mahdavi, Mehdi

2015-01-01

Objective(s): Multi-epitopic protein vaccines and direction of vaccine delivery to dendritic cells (DCs) are promising approaches for enhancing immune responses against mutable pathogens. Escherichia coli is current host for expression of recombinant proteins, and it is important to optimize expression condition. The aim of this study was the optimization of multi-epitopic HIV-1 tat/pol/gag/env recombinant protein (HIVtop4) expression by E. coli and conjugation of purified protein to anti DEC-205 monoclonal antibody as candidate vaccine. Materials and Methods: In this study, expression was induced in BL21 (DE3) E. coli cells by optimization of induction condition, post induction incubation time, temperature and culture medium formula. Some culture mediums were used for cell culture, and isopropyl-beta-D-thiogalactopyranoside was used for induction of expression. Protein was purified by Ni-NTA column chromatography and confirmed against anti-His antibody in western-blotting. To exploit DCs properties for immunization purposes, recombinant protein chemically coupled to αDEC-205 monoclonal antibody and confirmed against anti-His antibody in western-blotting. Results: The optimum condition for expression was 1 mM IPTG during 4 hr cultures in 2XYT medium, and final protein produced in soluble form. Conjugation of purified protein to αDEC-205 antibody resulted in smears of protein: antibodies conjugate in different molecular weights. Conclusion: The best cultivation condition for production of HIVtop4 protein is induction by 1 mM IPTG during 4 hr in 2XYT medium. The final concentration of purified protein was 500 µg/ml. PMID:25810888

Human Anti-Plague Monoclonal Antibodies Protect Mice from Yersinia pestis in a Bubonic Plague Model

PubMed Central

Xiao, Xiaodong; Zhu, Zhongyu; Dankmeyer, Jennifer L.; Wormald, Michael M.; Fast, Randy L.; Worsham, Patricia L.; Cote, Christopher K.; Amemiya, Kei; Dimitrov, Dimiter S.

2010-01-01

Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr) V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs) against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252) and two anti-V-specific human mAb (m253, m254) by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans. PMID:20976274

Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model.

PubMed

Xiao, Xiaodong; Zhu, Zhongyu; Dankmeyer, Jennifer L; Wormald, Michael M; Fast, Randy L; Worsham, Patricia L; Cote, Christopher K; Amemiya, Kei; Dimitrov, Dimiter S

2010-10-13

Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr) V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs) against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252) and two anti-V-specific human mAb (m253, m254) by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans.

Construction and expression of hepatitis B surface antigen escape variants within the "a" determinant by site directed mutagenesis.

PubMed

Golsaz Shirazi, Forough; Amiri, Mohammad Mehdi; Mohammadi, Hamed; Bayat, Ali Ahmad; Roohi, Azam; Khoshnoodi, Jalal; Zarnani, Amir Hassan; Jeddi-Tehrani, Mahmood; Kardar, Gholam Ali; Shokri, Fazel

2013-09-01

The antibody response to hepatitis B surface antigen (HBsAg) controls hepatitis B virus infection. The "a" determinant of HBsAg is the most important target for protective antibody response, diagnosis and immunoprophylaxis. Mutations in this area may induce immune escape mutants and affect the performance of HBsAg assays. To construct clinically relevant recombinant mutant forms of HBsAg and assessment of their reactivity with anti-HBs monoclonal antibodies (MAbs). Wild type (wt) and mutant (mt) HBsAg genes were constructed by site directed mutagenesis and SEOing PCR. The amplified genes were inserted into pCMV6-neo plasmid and transfected in CHO cell line. The expression of wt- and mtHBsAg was assessed by commercial ELISA assays and stable cells were established and cloned by limiting dilution. The recombinant mutants were further characterized using a panel of anti-HBs monoclonal antibodies (MAbs) and the pattern of their reactivity was assessed by ELISA. Ten HBsAg mutants having single mutation within the "a" determinant including P120E, T123N, Q129H, M133L, K141E, P142S, D144A, G145R, N146S and C147S together with a wt form were successfully constructed and expressed in CHO cells. Reactivity of anti-HBs MAbs with mtHBsAgs displayed different patterns. The effect of mutations on antibody binding differed depending on the amino acid involved and its location within the ''a'' determinant. Mutation at amino acids 123 and 145 resulted in either complete loss or significant reduction of binding to all anti-HBs MAbs. Our panel of mtHBsAgs is a valuable tool for assessment of the antibody response to HBV escape mutants and may have substantial implications in HBV immunological diagnostics.

Platelet receptors for the Streptococcus sanguis adhesin and aggregation-associated antigens are distinguished by anti-idiotypical monoclonal antibodies.

PubMed Central

Gong, K; Wen, D Y; Ouyang, T; Rao, A T; Herzberg, M C

1995-01-01

Platelets aggregate in response to an adhesin and the platelet aggregation-associated protein (PAAP) expressed on the cell surfaces of certain strains of Streptococcus sanguis. We sought to identify the corresponding PAAP receptor and accessory adhesin binding sites on platelets. Since the adhesion(s) of S. sanguis for platelets has not been characterized, an anti-idiotype (anti-id) murine monoclonal antibody (MAb2) strategy was developed. First, MAb1s that distinguished the adhesin and PAAP antigens on the surface of S. sanguis I 133-79 were selected. Fab fragments of MAb1.2 (immunoglobulin G2b [IgG2b]; 70 pmol) reacted with 5 x 10(7) cells of S. sanguis to completely inhibit the aggregation of human platelets in plasma. Under similar conditions, MAb1.1 (IgG1) inhibited the adhesion of S. sanguis cells to platelets by a maximum of 34%, with a comparatively small effect on platelet aggregation. Together, these two MAb1s inhibited S. sanguis-platelet adhesion by 63%. In Western immunoblots, both MAb1s reacted with S. sanguis 133-79 87- and 150-kDa surface proteins and MAb1.2 also reacted with purified type I collagen. The hybridomas producing MAb1.1 and MAb1.2 were then injected into BALB/c mice. Enlarged spleens were harvested, and a panel of MAb2 hybridomas was prepared. To identify anti-ids against the specific MAb1s, the MAb2 panel was screened by enzyme-linked immunosorbent assay for reaction with rabbit polyclonal IgG antibodies against the 87- and 150-kDa antigens. The reactions between the specific rabbit antibodies and anti-ids were inhibited by the 87- and 150-kDa antigens. When preincubated with platelets, MAb2.1 (counterpart of MAb1.1) inhibited adhesion to platelets maximally by 46% and MAb2.2 (anti-MAb1.2) inhibited adhesion to platelets maximally by 35%. Together, both MAb2s inhibited the adhesion of S. sanguis to platelets by 81%. MAb2.2 also inhibited induction of platelet aggregation. MAb2.2 immunoprecipitated a biotinylated platelet membrane antigen of 170 kDa (unreduced); MAb2.1 precipitated membrane antigens of 175- and 230-kDa (unreduced). Therefore, platelet binding sites and the receptor for the S. sanguis adhesin and PAAP, respectively, are distinguished by the anti-id MAb2s. PMID:7642300

Reactivity of human monoclonal IgM with nerve glycosphingolipids.

PubMed Central

Hauttecoeur, B; Schmitt, C; Dubois, C; Danon, F; Brouet, J C

1990-01-01

We examined the reactivity of monoclonal IgM of sera from patients with neuropathy and monoclonal IgM, with or without antibody activity to myelin-associated glycoprotein (MAG), as well as sera from non-neurologic patients with Waldenström's macroglobulinaemia, with various nerve glycolipids extracts or with purified gangliosides. As expected from previous studies, all (five cases) anti-MAG IgM stained two glycolipids, the chemical characteristics of which corresponded to sulphated glucuronyl-paragloboside (SGPG) and sulphated glucuronyl-lactosaminyl-paragloboside (SGLPG). Five of 12 sera from patients with neuropathy whose IgM was devoid of anti-MAG reactivity stained nerve extracts greatly enriched (98%) with SGPG and SGLPG. Three of these five sera reacted with additional glycolipids and/or gangliosides. Two of 16 sera from patients with macroglobulinaemia without neuropathy reacted strongly with both SGPG and SGLPG. The latter finding as well as the detection of low titre of anti-sphingolipid antibodies in normal sera may cast a doubt on the pathogenetic significance of this antibody activity. Images Fig. 1 Fig. 2 Fig. 3 PMID:1694117

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ratcliffe, D.R.

This study on passive immunity dealt principally with the following topics: pathogenesis of the pneumonia produced by influenza virus (PR8) in CF-1 mice; the distribution and retention of monoclonal antibody instilled intratracheally (IT) into the lung; and prophylaxis and therapy of influenza pneumonia using specific monoclonal antibody (IgG 2a/k anti-HA). The fate of a single 50 ul bolus of antibody instilled IT was determined by monitoring the activity of /sup 125/I-labelled monoclonal IgG in the lungs and by lavage recovery of functional antibody.Antibody was demonstrated in high concentrations for the first 3 days and was present in the lungs formore » a period of 7 days. For prophylaxis several trials indicated that monoclonal antibody provided significant protection from lethal effects of the virus. Antibody given to clinically ill mice on day 3 produced a highly significant reduction in mortality (P < 0.001) when compared to control mice. The treatment reversed the weight loss and apparently arrested the development of lesions in most of the mice within 2 days following antibody administration.« less

Production and Characterization of Monoclonal Antibody against Recombinant Virus Coat Protein CP42.

PubMed

Shibaei, Naeimeh; Majidi, Jafar; Razavi, Khadijeh; Karkhane, Ali Asghar; Sokhandan-Bashir, Nemat; Aghebati-Maleki, Leili

2017-02-01

There are many studies related to the production of a ELISA kit for diagnosing virus infections. However, production of most kits depends on purification of whole virus particles, which involves the use of costly equipment and reagents. The purpose of this study was to check out if the anti-CP42 antibodies could be used as a diagnostic assay for detection of Grapevine fanleaf Virus (GFLV). In this study, recombinant GFLV coat protein gene related to selected antigenic determinants was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain (DE3). Expressed protein was verified with western blotting assay by the use of commercially available anti-GFLV antibody. The recombinant protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) resin. Balb/c mice were immunized with purified protein and splenocytes of hyperimmunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by ELISA using CP42 as coating antigen. The results showed that monoclonal antibody (MAb) specific to CP42 has been successfully generated. Efficiency of produced antibody was analyzed by ELISA and western blotting assay using some confirmed grapevine samples. The infection was confirmed previously based on morphological features and ELISA assay, performed using commercial anti-GFLV antibody. The monoclonal antibody reacted with antigen in ELISA and immunoblot method. Our results demonstrated that anti recombinant CP42 monoclonal antibodies are able to diagnose whole virus in infected grapevine sample using ELISA test.

Targeting to cells of fluorescent liposomes covalently coupled with monoclonal antibody or protein A

NASA Astrophysics Data System (ADS)

Leserman, Lee D.; Barbet, Jacques; Kourilsky, François; Weinstein, John N.

1980-12-01

Many applications envisioned for liposomes in cell biology and chemotherapy require their direction to specific cellular targets1-3. The ability to use antibody as a means of conferring specificity to liposomes would markedly increase their usefulness. We report here a method for covalently coupling soluble proteins, including monoclonal antibody and Staphylococcus aureus protein A (ref. 4), to small sonicated liposomes, by using the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate (SPDP, Pharmacia). Liposomes bearing covalently coupled mouse monoclonal antibody against human β2-microglobulin [antibody B1.1G6 (IgG2a, κ) (B. Malissen et al., in preparation)] bound specifically to human, but not to mouse cells. Liposomes bearing protein A became bound to human cells previously incubated with the B1.1G6 antibody, but not to cells incubated without antibody. The coupling method results in efficient binding of protein to the liposomes without aggregation and without denaturation of the coupled ligand; at least 60% of liposomes bound functional protein. Further, liposomes did not leak encapsulated carboxyfluorescein (CF) as a consequence of the reaction.

Generation and characterization of monoclonal antibodies against Giardia muris trophozoites.

PubMed Central

Heyworth, M F; Ho, K E; Pappo, J

1989-01-01

Mouse monoclonal antibodies (mAb) were produced against Giardia muris trophozoite surface antigens. To generate B-cell hybridomas, P3/NS1/1-Ag4-1 myeloma cells were fused with splenic lymphocytes from BALB/c mice that had been immunized parenterally with G. muris trophozoites. Hybridoma culture supernatants were screened for mAb by flow cytometry of G. muris trophozoites incubated with culture supernatant followed by fluorescein-conjugated anti-mouse IgG and IgM. Flow cytometry showed three types of trophozoite staining by mAb: (i) bright staining of greater than 90% of trophozoites, with aggregation of the organisms; (ii) bright staining of approximately 90% of trophozoites, with little or no aggregation; (iii) dull staining of approximately 20% of trophozoites, without aggregation. Western blotting of mAb on G. muris trophozoite antigens separated by polyacrylamide gel electrophoresis showed that a mAb exhibiting the third of these flow cytometry staining patterns recognized trophozoite antigens of MW approximately 31,000 and 35,000. Immunoprecipitation studies indicated that the same mAb specifically precipitated two 125I-labelled trophozoite surface antigens of MW approximately 30,000. Monoclonal antibodies generated in this study may facilitate the purification and biochemical characterization of trophozoite antigens that are targets for protective intestinal antibody in G. muris-infected mice. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:2592009

Generation of anti-azoxystrobin monoclonal antibodies from regioisomeric haptens functionalized at selected sites and development of indirect competitive immunoassays.

PubMed

Parra, Javier; Mercader, Josep V; Agulló, Consuelo; Abad-Somovilla, Antonio; Abad-Fuentes, Antonio

2012-02-17

Azoxystrobin is a modern strobilurin fungicide used around the world to combat prime diseases affecting highly valuable crops. Accordingly, residues of this chemical are frequently found in food, even though mostly under maximum tolerated levels. We herein describe the development of an indirect competitive immunoassay for the determination of azoxystrobin residues. A panel of monoclonal antibodies displaying subnanomolar affinity to azoxystrobin was generated using, as immunizing haptens in mice, four functionalized derivatives carrying the same spacer arm located at different rationally chosen positions. This collection of antibodies was thoroughly characterized with homologous and heterologous antigens, and the immunoassay consisting of monoclonal antibody AZo6#49 and the coating conjugate OVA-AZb6, which displayed an IC(50) value of 0.102 μg L(-1) and a LOD of 0.017 μg L(-1), was eventually optimized. The response to different pH and ionic strength conditions of the specific assay was studied using a biparametric approach. In addition, the influence of Tween 20 and organic solvents over the assay parameters was also evaluated. After optimization, the developed immunochemical assay was applied to the analysis of azoxystrobin in spiked juices of relevant fruits and vegetables, showing excellent recoveries between 2 and 500 μg L(-1). Copyright © 2011 Elsevier B.V. All rights reserved.

Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies.

PubMed

Corrêa, Arthur Luiz; Senna, José Procópio Moreno; de Sousa, Álvaro Paiva Braga

2016-05-01

Monoclonal antibodies (mAb) are high added value glycoproteins recommended for immunotherapy, diagnosis, and also for the treatment of bacterial infections resistant to multiple drugs such as Methicillin Resistant Staphylococcus aureus (MRSA). In addition to environmental conditions related to cell cultures, the intrinsic characteristics of hybridoma cells, like the secretion stability of monoclonal antibodies by the cells through successive subcultures, are relevant for the characterization of cell lines related to the productivity of mAb. The rate of mAb production differs significantly between different cell lines and different passage numbers, and it is an important variable in characterization of cell lines. In order to find a more robust, faster-growing, and higher-productivity cell line of hybridoma, cultivations in 24-well plates were performed in different subculture periods, or cell passages (P), of hybridoma cells producing MRSA anti-PBP2a monoclonal antibodies [MRSA-antiPBP2a (mAb)]. The objective of this study was to study the effects of cell growth and production of MRSA-antiPBP2a mAb secreted by murine hybridoma cells grown in different passages as well as determine the which passages the hybridomas can be cultivated without harming their growth and productivity. So, cell growth profiles of hybridomas secreting MRSA-antiPBP2a (mAb) and the production of MRSA-antiPBP2a mAb in different subculture periods or cell passages (P) were studied. Cell growth tests, monoclonal antibody productivity, and metabolite characteristics revealed substantial differences in those cells kept between P10 and P50. Similarities in the secretion of monoclonal antibody, growth, and metabolic profiles, were noted in the MRSA-antiPBP2a mAb producing hybridoma cells kept between P10 and P20. Also, glucose consumption (g/L) and lactate production (g/L) in the latter cell cultures were monitored daily through biochemical analyzer. As of P30, it was observed a 4.4 times reduction in productivity, a 13 % reduction in metabolic yield, and a significant change in cell growth. Secretion of MRSA-antiPBP2a mAb should be obtained through the culture of hybridomas up to P20 in order to keep its stability.

Inhibition of memory consolidation by antibodies against cell adhesion molecules after active avoidance conditioning in zebrafish.

PubMed

Pradel, G; Schachner, M; Schmidt, R

1999-05-01

Cell adhesion molecules are expected to play an important role in long-term storage of information in the central nervous system. Several of these glycoproteins, such as NCAM, L1, and the ependymins, express the HNK-1 carbohydrate structure, which is known to be involved in cell-cell and cell-matrix interactions. To investigate the contribution of the HNK-1 epitope and the secretory glycoproteins ependymins to memory formation in zebrafish (Brachydanio rerio), we developed an active avoidance conditioning paradigm. Zebrafish were trained in a shuttle-box to cross a hurdle, to avoid mild electric shocks following a conditioning light signal. One hour after acquisition of the task, zebrafish were injected intracerebroventricularly with monoclonal antibodies against the HNK-1 epitope or polyclonal antibodies against ependymins. Control fish received immunoglobulins G (IgGs) from nonimmune rat serum or the monoclonal antibody C183 against an unrelated cell-surface protein of the cyprinid brain. Two days later, injected zebrafish were tested for recall, and for quantitative evaluation a retention score (RS), ranging from 1.0 for immediate recall to 0.0, indicating no saving, was calculated. The average RS of anti-HNK-1-injected fish (RS = 0.30) and anti-ependymin-injected fish (0.24) were significantly different from the RS of uninjected fish (0.77), of controls injected with nonimmune serum IgGs (0.68), of C183-injected controls (0.78), and of overtrained fish injected with anti-HNK-1 antibodies (0.81). Anti-HNK-1 and anti-ependymin antibodies were characterized by Western blot analyses of subcellular brain fractions and immunohistochemical staining of the zebrafish optic tectum. Our data suggest that the antibodies influence cell recognition events at synaptic membranes and/or associated intracellular signaling cascades, and thereby block memory consolidation.

Characterization of antibodies directed against the immunoglobulin light kappa chain variable chain region (VK) of hepatitis C virus-related type-II mixed cryoglobulinemia and B-cell proliferations.

PubMed

de Re, Valli; Simula, Maria Paola; Pavan, Alessandro; Garziera, Marica; Marin, Dolores; Dolcetti, Riccardo; de Vita, Salvatore; Sansonno, Domenico; Geremia, Silvano; Toffoli, Giuseppe

2009-09-01

Autoimmune type-II cryoglobulinemia (II-MC) is sustained by hepatitis C virus (HCV) infection and B-cell (oligo)clones. This is the reason why the disease may be considered an "indolent B-cell lymphoma (NHL)." B clones show a restricted use of immunoglobulin variable genes (BCR), in particular in the use of the variable kappa (VK)3-20/15 light chain, and show a homology between their BCR functional regions and those of autoimmune rheumatoid factors. We underlined the BCR unique repertoire with frequent rheumatoid factor activity also observed in other autoimmune disorders associated with NHL. The immunoglobulin idiotype is a clonal B-cell marker and an ideal target for immunotherapy. Five monoclonal antibodies were produced in our laboratory toward the VK3-20 of a subject with HCV infection and a II-MC-associated NHL. Epitope determination was performed using the epitope excision approach. Monoclonal antibody reactivity was tested in vitro in ELISA, Western blot, and cytofluorimetry. Data confirmed that a panel of antibodies, reactive against shared idiotypes, can be produced from patients with HCV-associated B-cell lymphoproliferative diseases, thus obviating the need to produce an anti-idiotype antibody for each patient.

Anti-Semaphorin 3A neutralization monoclonal antibody prevents sepsis development in lipopolysaccharide-treated mice.

PubMed

Yamashita, Naoya; Jitsuki-Takahashi, Aoi; Ogawara, Miyuki; Ohkubo, Wataru; Araki, Tomomi; Hotta, Chie; Tamura, Tomohiko; Hashimoto, Shu-ichi; Yabuki, Takashi; Tsuji, Toru; Sasakura, Yukie; Okumura, Hiromi; Takaiwa, Aki; Koyama, Chika; Murakami, Koji; Goshima, Yoshio

2015-09-01

Semaphorin 3A (Sema3A), originally identified as a potent growth cone collapsing factor in developing sensory neurons, is now recognized as a key player in immune, cardiovascular, bone metabolism and neurological systems. Here we established an anti-Sema3A monoclonal antibody that neutralizes the effects of Sema3A both in vitro and in vivo. The anti-Sema3A neutralization chick IgM antibodies were screened by combining an autonomously diversifying library selection system and an in vitro growth cone collapse assay. We further developed function-blocking chick-mouse chimeric and humanized anti-Sema3A antibodies. We found that our anti-Sema3A antibodies were effective for improving the survival rate in lipopolysaccharide-induced sepsis in mice. Our antibody is a potential therapeutic agent that may prevent the onset of or alleviate symptoms of human diseases associated with Sema3A. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Developing injectable immunoglobulins to treat cognitive impairment in Alzheimer's disease.

PubMed

Steinitz, Michael

2008-05-01

Alzheimer's disease is a devastating disorder, clinically characterized by a comprehensive cognitive decline. The novel strategy of anti-amyloid-beta immunotherapy has been suggested following encouraging results obtained in murine models of Alzheimer's disease, in non-human primates, and in small-scale clinical trials. To examine the choice between active or passive anti-amyloid-beta immunization and the choice of the molecule to which the immune machinery should be targeted, which are central issues in future immune therapy of Alzheimer's disease. Research into the new area of Alzheimer's disease immune therapy is primarily based on in vivo and in vitro studies of murine models of Alzheimer's disease. The studies are hence limited to defined genetic deficiencies. In humans, infusion of anti-amyloid-beta antibodies is considered a safer approach than active anti-amyloid-beta vaccination. Alzheimer's-disease-protective anti-amyloid-beta monoclonal antibodies should target specific epitopes within the amyloid beta(1 42) peptide, avoiding possibly harmful binding to the ubiquitous normal amyloid precursor protein. Since Alzheimer's disease immunotherapy requires repeated infusion of antibodies over a prolonged period of time, Alzheimer's disease patients will tolerate such antibodies provided the latter are exclusively of human origin. Human monoclonal antibodies that correspond to ubiquitous anti-amyloid-beta, present in all healthy humans, might bear important protective characteristics.

Anti-B-Cell Therapies in Autoimmune Neurological Diseases: Rationale and Efficacy Trials.

PubMed

Alexopoulos, Harry; Biba, Angie; Dalakas, Marinos C

2016-01-01

B cells have an ever-increasing role in the etiopathology of a number of autoimmune neurological disorders, acting as antibody-producing cells and, most importantly, as sensors, coordinators, and regulators of the immune response. B cells, among other functions, regulate the T-cell activation process through their participation in antigen presentation and production of cytokines. The availability of monoclonal antibodies or fusion proteins against B-cell surface molecules or B-cell trophic factors bestows a rational approach for treating autoimmune neurological disorders, even when T cells are the main effector cells. This review summarizes basic aspects of B-cell biology, discusses the role(s) of B cells in neurological autoimmunity, and presents anti-B-cell drugs that are either currently on the market or are expected to be available in the near future for treating neurological autoimmune disorders.

A Rabbit Anti-pT1989 ATR Monoclonal Antibody for Use in Immunoassays | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at the National Cancer Institute (NCI) have developed a monoclonal antibody against ataxia telangiectasia-mutated and Rad3-related (ATR) kinase phosphorylated at threonine 1989. The antibody can be used for pharmacodynamic assays to quantify drug action on the ATR target.

Unusual Features of Vaccinia Virus Extracellular Virion Form Neutralization Resistance Revealed in Human Antibody Responses to the Smallpox Vaccine

PubMed Central

Benhnia, Mohammed Rafii-El-Idrissi; Maybeno, Matthew; Blum, David; Aguilar-Sino, Rowena; Matho, Michael; Meng, Xiangzhi; Head, Steven; Felgner, Philip L.; Zajonc, Dirk M.; Koriazova, Lilia; Kato, Shinichiro; Burton, Dennis R.; Xiang, Yan; Crowe, James E.; Peters, Bjoern

2013-01-01

The extracellular virion form (EV) of vaccinia virus (VACV) is essential for viral pathogenesis and is difficult to neutralize with antibodies. Why this is the case and how the smallpox vaccine overcomes this challenge remain incompletely understood. We previously showed that high concentrations of anti-B5 antibodies are insufficient to directly neutralize EV (M. R. Benhnia, et al., J. Virol. 83:1201–1215, 2009). This allowed for at least two possible interpretations: covering the EV surface is insufficient for neutralization, or there are insufficient copies of B5 to allow anti-B5 IgG to cover the whole surface of EV and another viral receptor protein remains active. We endeavored to test these possibilities, focusing on the antibody responses elicited by immunization against smallpox. We tested whether human monoclonal antibodies (MAbs) against the three major EV antigens, B5, A33, and A56, could individually or together neutralize EV. While anti-B5 or anti-A33 (but not anti-A56) MAbs of appropriate isotypes were capable of neutralizing EV in the presence of complement, a mixture of anti-B5, anti-A33, and anti-A56 MAbs was incapable of directly neutralizing EV, even at high concentrations. This remained true when neutralizing the IHD-J strain, which lacks a functional version of the fourth and final known EV surface protein, A34. These immunological data are consistent with the possibility that viral proteins may not be the active component of the EV surface for target cell binding and infectivity. We conclude that the protection afforded by the smallpox vaccine anti-EV response is predominantly mediated not by direct neutralization but by isotype-dependent effector functions, such as complement recruitment for antibodies targeting B5 and A33. PMID:23152530

Deciphering the Role of B Cells in Multiple Sclerosis—Towards Specific Targeting of Pathogenic Function

PubMed Central

Lehmann-Horn, Klaus; Kinzel, Silke; Weber, Martin S.

2017-01-01

B cells, plasma cells and antibodies may play a key role in the pathogenesis of multiple sclerosis (MS). This notion is supported by various immunological changes observed in MS patients, such as activation and pro-inflammatory differentiation of peripheral blood B cells, the persistence of clonally expanded plasma cells producing immunoglobulins in the cerebrospinal fluid, as well as the composition of inflammatory central nervous system lesions frequently containing co-localizing antibody depositions and activated complement. In recent years, the perception of a respective pathophysiological B cell involvement was vividly promoted by the empirical success of anti-CD20-mediated B cell depletion in clinical trials; based on these findings, the first monoclonal anti-CD20 antibody—ocrelizumab—is currently in the process of being approved for treatment of MS. In this review, we summarize the current knowledge on the role of B cells, plasma cells and antibodies in MS and elucidate how approved and future treatments, first and foremost anti-CD20 antibodies, therapeutically modify these B cell components. We will furthermore describe regulatory functions of B cells in MS and discuss how the evolving knowledge of these therapeutically desirable B cell properties can be harnessed to improve future safety and efficacy of B cell-directed therapy in MS. PMID:28946620

Blocking FGF2 with a new specific monoclonal antibody impairs angiogenesis and experimental metastatic melanoma, suggesting a potential role in adjuvant settings.

PubMed

de Aguiar, Rodrigo Barbosa; Parise, Carolina Bellini; Souza, Carolina Rosal Teixeira; Braggion, Camila; Quintilio, Wagner; Moro, Ana Maria; Navarro Marques, Fabio Luiz; Buchpiguel, Carlos Alberto; Chammas, Roger; de Moraes, Jane Zveiter

2016-02-28

Compelling evidence suggests that fibroblast growth factor 2 (FGF2), overexpressed in melanomas, plays an important role in tumor growth, angiogenesis and metastasis. In this study, we evaluated the therapeutic use of a new anti-FGF2 monoclonal antibody (mAb), 3F12E7, using for that the B16-F10 melanoma model. The FGF2 neutralizing effect of this antibody was certified by in vitro assays, which allowed the further track of its possible in vivo application. 3F12E7 mAb could be retained in B16-F10 tumors, as shown by antibody low-pH elution and nuclear medicine studies, and also led to reduction in number and size of metastatic foci in lungs, when treatment starts one day after intravenous injection of B16-F10 cells. Such data were accompanied by decreased CD34(+) tumor vascular density and impaired subcutaneous tumor outgrowth. Treatments starting one week after melanoma cell intravenous injection did not reduce tumor burden, remaining the therapeutic effectiveness restricted to early-adopted regimens. Altogether, the presented anti-FGF2 3F12E7 mAb stands as a promising agent to treat metastatic melanoma tumors in adjuvant settings. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Anti-myeloperoxidase autoantibodies react with native but not denatured myeloperoxidase.

PubMed Central

Falk, R J; Becker, M; Terrell, R; Jennette, J C

1992-01-01

We wondered whether anti-myeloperoxidase (MPO) autoantibodies (MPO-ANCA) found in patients with systemic vasculitis react with a conformational epitope or epitopes on the MPO molecule. Sera from 15 human MPO-ANCA, and a polyclonal and a monoclonal anti-MPO antibodies were reacted with MPO in native and denatured states. Human MPO-ANCA and mouse monoclonal anti-MPO reacted with native MPO, and a 120-kD band representing the MPO hologenzyme, but not with denatured MPO fragments; however, MPO-ANCA and mouse anti-MPO did not demonstrate competitive inhibition of binding to MPO. Polyclonal rabbit anti-MPO reacted with both native and denatured MPO. All MPO-ANCA tested showed the same patterns of reactivity with native and denatured MPO in dot blot and Western blot analyses. Both polyclonal and monoclonal anti-MPO antibodies inhibited MPO's protein iodination by over 90%, whereas MPO-ANCA IgGs, normal IgGs and disease control IgGs did not. These data suggest that (i) MPO-ANCA interact with a conformational epitope on the MPO molecule; and (ii) MPO-ANCA from different patients have similar reactivity with native versus denatured MPO. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:1379133

Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques

DOE PAGES

Santra, Sampa; Tomaras, Georgia D.; Warrier, Ranjit; ...

2015-08-03

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4⁺ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant regionmore » of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.« less

Human Non-neutralizing HIV-1 Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques

PubMed Central

Liao, Hua-Xin; Pollara, Justin; Liu, Pinghuang; Alam, S. Munir; Zhang, Ruijun; Cocklin, Sarah L.; Shen, Xiaoying; Duffy, Ryan; Xia, Shi-Mao; Schutte, Robert J.; Pemble IV, Charles W.; Dennison, S. Moses; Li, Hui; Chao, Andrew; Vidnovic, Kora; Evans, Abbey; Klein, Katja; Kumar, Amit; Robinson, James; Landucci, Gary; Forthal, Donald N.; Montefiori, David C.; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Rerks-Ngarm, Supachai; Robb, Merlin L.; Michael, Nelson L.; Kim, Jerome H.; Soderberg, Kelly A.; Giorgi, Elena E.; Blair, Lily; Korber, Bette T.; Moog, Christiane; Shattock, Robin J.; Schmitz, Joern E.; Moody, M. A.; Gao, Feng; Ferrari, Guido; Shaw, George M.; Haynes, Barton F.

2015-01-01

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses. PMID:26237403

Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Santra, Sampa; Tomaras, Georgia D.; Warrier, Ranjit

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4⁺ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant regionmore » of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.« less

Treatment of non-Hodgkin's lymphoma xenografts with the HB22.7 anti-CD22 monoclonal antibody and phosphatase inhibitors improves efficacy.

PubMed

O'Donnell, Robert T; Pearson, David; McKnight, Hayes C; Ma, Ya Peng; Tuscano, Joseph M

2009-10-01

To examine the role of phosphatase inhibition on anti-CD22, HB22.7-mediated lymphomacidal effects. CD22 is a cell-surface molecule expressed on most B cell lymphomas (NHL). HB22.7 is an anti-CD22 monoclonal antibody that binds a unique CD22-epitope, blocks ligand binding, initiates signaling, and has demonstrated lymphomacidal activity. The SHP-1 tyrosine phosphatase is associated with the cytoplasmic domain of CD22. Sodium orthovanadate (NaV) is a phosphatase inhibitor. The SHP-1-CD22 interaction presents an opportunity to manipulate CD22-mediated signaling effects. In vitro cell culture assays and in vivo human NHL xenograft studies were used to assess the effects of phosphatase inhibition. NaV caused dose dependent killing of NHL cells in vitro; when HB22.7 was given with NaV, antibody-mediated cell death was augmented. Flow cytometry showed that NaV-pretreatment resulted in less CD22 internalization after ligation with HB22.7 than did control cells. Studies in mice bearing Raji NHL xenografts showed that the combination of NaV and HB22.7 shrank NHL tumors more rapidly, had a higher complete response rate (80%), and produced the best survival compared to controls; no toxicity was detected. Studies using Raji cells stably transfected with SHP-1DN confirmed that these observations were due to SHP-1 inhibition. The relatively specific association of SHP-1 with CD22 suggests that CD22-specific signal augmentation by phosphatase inhibitors can improve the clinical outcome of anti-CD22 based immunotherapy.

Bone marrow transplantation across major histocompatibility barriers. V. Protection of mice from lethal graft-vs. -host disease by pretreatment of donor cells with monoclonal anti-Thy-1. 2 coupled to the toxin ricin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vallera, D.A.; Youle, R.J.; Neville, D.M. Jr.

1982-03-01

A new method has been devised to eliminate T cells from murine bone marrow grafts across major histocompatibility barriers and thus prevent graft-vs.-host disease (GVHD). The method utilizes a monoclonal antibody directed at the Thy-1.2 antigen but is complement independent. To make anti-Thy-1.2 toxic, the antibody is covalently linked to the toxin ricin. Ricin ordinarily binds, enters, and kills cells through receptors containing galactose. The hybrid protein, anti-Thy-1.2-ricin, can enter and kill cells via the Thy-1.2 receptor. In the presence of lactose the usual entry route for ricin is largely blocked and the hybrid is shown to be a highlymore » selective reagent that is T cell specific in its inhibition of mitogen-stimulated splenocytes. We have used a model of severe and fatal GVHD where BALB/c splenocytes and bone marrow cells are given to irradiated C57BL/6 recipients. Over 90% of these mice die by day 70, exhibiting signs of GVHD. When donor cells are pretreated with 0.5 microgram/ml of anti-Thy-1.2-ricin plus 200 mM lactose before injection, 10 of 11 animals survive through day 70 without signs of GVHD. These studies demonstrate that ricin linked to monoclonal antibodies may have utility related to the prevention of GVHD in human bone marrow transplantation.« less

A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

PubMed

Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

2012-12-01

Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

The anticancer immune response of anti-PD-1/PD-L1 and the genetic determinants of response to anti-PD-1/PD-L1 antibodies in cancer patients

PubMed Central

Han, Weidong; Wang, Xian; Fang, Yong; Li, Da; Pan, Hongming; Zhang, Li

2015-01-01

The programmed death-1 (PD-1), a coinhibitory receptor expressed on activated T cells and B cells, is demonstrated to induce an immune-mediated response and play a critical role in tumor initiation and development. The cancer patients harboring PD-1 or PD ligand 1 (PD-L1) protein expression have often a poor prognosis and clinical outcome. Currently, targeting PD-1 pathway as a potential new anticancer strategy is attracting more and more attention in cancer treatment. Several monoclonal antibodies against PD-1 or PD-L1 have been reported to enhance anticancer immune responses and induce tumor cell death. Nonetheless, the precise molecular mechanisms by which PD-1 affects various cancers remain elusive. Moreover, this therapy is not effective for all the cancer patients and only a fraction of patients respond to the antibodies targeting PD-1 or PD-L1, indicating these antibodies may only works in a subset of certain cancers. Thus, understanding the novel function of PD-1 and genetic determinants of response to anti-PD-1 therapy will allow us to develop a more effective and individualized immunotherapeutic strategy for cancer. PMID:26305724

Specific binding of TES-23 antibody to tumour vascular endothelium in mice, rats and human cancer tissue: a novel drug carrier for cancer targeting therapy

PubMed Central

Tsunoda, S; Ohizumi, I; Matsui, J; Koizumi, K; Wakai, Y; Makimoto, H; Tsutsumi, Y; Utoguchi, N; Taniguchi, K; Saito, H; Harada, N; Ohsugi, Y; Mayumi, T

1999-01-01

The tissue distribution of anti-tumour vascular endothelium monoclonal antibody (TES-23) produced by immunizing with plasma membrane vesicles from isolated rat tumour-derived endothelial cells (TECs) was assessed in various tumour-bearing animals. Radiolabelled TES-23 dramatically accumulated in KMT-17 fibrosarcoma, the source of isolated TECs after intravenous injection. In Meth-A fibrosarcoma, Colon-26 adenocarcinoma in BALB/c mice and HT-1080 human tumour tissue in nude mice, radioactivities of 125I-labelled TES-23 were also up to 50 times higher than those of control antibody with little distribution to normal tissues. The selective recognition of TES-23 to TECs was competitively blocked by preadministration of unlabelled TES-23 in vivo. Furthermore, immunostaining of human tissue sections showed specific binding of TES-23 on endothelium in oesophagus cancers. These results indicate that tumour vascular endothelial cells express common antigen in different tumour types of various animal species. In order to clarify the efficacy of TES-23 as a drug carrier, an immunoconjugate, composed of TES-23 and neocarzinostatin, was tested for its anti-tumour effect in rats bearing KMT-17 fibrosarcomas. The immunoconjugate (TES-23-NCS) caused marked regression of the tumour, accompanied by haemorrhagic necrosis. Thus, from a clinical view, TES-23 would be a novel drug carrier because of its high specificity to tumour vascular endothelium and its application to many types of cancer. © 1999 Cancer Research Campaign PMID:10584876

[Morphological substrate and pathogenetic mechanisms of pelvic pain syndrome in endometriosis. Part II. Peripheral nerve tissue remodeling in the foci of endometriosis].

PubMed

Kogan, E A; Ovakimyan, A S; Paramonova, N B; Faizullina, N M; Kazachenko, I F; Adamyan, L V

2016-01-01

Endometriosis (EM) is morphologically characterized by the development of extrauterine endometrioid heterotopies, the major clinical symptoms of which is chronic pelvic pain, which is a serious problem not only in modern gynecology, but also in public health as a whole. to investigate neurogenic markers in the foci of EM of various sites and histological structure in women with and without pain syndrome. The investigation was performed using the operative material (resected segments of the intestine, bladder, rectovaginal septum, and small pelvic peritoneum) obtained from 52 women with an intraoperative and morphologically verified diagnosis of EM and (Group 1) and without (Group 2) pain syndrome. Immunohistochemical examination was made on paraffin-embedded tissue sections in accordance with the standard protocols, by using the antibodies: 1) anti-PGP 9.5 polyclonal rabbit antibodies; 2) mouse anti-human neurofilament (NF) protein monoclonal antibodies (Clone 2F1); 3) mouse anti-nerve growth factor (NGF) monoclonal antibodies; 4) monoclonal mouse anti-human NGF receptor p75 (NGFRp75) antibodies (Dako, Denmark). Our findings demonstrate differences in the expression of PGP 9.5, NFs, NGF, and NGFRp75 in the foci and adjacent tissue in painful and painless EM irrespective of the locations of heterotopies. The found molecular features are a manifestation of the remodeling of nerve fibers and nerve endings in the foci of EM and PGP9.5, NGF, and NGFRp75 give rise to nerve fiber neoformation and pain syndrome in EM. At the same time, the immunohistochemical phenotype of EM foci does not depend on their site and reflects the presence or absence of pain syndrome.

Optimization of pore structure and particle morphology of mesoporous silica for antibody adsorption for use in affinity chromatography

NASA Astrophysics Data System (ADS)

Hikosaka, Ryouichi; Nagata, Fukue; Tomita, Masahiro; Kato, Katsuya

2016-10-01

Antibodies have received significant attention for use as antibody drugs, because they bind the objective protein (antigen) via antigen-antibody reactions. Recently, many reports have appeared on various monoclonal antibodies that recognize a single antigen. In this study, monoclonal antibodies are used as adsorbates on mesoporous silica (MPS) for affinity chromatography. MPS has high surface area and large pore volume; moreover, pore diameter, pore structure, and particle morphology are relatively easy to tune by adjusting the conditions of synthesis. The pore structure (two-dimensional (2D) hexagonal and three-dimensional cubic) and particle morphology (spherical and polyhedral) of MPS are optimized for use in a monoclonal antibody/MPS composite. When anti-IgG (one of the monoclonal antibodies) adsorbs on the MPS material and IgG (antigen) binds to anti-IgG/MPS composites, MCM-41p with a 2D-hexagonal pore structure and polyhedral particle morphology has the highest IgG binding efficiency. In addition, the antibody/MPS composites remain stable in chaotropic and low-pH solutions and can be cycled at least five times without decreasing IgG elution. In purification and removal tests, the use of the antibody/MPS composites allows only the objective protein from protein mixtures to be bound and eluted.

Prevalence of factor H-binding protein variants and NadA among meningococcal group B isolates from the United States: implications for the development of a multicomponent group B vaccine.

PubMed

Beernink, Peter T; Welsch, Jo Anne; Harrison, Lee H; Leipus, Arunas; Kaplan, Sheldon L; Granoff, Dan M

2007-05-15

Two promising recombinant meningococcal protein vaccines are in development. One contains factor H-binding protein (fHBP) variants (v.) 1 and 2, whereas the other contains v.1 and 4 other antigens discovered by genome mining (5 component [5C]). Antibodies against fHBP are bactericidal against strains within a variant group. There are limited data on the prevalence of strains expressing different fHBP variants in the United States. A total of 143 group B isolates from patients hospitalized in the United States were tested for fHBP variant by quantitative polymerase chain reaction, for reactivity with 6 anti-fHBP monoclonal antibodies (MAb) by dot immunoblotting, and for susceptibility to bactericidal activity of mouse antisera. fHBP v.1 isolates predominated in California (83%), whereas isolates expressing v.1 (53%) or v.2 (42%) were common in 9 other states. Isolates representative of 5 anti-fHBP MAb-binding phenotypes (70% of isolates) were highly susceptible to anti-fHBP v.1 or v.2 bactericidal activity, whereas 3 phenotypes were approximately 50% susceptible. Collectively, antibodies against the fHBP v.1 and v.2 vaccine and the 5C vaccine killed 76% and 83% of isolates, respectively. Susceptibility to bactericidal activity can be predicted, in part, on the basis of fHBP phenotypes. Both vaccines have the potential to prevent most group B disease in the United States.

Increased T cell proliferative responses to islet antigens identify clinical responders to anti-CD20 monoclonal antibody (rituximab) therapy in type 1 diabetes.

PubMed

Herold, Kevan C; Pescovitz, Mark D; McGee, Paula; Krause-Steinrauf, Heidi; Spain, Lisa M; Bourcier, Kasia; Asare, Adam; Liu, Zhugong; Lachin, John M; Dosch, H Michael

2011-08-15

Type 1 diabetes mellitus is believed to be due to the autoimmune destruction of β-cells by T lymphocytes, but a single course of rituximab, a monoclonal anti-CD20 B lymphocyte Ab, can attenuate C-peptide loss over the first year of disease. The effects of B cell depletion on disease-associated T cell responses have not been studied. We compare changes in lymphocyte subsets, T cell proliferative responses to disease-associated target Ags, and C-peptide levels of participants who did (responders) or did not (nonresponders) show signs of β-cell preservation 1 y after rituximab therapy in a placebo-controlled TrialNet trial. Rituximab decreased B lymphocyte levels after four weekly doses of mAb. T cell proliferative responses to diabetes-associated Ags were present at baseline in 75% of anti-CD20- and 82% of placebo-treated subjects and were not different over time. However, in rituximab-treated subjects with significant C-peptide preservation at 6 mo (58%), the proliferative responses to diabetes-associated total (p = 0.032), islet-specific (p = 0.048), and neuronal autoantigens (p = 0.005) increased over the 12-mo observation period. This relationship was not seen in placebo-treated patients. We conclude that in patients with type 1 diabetes mellitus, anti-B cell mAb causes increased proliferative responses to diabetes Ags and attenuated β-cell loss. The way in which these responses affect the disease course remains unknown.

[Diagnostic and therapeutic use of human anti-D (Rho) monoclonal antibodies. Evaluation and perspectives].

PubMed

Rouger, P; Goossens, D; Champomier, F; Tsikas, G; Liberge, G; Leblanc, J; Richard, C; Bailleul, C; Salmon, C

1985-12-01

Human monoclonal antibodies will be essential in medicine. They are valuable tools for biological diagnosis and therapeutics. Our model, human monoclonal antibodies directed against the Rhesus D antigen can be used for the determination of the Rhesus D phenotype and for the suppression of Rh(D) immunisation in women. These new products require new procedures of preparation, new regulations for the quality controls, which will be discussed in this paper.

Plasma exchanges for severe acute neurological deterioration in patients with IgM anti-myelin-associated glycoprotein (anti-MAG) neuropathy.

PubMed

Baron, M; Lozeron, P; Harel, S; Bengoufa, D; Vignon, M; Asli, B; Malphettes, M; Parquet, N; Brignier, A; Fermand, J P; Kubis, N; Arnulf, Bertrand

2017-06-01

Monoclonal IgM anti-myelin-associated glycoprotein (MAG) antibody-related peripheral neuropathy (anti-MAG neuropathy) is predominantly a demyelinating sensory neuropathy with ataxia and distal paresthesia. The clinical course of anti-MAG neuropathy is usually slowly progressive making difficult the identification of clear criteria to start a specific treatment. Although no consensus treatment is yet available, a rituximab-based regimen targeting the B-cell clone producing the monoclonal IgM may be proposed, alone or in combination with alkylating agents or purine analogs. However, in some rare cases, an acute and severe neurological deterioration can occur in few days leading to a rapid loss of autonomy. In these cases, a treatment rapidly removing the monoclonal IgM from the circulation might be useful before initiating a specific therapy. We report successful treatment with plasma exchanges (PE) in four patients presenting with acute neurological deterioration. PE allowed a dramatic and rapid neurological improvement in all patients. PE are safe and may be useful at the initial management of these cases of anti-MAG neuropathy.

Anti-influenza M2e antibody

DOEpatents

Bradbury, Andrew M.

2013-04-16

Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

Anti-influenza M2e antibody

DOEpatents

Bradbury, Andrew M [Santa Fe, NM

2011-12-20

Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

A monoclonal antibody against PDGF B-chain inhibits PDGF-induced DNA synthesis in C3H fibroblasts and prevents binding of PDGF to its receptor.

PubMed

Vassbotn, F S; Langeland, N; Hagen, I; Holmsen, H

1990-09-01

A monoclonal antibody (MAb 6D11) against platelet-derived growth factor (PDGF) was studied. We found that the MAb 6D11 in concentrations equimolar to PDGF blocked the [3H]thymidine incorporation in C3H/10T1/2 C18 fibroblasts stimulated by PDGF B-B and PDGF A-B. This inhibition was overcome by high doses of PDGF. The [3H]thymidine incorporation stimulated by other growth factors (aFGF, bFGF and bombesin) was not inhibited by the antibody. The MAb 6D11 blocked receptor binding of PDGF B-B, but not PDGF A-A. These findings suggest that the MAb 6D11 abolishes PDGF-induced DNA synthesis by blocking PDGF receptor binding. In this communication we demonstrate an isoform-specific monoclonal antibody against PDGF.

Heterogeneity of Polyneuropathy Associated with Anti-MAG Antibodies

PubMed Central

Magy, Laurent; Kaboré, Raphaël; Mathis, Stéphane; Lebeau, Prisca; Ghorab, Karima; Caudie, Christiane; Vallat, Jean-Michel

2015-01-01

Polyneuropathy associated with IgM monoclonal gammopathy and anti-myelin associated glycoprotein (MAG) antibodies is an immune-mediated demyelinating neuropathy. The pathophysiology of this condition is likely to involve anti-MAG antibody deposition on myelin sheaths of the peripheral nerves and it is supposed to be distinct from chronic inflammatory demyelinating neuropathy (CIDP), another immune-mediated demyelinating peripheral neuropathy. In this series, we have retrospectively reviewed clinical and laboratory findings from 60 patients with polyneuropathy, IgM gammopathy, and anti-MAG antibodies. We found that the clinical picture in these patients is highly variable suggesting a direct link between the monoclonal gammopathy and the neuropathy. Conversely, one-third of patients had a CIDP-like phenotype on electrodiagnostic testing and this was correlated with a low titer of anti-MAG antibodies and the absence of widening of myelin lamellae. Our data suggest that polyneuropathy associated with anti-MAG antibodies is less homogeneous than previously said and that the pathophysiology of the condition is likely to be heterogeneous as well with the self-antigen being MAG in most of the patients but possibly being another component of myelin in the others. PMID:26065001

Structural analysis of a novel rabbit monoclonal antibody R53 targeting an epitope in HIV-1 gp120 C4 region critical for receptor and co-receptor binding

DOE PAGES

Pan, Ruimin; Chen, Yuxin; Vaine, Michael; ...

2015-07-15

The fourth conserved region (C4) in the HIV-1 envelope glycoprotein (Env) gp120 is a structural element that is important for its function, as it binds to both the receptor CD4 and the co-receptor CCR5/CXCR4. It has long been known that this region is highly immunogenic and that it harbors B-cell as well as T-cell epitopes. It is the target of a number of antibodies in animal studies, which are called CD4-blockers. However, the mechanism by which the virus shields itself from such antibody responses is not known. Here, we determined the crystal structure of R53 in complex with its epitopemore » peptide using a novel anti-C4 rabbit monoclonal antibody R53. Our data show that although the epitope of R53 covers a highly conserved sequence 433AMYAPPI 439, it is not available in the gp120 trimer and in the CD4-bound conformation. Our results suggest a masking mechanism to explain how HIV-1 protects this critical region from the human immune system.« less

Structural analysis of a novel rabbit monoclonal antibody R53 targeting an epitope in HIV-1 gp120 C4 region critical for receptor and co-receptor binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pan, Ruimin; Chen, Yuxin; Vaine, Michael

The fourth conserved region (C4) in the HIV-1 envelope glycoprotein (Env) gp120 is a structural element that is important for its function, as it binds to both the receptor CD4 and the co-receptor CCR5/CXCR4. It has long been known that this region is highly immunogenic and that it harbors B-cell as well as T-cell epitopes. It is the target of a number of antibodies in animal studies, which are called CD4-blockers. However, the mechanism by which the virus shields itself from such antibody responses is not known. Here, we determined the crystal structure of R53 in complex with its epitopemore » peptide using a novel anti-C4 rabbit monoclonal antibody R53. Our data show that although the epitope of R53 covers a highly conserved sequence 433AMYAPPI 439, it is not available in the gp120 trimer and in the CD4-bound conformation. Our results suggest a masking mechanism to explain how HIV-1 protects this critical region from the human immune system.« less

Detection of anti-Yta antibodies using a sensitive and specific enzyme-linked immunosorbent assay.

PubMed

Geen, J; Hullin, D A; Hogg, S I

1999-01-01

A specific, sensitive and semi-quantitative enzyme-linked immunosorbent assay (ELISA) is described to detect anti-Yta antibodies in human serum. Recombinant acetylcholinesterase (AChE E.C.3.1.1.7) was employed as the coating antigen in the microtitre plate and horseradish peroxidase (HRP)-conjugated specific antibody (IgG) was used as the secondary antibody. The method developed showed excellent sensitivity, detecting a titre > 1 in 600,000 (3.5 ng/mL mouse IgG protein) for mouse monoclonal (mMAb) anti-AChE antibody. No cross-reaction was seen with other common blood group antibodies, confirming the specificity of the method. The recombinant antigen's AChE phenotype was confirmed as Yta, as no reaction was detected with anti-Ytb-positive sera. The ELISA method correlated closely with the established serological grading system used routinely in blood transfusion laboratories.

Demonstration of monoclonal anti-carcinoembryonic antigen (CEA) antibody internalization by electron microscopy, western blotting and radioimmunoassay.

PubMed

Tsaltas, G; Ford, C H; Gallant, M

1992-01-01

One of the important factors affecting the action of monoclonal antibodies (Mabs) or immunoconjugates on tumour sites depends on whether the Mab is internalized by the cancer cells in question. The underexplored subject of internalization is discussed in this paper, and a number of in vitro techniques for investigating internalization are evaluated, using a model which consists of a well characterized anti-carcinoembryonic antigen (anti-CEA) Mab and a number of CEA expressing human cancer cell lines. Employing two alternative radiolabeling assays, evidence for internalization of the anti-CEA Mab by a CEA-positive colorectal cancer cell line (LS174T) was obtained throughout the time intervals examined (5 min to 150 min). Electronmicroscopy employing horseradish-peroxidase labeled anti-CEA Mab and control antibody permitted direct visualization of anti-CEA Mab-related staining in intracellular compartments of a high CEA-expressor human colorectal cell line (SKCO1). Finally Western blots of samples derived from cytosolic and membrane components of solubilized cells from lung and colonic cancer cell lines provided evidence for internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 minutes. Internalized anti-CEA was detected in all CEA expressing cell lines (LS174T, SKCO1, BENN) but not in the case of a very low CEA expressor line (COLO 320).

Method of rapid production of hybridomas expressing monoclonal antibodies on the cell surface

DOEpatents

Meagher, Richard B.; Laterza, Vince

2006-12-12

The present invention relates to genetically altered hybridomas, myelomas and B cells. The invention also relates to utilizing genetically altered hybridomas, myelomas and B cells in methods of making monoclonal antibodies. The present invention also provides populations of hybridomas and B cells that can be utilized to make a monoclonal antibody of interest.

[Establishment of A1E3 and B1C4 monoclonal antibody-based ELISA for detecting circulating antigen of Schistosoma japonicum and its preliminary application].

PubMed

Cai, Yu-Chun; Chen, Shao-Hong; Tian, Li-Guang; Chu, Yan-Hong; Lu, Yan; Chen, Mu-Xin; Ai, Lin; Zhou, Yang; Chen, Jia-Xu

2014-02-01

To establish A1E3 and B1C4 monoclonal antibody-based ELISA for detecting circulating antigen of Schistosoma japonicum and explore its application value in the field. The characteristics of A1E3 and B1C4 monoclonal antibodies were analyzed by SDS-PAGE and Western blotting. The SEA-based ELISA was used to evaluate the titers of A1E3 and B1C4. The orthogonal test was used to determine the best concentration of coating antibody B1C4 and optimal working concentration of A1E3-HRP. Under the optimal conditions, the serum samples of 20 acute schistosomiasis cases, 46 chronic schistosomiasis cases, and 20 control sera were tested to evaluate its detection sensitivity and specificity. Seventy-two antibody positive serum samples from Jiangling County of Hubei Province were detected and compared to a commercially available ELISA kit, to evaluate the detection effects of this method. The results of SDS-PAGE demonstrated that the purified A1E3 and B1C4 contained a clear heavy chain with molecular weight of 88,000 and 52,000 respectively and had the same light chain with molecular weight of 20,000; while Western blotting demonstrated that A1E3 and B1C4 could be recognized by SEA and serum samples of acute schistosomiasis cases. The SEA-based ELISA demonstrated the titers of B1C4 and A1E3 were 1:10(5) and 1:30,000, respectively. The serum samples from all the acute cases and 86.9% of the chronic cases showed a positive reaction. All of the control sera from healthy persons gave a negative response. The positive rates of the double monoclonal antibody ELISA and commercial ELISA for detecting the circulating antigen were 45.8% and 43.1% respectively, and there was no significant difference between the results of the two methods. A1E3 and B1C4 monoclonal antibody-based ELISA is established successfully. It exhibits a high sensitivity and specificity in detecting circulating antigen of Schistosoma japonicum.

Characterization of digitalis-like factors in human plasma. Interactions with NaK-ATPase and cross-reactivity with cardiac glycoside-specific antibodies.

PubMed

Kelly, R A; O'Hara, D S; Canessa, M L; Mitch, W E; Smith, T W

1985-09-25

Much of the evidence for a physiologically important endogenous inhibitor of the sodium pump has been either contradictory or indirect. We have identified three discrete fractions in desalted deproteinized plasma from normal humans that resemble the digitalis glycosides in that they: are of low molecular weight; are resistant to acid and enzymatic proteolysis; inhibit NaK-ATPase activity; inhibit Na+ pump activity in human erythrocytes; displace [3H]ouabain bound to the enzyme; and cross-react with high-affinity polyclonal and monoclonal digoxin-specific antibodies but not with anti-ouabain or anti-digitoxin antibodies. An additional fraction cross-reacted with digoxin-specific antibodies but had no detectable activity against NaK-ATPase. The three inhibitory fractions differed from cardiac glycosides in that their concentration-effect curves in a NaK-ATPase inhibition and [3H]ouabain radioreceptor assays were steeper than unlabeled ouabain. This suggests that these inhibitors are not simple competitive ligands for binding to NaK-ATPase. In the presence of sodium, no fraction required ATP for binding to NaK-ATPase, and in the presence of potassium, only one fraction had the reduced affinity for the enzyme that is characteristic of cardiac glycosides. Unlike digitalis, all three NaK-ATPase inhibitory fractions stimulated the activity of skeletal muscle sarcoplasmic reticulum Ca-ATPase. The presence of at least three fractions in human plasma that inhibit NaK-ATPase and cross-react to a variable degree with different digoxin-specific antibody populations could explain much of the conflicting evidence for the existence of endogenous digitalis-like compounds in plasma.

Targeting CD22 with the monoclonal antibody epratuzumab modulates human B-cell maturation and cytokine production in response to Toll-like receptor 7 (TLR7) and B-cell receptor (BCR) signaling.

PubMed

Giltiay, Natalia V; Shu, Geraldine L; Shock, Anthony; Clark, Edward A

2017-05-15

Abnormal B-cell activation is implicated in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus (SLE). The B-cell surface molecule CD22, which regulates activation through the B-cell receptor (BCR), is a potential target for inhibiting pathogenic B cells; however, the regulatory functions of CD22 remain poorly understood. In this study, we determined how targeting of CD22 with epratuzumab (Emab), a humanized anti-CD22 IgG1 monoclonal antibody, affects the activation of human B-cell subsets in response to Toll-like receptor 7 (TLR7) and BCR engagement. B-cell subsets were isolated from human tonsils and stimulated with F(ab') 2 anti-human IgM and/or the TLR7 agonist R848 in the presence of Emab or a human IgG1 isotype control. Changes in mRNA levels of genes associated with B-cell activation and differentiation were analyzed by quantitative PCR. Cytokine production was measured by ELISA. Cell proliferation, survival, and differentiation were assessed by flow cytometry. Pretreatment of phenotypically naïve CD19 + CD10 - CD27 - cells with Emab led to a significant increase in IL-10 expression, and in some but not all patient samples to a reduction of IL-6 production in response to TLR7 stimulation alone or in combination with anti-IgM. Emab selectively inhibited the expression of PRDM1, the gene encoding B-lymphocyte-induced maturation protein 1 (Blimp-1) in activated CD10 - CD27 - B cells. CD10 - CD27 - IgD - cells were highly responsive to stimulation through TLR7 as evidenced by the appearance of blasting CD27 hi CD38 hi cells. Emab significantly inhibited the activation and differentiation of CD10 - CD27 - IgD - B cells into plasma cells. Emab can both regulate cytokine expression and block Blimp1-dependent B-cell differentiation, although the effects of Emab may depend on the stage of B-cell development or activation. In addition, Emab inhibits the activation of CD27 - IgD - tonsillar cells, which correspond to so-called double-negative memory B cells, known to be increased in SLE patients with more active disease. These data may be relevant to the therapeutic effect of Emab in vivo via modulation of the production of pro-inflammatory and anti-inflammatory cytokines by B cells. Because Blimp-1 is required by B cells to mature into antibody-producing cells, inhibition of Blimp1 may reduce autoantibody production.

Use of hybridoma immunoglobulin switch variants in the analysis of the protective properties of anti-lipopolysaccharide antibodies in Escherichia coli K1 infection.

PubMed

Pelkonen, S; Pluschke, G

1989-10-01

Functional properties of rat immunoglobulins obtained from hybridoma isotype switch variants were studied in vivo in a rat model for neonatal bacterial sepsis. Escherichia coli 018:K1, a common cause of human neonatal sepsis and meningitis, was injected intravenously into 6-day-old rats after incubation with 018-specific antibodies IgM, IgG1, IgG2a, IgG2b, IgG2c, IgE and IgA. The clearance of bacteria treated with saline or IgE was low, whereas monoclonal antibodies of other isotypes triggered hepatic sequestration and killing of the K1 E. coli cells. All four IgG subclasses were more efficient than IgM and IgA. Comparable results were obtained upon injecting antibodies into rats with an established fulminating bacteraemia. IgM was inactive in animals depleted of complement with cobra-venom factor (CVF), whereas IgG2b was able to trigger hepatic clearance independently of complement.

The abundance and organization of polypeptides associated with antigens of the Rh blood group system.

PubMed

Gardner, B; Anstee, D J; Mawby, W J; Tanner, M J; von dem Borne, A E

1991-06-01

Twelve murine monoclonal antibodies, which react with human red cells of common Rh phenotype but give weak or negative reactions with Rh null erythrocytes, were used in quantitative binding assays and competitive binding assays to investigate the abundance and organization of polypeptides involved in the expression of antigens of the Rh blood group system. Antibodies of the R6A-type (R6A, BRIC-69, BRIC-207) and the 2D10-type (MB-2D10, LA18.18, LA23.40) recognize related structures and 100,000-200,000 molecules of each antibody bind maximally to erythrocytes of common Rh phenotype. Antibodies of the BRIC-125 type (BRICs 32, 122, 125, 126, 168, 211) recognize structures that are unrelated to those recognized by R6A-type and 2D10-type antibodies and between 10,000 and 50,000 antibody molecules bind maximally to erythrocytes of the common Rh phenotype. The binding of antibodies of the R6A-type and the 2D10-type, but not of antibodies of the BRIC-125-type could be partially inhibited by human anti-D antibodies (polyclonal and monoclonal) and a murine anti-e-like antibody. These results are consistent with evidence (Moore & Green 1987; Avent et al., 1988b) that the Rh blood group antigens are associated with a complex that comprises two groups of related polypeptides of M(r) 30,000 and M(r) 35,000-100,000, respectively, and suggest that there are 1-2 x 10(5) copies of this complex per erythrocyte. The polypeptide recognized by antibodies of the BRIC-125 type is likely to be associated with this complex.

The application of digital image analysis for blood typing: the comparison of anti-A and anti-B monoclonal antibodies activity with standard hemagglutinating sera

NASA Astrophysics Data System (ADS)

Medvedeva, Maria F.; Doubrovski, Valery A.

2017-03-01

The resolution of the acousto-optical method for blood typing was estimated experimentally by means of two types of reagents: monoclonal antibodies and standard hemagglutinating sera. The peculiarity of this work is the application of digital photo images processing by pixel analysis previously proposed by the authors. The influence of the concentrations of reagents, of blood sample, which is to be tested, as well as of the duration of the ultrasonic action on the biological object upon the resolution of acousto-optical method were investigated. The optimal experimental conditions to obtain maximum of the resolution of the acousto-optical method were found, it creates the prerequisites for a reliable blood typing. The present paper is a further step in the development of acousto-optical method for determining human blood groups.

Targeting connective tissue growth factor (CTGF) in acute lymphoblastic leukemia preclinical models: anti-CTGF monoclonal antibody attenuates leukemia growth.

PubMed

Lu, Hongbo; Kojima, Kensuke; Battula, Venkata Lokesh; Korchin, Borys; Shi, Yuexi; Chen, Ye; Spong, Suzanne; Thomas, Deborah A; Kantarjian, Hagop; Lock, Richard B; Andreeff, Michael; Konopleva, Marina

2014-03-01

Connective tissue growth factor (CTGF/CCN2) is involved in extracellular matrix production, tumor cell proliferation, adhesion, migration, and metastasis. Recent studies have shown that CTGF expression is elevated in precursor B-acute lymphoblastic leukemia (ALL) and that increased expression of CTGF is associated with inferior outcome in B-ALL. In this study, we characterized the functional role and downstream signaling pathways of CTGF in ALL cells. First, we utilized lentiviral shRNA to knockdown CTGF in RS4;11 and REH ALL cells expressing high levels of CTGF mRNA. Silencing of CTGF resulted in significant suppression of leukemia cell growth compared to control vector, which was associated with AKT/mTOR inactivation and increased levels of cyclin-dependent kinase inhibitor p27. CTGF knockdown sensitized ALL cells to vincristine and methotrexate. Treatment with an anti-CTGF monoclonal antibody, FG-3019, significantly prolonged survival of mice injected with primary xenograft B-ALL cells when co-treated with conventional chemotherapy (vincristine, L-asparaginase and dexamethasone). Data suggest that CTGF represents a targetable molecular aberration in B-ALL, and blocking CTGF signaling in conjunction with administration of chemotherapy may represent a novel therapeutic approach for ALL patients.

Application of boronated anti-CEA immunoliposome to tumour cell growth inhibition in in vitro boron neutron capture therapy model.

PubMed Central

Yanagië, H.; Tomita, T.; Kobayashi, H.; Fujii, Y.; Takahashi, T.; Hasumi, K.; Nariuchi, H.; Sekiguchi, M.

1991-01-01

An immunoliposome containing a 10B-compound has been examined as a selective drug delivery system in boron neutron-capture therapy. Liposomes, conjugated with monoclonal antibodies specific for carcinoembryonic antigen (CEA) were shown to bind selectively to cells bearing CEA on their surface. The immunoliposomes attached to tumour cells suppressed growth in vitro upon thermal neutron irradiation and suppression was dependent upon the concentration of the 10B-compound in the liposomes and on the density of antibody conjugated to the liposomes. The results suggest that immunoliposomes containing the 10B-compound could act as a selective and efficient carrier of 10B atoms to target tumour cells in boron neutron-capture therapy. Images Figure 1 PMID:2021537

Potential future clinical applications for the GPIIb/IIIa antagonist, abciximab in thrombosis, vascular and oncological indications.

PubMed

Cohen, S A; Trikha, M; Mascelli, M A

2000-01-01

Abciximab (ReoPro) is a mouse-human chimeric monoclonal antibody Fab fragment of the parent murine monoclonal antibody 7E3, and was the first of these agents approved for use as adjunct therapy for the prevention of cardiac ischemic complications in patients undergoing percutaneous coronary intervention (PCI). Abciximab binds with high avidity to both the non-activated and activated form of the GPIIb/IIIa receptor of platelets, the major adhesion receptor involved in aggregation. Additional cardiovascular indications for abciximab are unstable angina, carotid stenting, ischemic stroke and peripheral vascular diseases. Abciximab also interacts with two other integrin receptors; the a av b b3 receptor, which is present in low numbers on platelets but in high density on activated endothelial and smooth muscle cells, and a aMb b2 integrin which is present on activated leukocytes. Cell types that express integrins GPIIb/IIIa and a av b b3 such as platelets, endothelial and tumor cells have been implicated in angiogenesis, tumor growth and metastasis. Since abciximab interacts with high avidity to integrins GPIIb/IIIa and a av b b3, it is reasonable to assume that it may possess anti-angiogenic properties in angiogenesis-related diseases, as well as anti-metastastatic properties in case of disseminating tumors expressing the target integrin receptors.

Anti-EGFR monoclonal antibody in cancer treatment: in vitro and in vivo evidence

PubMed

Quatrale, Anna Elisa; Petriella, Daniela; Porcelli, Letizia; Tommasi, Stefania; Silvestris, Nicola; Colucci, Giuseppe; Angelo, Angelo; Azzariti, Amalia

2011-01-01

The complexity of EGFR signaling network suggests that the receptor could be promising targets for new personalised therapy. In clinical practice two strategies targeting the receptor are available; they utilise monoclonal antibodies, directed towards the extracellular domain of EGFR, and small molecule tyrosine kinase inhibitors, which bind the catalytic kinase domain of the receptor. In this review, we summarise currently known pre-clinical data on the antitumor effects of monoclonal antibodies, which bind to EGFR in its inactive configuration, competing for ligand binding and thereby blocking ligand-induced EGFR tyrosine kinase activation. As a consequence of treatment, key EGFR-dependent intracellular signals in cancer cells are affected. Data explaining the mechanisms of action of anti-EGFR monoclonal antibodies, currently used in clinical setting and under development for the treatment of solid tumors, are revised with the aim to provide an overview of the most important preclinical studies showing the impact of this class of EGFR targeted agents on tumor biology.

Monoclonal antibodies to molluskan hemocyanin from Concholepas concholepas demonstrate common and specific epitopes among subunits.

PubMed

Oliva, Harold; Moltedo, Bruno; De Ioannes, Pablo; Faunes, Fernando; De Ioannes, Alfredo E; Becker, María Inés

2002-10-01

We studied the reactivity of mouse monoclonal antibodies (MAbs) against the hemocyanin from the Chilean marine gastropod Concholepas concholepas (CCH). This protein has been successfully used as a carrier to produce antibodies to haptens and peptides. All MAbs (13) belonging to IgG subclass exhibit dissociation constants (K(d)) from 1 x 10(-7) M to 1 x 10(-9) M. MAbs were characterized by enzyme-linked immunosorbant assay (ELISA) using CCH treated with different procedures, including dissociation into CCH-A and CCH-B subunits, Western blot, enzymatic digestion, chemical deglycosylation, and thermal denaturation. MAbs were classified into three categories, according to subunit specificity by ELISA. The epitope distribution shows that CCH subunits display common epitopes (group I, 5 MAbs, 1H5, 2A8, 3A5, 3B3, and 3E3), as well as specific epitopes for CCH-A subunits (group II, 3 MAbs, 1B8, 4D8, and 8E5) and for CCH-B subunits (group III, 5 MAbs, 1A4, 1E4, 2H10, 3B7, and 7B4). The results can be summarized as follows: (1). six antibodies react with thermal denatured CCH, suggesting that they recognize linear epitopes, whereas seven recognize conformational epitopes; (2). oxidation of carbohydrate moieties does not affect the binding of the MAbs; (3). enzymatic digestion of CCH decreases the reactivity of all antibodies irrespective of the protease used (elastase or trypsin); (4). bringing together the above data, in addition to epitopic complementarity analysis, we identified 12 different epitopes on the CCH molecule recognized by these MAbs. The anti-CCH MAbs presented here can be useful tools to understand the subunit organization of the CCH and its complex structure, which can explain its immunogenic and immunostimulating properties in mammals.

G-CSF/anti-G-CSF antibody complexes drive the potent recovery and expansion of CD11b+Gr-1+ myeloid cells without compromising CD8+ T cell immune responses

PubMed Central

2013-01-01

Background Administration of recombinant G-CSF following cytoreductive therapy enhances the recovery of myeloid cells, minimizing the risk of opportunistic infection. Free G-CSF, however, is expensive, exhibits a short half-life, and has poor biological activity in vivo. Methods We evaluated whether the biological activity of G-CSF could be improved by pre-association with anti-G-CSF mAb prior to injection into mice. Results We find that the efficacy of G-CSF therapy can be enhanced more than 100-fold by pre-association of G-CSF with an anti-G-CSF monoclonal antibody (mAb). Compared with G-CSF alone, administration of G-CSF/anti-G-CSF mAb complexes induced the potent expansion of CD11b+Gr-1+ myeloid cells in mice with or without concomitant cytoreductive treatment including radiation or chemotherapy. Despite driving the dramatic expansion of myeloid cells, in vivo antigen-specific CD8+ T cell immune responses were not compromised. Furthermore, injection of G-CSF/anti-G-CSF mAb complexes heightened protective immunity to bacterial infection. As a measure of clinical value, we also found that antibody complexes improved G-CSF biological activity much more significantly than pegylation. Conclusions Our findings provide the first evidence that antibody cytokine complexes can effectively expand myeloid cells, and furthermore, that G-CSF/anti-G-CSF mAb complexes may provide an improved method for the administration of recombinant G-CSF. PMID:24279871

Monoclonal Antibodies to Ferric Pseudobactin, the Siderophore of Plant Growth-Promoting Pseudomonas putida B10

PubMed Central

Buyer, Jeffrey S.; Sikora, Lawrence J.; Kratzke, Marian G.

1990-01-01

Monoclonal antibodies to ferric pseudobactin, the siderophore (microbial iron transport agent) of plant growth-promoting Pseudomonas putida B10, have been developed. Three immunoglobulin G subclass 1-type monoclonal antibodies have been characterized. Each antibody appears to be unique on the basis of their reactions with ferric pseudobactin and with culture supernatants from other pseudomonads. None of the three cross-reacts with ferric pseudobactin-type siderophores produced by seven other pseudomonads. However, P. aeruginosa ATCC 15692 and P. fluorescens ATCC 17400 produced relatively high-molecular-mass compounds (mass greater than approximately 30,000 daltons) that did react with the antibodies. The compound from P. aeruginosa was not iron regulated, while the compound from P. fluorescens was produced only under iron-limiting conditions. A competitive assay using these antibodies has a detection limit of 5 × 10−12 mol of ferric pseudobactin. This is, to our knowledge, the first report of monoclonal antibodies reactive with siderophores. PMID:16348116

Recent advances in understanding and treating vasculitis

PubMed Central

Koster, Matthew J.; Warrington, Kenneth J.

2016-01-01

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs) are near universally fatal conditions if untreated. Although effective therapeutic options are available for these diseases, treatment regimens are associated with both short- and long-term adverse effects. The recent identification of effective B-cell-targeted therapy with an anti-CD20 monoclonal antibody has transformed the treatment landscape of AAV. Questions, nevertheless, remain regarding the appropriate timing, dose, frequency, duration, and long-term effects of treatment. The aim of this article is to provide an overview of the current information, recent advances, ongoing clinical trials, and future treatment possibilities in AAV. PMID:27347395

Role of T cells in the B-cell response: glutaraldehyde-fixed T-helper hybridoma cells synergize with the lymphokine IL-4 to induce B-cell activation and proliferation.

PubMed

Kubota, E; McKenzie, D T; Dutton, R W; Swain, S L

1991-01-01

Antigen-unselected helper T-cell hybridomas (Th) which activate normal resting B cells to RNA synthesis and proliferation in the presence of concanavalin A (Con A) have been developed. The response is completely Th cell dependent, and not restricted by the haplotype of the B-cell major histocompatibility complex (MHC). Culture supernatants from the Con A-stimulated Th hybridomas contain interleukin-4 (IL-4) and IL-2, but undetectable level of IL-5. The supernatant alone, however, does not induce B-cell activation or proliferation. Although the Con A-mediated Th cell-dependent B-cell response occurs in an MHC-unrestricted manner, the response of resting B cells can be blocked by monoclonal Ia antibody specific for the surface class II molecules of the responding B cell. The response is also blocked by monoclonal antibody to L3T4. Significant activation and proliferation of resting B cells can also be triggered by glutaraldehyde-fixed Th hybridomas and Con A when exogenous IL-4 is added. The stimulation with fixed Th hybridomas plus IL-4 can be inhibited by monoclonal anti-L3T4 or anti-Ia. These results suggest that maximal B-cell activation requires a direct helper T cell-B cell interaction which depends on availability of Ia on the B cell and L3T4 on the T cell, even when Con A overcomes the requirement for MHC-restricted T-cell recognition. We suggest that this signal, in conjunction with T-cell produced lymphokine IL-4, is responsible for the activation and subsequent proliferation of the B cells which occurs following interaction with T cells.

Monoclonal antibodies against GARP/TGF-β1 complexes inhibit the immunosuppressive activity of human regulatory T cells in vivo.

PubMed

Cuende, Julia; Liénart, Stéphanie; Dedobbeleer, Olivier; van der Woning, Bas; De Boeck, Gitte; Stockis, Julie; Huygens, Caroline; Colau, Didier; Somja, Joan; Delvenne, Philippe; Hannon, Muriel; Baron, Frédéric; Dumoutier, Laure; Renauld, Jean-Christophe; De Haard, Hans; Saunders, Michael; Coulie, Pierre G; Lucas, Sophie

2015-04-22

Regulatory T cells (Tregs) are essential to prevent autoimmunity, but excessive Treg function contributes to cancer progression by inhibiting antitumor immune responses. Tregs exert contact-dependent inhibition of immune cells through the production of active transforming growth factor-β1 (TGF-β1). On the Treg cell surface, TGF-β1 is in an inactive form bound to membrane protein GARP and then activated by an unknown mechanism. We demonstrate that GARP is involved in this activation mechanism. Two anti-GARP monoclonal antibodies were generated that block the production of active TGF-β1 by human Tregs. These antibodies recognize a conformational epitope that requires amino acids GARP137-139 within GARP/TGF-β1 complexes. A variety of antibodies recognizing other GARP epitopes did not block active TGF-β1 production by Tregs. In a model of xenogeneic graft-versus-host disease in NSG mice, the blocking antibodies inhibited the immunosuppressive activity of human Tregs. These antibodies may serve as therapeutic tools to boost immune responses to infection or cancer via a mechanism of action distinct from that of currently available immunomodulatory antibodies. Used alone or in combination with tumor vaccines or antibodies targeting the CTLA4 or PD1/PD-L1 pathways, blocking anti-GARP antibodies may improve the efficiency of cancer immunotherapy. Copyright © 2015, American Association for the Advancement of Science.

Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity.

PubMed

Prendergast, Jillian M; Galvao da Silva, Ana Paula; Eavarone, David A; Ghaderi, Darius; Zhang, Mai; Brady, Dane; Wicks, Joan; DeSander, Julie; Behrens, Jeff; Rueda, Bo R

Targeted therapeutics that can differentiate between normal and malignant tumor cells represent the ideal standard for the development of a successful anti-cancer strategy. The Sialyl-Thomsen-nouveau antigen (STn or Sialyl-Tn, also known as CD175s) is rarely seen in normal adult tissues, but it is abundantly expressed in many types of human epithelial cancers. We have identified novel antibodies that specifically target with high affinity the STn glycan independent of its carrier protein, affording the potential to recognize a wider array of cancer-specific sialylated proteins. A panel of murine monoclonal anti-STn therapeutic antibodies were generated and their binding specificity and efficacy were characterized in vitro and in in vivo murine cancer models. A subset of these antibodies were conjugated to monomethyl auristatin E (MMAE) to generate antibody-drug conjugates (ADCs). These ADCs demonstrated in vitro efficacy in STn-expressing cell lines and significant tumor growth inhibition in STn-expressing tumor xenograft cancer models with no evidence of overt toxicity.

Development of an animal model of progressive vaccinia in nu/nu mice and the use of bioluminescence imaging for assessment of the efficacy of monoclonal antibodies against vaccinial B5 and L1 proteins.

PubMed

Zaitseva, Marina; Thomas, Antonia; Meseda, Clement A; Cheung, Charles Y K; Diaz, Claudia G; Xiang, Yan; Crotty, Shane; Golding, Hana

2017-08-01

Bioluminescence imaging (BLI) was used to follow dissemination of recombinant vaccinia virus (VACV) expressing luciferase (IHD-J-Luc) in BALB/c nu/nu mice treated post-challenge with monoclonal antibodies (MAbs) against L1 and B5 VACV proteins in a model of Progressive Vaccinia (PV). Areas Under the flux Curve (AUC) were calculated for viral loads in multiple organs in individual mice. Following scarification with 10 5  pfu, IHD-J-Luc VACV undergoes fast replication at the injection site and disseminates rapidly to the inguinal lymph nodes followed by spleen, liver, and axillary lymph nodes within 2-3 days and before primary lesions are visible at the site of scarification. Extension of survival in nude mice treated with a combination of anti-B5 and anti-L1 MAbs 24 h post challenge correlated with a significant reduction in viral load at the site of scarification and delayed systemic dissemination. Nude mice reconstituted with 10 4  T cells prior to challenge with IHD-J-Luc, and treated with MAbs post-challenge, survived infection, cleared the virus from all organs and scarification site, and developed anti-VACV IgG and VACV-specific polyfunctional CD8 + T cells that co-expressed the degranulation marker CD107a, and IFNγ and TNFα cytokines. All T cell reconstituted mice survived intranasal re-challenge with IHD-J-Luc (10 4  pfu) two months after the primary infection. Thus, using BLI to monitor VACV replication in a PV model, we showed that anti-VACV MAbs administered post challenge extended survival of nude mice and protected T cell reconstituted nude mice from lethality by reducing replication at the site of scarification and systemic dissemination of VACV. Published by Elsevier B.V.

Caspr2-reactive antibody cloned from a mother of an ASD child mediates an ASD-like phenotype in mice.

PubMed

Brimberg, L; Mader, S; Jeganathan, V; Berlin, R; Coleman, T R; Gregersen, P K; Huerta, P T; Volpe, B T; Diamond, B

2016-12-01

Autism spectrum disorder (ASD) occurs in 1 in 68 births, preferentially affecting males. It encompasses a group of neurodevelopmental abnormalities characterized by impaired social interaction and communication, stereotypic behaviors and motor dysfunction. Although recent advances implicate maternal brain-reactive antibodies in a causative role in ASD, a definitive assessment of their pathogenic potential requires cloning of such antibodies. Here, we describe the isolation and characterization of monoclonal brain-reactive antibodies from blood of women with brain-reactive serology and a child with ASD. We further demonstrate that male but not female mice exposed in utero to the C6 monoclonal antibody, binding to contactin-associated protein-like 2 (Caspr2), display abnormal cortical development, decreased dendritic complexity of excitatory neurons and reduced numbers of inhibitory neurons in the hippocampus, as well as impairments in sociability, flexible learning and repetitive behavior. Anti-Caspr2 antibodies are frequent in women with brain-reactive serology and a child with ASD. Together these studies provide a methodology for obtaining monclonal brain-reactive antibodies from blood B cells, demonstrate that ASD can result from in utero exposure to maternal brain-reactive antibodies of single specificity and point toward the exciting possibility of prognostic and protective strategies.

A Novel Blocking ELISA for Detection of Antibodies against Hepatitis E Virus in Domestic Pigs.

PubMed

Chen, Yiyang; Zhao, Qin; Liu, Baoyuan; Wang, Lizhen; Sun, Yani; Li, Huixia; Wang, Xinjie; Syed, Shahid Faraz; Zhang, Gaiping; Zhou, En-Min

2016-01-01

Hepatitis E virus (HEV) infects both humans and animals, with an overall human mortality rate generally less than 1%, but as high as 20% among pregnant women. HEV strains fall into 4 major genotypes. Zoonotic genotypes 3 and 4 associate with sporadic human and animal HEV cases in many industrialized countries. To date, collective evidence implicates pigs as the main HEV reservoir, justifying the importance of monitoring HEV infection rates in pig herds to prevent human illness. Due to the lack of a robust in vitro cell culture system for viral propagation, no "gold standard" assay has yet been developed to detect HEV infection in domestic pigs. 1E4, a monoclonal antibody (mAb) specific for the C-terminal 268 amino acids of HEV genotype 4 ORF2 capsid protein (sORF2-C), was generated and conjugated to horseradish peroxidase (HRP) for use in a blocking ELISA (bELISA). Optimal sORF2-C coating antigen concentration (8 μg/ml), HRP-1E4 dilution (1:1000), and test pig serum dilution (1:20) were determined using a checkerboard titration test. A cut-off value of 16.9% was chosen to differentiate between positive vs. negative sera after mean percent inhibition (PI) testing of 230 negative pig sera. Compared with the indirect ELISA (iELISA), western blot, and a commercial ELISA kit for detecting anti-HEV antibodies in human sera, the bELISA showed no statistical differences and statistically high coincidence of 93.23%, 92%, and 95% with the other tests, respectively. A blocking ELISA (bELISA) for detecting anti-HEV antibodies in pig serum samples was developed with high sensitivity and high specificity comparable to that of the indirect ELISA. The bELISA results exhibited high agreement with iELISA, western blot, and a commercial ELISA kit designed to detect human anti-HEV antibodies. Therefore, bELISA should serve as an ideal method for large-scale serological investigation of anti-HEV antibodies in domestic pigs.

Comparison of antibody and cytokine responses to primary Giardia muris infection in H-2 congenic strains of mice.

PubMed

Venkatesan, P; Finch, R G; Wakelin, D

1996-11-01

The course of primary infections with Giardia muris differs between BALB and B10 H-2 congenic strains of mice. In the first 3 weeks of infection, there is a more rapid decline in intestinal trophozoite and fecal cyst counts in B10 strains than in BALB strains. To determine whether this difference could be explained by variation in specific antibody responses, both secretory immunoglobulin A (IgA) and serum antibody responses were compared between these strains. No significant differences in the timing, titer, or specificity of secretory or serum antibodies were found. However, on comparing specific anti-G. muris serum IgG subclass responses, we found that B10 strains produced IgG2a while BALB strains produced IgG1, suggesting differential involvement of T helper 1 and 2 subsets of lymphocytes. When cells harvested from mesenteric lymph nodes were stimulated with concanavalin A in vitro, both gamma interferon and interleukin-5 were secreted by cells from B10 mice, but only interleukin-5 was secreted by cells from BALB/c mice. Specific blockade of gamma interferon by monoclonal antibody administered to B10 mice resulted in an enhanced intensity of infection.

The Case for Adjunctive Monoclonal Antibody Immunotherapy in Schizophrenia.

PubMed

Miller, Brian J; Buckley, Peter F

2016-06-01

This article presents the case in favor of clinical trials of adjunctive monoclonal antibody immunotherapy in schizophrenia. Evidence for prenatal and premorbid immune risk factors for the development of schizophrenia in the offspring is highlighted. Then key evidence for immune dysfunction in patients with schizophrenia is considered. Next, previous trials of adjunctive anti-inflammatory or other immunotherapy in schizophrenia are discussed. Then evidence for psychosis as a side effect of immunotherapy for other disorders is discussed. Also presented is preliminary evidence for adjunctive monoclonal antibody immunotherapy in psychiatric disorders. Finally, important considerations in the design and implementation of clinical trials of adjunctive monoclonal antibody immunotherapy in schizophrenia are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Seeing is believing: anti-PD-1/PD-L1 monoclonal antibodies in action for checkpoint blockade tumor immunotherapy

PubMed Central

Tan, Shuguang; Zhang, Catherine W-H; Gao, George F

2016-01-01

Structural immunology, focusing on structures of host immune related molecules, enables the immunologists to see what the molecules look like, and more importantly, how they work together. Antibody-based PD-1/PD-L1 blockade therapy has achieved brilliant successes in clinical applications. The recent breakthrough of the complex structures of checkpoint blockade antibodies with their counterparts, pembrolizumab with PD-1 and avelumab with PD-L1, have made it clear how these monoclonal antibodies compete the binding of PD-1/PD-L1 and function to blockade the receptor-ligand interaction. Herein, we summarize the structural findings of these two reports and look into the future for how this information would facilitate the development of more efficient PD-1/PD-L1 targeting antibodies, small molecule drugs, and other protein or non-protein inhibitors. PMID:29263905

Bioconjugated fluorescent silica nanoparticles for the rapid detection of Entamoeba histolytica.

PubMed

Hemadi, Ahmad; Ekrami, Alireza; Oormazdi, Hormozd; Meamar, Ahmad Reza; Akhlaghi, Lame; Samarbaf-Zadeh, Ali Reza; Razmjou, Elham

2015-05-01

Rapid detection of Entamoeba histolytica based on fluorescent silica nanoparticle (FSNP) indirect immunofluorescence microscopy was evaluated. Silica nanoparticles were synthesized using Stöber's method, with their surface activated to covalently bind to, and immobilize, protein A. For biolabeling, FSNP was added to conjugated E. histolytica trophozoites with monoclonal anti-E. histolytica IgG1 for microscopic observation of fluorescence. Fluorescent silica nanoparticle sensitivity was determined with axenically cultured E. histolytica serially diluted to seven concentrations. Specificity was evaluated using other intestinal protozoa. Fluorescent silica nanoparticles detected E. histolytica at the lowest tested concentration with no cross-reaction with Entamoeba dispar, Entamoeba moshkovskii, Blastocystis sp., or Giardia lamblia. Visualization of E. histolytica trophozoites with anti-E. histolytica antibody labeled with fluorescein isothiocyanate (FITC) was compared with that using anti-E. histolytica antibody bioconjugated FSNP. Although FITC and FSNP produced similar results, the amount of specific antibody required for FITC to induce fluorescence of similar intensity was fivefold that for FSNP. Fluorescent silica nanoparticles delivered a rapid, simple, cost-effective, and highly sensitive and specific method of detecting E. histolytica. Further study is needed before introducing FSNP for laboratory diagnosis of amoebiasis. Copyright © 2015 Elsevier B.V. All rights reserved.

Evidence for specific annexin I-binding proteins on human monocytes.

PubMed Central

Goulding, N J; Pan, L; Wardwell, K; Guyre, V C; Guyre, P M

1996-01-01

Recombinant human annexin I and a monoclonal antibody specific for this protein (mAb 1B) were used to investigate surface binding of this member of the annexin family of proteins to peripheral blood monocytes. Flow cytometric analysis demonstrated trypsin-sensitive, saturable binding of annexin I to human peripheral blood monocytes but not to admixed lymphocytes. A monoclonal antibody that blocks the anti-phospholipase activity of annexin I also blocked its binding to monocytes. These findings suggest the presence of specific binding sites on monocytes. Furthermore, surface iodination, immunoprecipitation and SDS/PAGE analysis were used to identify two annexin I-binding proteins on the surface of monocytes with molecular masses of 15 kDa and 18 kDa respectively. The identification and characterization of these annexin I-binding molecules should help us to better understand the specific interactions of annexin I with monocytes that lead to down-regulation of pro-inflammatory cell functions. PMID:8687405

Assessment of ability of murine and human anti-lipid A monoclonal antibodies to bind and neutralize lipopolysaccharide

PubMed Central

1993-01-01

The use of monoclonal antibodies (mAbs) directed to lipid A for the therapy of gram-negative sepsis is controversial. In an attempt to understand their biologic basis of action, we used a fluid-phase radioimmunoassay to measure binding between bacterial lipopolysaccharide (LPS) and two IgM mAbs directed to lipid A that are being evaluated for the treatment of gram-negative bacterial sepsis. Both antibodies bound 3H-LPS prepared from multiple strains of gram- negative bacteria when large excesses of antibody were used, although binding was modest and only slightly greater than control preparations. We also studied the ability of each anti-lipid A antibody to neutralize some of the biological effects of LPS in vitro. Despite large molar excesses, neither antibody neutralized LPS as assessed by the limulus lysate test, by a mitogenic assay for murine splenocytes, or by the production of cytokines interleukin (IL)-1, IL-6, or tumor necrosis factor from human monocytes in culture medium or in whole blood. Our experiments do not support the hypothesis that either of these anti- lipid A mAbs function by neutralizing the toxic effects of LPS. PMID:8418211

Development of a serotype colloidal gold strip using monoclonal antibody for rapid detection type Asia1 foot-and-mouth disease

PubMed Central

2011-01-01

Background In this study, we developed a rapid, one step colloid gold strip (CGS) capable of specifically detecting type Asia1 foot-and-mouth disease virus (FMDV). We have produced two monoclonal antibodies (mAb) to type Asia1 FMD (named 1B8 and 5E2). On the test strip, the purified 1B8 labelled with the colloidal gold was used as the detector, and the purified 5E2 and goat anti-mouse antibodies were wrapped onto nitrocellulose (NC) membranes as the test and the control line, respectively. The rapid colloidal gold stereotype diagnostic strip was housed in a plastic case. Results In specificity and sensitivity assay, there was no cross-reaction of the antigen with the other type of FMD and SVDV. The detection sensitivity was found to be as high as 10-5 dilution of Asia1/JSL/05 (1 × 107.2TCID50/50 μL). There was excellent agreement between the results obtained by CGS and reverse indirect hemagglutination assay (RIHA), and the agreement can reach to 98.75%. Conclusion We developed colloidal gold strips that have good qualities and does not require specialized equipment or technicians. This method provided a feasible, convenient, rapid, and effective for detecting type Asia1 FMDV in the fields. PMID:21880157

Development of a serotype colloidal gold strip using monoclonal antibody for rapid detection type Asia1 foot-and-mouth disease.

PubMed

Lin, Tong; Shao, Jun-jun; Du, Jun-zheng; Cong, Guo-zheng; Gao, Shan-dian; Chang, Huiyun

2011-09-01

In this study, we developed a rapid, one step colloid gold strip (CGS) capable of specifically detecting type Asia1 foot-and-mouth disease virus (FMDV). We have produced two monoclonal antibodies (mAb) to type Asia1 FMD (named 1B8 and 5E2). On the test strip, the purified 1B8 labelled with the colloidal gold was used as the detector, and the purified 5E2 and goat anti-mouse antibodies were wrapped onto nitrocellulose (NC) membranes as the test and the control line, respectively. The rapid colloidal gold stereotype diagnostic strip was housed in a plastic case. In specificity and sensitivity assay, there was no cross-reaction of the antigen with the other type of FMD and SVDV. The detection sensitivity was found to be as high as 10(-5) dilution of Asia1/JSL/05 (1 × 10(7.2)TCID(50)/50 μL). There was excellent agreement between the results obtained by CGS and reverse indirect hemagglutination assay (RIHA), and the agreement can reach to 98.75%. We developed colloidal gold strips that have good qualities and does not require specialized equipment or technicians. This method provided a feasible, convenient, rapid, and effective for detecting type Asia1 FMDV in the fields.

Molecular mechanism of PD-1/PD-L1 blockade via anti-PD-L1 antibodies atezolizumab and durvalumab.

PubMed

Lee, Hyun Tae; Lee, Ju Yeon; Lim, Heejin; Lee, Sang Hyung; Moon, Yu Jeong; Pyo, Hyo Jeong; Ryu, Seong Eon; Shin, Woori; Heo, Yong-Seok

2017-07-17

In 2016 and 2017, monoclonal antibodies targeting PD-L1, including atezolizumab, durvalumab, and avelumab, were approved by the FDA for the treatment of multiple advanced cancers. And many other anti-PD-L1 antibodies are under clinical trials. Recently, the crystal structures of PD-L1 in complex with BMS-936559 and avelumab have been determined, revealing details of the antigen-antibody interactions. However, it is still unknown how atezolizumab and durvalumab specifically recognize PD-L1, although this is important for investigating novel binding sites on PD-L1 targeted by other therapeutic antibodies for the design and improvement of anti-PD-L1 agents. Here, we report the crystal structures of PD-L1 in complex with atezolizumab and durvalumab to elucidate the precise epitopes involved and the structural basis for PD-1/PD-L1 blockade by these antibodies. A comprehensive comparison of PD-L1 interactions with anti-PD-L1 antibodies provides a better understanding of the mechanism of PD-L1 blockade as well as new insights into the rational design of improved anti-PD-L1 therapeutics.

Immunoassays for Identification of Biological Agents in Sample Unknowns: NATO SlBCA Exercise VI

DTIC Science & Technology

2005-12-01

Yersiniapestis 103 cfu/mL Enzyme-linked immunosorbent assays Antibodies Unlabelled antibodies Antibody stocks developed under DRES contract by SciLab Consulting...goat anti-rabbit IgG, (whole molecule, lot no. 90H8990). Antibody purification Antibodies produced by Scilab Consulting Inc. were purified on a Bio...No. W7702-4-R430, Final Report. Scilab Consulting Inc. DRDC Suffield TM 2005-223 17 14. Fulton, R.E. and Thompson, H.G. Evaluation of the Rapid

Analysis of the antigen recognition sites of anti-methamphetamine monoclonal antibodies (II): unique feature of MA-3 antibody.

PubMed

Ishimaru, M; Morikawa, K; Hifumi, E; Itoh, T; Uda, T

2000-01-01

A monoclonal antibody against methamphetamine (MA-3 mAb) was found to be strongly bound to ephedrine. This feature was quite different from that of other fourteen mAbs against MA. Analyses of cDNA sequence and steric conformation by molecular modeling revealed that one hydrophilic pocket was generated in the heavy chain of MA-3 mAb involving CDRH-1 and CDRH-2. Asn33, Asn35, Asn50 and Asp52 were the main components of the unique pocket capable of binding to the hydroxyl group of ephedrine.

In vitro photothermal destruction of neuroblastoma cells using carbon nanotubes conjugated with GD2 monoclonal antibody

NASA Astrophysics Data System (ADS)

Wang, Chung-Hao; Huang, Yao-Jhang; Chang, Chia-Wei; Hsu, Wen-Ming; Peng, Ching-An

2009-08-01

Despite aggressive multimodality therapy, most neuroblastoma-bearing patients relapse and survival rate remains poor. Exploration of alternative therapeutic modalities is needed. Carbon nanotubes (CNTs), revealing optical absorbance in the near-infrared region, warrant their merits in photothermal therapy. In order to specifically target disialoganglioside (GD2) overexpressed on the surface of neuroblastoma stNB-V1 cells, GD2 monoclonal antibody (anti-GD2) was conjugated to acidified CNTs. To examine the fate of anti-GD2 bound CNTs after incubation with stNB-V1 cells, rhodamine B was labeled on carboxylated CNTs functionalized with and without anti-GD2. Our results illustrated that anti-GD2-linked CNTs were extensively internalized by neuroblastoma cells via GD2-mediated endocytosis. In addition, we showed that anti-GD2 bound CNTs were not ingested by PC12 cells without GD2 expression. After anti-GD2 conjugated CNTs were incubated with neuroblastoma cells for 6 h and endocytosed by the cells, CNT-laden neuroblastoma cells were further irradiated with an 808 nm near-infrared (NIR) laser with intensity ramping from 0.6 to 6 W cm-2 for 10 min which was then maintained at 6 W cm-2 for an additional 5 min. Post-NIR laser exposure, and after being examined by calcein-AM dye, stNB-V1 cells were all found to undergo necrosis, while non-GD2 expressing PC12 cells all remained viable. Based on the in vitro study, CNTs bound with anti-GD2 have the potential to be utilized as a therapeutic thermal coupling agent that generates heat sufficient to selectively kill neuroblastoma cells under NIR laser light exposure.

Anti-IL-23 receptor monoclonal antibody prevents CD4+ T cell-mediated colitis in association with decreased systemic Th1 and Th17 responses.

PubMed

Imamura, Emiko; Taguchi, Katsunari; Sasaki-Iwaoka, Haruna; Kubo, Satoshi; Furukawa, Shigetada; Morokata, Tatsuaki

2018-04-05

Experimental colitis studies, including T cell-mediated colitis, indicate that IL-23 rather than IL-12 orchestrates intestinal inflammation in inflammatory bowel disease (IBD). Previous studies have identified the roles of IL-12 and IL-23 using mice deficient for their specific subunits, p35 and p19, respectively. However, these studies do not completely reflect the difference in roles between IL-12 and IL-23, especially since the discovery of novel IL-12 family cytokines, which also include p35 or p19 subunits. Here, to clarify the contribution of IL-12 and IL-23 in T cell-mediated colitis, we compared the efficacy of a monoclonal antibody (mAb) to an IL-23-specific receptor subunit with that of an anti-IL-12/23p40 mAb in a naive CD4 + T cell transfer model of experimental colitis, which is associated with enhanced Th1 and Th17 responses. Both antibodies almost completely prevented the development of colitis and showed reduced associated histological changes, including mucosal hyperplasia, infiltration of inflammatory cells and loss of goblet cells. The anti-IL-23 receptor mAb inhibited not only the systemic Th17-response but also the Th1-response, both of which were up-regulated in this model. These results suggest that IL-23, but not IL-12, signaling is critical for the development of colitis. Blockade of IL-23 signaling is a promising therapeutic approach for IBD. Copyright © 2018 Elsevier B.V. All rights reserved.

Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model.

PubMed

Duehr, James; Wohlbold, Teddy John; Oestereich, Lisa; Chromikova, Veronika; Amanat, Fatima; Rajendran, Madhusudan; Gomez-Medina, Sergio; Mena, Ignacio; tenOever, Benjamin R; García-Sastre, Adolfo; Basler, Christopher F; Munoz-Fontela, Cesar; Krammer, Florian

2017-08-15

Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2 -/- mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro , suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity. IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two antibodies with broad cross-reactivity to all known ebolavirus species. The antibodies were raised using a heterologous DNA-viral vector prime-boost regimen, resulting in a high proportion of cross-reactive antibodies (25%). Similar vaccination regimens have been used successfully to induce broad protection against influenza viruses in humans, and our limited data indicate that this might be a useful strategy for filovirus vaccines as well. Several of our antibodies showed protective efficacy when tested in a novel murine challenge model and may be developed into future therapeutics. Copyright © 2017 American Society for Microbiology.

Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model

PubMed Central

Duehr, James; Wohlbold, Teddy John; Oestereich, Lisa; Chromikova, Veronika; Amanat, Fatima; Gomez-Medina, Sergio; Mena, Ignacio; tenOever, Benjamin R.; García-Sastre, Adolfo; Basler, Christopher F.

2017-01-01

ABSTRACT Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2−/− mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro, suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro. Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity. IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two antibodies with broad cross-reactivity to all known ebolavirus species. The antibodies were raised using a heterologous DNA-viral vector prime-boost regimen, resulting in a high proportion of cross-reactive antibodies (25%). Similar vaccination regimens have been used successfully to induce broad protection against influenza viruses in humans, and our limited data indicate that this might be a useful strategy for filovirus vaccines as well. Several of our antibodies showed protective efficacy when tested in a novel murine challenge model and may be developed into future therapeutics. PMID:28592526

Broadly-reactive human monoclonal antibodies elicited following pandemic H1N1 influenza virus exposure protect mice from highly pathogenic H5N1 challenge.

PubMed

Nachbagauer, Raffael; Shore, David; Yang, Hua; Johnson, Scott K; Gabbard, Jon D; Tompkins, S Mark; Wrammert, Jens; Wilson, Patrick C; Stevens, James; Ahmed, Rafi; Krammer, Florian; Ellebedy, Ali H

2018-06-13

Broadly cross-reactive antibodies that recognize conserved epitopes within the influenza virus hemagglutinin (HA) stalk domain are of particular interest for their potential use as therapeutic and prophylactic agents against multiple influenza virus subtypes including zoonotic virus strains. Here, we characterized four human HA stalk-reactive monoclonal antibodies (mAbs) for their binding breadth and affinity, in vitro neutralization capacity, and in vivo protective potential against an highly pathogenic avian influenza virus. The monoclonal antibodies were isolated from individuals shortly following infection with (70-1F02 and 1009-3B05) or vaccination against (05-2G02 and 09-3A01) A(H1N1)pdm09. Three of the mAbs bound HAs from multiple strains of group 1 viruses, and one mAb, 05-2G02, bound to both group 1 and group 2 influenza A HAs. All four antibodies prophylactically protected mice against a lethal challenge with the highly pathogenic A/Vietnam/1203/04 (H5N1) strain. Two mAbs, 70-1F02 and 09-3A01, were further tested for their therapeutic efficacy against the same strain and showed good efficacy in this setting as well. One mAb, 70-1F02, was co-crystallized with H5 HA and showed similar heavy chain only interactions as a the previously described anti-stalk antibody CR6261. Finally, we showed that antibodies that compete with these mAbs are prevalent in serum from an individual recently infected with A(H1N1)pdm09 virus. The antibodies described here can be developed into broad-spectrum antiviral therapeutics that could be used to combat infections with zoonotic or emerging pandemic influenza viruses. IMPORTANCE The rise in zoonotic infections of humans with emerging influenza viruses is a worldwide public health concern. The majority of recent zoonotic human influenza cases were caused by H7N9 and H5Nx viruses and were associated with high morbidity and mortality. In addition, seasonal influenza viruses are estimated to cause up to 650,000 deaths annually worldwide. Currently available anti-viral treatment options include only neuraminidase inhibitors, but some influenza viruses are naturally resistant to these drugs, and others quickly develop resistance-conferring mutations. Alternative therapeutics are urgently needed. Broadly protective antibodies that target the conserved 'stalk' domain of the hemagglutinin represent potential potent antiviral prophylactic and therapeutic agents that can assist pandemic preparedness. Here, we describe four human monoclonal antibodies that target conserved regions of influenza HA and characterize their binding spectrum, as well as their protective capacity in prophylactic and therapeutic settings against a lethal challenge with a zoonotic influenza virus. Copyright © 2018 American Society for Microbiology.

Monoclonal Antibody Fragments for Targeting Therapeutics to Growth Plate Cartilage | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at The Eunice Kennedy Shriver National Institute on Child Health and Human Development (NICHD) have discovered monoclonal antibodies that bind to matrilin-3, a protein specifically expressed in cartilage tissue, that could be used for treating or inhibiting growth plate disorders, such as a skeletal dysplasia or short stature. The monoclonal antibodies can also be used to target therapeutic agents, such as anti-arthritis agents, to cartilage tissue. NICHD seeks statements of capability or interest from parties interested in collaborative research to co-develop, evaluate, or commercialize treatment of skeletal disorders using targeting antibodies.

The form variation of the capsular polysaccharide K1 is not a critical virulence factor of Escherichia coli in a neonatal mouse model of infection.

PubMed

Colino, J; Outschoorn, I

1999-10-01

Escherichia coli K1 is a prevalent cause of Gram-negative neonatal bacteraemia and meningitis in humans. Its capsular polysaccharide K1 (CpsK1) has been identified as an important virulence factor. Nevertheless, the biological and pathogenic implications of its O-acetylated and non-O-acetylated forms are poorly understood. In an attempt to address this, we monitored the expression of both CpsK1 form variants in a neonatal mouse infection model. In the absence of anti-CpsK1 antibodies, no CpsK1 form variant selection was observed during the course of infection. The administration of monoclonal antibodies specific for CpsK1 provided a high level of protection. The monoclonal antibodies that recognized both CpsK1 forms (MGB12) provided protection from up to 850 LD(50). By contrast, the administration of the monoclonal antibodies (MGB15) specific for non-O-acetylated CpsK1 cleared only bacteria expressing this CpsK1 form; a few mouse pups remained bacteraemic, and the bacteria in the blood had O-acetylated CpsK1. In those pups, the infection progressed in a similar fashion to that in mice not treated with monoclonal antibody. Moreover, when the number of bacteria expressing the O-acetylated CpsK1 in the inoculated dose is considered independently, the LD(50)was similar to that for the original strain in pups that had not been treated with monoclonal antibodies (35 CFU). These results suggest that whereas variation in acetylation form per se does not reinforce virulence, it could enable E. coli to avoid immune defenses. This highlights the importance of using highly specific monoclonal antibodies in immunotherapeutic approaches to E. coli K1 neonatal meningitis. Copyright 1999 Academic Press.

Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen.

PubMed

Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette; Brandt, Jette; Kliem, Anette; Skjødt, Karsten; Koch, Claus; Teisner, Børge

2004-01-01

This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious advantages using this assay, are that it can be performed directly on culture supernatants in the early phase of monoclonal antibody production, and also works for antigens with repetitive epitopes. Moreover, the bonus effect, i.e., a signal in excess of the reference signal when sets of monoclonal antibodies with different epitope specificity are compared, gives a relative measure of affinity.

Monoclonal antibody anti-MPO is useful in recognizing minimally differentiated acute myeloid leukaemia.

PubMed

Praxedes, M K; De Oliveira, L Z; Pereira, W da V; Quintana, I Z; Tabak, D G; De Oliveira, M S

1994-01-01

The enzyme myeloperoxidase (MPO) is the most specific marker of myeloid lineage. The recognition of acute myeloid leukaemia (AML) with minimally differentiation (AML-M0) is established with methods that include myeloid markers CD13/CD33 and detection of MPO in blast cells by immunological techniques or electron microscopy cytochemistry (EM). We have analysed the presence of MPO in leukaemic blast cells by conventional cytochemistry and immunological methods using a monoclonal antibody anti-MPO (CLB-MPO1) in 121 cases of acute leukaemia. The aim of the study was to investigate the sensitivity of this McAb to identify AML-M0, as CD13/CD33 can be expressed in some cases of acute lymphoblastic leukaemia (ALL) and EM cytochemistry is not always available in many laboratories. Anti-MPO was positive in all cases of AML (M1-M5) which were positive by Sudan Black B reaction in similar or higher percentage ratio for each case, although in some of them did not label with CD13/CD33 tested by IF and IPc techniques. Based on the anti-MPO positivity, 5 out of 10 cases called undifferentiated leukaemia (AUL) were reclassified as AML-M0, though 4 cases were CD13/CD33 negative. Furthermore, after analysing the anti-MPO expression among 32 cases of ALL, we had to reclassify four of them as acute biphenotypic leukaemia. We conclude that anti-MPO is a very sensitive and reliable tool in AML diagnosis and has an important role in distinguishing minimally differentiated AML and biphenotypic acute leukaemia from AUL and ALL.

AAVrh.10-mediated expression of an anti-cocaine antibody mediates persistent passive immunization that suppresses cocaine-induced behavior.

PubMed

Rosenberg, Jonathan B; Hicks, Martin J; De, Bishnu P; Pagovich, Odelya; Frenk, Esther; Janda, Kim D; Wee, Sunmee; Koob, George F; Hackett, Neil R; Kaminsky, Stephen M; Worgall, Stefan; Tignor, Nicole; Mezey, Jason G; Crystal, Ronald G

2012-05-01

Cocaine addiction is a major problem affecting all societal and economic classes for which there is no effective therapy. We hypothesized an effective anti-cocaine vaccine could be developed by using an adeno-associated virus (AAV) gene transfer vector as the delivery vehicle to persistently express an anti-cocaine monoclonal antibody in vivo, which would sequester cocaine in the blood, preventing access to cognate receptors in the brain. To accomplish this, we constructed AAVrh.10antiCoc.Mab, an AAVrh.10 gene transfer vector expressing the heavy and light chains of the high affinity anti-cocaine monoclonal antibody GNC92H2. Intravenous administration of AAVrh.10antiCoc.Mab to mice mediated high, persistent serum levels of high-affinity, cocaine-specific antibodies that sequestered intravenously administered cocaine in the blood. With repeated intravenous cocaine challenge, naive mice exhibited hyperactivity, while the AAVrh.10antiCoc.Mab-vaccinated mice were completely resistant to the cocaine. These observations demonstrate a novel strategy for cocaine addiction by requiring only a single administration of an AAV vector mediating persistent, systemic anti-cocaine passive immunity.

Evaluation of immobilized metal-ion affinity chromatography (IMAC) as a technique for IgG(1) monoclonal antibodies purification: the effect of chelating ligand and support.

PubMed

Bresolin, I T L; Borsoi-Ribeiro, M; Tamashiro, W M S C; Augusto, E F P; Vijayalakshmi, M A; Bueno, S M A

2010-04-01

Monoclonal antibodies (MAbs) have been used for therapies and some analytical procedures as highly purified molecules. Many techniques have been applied and studied, focusing on monoclonal antibodies purification. In this study, an immobilized metal affinity chromatography membrane was developed and evaluated for the purification of anti-TNP IgG(1) mouse MAbs from cell culture supernatant after precipitation with a 50% saturated ammonium sulfate solution. The chelating ligands iminodiacetic acid, carboxymethylated aspartic acid (CM-Asp), nitrilotriacetic acid, and tris (carboxymethyl) ethylenediamine in agarose gels with immobilized Ni(II) and Zn(II) ions were compared for the adsorption and desorption of MAbs. The most promising chelating ligand--CM-Asp--was then coupled to poly(ethylene vinyl alcohol) (PEVA) hollow fiber membranes. According to SDS-PAGE and ELISA analyses, a higher selectivity and a purification factor of 85.9 (fraction eluted at 500 mM Tris) were obtained for IgG(1) using PEVA-CM-Asp-Zn(II). The anti-TNP MAb could be eluted under mild pH conditions causing no loss of antigen binding capacity.

Recombinant Hepatitis C Virus Envelope Glycoprotein Vaccine Elicits Antibodies Targeting Multiple Epitopes on the Envelope Glycoproteins Associated with Broad Cross-Neutralization

PubMed Central

Wong, Jason Alexander Ji-Xhin; Bhat, Rakesh; Hockman, Darren; Logan, Michael; Chen, Chao; Levin, Aviad; Frey, Sharon E.; Belshe, Robert B.; Tyrrell, D. Lorne

2014-01-01

ABSTRACT Although effective hepatitis C virus (HCV) antivirals are on the horizon, a global prophylactic vaccine for HCV remains elusive. The diversity of the virus is a major concern for vaccine development; there are 7 major genotypes of HCV found globally. Therefore, a successful vaccine will need to protect against HCV infection by all genotypes. Despite the diversity, many monoclonal antibodies (MAbs) with broadly cross-neutralizing activity have been described, suggesting the presence of conserved epitopes that can be targeted to prevent infection. Similarly, a vaccine comprising recombinant envelope glycoproteins (rE1E2) derived from the genotype 1a HCV-1 strain has been shown to be capable of eliciting cross-neutralizing antibodies in guinea pigs, chimpanzees, and healthy human volunteers. In order to investigate the basis for this cross-neutralization, epitope mapping of anti-E1E2 antibodies present within antisera from goats and humans immunized with HCV-1 rE1E2 was conducted through peptide mapping and competition studies with a panel of cross-neutralizing MAbs targeting various epitopes within E1E2. The immunized-goat antiserum was shown to compete with the binding of all MAbs tested (AP33, HC33.4, HC84.26, 1:7, AR3B, AR4A, AR5A, IGH526, and A4). Antisera showed the best competition against HC84.26 and AR3B and the weakest competition against AR4A. Furthermore, antisera from five immunized human vaccinees were shown to compete with five preselected MAbs (AP33, AR3B, AR4A, AR5A, and IGH526). These data show that immunization with HCV-1 rE1E2 elicits antibodies targeting multiple cross-neutralizing epitopes. Our results further support the use of such a vaccine antigen to induce cross-genotype neutralization. IMPORTANCE An effective prophylactic vaccine for HCV is needed for optimal control of the disease burden. The high diversity of HCV has posed a challenge for developing vaccines that elicit neutralizing antibodies for protection against infection. Despite this, we have previously shown that a vaccine comprising recombinant envelope glycoproteins derived from a single genotype 1a strain was capable of eliciting a cross-neutralizing antibody response in human volunteers. Here, we have used competition binding assays and peptide binding assays to show that antibodies present in the antisera from vaccinated goats and humans bind epitopes overlapping with those of a variety of well-characterized cross-neutralizing monoclonal antibodies. This provides a mechanism for the cross-neutralizing human antisera: antibodies present in the antisera bind to conserved regions associated with cross-neutralization. Importantly, this work provides further support for a vaccine comprising recombinant envelope glycoproteins, perhaps in a formulation with a vaccine component eliciting strong anti-HCV CD4+ and CD8+ T cell responses. PMID:25275133

Ligand-induced Epitope Masking: DISSOCIATION OF INTEGRIN α5β1-FIBRONECTIN COMPLEXES ONLY BY MONOCLONAL ANTIBODIES WITH AN ALLOSTERIC MODE OF ACTION.

PubMed

Mould, A Paul; Askari, Janet A; Byron, Adam; Takada, Yoshikazu; Jowitt, Thomas A; Humphries, Martin J

2016-09-30

We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5β1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-β1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-β1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5β1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular cloning and characterization of markers and cytokines for equid myeloid cells.

PubMed

Steinbach, Falko; Stark, Robert; Ibrahim, Sherif; Gawad, Eman Abd-El; Ludwig, Hanns; Walter, Jakob; Commandeur, Ulrich; Mauel, Susanne

2005-10-18

The myeloid cell system comprises of monocytes, macrophages (MPhi), dendritic cells (DC), Kupffer cells, osteoclasts or microglia and is also known as the mononuclear phagocytic system (MPS). Essential cytokines to differentiate or activate these cells include GM-CSF or IL-4. Important markers for characterization include CD1, CD14, CD68, CD163 and CD206. All these markers, however, were not cloned or further characterized in equids by use of monoclonal antibodies earlier. To overcome this problem with the present study, two approaches were used. First, we cloned equine cytokines and markers, and second we analyzed cross-reactivity of human homologues or anti-human monoclonal antibodies. For cloning of equine cytokines and markers, we used degenerate primers delineated from other species, or equine-specific primers based on previous information in Genbank. Flow cytometry was used to determine the expression of markers on myeloid cells. Cross-reactivity could be shown for anti-human CD14, CD163 and mannose receptor (CD206) mAbs. Surface markers such as CD1 and CD68 that distinguish MPhi and DC were cloned and sequenced. According to blast homology, equine CD1a and CD1b could be identified and distinguished. With the resulting information, dendritic cells and macrophages of horses may be characterized.

Generation of Monoclonal Antibodies against Immunoglobulin Proteins of the Domestic Ferret (Mustela putorius furo)

PubMed Central

2017-01-01

The domestic ferret (Mustela putorius furo) serves as an animal model for the study of several viruses that cause human disease, most notably influenza. Despite the importance of this animal model, characterization of the immune response by flow cytometry (FCM) is severely hampered due to the limited number of commercially available reagents. To begin to address this unmet need and to facilitate more in-depth study of ferret B cells including the identification of antibody-secreting cells, eight unique murine monoclonal antibodies (mAb) with specificity for ferret immunoglobulin (Ig) were generated using conventional B cell hybridoma technology. These mAb were screened for reactivity against ferret peripheral blood mononuclear cells by FCM and demonstrate specificity for CD79β+ B cells. Several of these mAb are specific for the light chain of surface B cell receptor (BCR) and enable segregation of kappa and lambda B cells. Additionally, a mAb that yielded surface staining of nearly all surface BCR positive cells (i.e., pan ferret Ig) was generated. Collectively, these MαF-Ig mAb offer advancement compared to the existing portfolio of polyclonal anti-ferret Ig detection reagents and should be applicable to a wide array of immunologic assays including the identification of antibody-secreting cells by FCM. PMID:28286781

Monoclonal antibodies against human angiotensinogen, their characterization and use in an angiotensinogen enzyme linked immunosorbent assay.

PubMed

Rubin, I; Lykkegaard, S; Olsen, A A; Selmer, J; Ballegaard, M

1988-01-01

Monoclonal antibodies were produced against human angiotensinogen. An enzyme linked immunosorbent assay (ELISA) was developed using a high affinity monoclonal antibody as catching antibody and a polyclonal rabbit anti human angiotensinogen antibody as detecting antibody in a "sandwich" ELISA. Linear range of the ELISA was 15-450 pmol/l of human angiotensinogen. Intra- and inter- assay variation coefficients were in the range of 2% to 8%. A correlation coefficient, r = 0.97, (n = 20), with values obtained by radioimmunoassay. This correlation coefficient, obtained by using both normal and pregnant sera, confirmed that the ELISA fulfill the requirements for clinical useful assay. Characterization of the antibodies were performed with respect to affinity constant and epitopes.

The distribution of the therapeutic monoclonal antibodies cetuximab and trastuzumab within solid tumors.

PubMed

Lee, Carol M; Tannock, Ian F

2010-06-03

Poor distribution of some anticancer drugs in solid tumors may limit their anti-tumor activity. Here we used immunohistochemistry to quantify the distribution of the therapeutic monoclonal antibodies cetuximab and trastuzumab in relation to blood vessels and to regions of hypoxia in human tumor xenografts. The antibodies were injected into mice implanted with human epidermoid carcinoma A431 or human breast carcinoma MDA-MB-231 transfected with ERBB2 (231-H2N) that express high levels of ErbB1 and ErbB2 respectively, or wild-type MDA-MB-231, which expresses intermediate levels of ErbB1 and low levels of ErbB2. The distribution of cetuximab in A431 xenografts and trastuzumab in 231-H2N xenografts was time and dose dependent. At early intervals after injection of 1 mg cetuximab into A431 xenografts, the concentration of cetuximab decreased with increasing distance from blood vessels, but became more uniformly distributed at later times; there remained however limited distribution and binding in hypoxic regions of tumors. Injection of lower doses of cetuximab led to heterogeneous distributions. Similar results were observed with trastuzumab in 231-H2N xenografts. In MDA-MB-231 xenografts, which express lower levels of ErbB1, homogeneity of distribution of cetuximab was achieved more rapidly. Cetuximab and trastuzumab distribute slowly, but at higher doses achieve a relatively uniform distribution after about 24 hours, most likely due to their long half-lives in the circulation. There remains poor distribution within hypoxic regions of tumors.

Neutralizing antibodies decrease the envelope fluidity of HIV-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harada, Shinji; Monde, Kazuaki; Tanaka, Yuetsu

2008-01-05

For successful penetration of HIV-1, the formation of a fusion pore may be required in order to accumulate critical numbers of fusion-activated gp41 with the help of fluidization of the plasma membrane and viral envelope. An increase in temperature to 40 {sup o}C after viral adsorption at 25 {sup o}C enhanced the infectivity by 1.4-fold. The enhanced infectivity was inhibited by an anti-CXCR4 peptide, T140, and anti-V3 monoclonal antibodies (0.5{beta} and 694/98-D) by post-attachment neutralization, but not by non-neutralizing antibodies (670-30D and 246-D) specific for the C5 of gp120 and cluster I of gp41, respectively. Anti-HLA-II and an anti-HTLV-I gp46more » antibody, LAT27, neutralized the molecule-carrying HIV-1{sub C-2(MT-2)}. The anti-V3 antibodies suppressed the fluidity of the HIV-1{sub C-2} envelope, whereas the non-neutralizing antibodies did not. The anti-HLA-II antibody decreased the envelope fluidity of HIV-1{sub C-2(MT-2)}, but not that of HIV-1{sub C-2}. Therefore, fluidity suppression by these antibodies represents an important neutralization mechanism, in addition to inhibition of viral attachment.« less

Production of anti-CD14 monoclonal antibody using synthetic peptide of human CD14 as immunizing antigen.

PubMed

Maleki, Leili Aghebati; Shanehbandi, Dariush; Majidi, Jafar; Yusefi, Mehdi; Abdolalizadeh, Jalal; Orangi, Mona; Baradaran, Behzad

2013-01-01

CD14 is a myeloid differentiation antigen expressed primarily on peripheral blood monocytes, dendritic cells and macrophages. It is a key regulator of inflammatory responses to gram-negative bacteria, oxidative burst and septic shock. The aim of this study was to produce and characterize monoclonal antibody against CD14 for use in detection and diagnosis of monocytes. To produce MAb against CD14 protein, mice were immunized with two KLH-conjugated CD14 peptides. The spleen cells of the immunized mice were then fused with SP2/0 by hybridoma technique. Fused cells were grown in selective medium and cloned by limiting dilution method. The desired clones were selected and supernatants of hybridoma cells were screened by ELISA for antibody. Monoclonal antibody was purified by chromatography and confirmed by SDS-PAGE. Finally, immunoblotting and flowcytometry were recruited to explore the specificity of the MAb. Our results showed successful production and characterization of anti CD14 monoclonal antibody. The MAb was IgG2a with Kappa light chain and immunobloting and flowcytometry results demonstrated specific reactivity of this MAb with CD14. The results show that, the produced anti- CD14 MAb is highly specific and functional in biomedical applications such as flow cytometry and western blotting and could be utilized for identification of monocytes.

Potent Inhibition of Human Immunodeficiency Virus Type 1 Replication by an Intracellular Anti-Rev Single-Chain Antibody

NASA Astrophysics Data System (ADS)

Duan, Lingxun; Bagasra, Omar; Laughlin, Mark A.; Oakes, Joseph W.; Pomerantz, Roger J.

1994-05-01

Human immunodeficiency virus type 1 (HIV-1) has a complex life cycle, which has made it a difficult target for conventional therapeutic modalities. A single-chain antibody moiety, directed against the HIV-1 regulatory protein Rev, which rescues unspliced viral RNA from the nucleus of infected cells, has now been developed. This anti-Rev single-chain construct (SFv) consists of both light and heavy chain variable regions of an anti-Rev monoclonal antibody, which, when expressed intracellularly within human cells, potently inhibits HIV-1 replication. This intracellular SFv molecule is demonstrated to specifically antagonize Rev function. Thus, intracellular SFv expression, against a retroviral regulatory protein, may be useful as a gene therapeutic approach to combat HIV-1 infections.

Antitumour effects of single or combined monoclonal antibodies directed against membrane antigens expressed by human B cells leukaemia

PubMed Central

2011-01-01

Background The increasing availability of different monoclonal antibodies (mAbs) opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL) respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells. Results The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to longer mouse survival, as compared to single mAb injection, up to complete inhibition of tumour growth in 100% mice treated with both anti-HLA-DR and anti-CD5. Conclusions Altogether these data suggest that the combined use of two mAbs, such as anti-HLA-DR and anti-CD5, may significantly enhance their therapeutic potential. PMID:21504579

The role of macrophages in the regulation of erythroid colony growth in vitro.

PubMed

Wang, C Q; Udupa, K B; Lipschitz, D A

1992-10-01

Depletion of macrophages from murine marrow by the use of a monoclonal anti-macrophage antibody resulted in a significant increase in the number of erythroid burst forming units (BFU-E). This increase could be neutralized by the addition back to culture of macrophages or macrophage conditioned medium indicating that the suppression was mediated by soluble factors. To further characterize this effect, the addition to culture, either alone or in combination, of interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the growth of BFU-E and the colony-forming unit granulocyte-macrophage (CFU-GM) was examined in macrophage-containing and macrophage-depleted cultures. The addition of IL-1 alpha to culture stimulated the release of both TNF alpha and GM-CSF and acted synergistically with both cytokines, resulting in a dose-dependent suppression of BFU-E and stimulation of CFU-GM growth. The increase in CFU-GM caused by the addition of IL-1 alpha was mediated by GM-CSF but not by TNF alpha as the increase was prevented by the addition of a monoclonal anti-GM-CSF antibody but not by anti-TNF alpha. When either TNF alpha or GM-CSF was neutralized by monoclonal antibodies the addition of IL-1 alpha resulted in a significant increase in BFU-E growth. The addition of GM-CSF to culture caused a dose-dependent suppression of BFU-E that was mediated by TNF alpha, as colony number was not reduced when GM-CSF and a monoclonal anti-TNF alpha antibody were simultaneously added to culture. TNF alpha-induced suppression of BFU-E only occurred in the presence of macrophages. In macrophage-depleted cultures, a dose-dependent suppression of BFU-E could be induced if subinhibitory concentrations of IL-1 alpha or GM-CSF were simultaneously added with increasing concentrations of TNF alpha. The effects of IL-1 alpha or GM-CSF and TNF alpha were markedly synergistic so that the doses required to induce suppression when added simultaneously was only 10% of that required when either were added to culture alone. Suppression of BFU-E by GM-CSF or the combined addition of GM-CSF and TNF alpha did not require IL-1 alpha because inhibition was not neutralized by the addition of anti-IL-1 alpha antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Unexpected cross-reactivity of anti-cathepsin B antibodies leads to uncertainties regarding the mechanism of action of anti-CD20 monoclonal antibody GA101.

PubMed

Chien, Wei Wen; Niogret, Charlène; Jugé, Romain; Lionnard, Loïc; Cornut-Thibaut, Aurélie; Kucharczak, Jérôme; Savina, Ariel; Salles, Gilles; Aouacheria, Abdel

2017-04-01

GA101, also known as obinutuzumab or Gazyva (Gazyvaro), is a glycoengineered type II humanized antibody that targets the CD20 antigen expressed at the surface of B-cells. This novel anti-CD20 antibody is currently assessed in clinical trials with promising results as a single agent or as part of therapeutic combinations for the treatment of B-cell malignancies. Detailed understanding of the mechanisms of GA101-induced cell death is needed to get insight into possible resistance mechanisms occurring in patients. Although multiple in vitro and in vivo mechanisms have been suggested to describe the effects of GA101 on B-cells, currently available data are ambiguous. The aim of our study was to clarify the cellular mechanisms involved in GA101-induced cell death in vitro, and more particularly the respective roles played by lysosomal and mitochondrial membrane permeabilization. Our results confirm previous reports suggesting that GA101 triggers homotypic adhesion and caspase-independent cell death, two processes that are dependent on actin remodeling and involve the production of reactive oxygen species. With respect to lysosomal membrane permeabilization (LMP), our data suggest that lack of specificity of available antibodies directed against cathepsin B may have confounded previously published results, possibly challenging current LMP-driven model of GA101 action mode. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of protection by two endotoxin-neutralizing IgM monoclonal antibodies in different peritonitis models.

PubMed

Hustinx, W N; Benaissa-Trouw, B J; Harmsen, T; Klein, S; Verhoef, J; Hoepelman, A I; Kraaijeveld, K

1997-10-01

Two anti-core glycolipid (CGL) IgM monoclonal antibodies (mAbs 8-2 and 26-20), previously shown to display cross-reactivity with heterologous lipopolysaccharide (LPS) in vitro and to provide cross-protectivity against endotoxin challenge in vivo, were evaluated for their potential to protect mice against death from peritonitis caused by heterologous bacterial challenge. Without concurrent antibiotic treatment neither antibody was protective. Compared with a control mAb, prophylactic treatment with mAb 8-2 significantly increased the survival of gentamicin-treated mice challenged with the rough strain Salmonella minnesota Re595. Both mAb 8-2 and a control mAb, in combination with a suboptimal dose of ceftazidime, increased survival following challenge with the clinical isolate Escherichia coli O7:K1. In a model of mucin-enhanced peritonitis, neither mAb was protective against challenge with inocula of E. coli O7:K1, ranging from 10(2) to 10(4) bacteria. We conclude that protection of mice by anti-CGL mAb 8-2 against heterologous challenge is vitally dependent on concurrent treatment with antibiotics and that protection may not be attributable to the anti-CGL specificity of these antibodies.

ERBB oncogene proteins as targets for monoclonal antibodies.

PubMed

Polanovski, O L; Lebedenko, E N; Deyev, S M

2012-03-01

General properties of the family of tyrosine kinase ERBB receptors are considered in connection with their role in the generation of cascades of signal transduction in normal and tumor cells. Causes of acquisition of oncogene features by genes encoding these receptors and their role in tumorigenesis are analyzed. Anti-ERBB monoclonal antibodies approved for therapy are described in detail, and mechanisms of their antitumor activity and development of resistance to them are reviewed. The existing and the most promising strategies for creating and using monoclonal antibodies and their derivatives for therapy of cancer are discussed.

Emerging monoclonal antibodies for the treatment of renal cell carcinoma (RCC).

PubMed

Atkins, Michael B; Philips, George K

2016-09-01

Advanced renal cell carcinoma (RCC) was considered refractory to most cancer therapies until the 1980s, after which immune modulating agents and targeted agents were developed. Recently the rapid development of therapeutic monoclonal antibodies targeting immune checkpoint pathways has provided significant clinical benefit in patients with many distinct cancer types. Nivolumab, an anti-PD1 monoclonal antibody showed improvement in response rate and overall survival in patients with previously treated RCC and received US FDA approval in late 2015. Current efforts with anti-PD1-based therapy include combinations with ipilimumab and with VEGF pathway blockers in the hopes on building on the activity of single agent therapy. We describe our current understanding of tumor immunology including the basis of the tumor-specific immune response and the adaptive mechanisms used by the tumor for immune escape. We describe the mechanisms of action as well as the therapeutic application of the antibodies, ipilimumab, nivolumab and atezolizumab in patients with RCC. We identify key areas of active research in biomarker development and combination therapies. Clinical trials and the field of RCC therapeutics are expected to move in the direction of combination therapies using immune checkpoint inhibitors, extending overall survival as a benchmark for new drug approvals, and biomarker validation for improved selection of patients for specific therapies.

Progression and Metastasis of Mammary Carcinomas: Potential Role of the Muc1 Glycoprotein

DTIC Science & Technology

1998-09-01

polyclonal antibody to the cytoplasmic tail of Mucl or antibody previously blocked with 5mg/ml of synthetic peptide (diluted 1:50) for 1 hr at 25°C...Following three 5 minute washes with PBS pH 7.4, the sections were incubated with fluorescein isothiocyanate-conjugated swine anti-rabbit antibody ...stained with Ab-3 murine monoclonal antibody that recognizes the neu oncogene (Oncogene Science,Uniondale, NY; diluted 1:100), for 1 hr at 25°C

Detection of Signal Regulatory Protein α in Saimiri sciureus (Squirrel Monkey) by Anti-Human Monoclonal Antibody

PubMed Central

de Souza, Hugo Amorim dos Santos; Costa-Correa, Edmar Henrique; Bianco-Junior, Cesare; Andrade, Márcia Cristina Ribeiro; Lima-Junior, Josué da Costa; Pratt-Riccio, Lilian Rose; Daniel-Ribeiro, Cláudio Tadeu; Totino, Paulo Renato Rivas

2017-01-01

Non-human primates (NHP) are suitable models for studying different aspects of the human system, including pathogenesis and protective immunity to many diseases. However, the lack of specific immunological reagents for neo-tropical monkeys, such as Saimiri sciureus, is still a major factor limiting studies in these models. An alternative strategy to circumvent this obstacle has been the selection of immunological reagents directed to humans, which present cross-reactivity with NHP molecules. In this context and considering the key role of inhibitory immunoreceptors—such as the signal regulatory protein α (SIRPα)—in the regulation of immune responses, in the present study, we attempted to evaluate the ability of anti-human SIRPα monoclonal antibodies to recognize SIRPα in antigen-presenting S. sciureus peripheral blood mononuclear cells (PBMC). As shown by flow cytometry analysis, the profile of anti-SIRPα staining as well as the levels of SIRPα-positive cells in PBMC from S. sciureus were similar to those observed in human PBMC. Furthermore, using anti-SIRPα monoclonal antibody, it was possible to detect a decrease of the SIRPα levels on surface of S. sciureus cells after in vitro stimulation with lipopolysaccharides. Finally, using computed-based analysis, we observed a high degree of conservation of SIRPα across six species of primates and the presence of shared epitopes in the extracellular domain between humans and Saimiri genus that could be targeted by antibodies. In conclusion, we have identified a commercially available anti-human monoclonal antibody that is able to detect SIRPα of S. sciureus monkeys and that, therefore, can facilitate the study of the immunomodulatory role of SIRPα when S. sciureus is used as a model. PMID:29312325

Therapeutic Administration of a Monoclonal Anti-Il-1β Antibody Protects Against Experimental Melioidosis.

PubMed

Weehuizen, Tassili A F; Lankelma, Jacqueline M; De Jong, Hanna K; De Boer, Onno J; Roelofs, Joris J T H; Day, Nicholas P; Gram, Hermann; De Vos, Alex F; Wiersinga, W Joost

2016-11-01

Melioidosis, caused by the gram-negative bacterium Burkholderia pseudomallei, is a common cause of community-acquired sepsis in Southeast Asia and Northern Australia. The NLRP3 inflammasome and its downstream product interleukin-1 beta (IL-1β) have been proposed to play crucial roles in melioidosis. In this study, we characterized the role of IL-1β more closely and we assessed its therapeutic potential. mRNA expression of inflammasome components was determined in isolated leukocytes of 32 healthy controls and 34 patients with sepsis caused by B pseudomallei.Wild-type (WT), NLRP3-deficient (Nlrp3), and Asc mice were infected with B pseudomallei. In additional experiments, infected WT mice were treated with an anti-IL-1β antibody. After 24, 48, and 72 hours (h) mice were sacrificed and organs were harvested. Furthermore, survival studies were performed. Patients with melioidosis exhibited lower mRNA levels of caspase-1, NLRP3, and ASC. Bacterial dissemination and organ damage were increased in B pseudomallei-infected Nlrp3 and Asc mice, together with a reduced pulmonary cell influx. Anti-IL-1β treatment of B pseudomallei challenged mice resulted in strongly reduced bacterial counts, organ damage, and pulmonary granulocyte influx together with reduced mortality. Postponement of anti-IL-1β treatment for 24 h postinfection still protected mice during melioidosis. Expression of caspase-1, NLRP3, and ASC is altered in melioidosis patients. In mice, both NLRP3 and ASC contribute to the host defense against melioidosis. Anti-IL-1β treatment protects mice against B pseudomallei infection and might be a novel treatment strategy in melioidosis.

Single-Molecule Interactions of a Monoclonal Anti-DNA Antibody with DNA

PubMed Central

Nevzorova, Tatiana A.; Zhao, Qingze; Lomakin, Yakov A.; Ponomareva, Anastasia A.; Mukhitov, Alexander R.; Purohit, Prashant K.; Weisel, John W.; Litvinov, Rustem I.

2017-01-01

Interactions of DNA with proteins are essential for key biological processes and have both a fundamental and practical significance. In particular, DNA binding to anti-DNA antibodies is a pathogenic mechanism in autoimmune pathology, such as systemic lupus erythematosus. Here we measured at the single-molecule level binding and forced unbinding of surface-attached DNA and a monoclonal anti-DNA antibody MRL4 from a lupus erythematosus mouse. In optical trap-based force spectroscopy, a microscopic antibodycoated latex bead is trapped by a focused laser beam and repeatedly brought into contact with a DNA-coated surface. After careful discrimination of non-specific interactions, we showed that the DNA-antibody rupture force spectra had two regimes, reflecting formation of weaker (20–40 pN) and stronger (>40 pN) immune complexes that implies the existence of at least two bound states with different mechanical stability. The two-dimensional force-free off-rate for the DNA-antibody complexes was ~2.2 × 10−3 s−1, the transition state distance was ~0.94 nm, the apparent on-rate was ~5.26 s−1, and the stiffness of the DNA-antibody complex was characterized by a spring constant of 0.0021 pN/nm, suggesting that the DNA-antibody complex is a relatively stable, but soft and deformable macromolecular structure. The stretching elasticity of the DNA molecules was characteristic of single-stranded DNA, suggesting preferential binding of the MRL4 antibody to one strand of DNA. Collectively, the results provide fundamental characteristics of formation and forced dissociation of DNA-antibody complexes that help to understand principles of DNA-protein interactions and shed light on the molecular basis of autoimmune diseases accompanied by formation of anti-DNA antibodies. PMID:29104846

C1q-targeted inhibition of the classical complement pathway prevents injury in a novel mouse model of acute motor axonal neuropathy.

PubMed

McGonigal, Rhona; Cunningham, Madeleine E; Yao, Denggao; Barrie, Jennifer A; Sankaranarayanan, Sethu; Fewou, Simon N; Furukawa, Koichi; Yednock, Ted A; Willison, Hugh J

2016-03-02

Guillain-Barré syndrome (GBS) is an autoimmune disease that results in acute paralysis through inflammatory attack on peripheral nerves, and currently has limited, non-specific treatment options. The pathogenesis of the acute motor axonal neuropathy (AMAN) variant is mediated by complement-fixing anti-ganglioside antibodies that directly bind and injure the axon at sites of vulnerability such as nodes of Ranvier and nerve terminals. Consequently, the complement cascade is an attractive target to reduce disease severity. Recently, C5 complement component inhibitors that block the formation of the membrane attack complex and subsequent downstream injury have been shown to be efficacious in an in vivo anti-GQ1b antibody-mediated mouse model of the GBS variant Miller Fisher syndrome (MFS). However, since gangliosides are widely expressed in neurons and glial cells, injury in this model was not targeted exclusively to the axon and there are currently no pure mouse models for AMAN. Additionally, C5 inhibition does not prevent the production of early complement fragments such as C3a and C3b that can be deleterious via their known role in immune cell and macrophage recruitment to sites of neuronal damage. In this study, we first developed a new in vivo transgenic mouse model of AMAN using mice that express complex gangliosides exclusively in neurons, thereby enabling specific targeting of axons with anti-ganglioside antibodies. Secondly, we have evaluated the efficacy of a novel anti-C1q antibody (M1) that blocks initiation of the classical complement cascade, in both the newly developed anti-GM1 antibody-mediated AMAN model and our established MFS model in vivo. Anti-C1q monoclonal antibody treatment attenuated complement cascade activation and deposition, reduced immune cell recruitment and axonal injury, in both mouse models of GBS, along with improvement in respiratory function. These results demonstrate that neutralising C1q function attenuates injury with a consequent neuroprotective effect in acute GBS models and promises to be a useful new target for human therapy.

Enzyme-linked immunosorbent assay using a recombinant baculovirus-expressed Bacillus anthracis protective antigen (PA): measurement of human anti-PA antibodies.

PubMed Central

Iacono-Connors, L C; Novak, J; Rossi, C; Mangiafico, J; Ksiazek, T

1994-01-01

We developed an antigen capture enzyme-linked immunosorbent assay (ELISA) which does not require purified protective antigen (PA) for detection of human antibodies to Bacillus anthracis PA. Lysates of Spodoptera frugiperda (Sf-9) cells infected with recombinant baculovirus containing the PA gene were used as the source of PA to develop the ELISA. Recombinant PA from crude Sf-9 cell lysates or PA purified from B. anthracis Sterne strain was captured by an anti-PA monoclonal antibody coated onto microtiter plates. We demonstrated that human serum antibody titers to PA were identical in the ELISA whether we used crude Sf-9 cell lysates containing recombinant baculovirus-expressed PA or purified Sterne PA. Finally, false-positive results observed in a direct ELISA were eliminated with this antigen capture ELISA. Thus, the antigen capture ELISA with crude preparations of baculovirus-expressed PA is reliable, safe, and inexpensive for determining anti-PA antibody levels in human sera. PMID:7496927

Lea blood group antigen on human platelets.

PubMed

Dunstan, R A; Simpson, M B; Rosse, W F

1985-01-01

One- and two-stage radioligand assays were used to determine if human platelets possess the Lea antigen. Goat IgG anti-Lea antibody was purified by multiple adsorptions with Le(a-b-) human red blood cells, followed by affinity chromatography with synthetic Lea substance and labeling with 125I. Human IgG anti-Lea antibody was used either in a two stage radioassay with 125I-labeled mouse monoclonal IgG anti-human IgG as the second antibody or, alternatively, purified by Staph protein A chromatography, labeled with 125I, and used in a one-stage radioassay. Platelets from donors of appropriate red blood cell phenotypes were incubated with the antisera, centrifuged through phthalate esters, and assayed in a gamma scintillation counter. Dose response and saturation curve analysis demonstrate the presence of Lewis a antigen on platelets from Lea+ donors. Furthermore, platelets from an Le(a-b-) donor incubated in Le (a+b-) plasma adsorb Lea antigen in a similar manner to red blood cells. The clinical significance of these antigens in platelet transfusion remains undefined.

[Detection and analysis of anti-Rh blood group antibodies].

PubMed

Wu, Yuan-jun; Wu, Yong; Chen, Bao-chan; Liu, Yan

2008-06-01

To study the prevalence and distribution of anti-Rh blood group antibodies in Chinese population and its clinical significance. Irregular antibodies were screened and identified by Microcolum Gel Coomb's test. For those identified as positive anti-Rh samples, monoclonal antibodies (anti-D, -C, -c, -E and -e) were used to identify the specific antigen and confirm the accuracy of the irregular antibody tests. The titers, Ig-types and 37 Degrees Celsius-reactivity were tested to confirm its clinical significance. For evaluation of the origin of irregular antibodies, histories of pregnancy and transfusion were reviewed. For the newborns who had positive antibodies, their mothers were tested simultaneously to confirm the origin of the antibodies. 47 out of 54 000 (0.087%) patients were identified as positive with Rh blood group antibodies.Of them, 27 cases had history of pregnancy, 13 had transfusion and 1 had the histories of both. 6 newborns had antibodies derived form their mothers. The specificity of the antibody was as follows: 29 with anti-E (61.70%), 8 with anti-D (17.02%), anti-cE 5(10.64%), 4 with anti-c (8.51%) and 1 with anti-C (2.13%). All the 47 Rh blood group antibodies were IgG or IgG+IgM, and were reactive to red blood cells with corresponding antigens at 37 Degrees Celsius, with a highest titer of 1:4 096. The prevalence of Rh antibodies is lower in Chinese population as compared with that in White population.Of all the antibodies, anti-E is most frequently identified and anti-D was declining. Alloimmunization by pregnancy and transfusion is the major cause of Rh antibody production. Rh blood group antibodies derived from mothers are the major cause of Non-ABO-HDN.

HA Antibody-Mediated FcγRIIIa Activity Is Both Dependent on FcR Engagement and Interactions between HA and Sialic Acids.

PubMed

Cox, Freek; Kwaks, Ted; Brandenburg, Boerries; Koldijk, Martin H; Klaren, Vincent; Smal, Bastiaan; Korse, Hans J W M; Geelen, Eric; Tettero, Lisanne; Zuijdgeest, David; Stoop, Esther J M; Saeland, Eirikur; Vogels, Ronald; Friesen, Robert H E; Koudstaal, Wouter; Goudsmit, Jaap

2016-01-01

Interactions with receptors for the Fc region of IgG (FcγRs) have been shown to contribute to the in vivo protection against influenza A viruses provided by broadly neutralizing antibodies (bnAbs) that bind to the viral hemagglutinin (HA) stem. In particular, Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) has been shown to contribute to protection by stem-binding bnAbs. Fc-mediated effector functions appear not to contribute to protection provided by strain-specific HA head-binding antibodies. We used a panel of anti-stem and anti-head influenza A and B monoclonal antibodies with identical human IgG1 Fc domains and investigated their ability to mediate ADCC-associated FcγRIIIa activation. Antibodies which do not interfere with sialic acid binding of HA can mediate FcγRIIIa activation. However, the FcγRIIIa activation was inhibited when a mutant HA, unable to bind sialic acids, was used. Antibodies which block sialic acid receptor interactions of HA interfered with FcγRIIIa activation. The inhibition of FcγRIIIa activation by HA head-binding and sialic acid receptor-blocking antibodies was confirmed in plasma samples of H5N1 vaccinated human subjects. Together, these results suggest that in addition to Fc-FcγR binding, interactions between HA and sialic acids on immune cells are required for optimal Fc-mediated effector functions by anti-HA antibodies.

Indirect myosin immunocytochemistry for the identification of fibre types in equine skeletal muscle

NASA Technical Reports Server (NTRS)

Sinha, A. K.; Rose, R. J.; Pozgaj, I.; Hoh, J. F.

1992-01-01

The histochemical ATPase method for muscle fibre typing was first described by Brooke and Kaiser in 1970. However, problems have been found with the subdivision of type II fibres using this technique. To determine whether indirect myosin immunocytochemistry using anti-slow (5-4D), anti-fast (1A10) and anti-fast red (5-2B) monoclonal antibodies with cross reactivity for type I, II and IIa fibres, respectively, in a number of species, could identify three fibre types in equine skeletal muscle, data on fibre type composition and fibre size obtained using the two different techniques were compared. Results indicate that different myosin heavy chains can coexist in single equine muscle fibres. Type I and type II fibres were identified by immunocytochemistry, but subdivision of type II fibres was not possible. Although the percentage of type I and type II fibres was not significantly different for the two techniques, a few fibres reacted with both the 1A10 and 5-4D antibodies.

A Protein-Conjugate Approach to Develop a Monoclonal Antibody-Based Antigen Detection Test for the Diagnosis of Human Brucellosis

PubMed Central

Patra, Kailash P.; Saito, Mayuko; Atluri, Vidya L.; Rolán, Hortensia G.; Young, Briana; Kerrinnes, Tobias; Smits, Henk; Ricaldi, Jessica N.; Gotuzzo, Eduardo; Gilman, Robert H.; Tsolis, Renee M.; Vinetz, Joseph M.

2014-01-01

Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases. PMID:24901521

Monoclonal IgA Antibodies for Aflatoxin Immunoassays

PubMed Central

Ertekin, Özlem; Pirinçci, Şerife Şeyda; Öztürk, Selma

2016-01-01

Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2–50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort. PMID:27187470

[Antibody therapy for Alzheimer's disease].

PubMed

Tabira, Takeshi; Matsumoto, Shin-Ei; Jin, Haifeng

2011-11-01

In order to avoid Abeta-induced autoimmune encephalitis, several monoclonal and polyclonal antibodies are in clinical trials. These are bapineuzumab, solanezumab, ponezumab, gantenerumab, BAN2401, gammaguard and octagam. Since each antibody has a different antigen epitope of Abeta, anti-amyloid activities are different. It is unknown which antibody is effective for Alzheimer disease, and we must wait for the result of clinical trials. Some patients who developed tissue amyloid plaque immuno-reactive (TAPIR) antibody showed slower decline after AN-1792 vaccination. We developed TAPIR-like monoclonal antibody, which was found to react with Abeta oligomers preferentially.

Pembrolizumab and its role in relapsed/refractory classical Hodgkin's lymphoma: evidence to date and clinical utility.

PubMed

Shindiapina, Polina; Alinari, Lapo

2018-04-01

Immune evasion is a critical mechanism of malignant cell survival, and relies in part on molecular signaling through the programmed cell death 1 (PD-1)/PD-1 ligand (PD-L1) axis that contributes to T cell exhaustion. Immune modulatory therapy with monoclonal antibodies against PD-1 designed to enhance antitumor immune response have shown promise in the treatment of advanced solid tumors and hematologic malignancies. Classical Hodgkin's lymphoma (cHL), a unique B cell malignancy characterized by an extensive but ineffective immune cell infiltrate surrounding a small number of tumor cells, has shown significant response to anti-PD-1 directed therapy. The anti-PD-1 monoclonal antibodies nivolumab and pembrolizumab have shown similarly remarkable activity in relapsed/refractory cHL and have been approved by the Food and Drug Administration for treatment of this disease. In this article we review the rationale of targeting the PD-1/PD-L1 axis in cHL and the pharmacology of pembrolizumab, and summarize the data on activity and safety profile of this agent in the treatment of relapsed/refractory cHL. We also discuss the potential benefits and pitfalls of using PD-1 blockade in the setting of allogeneic stem-cell transplantation, and summarize ongoing prospective trials of single-agent pembrolizumab and combination strategies as well as future directions.

Pembrolizumab and its role in relapsed/refractory classical Hodgkin’s lymphoma: evidence to date and clinical utility

PubMed Central

Shindiapina, Polina; Alinari, Lapo

2018-01-01

Immune evasion is a critical mechanism of malignant cell survival, and relies in part on molecular signaling through the programmed cell death 1 (PD-1)/PD-1 ligand (PD-L1) axis that contributes to T cell exhaustion. Immune modulatory therapy with monoclonal antibodies against PD-1 designed to enhance antitumor immune response have shown promise in the treatment of advanced solid tumors and hematologic malignancies. Classical Hodgkin’s lymphoma (cHL), a unique B cell malignancy characterized by an extensive but ineffective immune cell infiltrate surrounding a small number of tumor cells, has shown significant response to anti-PD-1 directed therapy. The anti-PD-1 monoclonal antibodies nivolumab and pembrolizumab have shown similarly remarkable activity in relapsed/refractory cHL and have been approved by the Food and Drug Administration for treatment of this disease. In this article we review the rationale of targeting the PD-1/PD-L1 axis in cHL and the pharmacology of pembrolizumab, and summarize the data on activity and safety profile of this agent in the treatment of relapsed/refractory cHL. We also discuss the potential benefits and pitfalls of using PD-1 blockade in the setting of allogeneic stem-cell transplantation, and summarize ongoing prospective trials of single-agent pembrolizumab and combination strategies as well as future directions. PMID:29623180

Detection of anthrax protective antigen (PA) using europium labeled anti-PA monoclonal antibody and time-resolved fluorescence ◊

PubMed Central

Stoddard, Robyn A.; Quinn, Conrad P.; Schiffer, Jarad M.; Boyer, Anne E.; Goldstein, Jason; Bagarozzi, Dennis A.; Soroka, Stephen D.; Dauphin, Leslie A.; Hoffmaster, Alex R.

2015-01-01

Inhalation anthrax is a rare but acute infectious disease following adsorption of Bacillus anthracis spores through the lungs. The disease has a high fatality rate if untreated, but early and correct diagnosis has a significant impact on case patient recovery. The early symptoms of inhalation anthrax are, however, non-specific and current anthrax diagnostics are primarily dependent upon culture and confirmatory real-time PCR. Consequently, there may be a significant delay in diagnosis and targeted treatment. Rapid, culture-independent diagnostic tests are therefore needed, particularly in the context of a large scale emergency response. The aim of this study was to evaluate the ability of monoclonal antibodies to detect anthrax toxin proteins that are secreted early in the course of B. anthracis infection using a time-resolved fluorescence (TRF) immunoassay. We selected monoclonal antibodies that could detect protective antigen (PA), as PA83 and also PA63 and LF in the lethal toxin complex. The assay reliable detection limit (RDL) was 6.63 × 10−6 μM (0.551 ng/ml) for PA83 and 2.51 × 10−5 μM (1.58 ng/ml) for PA63. Despite variable precision and accuracy of the assay, PA was detected in 9 out of 10 sera samples from anthrax confirmed case patients with cutaneous (n=7), inhalation (n=2), and gastrointestinal (n=1) disease. Anthrax Immune Globulin (AIG), which has been used in treatment of clinical anthrax, interfered with detection of PA. This study demonstrates a culture-independent method of diagnosing anthrax through use of monoclonal antibodies to detect PA and LF in the lethal toxin complex. PMID:24857756

Epitope mapping of the variable repetitive region with the MB antigen of Ureaplasma urealyticum.

PubMed Central

Zheng, X; Lau, K; Frazier, M; Cassell, G H; Watson, H L

1996-01-01

One of the major surface structures of Ureaplasma urealyticum recognized by antibodies of patients during infection is the MB antigen. Previously, we showed by Western blot (immunoblot) analysis that any one of the anti-MB monoclonal antibodies (MAbs) 3B1.5, 5B1.1, and 10C6.6 could block the binding of patient antibodies to MB. Subsequent DNA sequencing revealed that a unique six-amino-acid direct tandem repeat region composed the carboxy two-thirds of this antigen. In the present study, using antibody-reactive peptide scanning of this repeat region, we demonstrated that the amino acids defining the epitopes for MAbs 3B1.5 5B1.1 and 10C6.6 are EQP, GK, and KEQPA, respectively. Peptide scanning analysis of an infected patient's serum antibody response showed that the dominant epitope was defined by the sequence PAGK. Mapping of these continuous epitopes revealed overlap between all MAb and patient polyclonal antibody binding sites, thus explaining the ability of a single MAb to apparently block all polyclonal antibody binding sites. We also show that a single amino acid difference in the sequence of the repeats of serovars 3 and 14 accounts for the lack of reactivity with serovar 14 of two of the serovar 3-specific MAbs. Finally, the data demonstrate the need to obtain the sequences of the mba genes of all serovars before an effective serovar-specific antibody detection method can be developed. PMID:8914774

The chemiluminescent response of human monocytes to red cells sensitized with monoclonal anti-Rh(D) antibodies.

PubMed

Hadley, A G; Kumpel, B M; Merry, A H

1988-01-01

Luminol-enhanced chemiluminescence (CL) was used to assess the metabolic response of human monocytes to red cells sensitized with known amounts of anti-Rh(D). Monoclonal antibodies were used to facilitate a comparison between the functional activities of IgG1 and IgG3 subclasses. The detection of CL provided a simple, rapid and semi-quantitative means of measuring monocyte response to sensitized red cells (IgG-RBC). Monocyte response to IgG3-RBC was quantitatively greater, more rapid and less susceptible to inhibition by fluid phase IgG than monocyte response to IgG1-RBC. The minimum levels of sensitization required to elicit CL from monocytes were approximately 2500 IgG3 molecules per red cell, or approximately 5000 IgG1 molecules per cell.

Mouse monoclonal antibodies against human c-Mpl and characterization for flow cytometry applications.

PubMed

Abbott, Christina; Huang, Guo; Ellison, Aaron R; Chen, Ching; Arora, Taruna; Szilvassy, Stephen J; Wei, Ping

2010-04-01

Mouse monoclonal antibodies (MAbs) against human c-Mpl, the cognate receptor for thrombopoietin (TPO), were generated using hybridoma technology and characterized by various assays to demonstrate their specificity and affinity. Two such MAbs, 1.6 and 1.75, were determined to be superior for flow cytometry studies and exhibited double-digit picomolar (pM) affinities to soluble human c-Mpl protein. Both MAbs specifically bound to cells engineered to overexpress human c-Mpl protein, immortalized human hematopoietic cell lines that express endogenous c-Mpl, primary human bone marrow and peripheral blood-derived CD34(+) cells, and purified human platelets. No binding was detected on cell lines that did not express c-Mpl. Receptor competition and siRNA knock-down studies further confirmed the specificity of antibodies 1.6 and 1.75 for human c-Mpl. In contrast to these newly generated MAbs, none of eight commercially available anti-c-Mpl antibodies tested were found to bind specifically to human c-Mpl and were thus shown to be unsuitable for flow cytometry studies. Monoclonal antibodies 1.6 and 1.75 will therefore be useful flow cytometry reagents to detect cell surface c-Mpl expression.

Production of anti-amoxicillin ScFv antibody and simulation studying its molecular recognition mechanism for penicillins.

PubMed

Liu, Jing; Zhang, Hui C; Duan, Chang F; Dong, Jun; Zhao, Guo X; Wang, Jian P; Li, Nan; Liu, Jin Z; Li, Yu W

2016-11-01

The molecular recognition mechanism of an antibody for its hapten is very interesting. The objective of this research was to study the intermolecular interactions of an anti-amoxicillin antibody with penicillin drugs. The single chain variable fragment (ScFv) antibody was generated from a hybridoma cell strain excreting the monoclonal antibody for amoxicillin. The recombinant ScFv antibody showed similar recognition ability for penicillins to its parental monoclonal antibody: simultaneous recognizing 11 penicillins with cross-reactivities of 18-107%. The three-dimensional structure of the ScFv antibody was simulated by using homology modeling, and its intermolecular interactions with 11 penicillins were studied by using molecular docking. Results showed that three CDRs are involved in antibody recognition; CDR L3 Arg 100, CDR H3 Tyr226, and CDR H3 Arg 228 were the key contact amino acid residues; hydrogen bonding was the main antibody-drug intermolecular force; and the core structure of penicillin drugs was the main antibody binding position. These results could explain the recognition mechanism of anti-amoxicillin antibody for amoxicillin and its analogs. This is the first study reporting the production of ScFv antibody for penicillins and stimulation studying its recognition mechanism.

Antibody Response to Serpin B13 Induces Adaptive Changes in Mouse Pancreatic Islets and Slows Down the Decline in the Residual Beta Cell Function in Children with Recent Onset of Type 1 Diabetes Mellitus.

PubMed

Kryvalap, Yury; Lo, Chi-Wen; Manuylova, Ekaterina; Baldzizhar, Raman; Jospe, Nicholas; Czyzyk, Jan

2016-01-01

Type 1 diabetes mellitus (T1D) is characterized by a heightened antibody (Ab) response to pancreatic islet self-antigens, which is a biomarker of progressive islet pathology. We recently identified a novel antibody to clade B serpin that reduces islet-associated T cell accumulation and is linked to the delayed onset of T1D. As natural immunity to clade B arises early in life, we hypothesized that it may influence islet development during that time. To test this possibility healthy young Balb/c male mice were injected with serpin B13 mAb or IgG control and examined for the number and cellularity of pancreatic islets by immunofluorescence and FACS. Beta cell proliferation was assessed by measuring nucleotide analog 5-ethynyl-2'-deoxyuridine (5-EdU) incorporation into the DNA and islet Reg gene expression was measured by real time PCR. Human studies involved measuring anti-serpin B13 autoantibodies by Luminex. We found that injecting anti-serpin B13 monoclonal Ab enhanced beta cell proliferation and Reg gene expression, induced the generation of ∼80 pancreatic islets per animal, and ultimately led to increase in the beta cell mass. These findings are relevant to human T1D because our analysis of subjects just diagnosed with T1D revealed an association between baseline anti-serpin activity and slower residual beta cell function decline in the first year after the onset of diabetes. Our findings reveal a new role for the anti-serpin immunological response in promoting adaptive changes in the endocrine pancreas and suggests that enhancement of this response could potentially help impede the progression of T1D in humans. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

In vivo Therapy with Monoclonal Anti-I-A Antibody Suppresses Immune Responses to Acetylcholine Receptor

NASA Astrophysics Data System (ADS)

Waldor, Matthew K.; Sriram, Subramaniam; McDevitt, Hugh O.; Steinman, Lawrence

1983-05-01

A monoclonal antibody to I-A gene products of the immune response gene complex attenuates both humoral and cellular responses to acetylcholine receptor and appears to suppress clinical manifestations of experimental autoimmune myasthenia gravis. This demonstrates that use of antibodies against immune response gene products that are associated with susceptibility to disease may be feasible for therapy in autoimmune conditions such as myasthenia gravis.

Novel immunoassay and rapid immunoaffinity chromatography method for the detection and selective extraction of naringin in Citrus aurantium.

PubMed

Qu, Huihua; Zhang, Yue; Qu, Baoping; Cheng, Jinjun; Liu, Shuchen; Feng, Shenglan; Wang, Qingguo; Zhao, Yan

2016-04-01

In this work, a novel monoclonal antibody specific for naringin was prepared and characterized. Subsequently, an indirect competitive enzyme-linked immunosorbent assay for naringin was developed, with an effective range from 4.8 to 156 ng/mL naringin. Next, an immunoaffinity column was obtained by coupling anti-naringin monoclonal antibodies to CNBr-activated Sepharose 4B and a rapid immunoaffinity chromatography assay for naringin was developed. The immunoaffinity column was used to separate naringin from Citrus aurantium. The results showed that 1 g of the dry Sepharose 4B can couple 10 mg of immunoglobulin G. And the immunoaffinity column can efficiently and specifically capture approximately 250 μg of naringin without cross reacting with its structurally similar compounds. Moreover, our results indicate that the application of immunoaffinity chromatography can simplify the pretreatment and the isolation process greatly compared to conventional methods, providing a potential method for extracting the target component from structurally similar compounds in natural products. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Simple Method to Avoid Nonspecific Signal When Using Monoclonal Anti-Tau Antibodies in Western Blotting of Mouse Brain Proteins.

PubMed

Petry, Franck R; Nicholls, Samantha B; Hébert, Sébastien S; Planel, Emmanuel

2017-01-01

In Alzheimer's disease and other tauopathies, tau displays several abnormal post-translation modifications such as hyperphosphorylation, truncation, conformation, and oligomerization. Mouse monoclonal antibodies have been raised against such tau modifications for research, diagnostic, and therapeutic purposes. However, many of these primary antibodies are at risk of giving nonspecific signals in common Western blotting procedures. Not because they are unspecific, but because the secondary antibodies used to detect them will also detect the heavy chain of endogenous mouse immunoglobulins (Igs), and give a nonspecific signal at the same molecular weight than tau protein (around 50 kDa). Here, we propose the use of anti-light chain secondary antibodies as a simple and efficient technique to prevent nonspecific Igs signals at around 50 kDa. We demonstrate the efficacy of this method by removing artifactual signals when using monoclonal antibodies directed at tau phosphorylation (AT100, 12E8, AT270), tau truncation (TauC3), tau oligomerization (TOMA), or tau abnormal conformation (Alz50), in wild-type, 3×Tg-AD, and tau knockout mice.

Antibody-Dependent Cell-Mediated Cytotoxicity Effector-Enhanced EphA2 Agonist Monoclonal Antibody Demonstrates Potent Activity against Human Tumors1

PubMed Central

Bruckheimer, Elizabeth M; Fazenbaker, Christine A; Gallagher, Sandra; Mulgrew, Kathy; Fuhrmann, Stacy; Coffman, Karen T; Walsh, William; Ready, Shannon; Cook, Kim; Damschroder, Melissa; Kinch, Michael; Kiener, Peter A; Woods, Rob; Gao, Changshou; Dall'Acqua, William; Wu, Herren; Coats, Steven

2009-01-01

EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC) activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated ∼80% tumor cell killing. In a dose-dependent manner, natural killer (NK) cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID) mice (which have functional NK cells and monocytes) and SCID nonobese diabetic (NOD) mice (which largely lack functional NK cells and monocytes). Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells. PMID:19484140

Improving the solubility of anti-LINGO-1 monoclonal antibody Li33 by isotype switching and targeted mutagenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pepinsky, R. Blake; Silvian, Laura; Berkowitz, Steven A.

2010-11-15

Monoclonal antibodies (Mabs) are a favorite drug platform of the biopharmaceutical industry. Currently, over 20 Mabs have been approved and several hundred others are in clinical trials. The anti-LINGO-1 Mab Li33 was selected from a large panel of antibodies by Fab phage display technology based on its extraordinary biological activity in promoting oligodendrocyte differentiation and myelination in vitro and in animal models of remyelination. However, the Li33 Fab had poor solubility when converted into a full antibody in an immunoglobulin G1 framework. A detailed analysis of the biochemical and structural features of the antibody revealed several possible reasons for itsmore » propensity to aggregate. Here, we successfully applied three molecular approaches (isotype switching, targeted mutagenesis of complementarity determining region residues, and glycosylation site insertion mutagenesis) to address the solubility problem. Through these efforts we were able to improve the solubility of the Li33 Mab from 0.3 mg/mL to >50 mg/mL and reduce aggregation to an acceptable level. These strategies can be readily applied to other proteins with solubility issues.« less

Structure of a Potential Therapeutic Antibody Bound to Interleukin-16 (IL-16)

PubMed Central

Hall, Gareth; Cullen, Eilish; Sawmynaden, Kovilen; Arnold, Joanne; Fox, Simon; Cowan, Richard; Muskett, Frederick W.; Matthews, David; Merritt, Andrew; Kettleborough, Catherine; Cruikshank, William; Taylor, Debra; Bayliss, Richard; Carr, Mark D.

2016-01-01

Interleukin-16 (IL-16) is reported to be a chemoattractant cytokine and modulator of T-cell activation, and has been proposed as a ligand for the co-receptor CD4. The secreted active form of IL-16 has been detected at sites of TH1-mediated inflammation, such as those seen in autoimmune diseases, ischemic reperfusion injury (IRI), and tissue transplant rejection. Neutralization of IL-16 recruitment to its receptor, using an anti-IL16 antibody, has been shown to significantly attenuate inflammation and disease pathology in IRI, as well as in some autoimmune diseases. The 14.1 antibody is a monoclonal anti-IL-16 antibody, which when incubated with CD4+ cells is reported to cause a reduction in the TH1-type inflammatory response. Secreted IL-16 contains a characteristic PDZ domain. PDZ domains are typically characterized by a defined globular structure, along with a peptide-binding site located in a groove between the αB and βB structural elements and a highly conserved carboxylate-binding loop. In contrast to other reported PDZ domains, the solution structure previously reported for IL-16 reveals a tryptophan residue obscuring the recognition groove. We have solved the structure of the 14.1Fab fragment in complex with IL-16, revealing that binding of the antibody requires a conformational change in the IL-16 PDZ domain. This involves the rotation of the αB-helix, accompanied movement of the peptide groove obscuring tryptophan residue, and consequent opening up of the binding site for interaction. Our study reveals a surprising mechanism of action for the antibody and identifies new opportunities for the development of IL-16-targeted therapeutics, including small molecules that mimic the interaction of the antibody. PMID:27231345

Use of AN Eosinophil Specific Monoclonal Antibody in Assessing Eosinophil Function.

NASA Astrophysics Data System (ADS)

Minkoff, Marjorie Sue

A monoclonal antibody to an eosinophil specific determinant is very important in assessing eosinophil function during helminthic infection. Eosinophils induced by Schistosoma mansoni infection in BALB/c mice were used to induce C57B1/6 immunocytes for production of hybridomas secreting eosinophil monoclonal antibodies. These antibodies were shown to react with an eosinophil surface epitope but not with neutrophils or macrophages as determined by ELISA, immunodiffusion, immunofluorescence, and immunoblot assay. Affinity chromatography with eosinophil chemotactic factor-sepharose consistently selected out a { rm M_ R} 67,000 protein from solubilized eosinophil membrane antigens but not from neutrophil and macrophage antigens. In vitro studies showed that the eosinophil-specific monoclonal antibodies abrogated antibody-dependent eosinophil -mediated killing of S. mansoni schistosomula using mouse, rat or human eosinophils. Neutrophil and macrophage killing activities were unaffected. The monoclonal antibodies effected complement-dependent lysis of mouse and rat eosinophils but not of human eosinophils. ECF-treated eosinophils showed enhanced killing of schistosomula which was blocked by the monoclonal antibody. Murine and human eosinophils preincubated with monoclonal antibody exhibited decreased chemotaxis to ECF at optimal chemotactic concentrations. The monoclonal antibody also blocked eosinophil binding to ECF- sepharose beads. In vivo induction of peripheral blood eosinophilia by injection of S. mansoni eggs was suppressed by injections of monoclonal antibodies 2CD13 and 2QD45 in mouse and rat experimental models. Eosinophilia induced by keyhole limpet hemocyanin- cyclophosphamide treatment was also suppressed by monoclonal antibody in both murine and rat systems. Pulmonary granulomas in mice given egg injection and monoclonal antibody were smaller and contained fewer eosinophils than those granulomas from mice given eggs only. In immuno-biochemical studies, the monoclonal antibody 2QD45 specifically immunoprecipitated the {rm M_ R} 67,000 ECF-binding protein from ^{125}{rm I}-labeled mouse, rat, and human eosinophils as assessed by SDS-PAGE and autoradiography. Two-dimensional gel electrophoresis showed that this ECF-binding protein has a lower PI point than either mouse or bovine albumin.

Generation of monoclonal antibodies to a human natural killer clone. Characterization of two natural killer-associated antigens, NKH1A and NKH2, expressed on subsets of large granular lymphocytes.

PubMed Central

Hercend, T; Griffin, J D; Bensussan, A; Schmidt, R E; Edson, M A; Brennan, A; Murray, C; Daley, J F; Schlossman, S F; Ritz, J

1985-01-01

The initial characterization of two monoclonal antibodies directed at antigens selectively expressed on large granular lymphocytes (LGL) is reported in the present paper. These two reagents, anti-natural killer (NK) H1A and anti-NKH2, were obtained following immunization of mouse spleen cells with a cloned human NK cell line termed JT3. In fresh human peripheral blood, both anti-NKH1A and anti-NKH2 selectively reacted with cells that appeared morphologically as large granular lymphocytes. However, complement lysis studies and two color fluorescence analysis demonstrated that some LGL express both antigens and other cells express only NKH1A or NKH2. Functional analysis of these subsets indicated that the population of NKH1A+ cells contains the entire pool of NK active lymphocytes, whereas expression of NKH2 antigen appeared to delineate a unique subpopulation of LGL which, in a resting state, display a low degree of spontaneous cytotoxicity. Expression of NKH1A and NKH2 was also investigated using a series of nine well characterized human NK clones. All NK clones were found to be NKH1A+ and four out of nine also expressed NKH2. These results strongly supported the view that NKH1A is a "pan-NK" associated antigen, and indicated that at least a fraction of cloned NKH2 + LGL are strongly cytotoxic. Anti-NKH1A was shown to have the same specificity as the previously described N901 antibody and was found here to precipitate a 200,000-220,000-mol wt molecule in SDS-polyacrylamide gel electrophoresis (PAGE) analysis. Anti-NKH2 was specific for a structure that migrates at 60,000 mol wt in SDS-PAGE analysis under reducing conditions. Two color immunofluorescence analysis of NKH1A, NKH2, and other NK-associated antigens (Leu7 and B73.1) demonstrated variable degrees of coexpression of these antigens, which confirmed that NKH1A and NKH2 define distinct cell surface structures. Anti-NKH1A and anti-NKH2 appear to be useful reagents for characterizing LGL present in human peripheral blood and for identifying functionally relevant subsets within this heterogeneous population of cytotoxic lymphocytes. Images PMID:3884668

Genetic modification of neurons to express bevacizumab for local anti-angiogenesis treatment of glioblastoma.

PubMed

Hicks, Martin J; Funato, Kosuke; Wang, Lan; Aronowitz, Eric; Dyke, Jonathan P; Ballon, Douglas J; Havlicek, David F; Frenk, Esther Z; De, Bishnu P; Chiuchiolo, Maria J; Sondhi, Dolan; Hackett, Neil R; Kaminsky, Stephen M; Tabar, Viviane; Crystal, Ronald G

2015-01-01

The median survival of glioblastoma multiforme (GBM) is approximately 1 year. Following surgical removal, systemic therapies are limited by the blood-brain barrier. To circumvent this, we developed a method to modify neurons with the genetic sequence for therapeutic monoclonal antibodies using adeno-associated virus (AAV) gene transfer vectors, directing persistent, local expression in the tumor milieu. The human U87MG GBM cell line or patient-derived early passage GBM cells were administered to the striatum of NOD/SCID immunodeficient mice. AAVrh.10BevMab, an AAVrh.10-based vector coding for bevacizumab (Avastin), an anti-human vascular endothelial growth factor (VEGF) monoclonal antibody, was delivered to the area of the GBM xenograft. Localized expression of bevacizumab was demonstrated by quantitative PCR, ELISA and western blotting. Immunohistochemistry showed that bevacizumab was expressed in neurons. Concurrent administration of AAVrh.10BevMab with the U87MG tumor reduced tumor blood vessel density and tumor volume, and increased survival. Administration of AAVrh.10BevMab 1 week after U87MG xenograft reduced growth and increased survival. Studies with patient-derived early passage GBM primary cells showed a reduction in primary tumor burden with an increased survival. These data support the strategy of AAV-mediated central nervous system gene therapy to treat GBM, overcoming the blood-brain barrier through local, persistent delivery of an anti-angiogenesis monoclonal antibody.

Differential clinical efficacy of anti-CD4 monoclonal antibodies in rat adjuvant arthritis is paralleled by differential influence on NF-κB binding activity and TNF-α secretion of T cells

PubMed Central

Pohlers, Dirk; Schmidt-Weber, Carsten B; Franch, Angels; Kuhlmann, Jürgen; Bräuer, Rolf; Emmrich, Frank; Kinne, Raimund W

2002-01-01

The aim of this study was to analyze the differential effects of three anti-CD4 monoclonal antibodies (mAbs) (with distinct epitope specifities) in the treatment of rat adjuvant arthritis (AA) and on T-cell function and signal transduction. Rat AA was preventively treated by intraperitoneal injection of the anti-CD4 mAbs W3/25, OX35, and RIB5/2 (on days -1, 0, 3, and 6, i.e. 1 day before AA induction, on the day of induction [day 0], and thereafter). The effects on T-cell reactivity in vivo (delayed-type hypersensitivity), ex vivo (ConA-induced proliferation), and in vitro (mixed lymphocyte culture) were assessed. The in vitro effects of anti-CD4 preincubation on T-cell receptor (TCR)/CD3-induced cytokine production and signal transduction were also analyzed. While preventive treatment with OX35 and W3/25 significantly ameliorated AA from the onset, treatment with RIB5/2 even accelerated the onset of AA by approximately 2 days (day 10), and ameliorated the arthritis only in the late phase (day 27). Differential clinical effects at the onset of AA were paralleled by a differential influence of the mAbs on T-cell functions, i.e. in comparison with OX35 and W3/25, the 'accelerating' mAb RIB5/2 failed to increase the delayed-type hypersentivity (DTH) to Mycobacterium tuberculosis, increased the in vitro tumor necrosis factor (TNF)-α secretion, and more strongly induced NF-κB binding activity after anti-CD4 preincubation and subsequent TCR/CD3-stimulation. Depending on their epitope specificity, different anti-CD4 mAbs differentially influence individual proinflammatory functions of T cells. This fine regulation may explain the differential efficacy in the treatment of AA and may contribute to the understanding of such treatments in other immunopathologies. PMID:12010568

Stable long-term cultures of self-renewing B cells and their applications.

PubMed

Kwakkenbos, Mark J; van Helden, Pauline M; Beaumont, Tim; Spits, Hergen

2016-03-01

Monoclonal antibodies are essential therapeutics and diagnostics in a large number of diseases. Moreover, they are essential tools in all sectors of life sciences. Although the great majority of monoclonal antibodies currently in use are of mouse origin, the use of human B cells to generate monoclonal antibodies is increasing as new techniques to tap the human B cell repertoire are rapidly emerging. Cloned lines of immortalized human B cells are ideal sources of monoclonal antibodies. In this review, we summarize our studies to the regulation of the replicative life span, differentiation, and maturation of B cells that led to the development of a platform that uses immortalization of human B cells by in vitro genetic modification for antibody development. We describe a number of human antibodies that were isolated using this platform and the application of the technique in other species. We also discuss the use of immortalized B cells as antigen-presenting cells for the discovery of tumor neoantigens. © 2016 The Authors. Immunological Reviews Published by John Wiley & Sons Ltd.

Generation and Characterization of Siglec-F-Specific Monoclonal Antibodies.

PubMed

Shahmohammadi-Farid, Sima; Ghods, Roya; Jeddi-Tehrani, Mahmood; Bayat, Ali-Ahmad; Mojtabavi, Nazanin; Razavi, Alireza; Zarnani, Amir-Hassan

2017-12-01

Siglec-F (SF) is a surface glycoprotein expressed by mouse eosinophils and induces caspase- and mitochondria-dependent apoptosis after engagement with its cognate ligand or specific antibodies. This targeting eosinophils by monoclonal antibodies may help diverse diseases associated with increased frequency of eosinophils including allergy and asthma. In this paper, production of murine and rat monoclonal antibodies (mAbs) against Siglec-F has been addressed. Balb/c mice were immunized with siglec-F1 (SF1) and siglec-F2 (SF2) synthetic peptides conjugated to a carrier protein. Rats were immunized with Chinese hamster ovary CHO cells overexpressing Siglec-F (CHO-SF) or with Siglec-F-human immunoglobulin FC fusion protein (CHO-SF-Ig). Hybridomas were produced by standard protocol and screened for their reactivity by enzyme-linked immunosorbent assay (ELISA), western blotting (WB), and flow cytometry. In parallel, polyclonal antibodies were generated in New Zealand White rabbits immunized with SF1 and SF2 peptides. Three mouse and three rat mAbs were generated against synthetic peptides and SF-Ig, respectively. All mouse monoclonal and rabbit polyclonal antibodies reacted well with immunizing molecules in ELISA and detected specific band of Siglec-F in WB. However, they failed to detect native molecule in flow cytometry analysis. Quite the contrary, rat mAbs did not reacted with the denatured protein in WB, instead exhibited significant reactivity with CHO-SF cells in flow cytometry. Based on the heavily glycosylated nature of Siglec-F, it seems that generation of anti-SF antibodies able to detect native protein needs a properly folded molecule for immunization. Monoclonal antibodies reported here are invaluable tools for studying linear and conformation epitopes of SF and tracing mouse eosinophils.

Structural Determination of the Broadly Reactive Anti-IGHV1-69 Anti-idiotypic Antibody G6 and Its Idiotope.

PubMed

Avnir, Yuval; Prachanronarong, Kristina L; Zhang, Zhen; Hou, Shurong; Peterson, Eric C; Sui, Jianhua; Zayed, Hatem; Kurella, Vinodh B; McGuire, Andrew T; Stamatatos, Leonidas; Hilbert, Brendan J; Bohn, Markus-Frederik; Kowalik, Timothy F; Jensen, Jeffrey D; Finberg, Robert W; Wang, Jennifer P; Goodall, Margaret; Jefferis, Roy; Zhu, Quan; Kurt Yilmaz, Nese; Schiffer, Celia A; Marasco, Wayne A

2017-12-12

The heavy chain IGHV1-69 germline gene exhibits a high level of polymorphism and shows biased use in protective antibody (Ab) responses to infections and vaccines. It is also highly expressed in several B cell malignancies and autoimmune diseases. G6 is an anti-idiotypic monoclonal Ab that selectively binds to IGHV1-69 heavy chain germline gene 51p1 alleles that have been implicated in these Ab responses and disease processes. Here, we determine the co-crystal structure of humanized G6 (hG6.3) in complex with anti-influenza hemagglutinin stem-directed broadly neutralizing Ab D80. The core of the hG6.3 idiotope is a continuous string of CDR-H2 residues starting with M53 and ending with N58. G6 binding studies demonstrate the remarkable breadth of binding to 51p1 IGHV1-69 Abs with diverse CDR-H3, light chain, and antigen binding specificities. These studies detail the broad expression of the G6 cross-reactive idiotype (CRI) that further define its potential role in precision medicine. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

B7-2 expressed on EL4 lymphoma suppresses antitumor immunity by an interleukin 4-dependent mechanism.

PubMed

Stremmel, C; Greenfield, E A; Howard, E; Freeman, G J; Kuchroo, V K

1999-03-15

For T cells to become functionally activated they require at least two signals. The B7 costimulatory molecules B7-1 and B7-2 provide the "second signal" pivotal for T cell activation. In this report, we studied the relative roles of B7-1 and B7-2 molecules in the induction of antitumor immunity to the T cell thymoma, EL4. We generated EL4 tumor cells that expressed B7-1, B7-2, and B7-1+B7-2 by transfecting murine cDNAs. Our results demonstrate that EL4-B7-1 cells are completely rejected in syngeneic mice. Unlike EL4-B7-1 cells, we find that EL4-B7-2 cells are not rejected but progressively grow in the mice. A B7-1- and B7-2-EL4 double transfectant was generated by introducing B7-2 cDNA into the EL4-B7-1 tumor line that regressed in vivo. The EL4-B7-1+B7-2 double transfectant was not rejected when implanted into syngeneic mice but progressively grew to produce tumors. The double transfectant EL4 cells could costimulate T cell proliferation that could be blocked by anti-B7-1 antibodies, anti-B7-2 antibodies, or hCTLA4 immunoglobulin, showing that the B7-1 and B7-2 molecules expressed on the EL4 cells were functional. In vivo, treatment of mice implanted with double-transfected EL4 cells with anti-B7-2 monoclonal antibody resulted in tumor rejection. Furthermore, the EL4-B7-2 and EL4-B7-1+B7-2 cells, but not the wild-type EL4 cells, were rejected in interleukin 4 (IL-4) knockout mice. Our data suggests that B7-2 expressed on some T cell tumors inhibits development of antitumor immunity, and IL-4 appears to play a critical role in abrogation of the antitumor immune response.

OVA-bound nanoparticles induce OVA-specific IgG1, IgG2a, and IgG2b responses with low IgE synthesis.

PubMed

Yanase, Noriko; Toyota, Hiroko; Hata, Kikumi; Yagyu, Seina; Seki, Takahiro; Harada, Mitsunori; Kato, Yasuki; Mizuguchi, Junichiro

2014-10-14

There is an urgent requirement for a novel vaccine that can stimulate immune responses without unwanted toxicity, including IgE elevation. We examined whether antigen ovalbumin (OVA) conjugated to the surface of nanoparticles (NPs) (OVA-NPs) with average diameter of 110nm would serve as an immune adjuvant. When BALB/c mice were immunized with OVA-NPs, they developed sufficient levels of OVA-specific IgG1 antibody responses with low levels of IgE synthesis, representing helper T (Th)2-mediated humoral immunity. OVA-specific IgG2a and IgG2b responses (i.e., Th1-mediated immunity) were also induced by secondary immunization with OVA-NPs. As expected, immunization with OVA in alum (OVA-alum) stimulated humoral immune responses, including IgG1 and IgE antibodies, with only low levels of IgG2a/IgG2b antibodies. CD4-positive T cells from mice primed with OVA-NPs produced substantial levels of IL-21 and IL-4, comparable to those from OVA-alum group. The irradiated mice receiving OVA-NPs-primed B cells together with OVA-alum-primed T cells exhibited enhanced anti-OVA IgG2b responses relative to OVA-alum-primed B cells and T cells following stimulation with OVA-NPs. Moreover, when OVA-NPs-primed, but not OVA-alum-primed, B cells were cultured in the presence of anti-CD40 monoclonal antibody, IL-4, and IL-21, or LPS plus TGF-β in vitro, OVA-specific IgG1 or IgG2b antibody responses were elicited, suggesting that immunization with OVA-NPs modulates B cells to generate IgG1 and IgG2b responses. Thus, OVA-NPs might exert their adjuvant action on B cells, and they represent a promising potential vaccine for generating both IgG1 and IgG2a/IgG2b antibody responses with low IgE synthesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

AAVrh.10-Mediated Expression of an Anti-Cocaine Antibody Mediates Persistent Passive Immunization That Suppresses Cocaine-Induced Behavior

PubMed Central

Rosenberg, Jonathan B.; Hicks, Martin J.; De, Bishnu P.; Pagovich, Odelya; Frenk, Esther; Janda, Kim D.; Wee, Sunmee; Koob, George F.; Hackett, Neil R.; Kaminsky, Stephen M.; Worgall, Stefan; Tignor, Nicole; Mezey, Jason G.

2012-01-01

Abstract Cocaine addiction is a major problem affecting all societal and economic classes for which there is no effective therapy. We hypothesized an effective anti-cocaine vaccine could be developed by using an adeno-associated virus (AAV) gene transfer vector as the delivery vehicle to persistently express an anti-cocaine monoclonal antibody in vivo, which would sequester cocaine in the blood, preventing access to cognate receptors in the brain. To accomplish this, we constructed AAVrh.10antiCoc.Mab, an AAVrh.10 gene transfer vector expressing the heavy and light chains of the high affinity anti-cocaine monoclonal antibody GNC92H2. Intravenous administration of AAVrh.10antiCoc.Mab to mice mediated high, persistent serum levels of high-affinity, cocaine-specific antibodies that sequestered intravenously administered cocaine in the blood. With repeated intravenous cocaine challenge, naive mice exhibited hyperactivity, while the AAVrh.10antiCoc.Mab-vaccinated mice were completely resistant to the cocaine. These observations demonstrate a novel strategy for cocaine addiction by requiring only a single administration of an AAV vector mediating persistent, systemic anti-cocaine passive immunity. PMID:22486244

Epitope mapping of PR81 anti-MUC1 monoclonal antibody following PEPSCAN and phage display techniques.

PubMed

Mohammadi, Mohammad; Rasaee, Mohammad Javad; Rajabibazl, Masoumeh; Paknejad, Malihe; Zare, Mehrak; Mohammadzadeh, Sara

2007-08-01

PR81 is an anti-MUC1 monoclonal antibody (MAb) which was generated against human MUC1 mucin that reacted with breast cancerous tissue, MUC1 positive cell line (MCF-7, BT-20, and T-4 7 D), and synthetic peptide, including the tandem repeat sequence of MUC1. Here we characterized the binding properties of PR81 against the tandem repeat of MUC1 by two different epitope mapping techniques, namely, PEPSCAN and phage display. Epitope mapping of PR81 MAb by PEPSCAN revealed a minimal consensus binding sequence, PDTRP, which is found on MUC1 peptide as the most important epitope. Using the phage display peptide library, we identified the motif PD(T/S/G)RP as an epitope and the motif AVGLSPDGSRGV as a mimotope recognized by PR81. Results of these two methods showed that the two residues, arginine and aspartic acid, have important roles in antibody binding and threonine can be substituted by either glycine or serine. These results may be of importance in tailor making antigens used in immunoassay.

Preparation of anti-CD4 monoclonal antibody-conjugated magnetic poly(glycidyl methacrylate) particles and their application on CD4+ lymphocyte separation.

PubMed

Pimpha, Nuttaporn; Chaleawlert-umpon, Saowaluk; Chruewkamlow, Nuttapol; Kasinrerk, Watchara

2011-03-15

Novel immunomagnetic particles have been prepared for separation of CD4(+) lymphocytes. The magnetic nanoparticles with a diameter of approximately 5-6 nm were first synthesized by co-precipitation from ferrous and ferric iron solutions and subsequently encapsulated with poly(glycidyl methacrylate) (PGMA) by precipitation polymerization. Monoclonal antibody specific to CD4 molecules expressed on CD4(+) lymphocytes was conjugated to the surface of magnetic PGMA particles through covalent bonding between epoxide functional groups on the particle surface and primary amine groups of the antibodies. The generated immunomagnetic particles have successfully separated CD4(+) lymphocytes from whole blood with over 95% purity. The results indicated that these particles can be employed for cell separation and provide a strong potential to be applied in various biomedical applications including diagnosis, and monitoring of human diseases. Copyright © 2010 Elsevier B.V. All rights reserved.

A novel antibody-drug conjugate targeting SAIL for the treatment of hematologic malignancies.

PubMed

Kim, S Y; Theunissen, J-W; Balibalos, J; Liao-Chan, S; Babcock, M C; Wong, T; Cairns, B; Gonzalez, D; van der Horst, E H; Perez, M; Levashova, Z; Chinn, L; D'Alessio, J A; Flory, M; Bermudez, A; Jackson, D Y; Ha, E; Monteon, J; Bruhns, M F; Chen, G; Migone, T-S

2015-05-29

Although several new therapeutic approaches have improved outcomes in the treatment of hematologic malignancies, unmet need persists in acute myeloid leukemia (AML), multiple myeloma (MM) and non-Hodgkin's lymphoma. Here we describe the proteomic identification of a novel cancer target, SAIL (Surface Antigen In Leukemia), whose expression is observed in AML, MM, chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). While SAIL is widely expressed in CLL, AML, MM, DLBCL and FL patient samples, expression in cancer cell lines is mostly limited to cells of AML origin. We evaluated the antitumor activity of anti-SAIL monoclonal antibodies, 7-1C and 67-7A, conjugated to monomethyl auristatin F. Following internalization, anti-SAIL antibody-drug conjugates (ADCs) exhibited subnanomolar IC50 values against AML cell lines in vitro. In pharmacology studies employing AML cell line xenografts, anti-SAIL ADCs resulted in significant tumor growth inhibition. The restricted expression profile of this target in normal tissues, the high prevalence in different types of hematologic cancers and the observed preclinical activity support the clinical development of SAIL-targeted ADCs.

Combined Effects of Primary and Tertiary Blast on Rat Brain: Characterization of a Model of Blast-induced Mild Traumatic Brain Injury

DTIC Science & Technology

2013-03-01

membranes and blocked with 4% non- fat dry milk for 1 h at room temperature. The blots were incubated with anti-mouse MPO monoclonal antibody (1:1000...Arun, M. Valiyaveettil, L. Biggemann, Y. Alamneh, Y. Wei, S. Oguntayo, Y. Wang, J.B. Long, M.P. Nambiar. Modulation of hearing related proteins in...skull with weight drop 30 sec post BOP, N=6-7 rats/gp) Fig 12. Changes in the neuron-specific cytoskeletal protein Microtubule-associated protein

Development and validation of an epitope-blocking ELISA using an anti-haemagglutinin monoclonal antibody for specific detection of antibodies in sheep and goat sera directed against peste des petits ruminants virus.

PubMed

Bodjo, Sanne Charles; Baziki, Jean-de-Dieu; Nwankpa, Nick; Chitsungo, Ethel; Koffi, Yao Mathurin; Couacy-Hymann, Emmanuel; Diop, Mariame; Gizaw, Daniel; Tajelser, Idris Badri Adam; Lelenta, Mamadou; Diallo, Adama; Tounkara, Karim

2018-07-01

Peste des petits ruminants (PPR) is a contagious and economically important disease affecting production of small ruminants (i.e., sheep and goats). Taking into consideration the lessons learnt from the Global Rinderpest Eradication Programme (GREP), PPR is now targeted by the international veterinary community as the next animal disease to be eradicated. To support the African continental programme for the control of PPR, the Pan African Veterinary Vaccine Centre of the African Union (AU-PANVAC) is developing diagnostics tools. Here, we describe the development of a blocking enzyme-linked immunosorbent assay (bELISA) that allows testing of a large number of samples for specific detection of antibodies directed against PPR virus in sheep and goat sera. The PPR bELISA uses an anti-haemagglutinin (H) monoclonal antibody (MAb) as a competitor antibody, and tests results are interpreted using the percentage of inhibition (PI) of MAb binding generated by the serum sample. PI values below or equal to 18% (PI ≤ 18%) are negative, PI values greater than or equal to 25% (PI ≥ 25%) are positive, and PI values greater than 18% and below 25% are doubtful. The diagnostic specificity (DSp) and diagnostic sensitivity (DSe) were found to be 100% and 93.74%, respectively. The H-based PPR-bELISA showed good correlation with the virus neutralization test (VNT), the gold standard test, with a kappa value of 0.947. The H-based PPR-bELISA is more specific than the commercial kit ID Screen® PPR Competition (N-based PPR-cELISA) from IDvet (France), but the commercial kit is slightly more sensitive than the H-based PPR-bELISA. The validation process also indicated good repeatability and reproducibility of the H-based PPR-bELISA, making this new test a suitable tool for the surveillance and sero-monitoring of the vaccination campaign.

Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

PubMed

Liao-Chan, Sindy; Daine-Matsuoka, Barbara; Heald, Nathan; Wong, Tiffany; Lin, Tracey; Cai, Allen G; Lai, Michelle; D'Alessio, Joseph A; Theunissen, Jan-Willem

2015-01-01

Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

Evaluation of the molecular recognition of monoclonal and polyclonal antibodies for sensitive detection of 2,4,6-trinitrotoluene (TNT) by indirect competitive surface plasmon resonance immunoassay.

PubMed

Shankaran, Dhesingh Ravi; Kawaguchi, Toshikazu; Kim, Sook Jin; Matsumoto, Kiyoshi; Toko, Kiyoshi; Miura, Norio

2006-11-01

Detection of TNT is an important environmental and security concern all over the world. We herein report the performance and comparison of four immunoassays for rapid and label-free detection of 2,4,6-trinitrotoluene (TNT) based on surface plasmon resonance (SPR). The immunosensor surface was constructed by immobilization of a home-made 2,4,6-trinitrophenyl-keyhole limpet hemocyanin (TNPh-KLH) conjugate onto an SPR gold surface by simple physical adsorption within 10 min. The immunoreaction of the TNPh-KLH conjugate with four different antibodies, namely, monoclonal anti-TNT antibody (M-TNT Ab), monoclonal anti-trinitrophenol antibody (M-TNP Ab), polyclonal anti-trinitrophenyl antibody (P-TNPh Ab), and polyclonal anti-TNP antibody (P-TNP Ab), was studied by SPR. The principle of indirect competitive immunoreaction was employed for quantification of TNT. Among the four antibodies, the P-TNPh Ab prepared by our group showed highest sensitivity with a detection limit of 0.002 ng/mL (2 ppt) TNT. The lowest detection limits observed with other commercial antibodies were 0.008 ng/mL (8 ppt), 0.25 ng/mL (250 ppt), and 40 ng/mL (ppb) for M-TNT Ab, P-TNP Ab, and M-TNP Ab, respectively, in the similar assay format. The concentration of the conjugate and the antibodies were optimized for use in the immunoassay. The response time for an immunoreaction was 36 s and a single immunocycle could be done within 2 min, including the sensor surface regeneration using pepsin solution. In addition to the quantification of TNT, all immunoassays were evaluated for robustness and cross-reactivity towards several TNT analogs.

Array-Based Rational Design of Short Peptide Probe-Derived from an Anti-TNT Monoclonal Antibody.

PubMed

Okochi, Mina; Muto, Masaki; Yanai, Kentaro; Tanaka, Masayoshi; Onodera, Takeshi; Wang, Jin; Ueda, Hiroshi; Toko, Kiyoshi

2017-10-09

Complementarity-determining regions (CDRs) are sites on the variable chains of antibodies responsible for binding to specific antigens. In this study, a short peptide probe for recognition of 2,4,6-trinitrotoluene (TNT), was identified by testing sequences derived from the CDRs of an anti-TNT monoclonal antibody. The major TNT-binding site in this antibody was identified in the heavy chain CDR3 by antigen docking simulation and confirmed by an immunoassay using a spot-synthesis based peptide array comprising amino acid sequences of six CDRs in the variable region. A peptide derived from heavy chain CDR3 (RGYSSFIYWF) bound to TNT with a dissociation constant of 1.3 μM measured by surface plasmon resonance. Substitution of selected amino acids with basic residues increased TNT binding while substitution with acidic amino acids decreased affinity, an isoleucine to arginine change showed the greatest improvement of 1.8-fold. The ability to create simple peptide binders of volatile organic compounds from sequence information provided by the immune system in the creation of an immune response will be beneficial for sensor developments in the future.

Therapeutic potential of an anti-high mobility group box-1 monoclonal antibody in epilepsy.

PubMed

Zhao, Junli; Wang, Yi; Xu, Cenglin; Liu, Keyue; Wang, Ying; Chen, Liying; Wu, Xiaohua; Gao, Feng; Guo, Yi; Zhu, Junming; Wang, Shuang; Nishibori, Masahiro; Chen, Zhong

2017-08-01

Brain inflammation is a major factor in epilepsy, and the high mobility group box-1 (HMGB1) protein is known to contribute significantly to the generation of seizures. Here, we investigated the therapeutic potential of an anti-HMGB1 monoclonal antibody (mAb) in epilepsy. anti-HMGB1 mAb attenuated both acute seizure models (maximal electroshock seizure, pentylenetetrazole-induced and kindling-induced), and chronic epilepsy model (kainic acid-induced) in a dose-dependent manner. Meanwhile, the anti-HMGB1 mAb also attenuated seizure activities of human brain slices obtained from surgical resection from drug-resistant epilepsy patients. The mAb showed an anti-seizure effect with a long-term manner and appeared to be minimal side effects at even very high dose (no disrupted physical EEG rhythm and no impaired basic physical functions, such as body growth rate and thermoregulation). This anti-seizure effect of mAb results from its inhibition of translocated HMGB1 from nuclei following seizures, and the anti-seizure effect was absent in toll-like receptor 4 knockout (TLR4 -/- ) mice. Interestingly, the anti-HMGB1 mAb also showed a disease-modifying anti-epileptogenetic effect on epileptogenesis after status epileptics, which is indicated by reducing seizure frequency and improving the impaired cognitive function. These results indicate that the anti-HMGB1 mAb should be viewed as a very promising approach for the development of novel therapies to treat refractory epilepsy. Copyright © 2017 Elsevier Inc. All rights reserved.

Development of a monoclonal-based enzyme-linked immunoassay for saxitoxin-induced protein.

PubMed

Smith, D S; Kitts, D D

1994-03-01

A monoclonal antibody was generated against saxitoxin-induced protein (SIP) from the small shore crab Hemigrapsus oregenesis. SIP was induced by saxitoxin injection and could be detected in the crude crab extracts with both polyclonal and monoclonal antibody preparations. On Western blots, the polyclonal serum reacted against several bands which were induced by saxitoxin in the crude extracts. These bands represented proteins related to SIP. The monoclonal (4G5), however, was specific for the 79,000 mol. wt subunit of SIP. A triple antibody sandwich ELISA was developed in which polyclonal anti-SIP IgG was used as a trapping layer and monoclonal 4G5 was used as the detection layer. This assay was shown to be more specific and more accurate than a direct bind assay which employed the polyclonal antiserum alone. Although the polyclonal serum was more sensitive than the monoclonal on Western blots, the triple antibody sandwich and direct bind ELISAs were of comparable sensitivity.

A monoclonal antibody against neem leaf glycoprotein recognizes carcinoembryonic antigen (CEA) and restricts CEA expressing tumor growth.

PubMed

Das, Arnab; Barik, Subhasis; Banerjee, Saptak; Bose, Anamika; Sarkar, Koustav; Biswas, Jaydip; Baral, Rathindranath; Pal, Smarajit

2014-10-01

Carcinoembryonic antigen (CEA) is one of the promising tumor antigens mainly associated with carcinoma of the colon, lung, breast, etc. and received wide attention for cancer immunotherapy. Neem leaf glycoprotein (NLGP), an effective immunomodulator, is able to generate humoral and cellular immune responses in murine tumor models. We have generated a monoclonal antibody (mAb) against NLGP by fusing NLGP-immunized mice splenocytes with nonsecretory myeloma cells. A highly anti-NLGP mAb secreting clone (1C8; IgG2a in nature) has been identified and propagated in culture. 1C8 recognizes human CEA as good as NLGP by enzyme linked immunosorbent assay, Western blotting, and immunoprecipitation. 1C8 detects CEA on colon cancer tissues by immunochistochemistry. By flow cytometry, 1C8 specifically reacts with CEA(+) human (Colo-205, HCT-116, and HT-29) and mouse (CT-26) colon cancer cells, but it showed minimum reactivity with CEA(-) human (MCF7, SiHa, and SCC084) and mouse (B16MelF10) cancer cells. This anti-NLGP 1C8 mAb revealed significant antitumor activity and better survivability in vivo in animals bearing mouse (CT-26 in BALB/c) and human (Colo-205 in athymic nude) CEA(+) cancer cells. 1C8 has no direct influence on proliferation and migration of CEA(+) cells, however, NK cell-dependent strong antibody-dependent cellular cytotoxicity reaction toward CEA(+) cells and normalization of angiogenesis are chiefly associated with tumor growth restriction. Obtained results provided a new immunotherapeutic approach for the effective management of CEA(+) tumors.

Binding specificity of anti-HNK-1 IgM M-protein in anti-MAG neuropathy: possible clinical relevance.

PubMed

Hamada, Yukihiro; Hirano, Makito; Kuwahara, Motoi; Samukawa, Makoto; Takada, Kazuo; Morise, Jyoji; Yabuno, Keiko; Oka, Shogo; Kusunoki, Susumu

2015-02-01

Anti-myelin-associated-glycoprotein (MAG) neuropathy is an intractable autoimmune polyneuropathy. The antigenic region of MAG is the human natural killer-1 (HNK-1) carbohydrate. We and others previously suggested that the extension of antibody reactivities to HNK-1-bearing proteins other than MAG was associated with treatment resistance, without statistical analyses. In this study, we established an ELISA method with recombinant proteins to test binding specificities of currently available monoclonal antibodies to MAG and another HNK-1-bearing protein, phosphacan. Using this system, we found the distinct binding specificities of anti-MAG antibody in 19 patients with anti-MAG neuropathy. Their clinical relevance was then determined retrospectively with the adjusted 10-points INCAT disability score (0 = normal and 10 = highly disable). The results showed that strong reactivities of anti-MAG antibodies to phosphacan were significantly associated with treatment resistance or progressive clinical courses, indicating a possible clinical relevance of the binding specificities. Copyright © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Pretargeted Molecular Imaging and Radioimmunotherapy

PubMed Central

Goldenberg, David M.; Chang, Chien-Hsing; Rossi, Edmund A.; J, William; McBride; Sharkey, Robert M.

2012-01-01

Pretargeting is a multi-step process that first has an unlabeled bispecific antibody (bsMAb) localize within a tumor by virtue of its anti-tumor binding site(s) before administering a small, fast-clearing radiolabeled compound that then attaches to the other portion of the bsMAb. The compound's rapid clearance significantly reduces radiation exposure outside of the tumor and its small size permits speedy delivery to the tumor, creating excellent tumor/nontumor ratios in less than 1 hour. Haptens that bind to an anti-hapten antibody, biotin that binds to streptavidin, or an oligonucleotide binding to a complementary oligonucleotide sequence have all been radiolabeled for use by pretargeting. This review will focus on a highly flexible anti-hapten bsMAb platform that has been used to target a variety of radionuclides to image (SPECT and PET) as well as treat tumors. PMID:22737190

Molecules mediating adhesion of T and B cells, monocytes and granulocytes to vascular endothelial cells.

PubMed Central

Prieto, J; Beatty, P G; Clark, E A; Patarroyo, M

1988-01-01

Leucocytes interact with vascular endothelial cells (EC), and adhesion between these two cell types in vitro is modulated by phorbol ester. Monocytes were found to display the highest basal adhesion to EC, followed by Epstein-Barr virus-immortalized normal B cells (EBV-B), T cells and granulocytes. Phorbol ester treatment increased the adhesion of all types of leucocytes, except monocytes. In the presence of this compound, monoclonal antibody 60.3 to GP90 (CD18, a leucocyte-adhesion protein which is non-covalently associated to either GP160, GP155, or GP130) was found to inhibit the adhesion of the four types of leucocytes to a considerable extent, while anti-lymphocyte function-associated antigen-1 (LFA-1) antibody to GP160 (CD11a) inhibited the adhesion of T and B cells only. Antibody 60.1 to GP155 (CD11b) had a major inhibitory activity exclusively on granulocytes, while antibody LB-2, which recognizes a distinct adhesion molecule (GP84) and, in contrast to the previous antibodies, reacts with EC, mainly inhibited adhesion of EBV-B and did not increase the inhibition obtained with antibody 60.3 alone. Fab fragments of antibody 60.3 inhibited leucocyte adhesion more efficiently, in either the absence or presence of phorbol ester, than the intact antibody molecule. It is concluded the GP90, either alone or associated to the larger glycoproteins, mediates the adhesion in all types of leucocytes, while GP84 mediates the adhesion of the activated B cells. Images Figure 2 PMID:3259203

Fully-human Monoclonal Antibodies Against Human EphrinB2 and EphB4 | NCI Technology Transfer Center | TTC

Cancer.gov

The National Cancer Institute's Cancer and Inflammation Program is seeking statements of capability or interest from parties interested in licensing fully-human monoclonal antibodies against human EphrinB2 and EphB4.

Characterization of Alpha-Toxin hla Gene Variants, Alpha-Toxin Expression Levels, and Levels of Antibody to Alpha-Toxin in Hemodialysis and Postsurgical Patients with Staphylococcus aureus Bacteremia

PubMed Central

Wu, Yuling; Tabor, David E.; Mok, Hoyin; Sellman, Bret R.; Jenkins, Amy; Yu, Li; Jafri, Hasan S.; Rude, Thomas H.; Ruffin, Felicia; Schell, Wiley A.; Park, Lawrence P.; Yan, Qin; Thaden, Joshua T.; Messina, Julia A.; Esser, Mark T.

2014-01-01

Alpha-toxin is a major Staphylococcus aureus virulence factor. This study evaluated potential relationships between in vitro alpha-toxin expression of S. aureus bloodstream isolates, anti-alpha-toxin antibody in serum of patients with S. aureus bacteremia (SAB), and clinical outcomes in 100 hemodialysis and 100 postsurgical SAB patients. Isolates underwent spa typing and hla sequencing. Serum anti-alpha-toxin IgG and neutralizing antibody levels were measured by using an enzyme-linked immunosorbent assay and a red blood cell (RBC)-based hemolysis neutralization assay. Neutralization of alpha-toxin by an anti-alpha-toxin monoclonal antibody (MAb MEDI4893) was tested in an RBC-based lysis assay. Most isolates encoded hla (197/200; 98.5%) and expressed alpha-toxin (173/200; 86.5%). In vitro alpha-toxin levels were inversely associated with survival (cure, 2.19 μg/ml, versus failure, 1.09 μg/ml; P < 0.01). Both neutralizing (hemodialysis, 1.26 IU/ml, versus postsurgical, 0.95; P < 0.05) and IgG (hemodialysis, 1.94 IU/ml, versus postsurgical, 1.27; P < 0.05) antibody levels were higher in the hemodialysis population. Antibody levels were also significantly higher in patients infected with alpha-toxin-expressing S. aureus isolates (P < 0.05). Levels of both neutralizing antibodies and IgG were similar among patients who were cured and those not cured (failures). Sequence analysis of hla revealed 12 distinct hla genotypes, and all genotypic variants were susceptible to a neutralizing monoclonal antibody in clinical development (MEDI4893). These data demonstrate that alpha-toxin is highly conserved in clinical S. aureus isolates. Higher in vitro alpha-toxin levels were associated with a positive clinical outcome. Although patients infected with alpha-toxin-producing S. aureus exhibited higher anti-alpha-toxin antibody levels, these levels were not associated with a better clinical outcome in this study. PMID:25392350

Preventative effect of OMZ-SPT on lipopolysaccharide-induced acute lung injury and inflammation via nuclear factor-kappa B signaling in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Ting; Department of Respiratory Medicine, Children's Hospital of Chongqing Medical University, Chongqing, 400014; Hou, Wanru

Acute lung injury (ALI) is an early pathophysiologic change in acute respiratory distress syndrome and its management can be challenging. Omalizumab (Xolair™) is a recombinant DNA-derived, humanized antibody. OMZ-SPT is a polypeptide on the heavy chain of omalizumab monoclonal antibody. Here, we found that intramuscular administration of OMZ-SPT significantly improved survival and attenuated lung inflammation in female C57BL/6 mice suffering from lipopolysaccharide (LPS)-induced ALI. We also demonstrated that OMZ-SPT can inhibit expression of the inflammatory cytokines tumor necrosis factor-α, interleukin-1β and interleukin-6 by ELISA in mice suffering from LPS-induced ALI and a mouse macrophage line (RAW264.7 cells). In addition, we showedmore » that OMZ-SPT inhibited LPS-induced activation of nuclear factor-kappa B (NF-κB) signaling and total expression of NF-κB by western blotting. These data suggest that OMZ-SPT could be a novel therapeutic choice for ALI. - Highlights: • OMZ-SPT is a polypeptide on the heavy chain of omalizumab monoclonal antibody. • Omalizumab (Xolair™) have anti-inflammatory effects. • OMZ-SPT can inhibit inflammatory responses and lung injury in LPS-induced ALI mice. • Protective effect of OMZ-SPT on ALI is due to inhibition of NF-κB signaling. • OMZ-SPT could be a novel therapeutic choice for ALI.« less

Monitoring the Systemic Human Memory B Cell Compartment of Melanoma Patients for Anti-Tumor IgG Antibodies

PubMed Central

Gilbert, Amy E.; Karagiannis, Panagiotis; Dodev, Tihomir; Koers, Alexander; Lacy, Katie; Josephs, Debra H.; Takhar, Pooja; Geh, Jenny L. C.; Healy, Ciaran; Harries, Mark; Acland, Katharine M.; Rudman, Sarah M.; Beavil, Rebecca L.; Blower, Philip J.; Beavil, Andrew J.; Gould, Hannah J.; Spicer, James; Nestle, Frank O.; Karagiannis, Sophia N.

2011-01-01

Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (P<0.0001). Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (P<0.0001). Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer. PMID:21559411

Anti-amyloid precursor protein antibodies inhibit amyloid-β production by steric hindrance

PubMed Central

Thomas, Rhian S.; Liddell, J. Eryl; Kidd, Emma J.

2015-01-01

Summary Cleavage of amyloid precursor protein (APP) by β- and γ-secretases results in the production of amyloid-β (Aβ) in Alzheimer’s disease (AD). We raised two monoclonal antibodies, 2B3 and 2B12, that recognise the β-secretase cleavage site on APP but not Aβ. We hypothesised that these antibodies would reduce Aβ levels via steric hindrance of β-secretase. Both antibodies decreased extracellular Aβ levels from astrocytoma cells, but 2B3 was more potent than 2B12. Levels of soluble sAPPα from the non-amyloidogenic α-secretase pathway and intracellular APP were not affected by either antibody nor were there any effects on cell viability. 2B3 exhibited a higher affinity for APP than 2B12 and its epitope appeared to span the cleavage site while 2B12 bound slightly upstream. Both of these factors probably contribute to its greater effect on Aβ levels. After 60 minutes incubation at pH 4.0, most 2B3 and 2B12 remained bound to their antigen, suggesting that the antibodies will remain bound to APP in the acidic endosomes where β-secretase cleavage probably occurs. Only 2B3 and 2B12, but not control antibodies, inhibited the cleavage of sAPPα by β-secretase in a cell-free assay where effects of antibody internalisation and intracellular degradation were excluded. 2B3 virtually abolished this cleavage. In addition, levels of C-terminal APP fragments, βCTF, generated following β-secretase cleavage, were significantly reduced in cells after incubation with 2B3. These results strongly suggest that anti-cleavage site antibodies can generically reduce Aβ levels via inhibition of β-secretase by steric hindrance and may provide a novel alternative therapy for AD. PMID:21122073

Imaging with indium111-labeled anticarcinoembryonic antigen monoclonal antibody ZCE-025 of recurrent colorectal or carcinoembryonic antigen-producing cancer in patients with rising serum carcinoembryonic antigen levels and occult metastases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patt, Y.Z.; Lamki, L.M.; Shanken, J.

1990-07-01

We tested whether nuclear imaging with indium111 (111In)-labeled murine monoclonal (MoAb) anticarcinoembryonic antigen (anti-CEA) ZCE-025 antibody could detect recurrent disease in patients with a rising serum CEA level but negative findings for computed tomographic (CT) scans of the abdomen and pelvis, chest radiograph, and colonoscopy or barium enema. Twenty patients with a history of completely resected CEA-producing adenocarcinoma and a rising serum CEA level were given an intravenous infusion of 2 mg of 111In-labeled ZCE-025 mixed with 38 mg of unlabeled ZCE-025. Planar and single-photon emission CT (SPECT) scans were acquired at 72 and 144 hours, and in 19 ofmore » the 20 patients these were positive. Of those 19, 13 underwent exploratory surgery, and cancer was found in 10, and two had a diagnostic biopsy, which confirmed cancer. Three patients who had negative laparotomies and all four patients who did not undergo surgery or biopsy were followed radiologically. In all seven, cancer was subsequently detected at the sites suggested by the ZCE-025 scan. Thus, tumor was confirmed in all 19 patients with positive scans. Five of 13 patients who were explored benefited from the study and the exploratory laparotomy, as disease was entirely resected in four or was subjected to definitive radiation therapy to the pelvis in the fifth. In two additional patients who were not explored, MoAb imaging resulted in definitive therapy to regionally confined recurrent disease. 111In-labeled anti-CEA MoAb ZCE-025 scanning in patients with rising CEA successfully imaged metastatic colorectal cancer that eluded detection by other methods and affected the care given to some. These results suggest an important role for 111In-labeled ZCE-025 scanning among patients with rising CEA and otherwise occult metastatic cancer.« less

Impact of tocilizumab therapy on antibody response to influenza vaccine in patients with rheumatoid arthritis.

PubMed

Mori, Shunsuke; Ueki, Yukitaka; Hirakata, Naoyuki; Oribe, Motohiro; Hidaka, Toshihiko; Oishi, Kazunori

2012-12-01

We assessed the influence of tocilizumab (TCZ), a humanised monoclonal anti-interleukin-6 receptor antibody, on antibody response following influenza vaccination in patients with rheumatoid arthritis (RA). A total of 194 RA patients received inactive trivalent influenza vaccination (A/H1N1, A/H3N2 and B/B1 strains). All patients were classified into the TCZ (n=62), TCZ+methotrexate (MTX) (n=49), MTX (n=65) and RA control (n=18) groups. Antibody titres were measured before and 4-6 weeks after vaccination using the haemagglutination inhibitory assay. For the A/H1N1 and A/H3N2 strains, the TCZ and TCZ+MTX groups achieved fold increases of 9.9-14.5, postvaccination seroprotection rates greater than 70% and seroresponse rates greater than 40%. For the B/B1 strain, seroresponse rates were approximately 30%, but fold increases and seroprotection rates were 5.0-5.4 and greater than 70%, respectively, in these treatment groups. MTX had a negative impact on vaccination efficacy, but adequate responses for protection were nevertheless demonstrated in the MTX group. Neither severe adverse effects nor RA flares were observed. TCZ does not hamper antibody response to influenza vaccine in RA patients. Influenza vaccination is considered effective in protecting RA patients receiving TCZ therapy with or without MTX.

Formation of infectious dengue virus-antibody immune complex in vivo in marmosets (Callithrix jacchus) after passive transfer of anti-dengue virus monoclonal antibodies and infection with dengue virus.

PubMed

Moi, Meng Ling; Ami, Yasushi; Shirai, Kenji; Lim, Chang-Kweng; Suzaki, Yuriko; Saito, Yuka; Kitaura, Kazutaka; Saijo, Masayuki; Suzuki, Ryuji; Kurane, Ichiro; Takasaki, Tomohiko

2015-02-01

Infection with a dengue virus (DENV) serotype induces cross-reactive, weakly neutralizing antibodies to different dengue serotypes. It has been postulated that cross-reactive antibodies form a virus-antibody immune complex and enhance DENV infection of Fc gamma receptor (FcγR)-bearing cells. We determined whether infectious DENV-antibody immune complex is formed in vivo in marmosets after passive transfer of DENV-specific monoclonal antibody (mAb) and DENV inoculation and whether infectious DENV-antibody immune complex is detectable using FcγR-expressing cells. Marmosets showed that DENV-antibody immune complex was exclusively infectious to FcγR-expressing cells on days 2, 4, and 7 after passive transfer of each of the mAbs (mAb 4G2 and mAb 6B6C) and DENV inoculation. Although DENV-antibody immune complex was detected, contribution of the passively transferred antibody to overall viremia levels was limited in this study. The results indicate that DENV cross-reactive antibodies form DENV-antibody immune complex in vivo, which is infectious to FcγR-bearing cells but not FcγR-negative cells. © The American Society of Tropical Medicine and Hygiene.

The binding property of a monoclonal antibody against the extracellular domains of aquaporin-4 directs aquaporin-4 toward endocytosis.

PubMed

Huang, Ping; Takai, Yoshiki; Kusano-Arai, Osamu; Ramadhanti, Julia; Iwanari, Hiroko; Miyauchi, Takayuki; Sakihama, Toshiko; Han, Jing-Yan; Aoki, Masashi; Hamakubo, Takao; Fujihara, Kazuo; Yasui, Masato; Abe, Yoichiro

2016-09-01

Neuromyelitis optica (NMO), an autoimmune disease of the central nervous system, is characterized by an autoantibody called NMO-IgG that recognizes the extracellular domains (ECDs) of aquaporin-4 (AQP4). In this study, monoclonal antibodies (mAbs) against the ECDs of mouse AQP4 were established by a baculovirus display method. Two types of mAb were obtained: one (E5415A) recognized both M1 and M23 isoforms, and the other (E5415B) almost exclusively recognized the square-array-formable M23 isoform. While E5415A enhanced endocytosis of both M1 and M23, followed by degradation in cells expressing AQP4, including astrocytes, E5415B did so to a much lesser degree, as determined by live imaging using fluorescence-labeled antibodies and by Western blotting of lysate of cells treated with these mAbs. E5415A promoted cluster formation of AQP4 on the cell surface prior to endocytosis as determined by immunofluorescent microscopic observation of bound mAbs to astrocytes as well as by Blue native PAGE analysis of AQP4 in the cells treated with the mAbs. These observations clearly indicate that an anti-AQP4-ECDs antibody possessing an ability to form a large cluster of AQP4 by cross-linking two or more tetramers outside the AQP4 arrays enhances endocytosis and the subsequent lysosomal degradation of AQP4.

Immuno capture PCR for rapid and sensitive identification of pathogenic Bacillus anthracis.

PubMed

Makam, Shivakiran S; Majumder, Saugata; Kingston, Joseph J; Urs, Radhika M; Tuteja, Urmil; Sripathi, Murali H; Batra, Harsh V

2013-12-01

Immuno capture PCR (IPCR) is a technique capable of detecting the pathogens with high specificity and sensitivity. Rapid and accurate detection of Bacillus anthracis was achieved using anti-EA1 antibodies to capture the cells and two primer sets targeting the virulence factors of the pathogen i.e., protective antigen (pag) and capsule (cap) in an IPCR format. Monoclonal antibodies specific to B. anthracis were generated against extractable antigen 1 protein and used as capture antibody onto 96 well polystyrene plates. Following the binding of the pathogen, the DNA extraction was carried out in the well itself and further processed for PCR assay. We compared IPCR described here with conventional duplex PCR using the same primers and sandwich ELISA using the monoclonal antibodies developed in the present study. IPCR was capable of detecting as few as 10 and 100 cfu ml⁻¹ of bacterial cells and spores, respectively. IPCR was found to be 2-3 logs more sensitive than conventional duplex PCR and the sandwich ELISA. The effect of other bacteria and any organic materials on IPCR was also analyzed and found that this method was robust with little change in the sensitivity in the presence of interfering agents. Moreover, we could demonstrate a simple process of microwave treatment for spore disruption which otherwise are resistant to chemical treatments. Also, the IPCR could clearly distinguish the pathogenic and nonpathogenic strains of B. anthracis in the same assay. This can help in saving resources on unnecessary decontamination procedures during false alarms.

Development and characterization of mouse monoclonal antibodies reactive with chicken IL-8

USDA-ARS?s Scientific Manuscript database

Interleukin-8 (IL-8) is a key mediator in neutrophil-mediated acute proinflammatory response and activates multiple signaling pathways downstream of two receptors (CXCR1 and CXCR2). However, there have not been any monoclonal antibodies which specifically detect chicken IL8 (chIL8) and define its b...

A cell sorter with modified bamboo charcoal for the efficient selection of specific antibody-producing hybridomas.

PubMed

Lin, Chien-Chen; Ni, Mei-Hui; Chang, Yu-Chung; Yeh, Hsiu-Lun; Lin, Feng-Huei

2010-11-01

Monoclonal antibodies (mAbs) have been proven useful in research and clinical applications. However, the generation of mAbs by conventional hybridoma technology is time-, cost- and labor-consuming. Here we developed a simplified procedure for efficient generation and selection of antibody-producing hybridomas within 1 h, using a particular cell sorter design, a cytoflow reactor-based cell sorter (CBCS) which consists mainly of the "cytoflow reactor" that comprises two components, a reaction chamber and a glass tubing for air and medium exchange by gravity, and the "sorting material", human EGFR-conjugated bamboo charcoal, for specific B-cell enrichment. The high surface area and porous structure of bamboo charcoal greatly increased cell density and protein production. Moreover, from Raman, FT-IR spectroscopy and IFA analysis, the carboxylation and immobilization of bamboo charcoal can be introduced easily by nitric acid treatment and conjugated handily with human EGFR using EDC/NHS. Other evidences, such as IFA, showed that the specific hybridomas generated in this study could secrete specific anti-human EGFR antibodies. Our design allows the production of mAbs while avoiding time-consuming steps, such as large numbers of limiting dilutions and screening assays, and demonstrates that the CBCS could be a powerful tool for monoclonal antibody production. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Detection of high PD-L1 expression in oral cancers by a novel monoclonal antibody L1Mab-4.

PubMed

Yamada, Shinji; Itai, Shunsuke; Kaneko, Mika K; Kato, Yukinari

2018-03-01

Programmed cell death-ligand 1 (PD-L1), which is a ligand of programmed cell death-1 (PD-1), is a type I transmembrane glycoprotein that is expressed on antigen-presenting cells and several tumor cells, including melanoma and lung cancer cells. There is a strong correlation between human PD-L1 (hPD-L1) expression on tumor cells and negative prognosis in cancer patients. In this study, we produced a novel anti-hPD-L1 monoclonal antibody (mAb), L 1 Mab-4 (IgG 2b , kappa), using cell-based immunization and screening (CBIS) method and investigated hPD-L1 expression in oral cancers. L 1 Mab-4 reacted with oral cancer cell lines (Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4) in flow cytometry and stained oral cancers in a membrane-staining pattern. L 1 Mab-4 stained 106/150 (70.7%) of oral squamous cell carcinomas, indicating the very high sensitivity of L 1 Mab-4. These results indicate that L 1 Mab-4 could be useful for investigating the function of hPD-L1 in oral cancers.

Characterization of the protective capacity and immunogenicity of the 69-kD outer membrane protein of Bordetella pertussis

PubMed Central

1990-01-01

Immunization with the 69-kD outer membrane protein (OMP) of Bordetella pertussis protected neonatal mice against lethal respiratory challenge with B. pertussis 18323. Active immunization elicited a serum IgG anti- 69-kD OMP response at the time of challenge, with IgG anti-69-kD OMP antibodies detected in bronchoalveolar lavage fluid after challenge. Intravenous administration of BPE8, a monoclonal IgG1 anti-69-kD OMP, also protected young mice against B. pertussis challenge. Intravenously injected BPE8 was detected in the lungs of mice at the time of aerosol challenge, suggesting that the presence of specific antibody in the lungs may mediate protection. Thus the 69-kD OMP of B. pertussis is a protective antigen in mice that elicits specific serum antibody that can transude to the lung. The 69-kD OMP was detected in a preparation of a Takeda acellular vaccine by immunoblot analysis and a serum antibody response to the 69-kD OMP was observed in 18-mo-old children boosted with this preparation of Japanese acellular vaccine. Our results demonstrate that the B. pertussis 69-kD OMP is a protective antigen in animals, is immunogenic in humans, and is present in a preparation of acellular pertussis vaccine that is widely used in Japan. These findings indicate that the 69-kD OMP should be seriously considered as a candidate for inclusion in new formulations of antigenically defined acellular pertussis vaccines. PMID:2295882

Common idiotypes expressed on human, monoclonal, abnormal immunoglobulins and cryoglobulins with polyreactive autoantibody activities.

PubMed Central

Barbouche, M R; Guilbert, B; Makni, S; Gorgi, Y; Ayed, K; Avrameas, S

1993-01-01

Several human monoclonal immunoglobulins with the same autoantibody activity have been shown to have cross-reactive idiotypes (CRI). In this study, using polyclonal anti-idiotypic antibodies, we found that 28% of human monoclonal immunoglobulins with polyreactive autoantibody activity from myeloma, Waldenström's macroglobulinaemia and cryoglobulinaemia patients shared common idiotype(s). Furthermore, the latter were expressed on human and murine natural MoAbs (respectively in 12% and 22% of the clones tested) and on human IgG preparations used for therapeutic intravenous injections (IVIg) and which contain natural antibodies. These findings suggest that monoclonal immunoglobulins could arise from the proliferation of a clone that normally produces a natural antibody. The existence of common idiotype(s) between monoclonal immunoglobulins and IVIg could be relevant to the improvement noted after treatment with IVIg in patients suffering from peripheral neuropathies associated with monoclonal gammopathy. PMID:8428386

Detecting fish parvalbumin with commercial mouse monoclonal anti-frog parvalbumin IgG.

PubMed

Chen, Lingyun; Hefle, Sue L; Taylor, Steve L; Swoboda, Ines; Goodman, Richard E

2006-07-26

Parvalbumin is a calcium-binding muscle protein that is highly conserved across fish species and amphibians. It is the major cross-reactive allergen associated with both fish and frog allergy. We used two-dimensional electrophoretic and immunoblotting techniques to investigate the utility of a commercial monoclonal anti-frog parvalbumin IgG for detecting parvalbumin present in some commonly consumed fish species. The 2D electrophoresis and immunoblots revealed species-specific differences in proteins that appear to represent various numbers of isoforms of parvalbumin in carp (5), catfish (3), cod (1) and tilapia (2). No parvalbumin was detected in yellowfin tuna. Based on minor differences in relative intensities of protein staining and immunodetection, parvalbumin isoforms may have slight differences in the epitope region recognized by the anti-frog parvalbumin antibody. These results suggest that the frog anti-parvalbumin antibody can be used as a valuable tool to detect parvalbumins from the fish tested in this study, except yellowfin tuna.

gp140, the EBV/C3d receptor (CR2) of human B lymphocytes, is involved in cell-free phosphorylation of p120, a nuclear ribonucleoprotein.

PubMed

Delcayre, A X; Fiandino, A; Barel, M; Frade, R

1987-12-01

gp140, the EB/C3d receptor (EBV/C3dR; CR2), is a membrane site involved in human B cell regulation. Cross-linking of this receptor on the cell surface by its specific ligands led to the enhancement of B cell proliferation in synergy with T cell factors. In vitro activation of human peripheral B lymphocytes by cross-linking membrane immunoglobulins with anti-mu antibody induced EBV/C3dR phosphorylation. These studies were pursued by analyzing cell-free phosphorylation of EBV/C3dR isolated from Raji cell fractions, and immobilized on OKB7, a monoclonal anti-EBV/C3dR antibody. Three EBV/C3dR-related antigens which could be cell-free phosphorylated were detected: gp140, the EBV/C3dR, p130 and p120. gp140, the mature form of EBV/C3dR, was isolated from plasma membrane and from purified nuclei. p130 was identified as an intracellular intermediate of EBV/C3dR glycosylation, localized in low-density microsomes. Phosphoamino acid analysis of EBV/C3dR allowed the detection of phosphotyrosine and phosphoserine residues. These data suggest that EBV/C3dR could carry an autophosphorylation activity and could be associated to serine kinases. Using polyclonal anti-p120 antibody and anti-120 kDa nuclear ribonucleoprotein monoclonal antibody (mAb), p120 was identified as a nuclear ribonucleoprotein antigenically not related to EBV/C3dR. Detection of p120 on EBV/C3dR, immobilized on OKB7, was due to interactions between both antigens, instead of anti-EBV/C3dR mAb cross-reactivity with p120. Cell-free phosphorylation of p120 was under the control of EBV/C3dR. However, it is not yet established whether other nuclear or membrane components were involved in the control of p120 cell-free phosphorylation by EBV/C3dR. From the data presented herein, we propose that phosphorylation of a 120-kDa nuclear ribonucleoprotein by EBV/C3dR-associated kinases could represent a crucial step in in vivo regulation of human B cell activation.

Antibody-dependent cellular cytotoxicity (ADCC) activity of a novel anti-PD-L1 antibody avelumab (MSB0010718C) on human tumor cells

PubMed Central

Fantini, Massimo; Heery, Christopher R.; Gulley, James L.; Tsang, Kwong Yok; Schlom, Jeffrey

2015-01-01

Several anti-PD1/PD-L1 monoclonal antibodies (MAb) are currently providing evidence of clinical benefit in subsets of cancer patients. The mode of action of these MAbs is to inhibit PD1 on immune cells interacting with PD-L1 on tumor cells. These MAbs are either designed or engineered to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), which, however, has been implicated as an important mechanism in several highly effective MAb-mediated cancer therapies. A fully human anti-PD-L1 MAb would potentially be able to block PD-L1/PD1 interactions and also mediate the ADCC lysis of tumor cells. MSB0010718C (designated avelumab) is a fully human IgG1 anti-PD-L1 MAb. The studies reported here demonstrate (a) the ability of avelumab to lyse a range of human tumor cells in the presence of PBMC or NK effectors; (b) IFNγ can enhance tumor cell PD-L1 expression and in some cases enhance ADCC tumor cell lysis; (c) purified NK cells are potent effectors for avelumab; (d) similar levels of avelumab-mediated ADCC lysis of tumor cells are seen using purified NK as effectors from either healthy donors or cancer patients; (e) very low levels of avelumab-mediated lysis are seen using whole PBMCs as targets; this finding complements results seen in analyses of PBMC subsets of patients receiving avelumab; and (f) the addition of IL12 to NK cells greatly enhances avelumab-mediated ADCC. These studies thus provide an additional mode of action for an anti-PD-L1 MAb and support the rationale for further studies to enhance avelumab-mediated ADCC activity. PMID:26014098

Production and characterization of monoclonal antibodies against horse immunoglobulins useful for the diagnosis of equine diseases.

PubMed

Di Febo, Tiziana; Luciani, Mirella; Ciarelli, Antonella; Bortone, Grazia; Di Pancrazio, Chiara; Rodomonti, Diamante; Teodori, Liana; Tittarelli, Manuela

2015-01-01

Monoclonal antibodies (MAbs) against horse IgG were produced by immunizing Balb/c mice with purified horse IgG and were characterized in indirect ELISA versus purified immunoglobulins from donkey, cow, buffalo, sheep, pig, and chicken. Three MAbs (1B10B6C9, 1B10B6C10, 1B10B6E9) reacted only with horse and donkey IgG and IgM and, in western blotting, were specific for the Fc fragment of equine IgG. MAb 1B10B6E9 was used in chemiluminescent immunoblotting assay for the diagnosis of dourine and in indirect immunofluorescence assay (IFA) for the diagnosis of African horse sickness and dourine.

Development of monoclonal antibodies to rohu [Labeo rohita] immunoglobulins for use in immunoassays.

PubMed

Rathore, Gaurav; Kumar, Gokhlesh; Sood, Neeraj; Kapoor, D; Lakra, W S

2008-12-01

Serum immunoglobulins [Ig] of rohu [Labeo rohita] were purified by affinity chromatography using bovine serum albumin as capture ligand. The purified rohu Ig [r-Ig] had a molecular weight [MW] of 880 kDa as determined with gel filtration chromatography. The heavy chain of r-Ig had an MW of 77.8 kDa and that of light chain was 26.4 kDa in SDS-PAGE. Purified r-Ig was used for the production of two anti-rohu Ig monoclonal antibodies [D7 and H4] that belonged to subclass IgG2b and IgG1, respectively. Both the MAbs were specific to heavy chain of r-Ig as seen in Western blotting. Anti-rohu Ig MAb was used as a diagnostic reagent in ELISA and immunocytochemical assays to demonstrate its application for sero-surveillance and for immunological studies in rohu. A competitive ELISA was used to demonstrate the antigenic relatedness of r-Ig with whole serum Ig of other fish species. Cross reactivity of anti-rohu Ig MAb was observed with serum Ig of Catla catla and Cirrihinus mrigala. No reactivity to serum Ig of Ophiocephalus striatus and Clarias gariepinus was seen. Anti-rohu Ig MAb was found to be suitable for the detection of pathogen specific [Edwardsiella tarda] antibodies in serum of immunized rohu by an indirect ELISA. In flow cytometry using D7 MAb, the mean percentage [+/-SE] of Ig positive cells in spleen and blood of rohu were found to be 64.85% [+/-2.34] and 51.84% [+/-2.55] of gated lymphocytes, respectively. Similarly, D7 MAb also stained 52.84% [+/-1.30] and 10.5% of gated lymphocytes in kidney and thymus, respectively. The anti-rohu Ig MAbs also showed specific staining of Ig bearing cells in spleen sections by the indirect immunoperoxidase test.

Conserved and Variant Epitopes of Plasmodium vivax Duffy Binding Protein as Targets of Inhibitory Monoclonal Antibodies

PubMed Central

Ntumngia, Francis B.; Schloegel, Jesse; Barnes, Samantha J.; McHenry, Amy M.; Singh, Sanjay; King, Christopher L.

2012-01-01

The Duffy binding protein (DBP) is a vital ligand for Plasmodium vivax blood-stage merozoite invasion, making the molecule an attractive vaccine candidate against vivax malaria. Similar to other blood-stage vaccine candidates, DBP allelic variation eliciting a strain-specific immunity may be a major challenge for development of a broadly effective vaccine against vivax malaria. To understand whether conserved epitopes can be the target of neutralizing anti-DBP inhibition, we generated a set of monoclonal antibodies to DBP and functionally analyzed their reactivity to a panel of allelic variants. Quantitative analysis by enzyme-linked immunosorbent assay (ELISA) determined that some monoclonal antibodies reacted strongly with epitopes conserved on all DBP variants tested, while reactivity of others was allele specific. Qualitative analysis characterized by anti-DBP functional inhibition using an in vitro erythrocyte binding inhibition assay indicated that there was no consistent correlation between the endpoint titers and functional inhibition. Some monoclonal antibodies were broadly inhibitory while inhibition of others varied significantly by target allele. These data demonstrate a potential for vaccine-elicited immunization to target conserved epitopes but optimization of DBP epitope target specificity and immunogenicity may be necessary for protection against diverse P. vivax strains. PMID:22215740

Conserved and variant epitopes of Plasmodium vivax Duffy binding protein as targets of inhibitory monoclonal antibodies.

PubMed

Ntumngia, Francis B; Schloegel, Jesse; Barnes, Samantha J; McHenry, Amy M; Singh, Sanjay; King, Christopher L; Adams, John H

2012-03-01

The Duffy binding protein (DBP) is a vital ligand for Plasmodium vivax blood-stage merozoite invasion, making the molecule an attractive vaccine candidate against vivax malaria. Similar to other blood-stage vaccine candidates, DBP allelic variation eliciting a strain-specific immunity may be a major challenge for development of a broadly effective vaccine against vivax malaria. To understand whether conserved epitopes can be the target of neutralizing anti-DBP inhibition, we generated a set of monoclonal antibodies to DBP and functionally analyzed their reactivity to a panel of allelic variants. Quantitative analysis by enzyme-linked immunosorbent assay (ELISA) determined that some monoclonal antibodies reacted strongly with epitopes conserved on all DBP variants tested, while reactivity of others was allele specific. Qualitative analysis characterized by anti-DBP functional inhibition using an in vitro erythrocyte binding inhibition assay indicated that there was no consistent correlation between the endpoint titers and functional inhibition. Some monoclonal antibodies were broadly inhibitory while inhibition of others varied significantly by target allele. These data demonstrate a potential for vaccine-elicited immunization to target conserved epitopes but optimization of DBP epitope target specificity and immunogenicity may be necessary for protection against diverse P. vivax strains.

Development of an enzyme-linked immunosorbent assay and immunoaffinity chromatography for glycyrrhizic acid using an anti-glycyrrhizic acid monoclonal antibody.

PubMed

Zhang, Yue; Qu, Huihua; Zeng, Wenhao; Zhao, Yan; Shan, Wenchao; Wang, Xueqian; Wang, Qingguo; Zhao, Yan

2015-07-01

In this work, a new monoclonal antibody specific for glycyrrhizic acid was prepared and characterized. A hybridoma secreting an anti-glycyrrhizic acid monoclonal antibody was produced by fusing splenocytes from a mouse immunized against a glycyrrhizic acid-bovine serum albumin conjugate with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma cell line (Sp2/0-Ag14). Subsequently, an indirect, competitive enzyme-linked immunosorbent assay for glycyrrhizic acid was developed using the monoclonal antibody. In this assay, we detected an effective measuring range of 78.12-2500 ng/mL. Both intra-assay and inter-assay repeatability and precision were achieved, with relative standard deviations lower than 10%. In addition, glycyrrhizic acid levels in both formulated Chinese medicines and biological samples were determined with high sensitivity and efficiency. We then successfully developed a reliable immunoaffinity chromatography to separate glycyrrhizic acid completely from its parent medicine. These methods will contribute to further research investigations to better understand the interactions of glycyrrhizic acid with other drugs in the complex system of traditional Chinese medicine. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[What's new in internal medicine?].

PubMed

Blétry, O; Sene, T; Kahn, J-E; Ackermann, F; Charles, P; Leport, J; Piette, A-M

2009-12-01

Among diagnostic progress over the last three years in internal medicine, Antisynthetase Syndrome is now more easily recognised with the diffusion of laboratory tests for research of antibodies against tRNA synthetases (Anti JO1, anti PL7, Anti PL12). In two third of cases, these antibodies are found despite absence of antinuclear antibodies. Hence, we have to search them specifically in patients with polyarthritis associated with myositis, cutaneous manifestations (Raynaud phenomenom and "mechanic'hands") and interstitial lung disease. Discovery of asymptomatic mutation in the L ferritin coding sequence help us to better understand the "unexplained" hyperferritinemia. Initially described by japonese gastroenterologists, auto immune pancreatitis in fact a part of a systemic sclerosing disease with a biochemical hallmark: in crease of a subclass of immunoglobulins G (IgG4). A new pediatric disease due to a deficiency of the interleukin1 receptor antagonist (multifocal aseptic osteitis, periostitis, stomatitis, disseminated pustulosis) help us to better understand unexplained auto inflammatory diseases. The therapeutic progress is primarily due to an explosion of biological therapies, particularly four of them very useful for internists (in an off label use) : Interleukin 1 inhibitors (anakinra, Canakinumab) to treat some auto inflammatory diseases (cryopirin associated periodic syndromes and deficency of interleukin 1 receptor antagonist), monoclonal antibody against interleukin 5 (mepolizumab) to treat some hypereosinophilic syndromes and Churg and Strauss angiitis, interleukin 6 inhibitiors to treat multifocal Castleman's disease and adult Still disease, a monoclonal antibody against vascular endothelial growth factor (Bevacizumab) to treat hereditary hemorrhagic telangiectasia. Copyright 2009 Elsevier Masson SAS. All rights reserved.

Evaluation of monoclonal anti-A and anti-B and affinity-purified Ulex europaeus lectin I for forensic blood grouping.

PubMed

Gaensslen, R E; Lee, H C; Carroll, J E

1984-01-01

Two different monoclonal anti-A and anti-B and several different affinity purified Ulex europaeus lectin I reagents were evaluated and compared with conventional anti-A and anti-B sera and Ulex anti-H for serologic properties, in inhibition tests with secretor salivas, and in elution tests with bloodstains. The monoclonal and purified reagents were found to be comparable to conventional ones, and accordingly suitable for forensic inhibition and elution procedures.

Novel monoclonal antibodies to study tissue regeneration in planarians.

PubMed

Ross, Kelly G; Omuro, Kerilyn C; Taylor, Matthew R; Munday, Roma K; Hubert, Amy; King, Ryan S; Zayas, Ricardo M

2015-01-21

Planarians are an attractive model organism for studying stem cell-based regeneration due to their ability to replace all of their tissues from a population of adult stem cells. The molecular toolkit for planarian studies currently includes the ability to study gene function using RNA interference (RNAi) and observe gene expression via in situ hybridizations. However, there are few antibodies available to visualize protein expression, which would greatly enhance analysis of RNAi experiments as well as allow further characterization of planarian cell populations using immunocytochemistry and other immunological techniques. Thus, additional, easy-to-use, and widely available monoclonal antibodies would be advantageous to study regeneration in planarians. We have created seven monoclonal antibodies by inoculating mice with formaldehyde-fixed cells isolated from dissociated 3-day regeneration blastemas. These monoclonal antibodies can be used to label muscle fibers, axonal projections in the central and peripheral nervous systems, two populations of intestinal cells, ciliated cells, a subset of neoblast progeny, and discrete cells within the central nervous system as well as the regeneration blastema. We have tested these antibodies using eight variations of a formaldehyde-based fixation protocol and determined reliable protocols for immunolabeling whole planarians with each antibody. We found that labeling efficiency for each antibody varies greatly depending on the addition or removal of tissue processing steps that are used for in situ hybridization or immunolabeling techniques. Our experiments show that a subset of the antibodies can be used alongside markers commonly used in planarian research, including anti-SYNAPSIN and anti-SMEDWI, or following whole-mount in situ hybridization experiments. The monoclonal antibodies described in this paper will be a valuable resource for planarian research. These antibodies have the potential to be used to better understand planarian biology and to characterize phenotypes following RNAi experiments. In addition, we present alterations to fixation protocols and demonstrate how these changes can increase the labeling efficiencies of antibodies used to stain whole planarians.

Computational structural analysis of an anti-l-amino acid antibody and inversion of its stereoselectivity

PubMed Central

Ranieri, Daniel I.; Hofstetter, Heike; Hofstetter, Oliver

2009-01-01

The binding site of a monoclonal anti-l-amino acid antibody was modeled using the program SWISS-MODEL. Docking experiments with the enantiomers of phenylalanine revealed that the antibody interacts with l-phenylalanine via hydrogen bonds and hydrophobic contacts, whereas the d-enantiomer is rejected due to steric hindrance. Comparison of the sequences of this antibody and an anti-d-amino acid antibody indicates that both immunoglobulins derived from the same germline progenitor. Substitution of four amino acids residues, three in the framework and one in the complementarity determining regions, allowed in silico conversion of the anti-l-amino acid antibody into an antibody that stereoselectively binds d-phenylalanine. PMID:19472280

[Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

PubMed

Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

2012-06-01

To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

Development and characterization of serotype-specific monoclonal antibodies against the dengue virus-4 (DENV-4) non-structural protein (NS1).

PubMed

Gelanew, Tesfaye; Hunsperger, Elizabeth

2018-02-06

Dengue, caused by one of the four serologically distinct dengue viruses (DENV-1 to - 4), is a mosquito-borne disease of serious global health significance. Reliable and cost-effective diagnostic tests, along with effective vaccines and vector-control strategies, are highly required to reduce dengue morbidity and mortality. Evaluation studies revealed that many commercially available NS1 antigen (Ag) tests have limited sensitivity to DENV-4 serotype compared to the other three serotypes. These studies indicated the need for development of new NS1 Ag detection test with improved sensitivity to DENV-4. An NS1 capture enzyme linked immunoassay (ELISA) specific to DENV-4 may improve the detection of DENV-4 cases worldwide. In addition, a serotype-specific NS1 Ag test identifies both DENV and the infecting serotype. In this study, we used a small-ubiquitin-like modifier (SUMO*) cloning vector to express a SUMO*-DENV-4 rNS1 fusion protein to develop NS1 DENV-4 specific monoclonal antibodies (MAbs). These newly developed MAbs were then optimized for use in an anti-NS1 DENV-4 capture ELISA. The serotype specificity and sensitivity of this ELISA was evaluated using (i) supernatants from DENV (1-4)-infected Vero cell cultures, (ii) rNS1s from all the four DENV (1-4) and, (iii) rNS1s of related flaviviruses (yellow fever virus; YFV and West Nile virus; WNV). From the evaluation studies of the newly developed MAbs, we identified three DENV-4 specific anti-NS1 MAbs: 3H7A9, 8A6F2 and 6D4B10. Two of these MAbs were optimal for use in a DENV-4 serotype-specific NS1 capture ELISA: MAb 8A6F2 as the capture antibody and 6D4B10 as a detection antibody. This ELISA was sensitive and specific to DENV-4 with no cross-reactivity to other three DENV (1-3) serotypes and other heterologous flaviviruses. Taken together these data indicated that our MAbs are useful reagents for the development of DENV-4 immunodiagnostic tests.

B-lymphoma cells escape rituximab-triggered elimination by NK cells through increased HLA class I expression.

PubMed

Borgerding, Andrea; Hasenkamp, Justin; Engelke, Michael; Burkhart, Nina; Trümper, Lorenz; Wienands, Jürgen; Glass, Bertram

2010-03-01

Antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells is a major effector mechanism of the monoclonal anti-CD20 antibody rituximab in eliminating B-cell lymphomas. Resistance to this treatment occurs, although CD20 antigen is expressed on the tumor cells. A model of ADCC was established by stimulating human bulk NK cells and inhibitory killer immunoglobulin receptor (KIR)-defined NK cells from human leukocyte antigen (HLA)-typed donors. NK-cell activation was triggered via stimulation of the Fc receptor with immunoglobulin G aggregates, rituximab-labeled HLA-defined CD20-positive B-lymphoblast cell lines or CD20-positive B-lymphoma cell lines. The effect of KIR ligation by anti-KIR antibodies and HLA, the HLA expression density and rituximab concentrations on the efficacy of ADCC were analyzed in granzyme B ELISPOT measuring NK-cell activation and fluorescein-activated cell sorting cytotoxicity assay. HLA, but not CD20 expression density correlated with NK-cell activity against rituximab-labeled targets. ADCC was increased or decreased following HLA shielding or KIR activation by anti-KIR antibodies, respectively. Herein we show that rituximab-induced ADCC is attenuated upon ligation of KIR by HLA molecules expressed on human B-lymphoma target cells. Moreover, anti-KIR antibodies do not only block KIR/HLA interactions, but display agonistic effects at the KIR, which has to be considered for therapeutical applications. KIR activation and HLA expression density are critical determinants for the efficacy of rituximab treatment. An explanation for the failure of rituximab treatment may be the protection of the tumor cells from ADCC by inhibiting NK-cell function with their surface HLA. Copyright 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Development of monoclonal antibodies bearing the internal image of the gizzerosine epitope and application in a competitive ELISA for fish meal.

PubMed

Manosalva, Headdy; De Ioannes, Alfredo E; Becker, María Inés

2004-02-01

Gizzerosine (GZ), a derivative of histamine, is a biogenic amine found in fish meal, and one of the causative agents of black vomit, a poultry disease. We describe here the preparation of anti-idiotype antibodies to the anti-GZ monoclonal antibody (anti-GZ 3H4) and their possible application to an immunoassay. BALB-c mice were immunized with anti-GZ 3H4 antibody coupled to hemocyanin from Concholepas concholepas. Using somatic cell fusion between NSO/2 cells and splenic lymphocytes from the immunized mice, we obtained 34 potential anti-idiotype antibodies. They were characterized by passive agglutination with supernatants from hybridoma cultures and latex particles conjugated to the idiotype. Anti-idiotype antibodies were analyzed by a competitive RIA, to determine their ability to dissociate the interaction between (125)I-GZ and the anti-GZ 3H4-idiotype antibody. They were also characterized by GZ inhibition of latex passive agglutination assay. Three anti-idiotypes named 2D11, 2H6, and 3A12, all of the IgG isotype, were obtained. They were evaluated by a competitive ELISA, in which GZ competes with the tracer (HRP-idiotype). All presented sensitivity in the range of 0.1-10 microg/mL of GZ; and the 3A12 anti-idiotype antibody showed the best performance. An ELISA was developed using the idiotype bound to the solid phase and the anti-idiotype 3A12-HRP as the tracer. The assay showed a similar sensitivity and cross-reactivity with histamine was only observed at concentrations over 10 microg/mL. Lysine and histidine did not interfere with the assay up to 500 microg/mL. An experiment was conducted with fish meal contaminated with synthetic GZ. The results are promising, and showed that no other compounds of the fish meal interfere with the ELISA system; however the extraction procedure of the sample needs to be improved. From the results presented here, we conclude that the idiotype anti-idiotype ELISA would be an appropriate method to determine GZ in fish meal.

Characterization of fimbriae of Actinomyces naeslundii N16 using monoclonal and polyclonal antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bragg, S.L.

1988-01-01

Two populations of fimbriae, which differ both in antigenicity and biological activity, have been identified on Actinomyces viscosus T14V cells. Although A. naeslundii serotype 1 isolates possess only one of these fimbrial populations (type 2 fimbriae), there was functional evidence to suggest that A. naeslundii serotype 3 strain N16 had both types of fimbriae. The purpose of this study was to characterize the fimbriae of A. naeslundii N16 immunologically by using both monoclonal and polyclonal antibodies. Three monoclonal antibodies (MAbs) to N16 were produced; all three bound to N16 fimbriae as determined by immunoelectron microscopy. In a solid-phase radioimmunoassay MAbmore » 3B5.A1 reacted with 100% of the A. naeslundii serotype 3 isolates tested, but it did not react with any heterologous isolates. Type 1 and type 2 fimbriae were detected in Lancefield extracts of N16 cells by crossed immunoelectrophoresis (XIEP) using rabbit antiserum against N16 whole cells. When {sup 125}I-MAb 3B5.A1 was also incorporated into the gel, autoradiography indicated that MAb 3B5.A1 was specific for type 2 fimbriae. The N16 type 2 fimbriae were purified by gel filtration and immunoaffinity chromatography on a MAb 3B5.A1 column. Fimbriae-specific polyclonal and monoclonal antibodies were used in various immunological assays to determine that (a) N16 type 1 fimbriae are not related antigenically to type 2 fimbriae, (b) each type of fimbriae has epitopes that are present on the corresponding fimbriae of certain heterologous strains, and (c) MAb 3B5.A1 recognizes a serotype-specific epitope residing on the type 2 fimbriae of A. naeslundii serotype 3 strains.« less

Stepwise Engineering of Heterodimeric Single Domain Camelid VHH Antibodies That Passively Protect Mice from Ricin Toxin*

PubMed Central

Vance, David J.; Tremblay, Jacqueline M.; Mantis, Nicholas J.; Shoemaker, Charles B.

2013-01-01

In an effort to engineer countermeasures for the category B toxin ricin, we produced and characterized a collection of epitopic tagged, heavy chain-only antibody VH domains (VHHs) specific for the ricin enzymatic (RTA) and binding (RTB) subunits. Among the 20 unique ricin-specific VHHs we identified, six had toxin-neutralizing activity: five specific for RTA and one specific for RTB. Three neutralizing RTA-specific VHHs were each linked via a short peptide spacer to the sole neutralizing anti-RTB VHH to create VHH “heterodimers.” As compared with equimolar concentrations of their respective monovalent monomers, all three VHH heterodimers had higher affinities for ricin and, in the case of heterodimer D10/B7, a 6-fold increase in in vitro toxin-neutralizing activity. When passively administered to mice at a 4:1 heterodimer:toxin ratio, D10/B7 conferred 100% survival in response to a 10 × LD50 ricin challenge, whereas a 2:1 heterodimer:toxin ratio conferred 20% survival. However, complete survival was achievable when the low dose of D10/B7 was combined with an IgG1 anti-epitopic tag monoclonal antibody, possibly because decorating the toxin with up to four IgGs promoted serum clearance. The two additional ricin-specific heterodimers, when tested in vivo, provided equal or greater passive protection than D10/B7, thereby warranting further investigation of all three heterodimers as possible therapeutics. PMID:24202178

Stepwise engineering of heterodimeric single domain camelid VHH antibodies that passively protect mice from ricin toxin.

PubMed

Vance, David J; Tremblay, Jacqueline M; Mantis, Nicholas J; Shoemaker, Charles B

2013-12-20

In an effort to engineer countermeasures for the category B toxin ricin, we produced and characterized a collection of epitopic tagged, heavy chain-only antibody VH domains (VHHs) specific for the ricin enzymatic (RTA) and binding (RTB) subunits. Among the 20 unique ricin-specific VHHs we identified, six had toxin-neutralizing activity: five specific for RTA and one specific for RTB. Three neutralizing RTA-specific VHHs were each linked via a short peptide spacer to the sole neutralizing anti-RTB VHH to create VHH "heterodimers." As compared with equimolar concentrations of their respective monovalent monomers, all three VHH heterodimers had higher affinities for ricin and, in the case of heterodimer D10/B7, a 6-fold increase in in vitro toxin-neutralizing activity. When passively administered to mice at a 4:1 heterodimer:toxin ratio, D10/B7 conferred 100% survival in response to a 10 × LD50 ricin challenge, whereas a 2:1 heterodimer:toxin ratio conferred 20% survival. However, complete survival was achievable when the low dose of D10/B7 was combined with an IgG1 anti-epitopic tag monoclonal antibody, possibly because decorating the toxin with up to four IgGs promoted serum clearance. The two additional ricin-specific heterodimers, when tested in vivo, provided equal or greater passive protection than D10/B7, thereby warranting further investigation of all three heterodimers as possible therapeutics.

In ovo injection of anti-chicken CD25 monoclonal antibodies depletes CD4+CD25+ T cells in chickens.

PubMed

Shanmugasundaram, Revathi; Selvaraj, Ramesh K

2013-01-01

The CD4(+)CD25(+) cells have T regulatory cell properties in chickens. This study investigated the effect of in ovo injection of anti-chicken CD25 monoclonal antibodies (0.5 mg/egg) on CD4(+)CD25(+) cell depletion and on amounts of interleukin-2 mRNA and interferon-γ mRNA in CD4(+)CD25(-) cells posthatch. Anti-chicken CD25 or PBS (control) was injected into 16-d-old embryos. Chicks hatched from eggs injected with anti-chicken CD25 antibodies had a lower CD4(+)CD25(+) cell percentage in the blood until 25 d posthatch. The anti-chicken CD25 antibody injection nearly depleted CD4(+)CD25(+) cells in the blood until 16 d posthatch. At 30 d posthatch, the CD4(+)CD25(+) cell percentage in the anti-CD25-antibody-injected group was comparable with the percentage in the control group. At 16 d posthatch, the anti-chicken CD25 antibody injection decreased CD4(+)CD25(+) cell percentages in the thymus, spleen, and cecal tonsils. Chickens hatched from anti-CD25-antibody-injected eggs had approximately 25% of CD4(+)CD25(+) cells in the cecal tonsils and thymus compared with those in the cecal tonsils and thymus of the control group. The CD4(+)CD25(-) cells from the spleen and cecal tonsils of chicks hatched from anti-chicken-CD25-injected eggs had higher amounts of interferon-γ and interleukin-2 mRNA than CD4(+)CD25(-) cells from the control group. It could be concluded that injecting anti-chicken CD25 antibodies in ovo at 16 d of incubation nearly depleted the CD4(+)CD25(+) cells until 25 d posthatch.

Evaluation of immobilized metal membrane affinity chromatography for purification of an immunoglobulin G1 monoclonal antibody.

PubMed

Serpa, Gisele; Augusto, Elisabeth Fátima Pires; Tamashiro, Wirla Maria Silva Cunha; Ribeiro, Mariana Borçoe; Miranda, Everson Alves; Bueno, Sônia Maria Alves

2005-02-25

The large scale production of monoclonal antibodies (McAbs) has gaining increased relevance with the development of the hybridoma cell culture in bioreactors creating a need for specific efficient bioseparation techniques. Conventional fixed bead affinity adsorption commonly applied for McAbs purification has the drawback of low flow rates and colmatage. We developed and evaluated a immobilized metal affinity chromatographies (IMAC) affinity membrane for the purification of anti-TNP IgG(1) mouse McAbs. We immobilized metal ions on a poly(ethylene vinyl alcohol) hollow fiber membrane (Me(2+)-IDA-PEVA) and applied it for the purification of this McAbs from cell culture supernatant after precipitation with 50% saturation of ammonium sulphate. The purity of IgG(1) in the eluate fractions was high when eluted from Zn(2+) complex. The anti-TNP antibody could be eluted under conditions causing no loss of antigen binding capacity. The purification procedure can be considered as an alternative to the biospecific adsorbent commonly applied for mouse IgG(1) purification, the protein G-Sepharose.

[Monoclonal antibodies against inflammatory mediators for the treatment of patients with sepsis].

PubMed

Matsubara, Tomoyo

2002-03-01

Sepsis is a common cause of morbidity and mortality, particularly in immunocompromised and critically ill patients. Recently, a new designation, systemic inflammatory response syndrome(SIRS), has been studied. When an abnormal generalized inflammatory reaction is due to an infection, the terms sepsis and SIRS are synonymous. The systemic response to infection is mediated via the macrophage-derived cytokines that target end organ receptors in response to injury or infection. One strategy used to perturb the septic cascade is to block a particular inflammatory molecule. Results have been published on clinical trials in sepsis patients treated with several monoclonal antibodies, such as antiendotoxin antibodies, anti-tumor necrosis factor antibodies, and anti CD14 antibodies. In this chapter, the results of clinical trials in patients and in vivo data from animal models of sepsis are summarized.

Modulation of thymus-leukemia antigens on mouse leukemia cells induced by IgG, but not IgM, antibody.

PubMed

Stackpole, C W

1980-04-01

Exposure of mouse leukemia cells bearing thymus-leukemia (TL) surface antigens to whole TL alloantiserum has previously been shown to desensitize the cells to subsequent lysis by guinea pig complement (C) and fresh antiserum (antigenic modulation) and to correlate with the ability of cells to escape immune destruction in mice immunized against TL antigens. Tested in vitro, IgG of TL.1,2,3,5 antiserum modulated RADA1 leukemia cells (TL.1,2,3,5) completely within 2 hours at 37 degrees C when fully sensitizing amounts were used, with normal mouse serum as a source of C3. Similar results were obtained with IgG1, IgG2a, and IgG2b fractions of TL antiserum. An IgG2a monoclonal TL.3 antibody also completely modulated TL.3 antigens and partially modulated all antigens detected with TL.1,2,3,5 antiserum. IgM anti-TL.1,2,3,5 failed to modulate RADA1 cells even after 6 hours in vitro when fully sensitizing amounts of antibody were used. An IgM monoclonal TL antibody also failed to induce modulation. Modulation did occur on cells incubated with fully sensitizing amounts of IgG and IgM TL.1,2,3,5 antibody simultaneously, and nearly all cell-bound immunoglobulins were IgG. In mice passively immunized with IgG TL antibody, RADA1 cells modulated completely within 24 hours, whereas no modulation occurred during 4 days in mice immunized with IgM antibody. However, in both instances, tumor cells grew actively, which indicated that tumor escape did not depend on achievement of a modulated state.

Investigation of a panel of monoclonal antibodies and polyclonal sera against anthrax toxins resulted in identification of an anti-lethal factor antibody with disease-enhancing characteristics.

PubMed

Kulshreshtha, Parul; Tiwari, Ashutosh; Priyanka; Joon, Shikha; Sinha, Subrata; Bhatnagar, Rakesh

2015-12-01

Hybridomas were created using spleen of mice that were actively immunized with rLFn (recombinant N-terminal domain of lethal factor). Later on, separate group of mice were immunized with rLFn to obtain a polyclonal control for passive immunization studies of monoclonal antibodies. This led to the identification of one cohort of rLFn-immnized mice that harboured disease-enhancing polyclonal antibodies. At the same time, the monoclonal antibodies secreted by all the hybridomas were being tested. Two hybridomas secreted monoclonal antibodies (H10 and H8) that were cross-reactive with EF (edema factor) and LF (lethal factor), while the other two hybridomas secreted LF-specific antibodies (H7 and H11). Single chain variable fragment (LETscFv) was derived from H10 hybridoma. H11 was found to have disease-enhancing property. Combination of H11 with protective monoclonal antibodies (H8 and H10) reduced its disease enhancing nature. This in vitro abrogation of disease-enhancement provides the proof of concept that in polyclonal sera the disease enhancing character of a fraction of antibodies is overshadowed by the protective nature of the rest of the antibodies generated on active immunization. Copyright © 2015. Published by Elsevier Ltd.

Characterization of a monoclonal antibody against P57, the C3/C3b-cleaving proteinase expressed in human erythrocyte membranes.

PubMed

Fiandino-Tirel, A; Barel, M; Lyamani, F; Gauffre, A; Hermann, J; Frade, R

1991-08-01

A monoclonal antibody was raised against p57, a serine proteinase, characterized by an apparent molecular weight of 57 kDa, and purified from human erythrocyte membranes. P57 proteinase cleaves the human third component of complement, C3. The antibody selected, MP1, of IgG2a isotype, precipitated specifically the p57 antigen which carried the C3/C3b-cleaving activity present in membrane crude extract of human erythrocytes. P57 proteinase eluted from MP1-sepharose was inhibited by 5 x 10(-4) M PMSF, enhanced by 0.5% SDS and generated C3 fragments identical to those generated by membrane crude extract of human erythrocytes. All these properties were identical to those of the p57 previously purified by biochemical procedures. In addition, 5000 binding sites were detected on cell surface. This MP1 monoclonal antibody will be helpful to analyse the role of p57 in human erythrocytes.

Lipopolysaccharide-Specific but Not Anti-Flagellar Immunoglobulin A Monoclonal Antibodies Prevent Salmonella enterica Serotype Enteritidis Invasion and Replication within HEp-2 Cell Monolayers

PubMed Central

Iankov, Ianko D.; Petrov, Dragomir P.; Mladenov, Ivan V.; Haralambieva, Iana H.; Mitov, Ivan G.

2002-01-01

The protective potential of immunoglobulin A (IgA) monoclonal antibodies (MAbs) directed against O and H antigens of Salmonella enterica serotype Enteritidis to prevent bacterial adhesion to and invasion of HEp-2 cells was evaluated. Although anti-flagellar IgA MAbs showed strong agglutinating capacities, they did not protect cell monolayers. In contrast, IgA MAbs specific for the O:9 epitope of Salmonella lipopolysaccharide antigen alone prevented S. enterica serotype Enteritidis entry and replication within HEp-2 cells, and the protection was not mediated by direct binding of antibodies to bacterial adhesins or by agglutination of microorganisms. PMID:11854252

B-Cell Depletion Promotes Aortic Infiltration of Immunosuppressive Cells and Is Protective of Experimental Aortic Aneurysm.

PubMed

Schaheen, Basil; Downs, Emily A; Serbulea, Vlad; Almenara, Camila C P; Spinosa, Michael; Su, Gang; Zhao, Yunge; Srikakulapu, Prasad; Butts, Cherié; McNamara, Coleen A; Leitinger, Norbert; Upchurch, Gilbert R; Meher, Akshaya K; Ailawadi, Gorav

2016-11-01

B-cell depletion therapy is widely used for treatment of cancers and autoimmune diseases. B cells are abundant in abdominal aortic aneurysms (AAA); however, it is unknown whether B-cell depletion therapy affects AAA growth. Using experimental models of murine AAA, we aim to examine the effect of B-cell depletion on AAA formation. Wild-type or apolipoprotein E-knockout mice were treated with mouse monoclonal anti-CD20 or control antibodies and subjected to an elastase perfusion or angiotensin II infusion model to induce AAA, respectively. Anti-CD20 antibody treatment significantly depleted B1 and B2 cells, and strikingly suppressed AAA growth in both models. B-cell depletion resulted in lower circulating IgM levels, but did not affect the levels of IgG or cytokine/chemokine levels. Although the total number of leukocyte remained unchanged in elastase-perfused aortas after anti-CD20 antibody treatment, the number of B-cell subtypes was significantly lower. Interestingly, plasmacytoid dendritic cells expressing the immunomodulatory enzyme indole 2,3-dioxygenase were detected in the aortas of B-cell-depleted mice. In accordance with an increase in indole 2,3-dioxygenase+ plasmacytoid dendritic cells, the number of regulatory T cells was higher, whereas the expression of proinflammatory genes was lower in aortas of B-cell-depleted mice. In a coculture model, the presence of B cells significantly lowered the number of indole 2,3-dioxygenase+ plasmacytoid dendritic cells without affecting total plasmacytoid dendritic cell number. The present results demonstrate that B-cell depletion protects mice from experimental AAA formation and promotes emergence of an immunosuppressive environment in aorta. © 2016 American Heart Association, Inc.

Imaging of myocardial infarction in dogs and humans using monoclonal antibodies specific for human myosin heavy chains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leger, J.; Chevalier, J.; Larue, C.

1991-08-01

The use of three different monoclonal antibodies specific for human ventricular myosin heavy chains in the visualization of the location and extent of necrosis in dogs with experimental acute myocardial infarction and in humans is described. Using a classic immunohistochemical method or ex vivo analysis of heart slices in dogs with acute myocardial infarction subjected to intravenous injection of unlabeled antimyosin antibodies or antimyosin antibodies labeled with indium-111, it was observed that all antibody fragments specifically reached the targeted necrotic zone less than 2 h after antibody injection and remained bound for up to 24 h. In a limited butmore » significant number of cases (5 of the 12 humans and 11 of 43 dogs), it was possible to image the necrotic zone in vivo as early as 2 to 4 h after antibody injection. In other cases, individual blood clearance variations retarded or even prevented in vivo necrosis detection. Higher antimyosin fixation values were obtained in the necrotic zones in dogs with a rapid blood clearance relative to that of the other dogs. It is concluded that antimyosin antibodies always reached necrotic areas within 2 h. If blood clearance was rapid, in vivo imaging of the necrotic area was possible 2 to 6 h after necrosis, even in humans. In some cases, however, uncontrolled individual variations in the timing required for sufficient blood clearance hampered this rapid in vivo detection of myocardial necrosis.« less

Anti-CXCL4 monoclonal antibody accelerates telogen to anagen transition and attenuates apoptosis of the hair follicle in mice

PubMed Central

Guan, Wen; Yu, Xiaolan; Li, Jingjing; Deng, Qing; Zhang, Yang; Gao, Jing; Xia, Peng; Yuan, Yunsheng; Gao, Jin; Zhou, Liang; Han, Wei; Yu, Yan

2017-01-01

Although hair loss or alopecia is a common disease, its exact mechanisms are not yet well understood. The present study investigated the hypothesis that the homeostatic regulation of genes during hair regeneration may participate in hair loss, based on the cyclicity of hair growth. A cluster of such genes was identified by an expression gene-array from the dorsal skin in a depilated mouse model, and CXCL4 was identified as a significantly regulated gene during the hair regeneration process. To elucidate the function of CXCL4 in hair growth, CXCL4 activity was blocked by the administration of an anti-CXCL4 monoclonal antibody (mAb). Histomorphometric analysis indicated that anti-CXCL4 mAb induced an earlier anagen phase and delayed hair follicle regression, in contrast with that in the control group. Moreover, CXCL4 mAb upregulated the transcription levels of several hair growth-related genes, including Lef1, Wnt10b, Bmp4 and Bmp2. In addition, CXCL4 mAb increased the levels of the proliferation-related protein PCNA and Bcl-2 during the anagen phase, while it reduced the expression of pro-apoptotic protein Bax and cleaved caspase-3 during the catagen phase. These findings reveal that CXCL4 plays an important role in hair growth, and that blockade of CXCL4 activity promotes hair growth. PMID:28810552

Anti-CXCL4 monoclonal antibody accelerates telogen to anagen transition and attenuates apoptosis of the hair follicle in mice.

PubMed

Guan, Wen; Yu, Xiaolan; Li, Jingjing; Deng, Qing; Zhang, Yang; Gao, Jing; Xia, Peng; Yuan, Yunsheng; Gao, Jin; Zhou, Liang; Han, Wei; Yu, Yan

2017-08-01

Although hair loss or alopecia is a common disease, its exact mechanisms are not yet well understood. The present study investigated the hypothesis that the homeostatic regulation of genes during hair regeneration may participate in hair loss, based on the cyclicity of hair growth. A cluster of such genes was identified by an expression gene-array from the dorsal skin in a depilated mouse model, and CXCL4 was identified as a significantly regulated gene during the hair regeneration process. To elucidate the function of CXCL4 in hair growth, CXCL4 activity was blocked by the administration of an anti-CXCL4 monoclonal antibody (mAb). Histomorphometric analysis indicated that anti-CXCL4 mAb induced an earlier anagen phase and delayed hair follicle regression, in contrast with that in the control group. Moreover, CXCL4 mAb upregulated the transcription levels of several hair growth-related genes, including Lef1, Wnt10b, Bmp4 and Bmp2. In addition, CXCL4 mAb increased the levels of the proliferation-related protein PCNA and Bcl-2 during the anagen phase, while it reduced the expression of pro-apoptotic protein Bax and cleaved caspase-3 during the catagen phase. These findings reveal that CXCL4 plays an important role in hair growth, and that blockade of CXCL4 activity promotes hair growth.

Promoting Cartilage Stem Cell Activity to Improve Recovery from Joint Fracture

DTIC Science & Technology

2012-03-01

Materials and Methods Bovine stifle joints from young adult cattle (15-24 months-old) were obtained from a local abattoir (Bud’s Custom Meats ...using a monoclonal anti-PCNA antibody (Abcam, Cambridge, MA) and a goat anti- mouse fluorescent secondary antibody (Alexafluor 488) from Jackson

The protective role of immunoglobulins in fungal infections and inflammation.

PubMed

Elluru, Sri Ramulu; Kaveri, Srini V; Bayry, Jagadeesh

2015-03-01

Increased incidence of fungal infections in the immunocompromised individuals and fungi-mediated allergy and inflammatory conditions in immunocompetent individuals is a cause of concern. Consequently, there is a need for efficient therapeutic alternatives to treat fungal infections and inflammation. Several studies have demonstrated that antibodies or immunoglobulins have a role in restricting the fungal burden and their clearance. However, based on the data from monoclonal antibodies, it is now evident that the efficacy of antibodies in fungal infections is dependent on epitope specificity, abundance of protective antibodies, and their isotype. Antibodies confer protection against fungal infections by multiple mechanisms that include direct neutralization of fungi and their antigens, inhibition of growth of fungi, modification of gene expression, signaling and lipid metabolism, causing iron starvation, inhibition of polysaccharide release, and biofilm formation. Antibodies promote opsonization of fungi and their phagocytosis, complement activation, and antibody-dependent cell toxicity. Passive administration of specific protective monoclonal antibodies could also prove to be beneficial in drug resistance cases, to reduce the dosage and associated toxic symptoms of anti-fungal drugs. The longer half-life of the antibodies and flexibilities to modify their structure/forms are additional advantages. The clinical data obtained with two monoclonal antibodies should incite interests in translating pre-clinical success into the clinics. The anti-inflammatory and immunoregulatory role of antibodies in fungal inflammation could be exploited by intravenous immunoglobulin or IVIg.

The EGF receptor family as targets for cancer therapy.

PubMed

Mendelsohn, J; Baselga, J

2000-12-27

Human carcinomas frequently express high levels of receptors in the EGF receptor family, and overexpression of at least two of these receptors, the EGF receptor (EGFr) and closely related ErbB2, has been associated with a more aggressive clinical behavior. Further, transfection or activation of high levels of these two receptors in nonmalignant cell lines can lead to a transformed phenotype. For these reasons therapies directed at preventing the function of these receptors have the potential to be useful anti-cancer treatments. In the last two decades monoclonal antibodies (MAbs) which block activation of the EGFr and ErbB2 have been developed. These MAbs have shown promising preclinical activity and 'chimeric' and 'humanized' MAbs have been produced in order to obviate the problem of host immune reactions. Clinical activity with these antibodies has been documented: trastuzumab, a humanized anti-ErbB2 MAb, is active and was recently approved in combination with paclitaxel for the therapy of patients with metastatic ErbB2-overexpressing breast cancer; IMC-C225, a chimeric anti-EGFr MAb, has shown impressive activity when combined with radiation therapy and reverses resistance to chemotherapy. In addition to antibodies, compounds that directly inhibit receptor tyrosine kinases have shown preclinical activity and early clinical activity has been reported. A series of phase III studies with these antibodies and direct tyrosine kinase inhibitors are ongoing or planned, and will further address the role of these active anti-receptor agents in the treatment of patients with cancer.

[Regulatory B cells activated by CpG-ODN combined with anti-CD40 monoclonal antibody inhibit CD4(+)T cell proliferation].

PubMed

Wang, Keng; Tao, Lei; Su, Jianbing; Zhang, Yueyang; Zou, Binhua; Wang, Yiyuan; Li, Xiaojuan

2016-09-01

Objective To observe the immunosuppressive function of regulatory B cells (Bregs) in vitro after activated by CpG oligodeoxynucleotide (CpG-ODN) and anti-CD40 mAb. Methods Mice splenic CD5(+)CD1d(high)B cells and CD5(-)CD1d(low)B cells were sorted by flow cytometry. These B cells were first stimulated with CpG-ODN combined with anti-CD40 mAb for 24 hours, and then co-cultured with purified CD4(+)T cells. The interleukin 10 (IL-10) expression in the activated Bregs and other B cell subset, as well as the proliferation and interferon γ (IFN-γ) expression in the CD4(+) T cells activated by anti-CD3 mAb plus anti-CD28 mAb were determined by flow cytometry. Results CD5(+)CD1d(high) B cells activated by CpG-ODN plus anti-CD40 mAb blocked the up-regulated CD4(+)T proliferation and significantly reduced the IFN-γ level. At the same time, activated CD5(-)CD1d(low)B cells showed no inhibitory effect on CD4(+)T cells. Further study revealed that IL-10 expression in the CD5(+)CD1d(high) B cells were much higher than that in the CD5(-)CD1d(low)B cells after stimulated with CpG-ODN combined with anti-CD40 mAb for 24 hours. Conclusion CD5(+)CD1d(high) B cells activated by CpG-ODN combined with anti-CD40 mAb have immune inhibitory effects on CD4(+)T cell activation in vitro , which possibly due to IL-10 secretion.

Alanine-170 and proline-172 are critical determinants for extracellular CD20 epitopes; heterogeneity in the fine specificity of CD20 monoclonal antibodies is defined by additional requirements imposed by both amino acid sequence and quaternary structure.

PubMed

Polyak, Maria J; Deans, Julie P

2002-05-01

In vivo ablation of malignant B cells can be achieved using antibodies directed against the CD20 antigen. Fine specificity differences among CD20 monoclonal antibodies (mAbs) are assumed not to be a factor in determining their efficacy because evidence from antibody-blocking studies indicates limited epitope diversity with only 2 overlapping extracellular CD20 epitopes. However, in this report a high degree of heterogeneity among antihuman CD20 mAbs is demonstrated. Mutation of alanine and proline at positions 170 and 172 (AxP) (single-letter amino acid codes; x indicates the identical amino acid at the same position in the murine and human CD20 sequences) in human CD20 abrogated the binding of all CD20 mAbs tested. Introduction of AxP into the equivalent positions in the murine sequence, which is not otherwise recognized by antihuman CD20 mAbs, fully reconstituted the epitope recognized by B1, the prototypic anti-CD20 mAb. 2H7, a mAb previously thought to recognize the same epitope as B1, did not recognize the murine AxP mutant. Reconstitution of the 2H7 epitope was achieved with additional mutations replacing VDxxD in the murine sequence for INxxN (positions 162-166 in the human sequence). The integrity of the 2H7 epitope, unlike that of B1, further depends on the maintenance of CD20 in an oligomeric complex. The majority of 16 antihuman CD20 mAbs tested, including rituximab, bound to murine CD20 containing the AxP mutations. Heterogeneity in the fine specificity of these antibodies was indicated by marked differences in their ability to induce homotypic cellular aggregation and translocation of CD20 to a detergent-insoluble membrane compartment previously identified as lipid rafts.

Immunoglobulin gene usage in the human anti-pathogen response.

PubMed

Newkirk, M M; Rioux, J D

1995-09-01

The human antibody response to foreign pathogens is generated to a relatively small number of target surface proteins and carbohydrates that nonetheless have an extensive array of epitopes. The study of human monoclonal antibodies to different pathogens shows that there are a diversity of mechanisms used to generate a sufficient repertoire of antibodies to combat the invading pathogens. Although many different immunoglobulin gene elements are used to construct the anti-pathogen response, some elements are used more often than would be expected if all elements were used randomly. For example, the immune response to Haemophilus influenzae polysaccharide appears to be quite narrow, being restricted primarily to a specific heavy-chain gene, 3-15, and a lambda light-chain family II member, 4A. In contrast, for the immune response to cytomegalovirus proteins, a wider group of gene elements is needed. It is also surprising that despite an investigator bias for IgG- rather than IgM-secreting immortal B cells (because of their high affinity and neutralizing abilities), 26% of light chains and 13% of heavy chains showed a very low level of somatic mutation, equivalent to an IgM molecule that has not undergone affinity maturation. Although some highly mutated IgG molecules are present in the anti-pathogen response, most of the monoclonal antibodies specific for viruses or bacteria have a level of somatic hypermutation similar to that of the adult IgM repertoire. A number of studies have shown that there are similarities in the antibody responses to pathogens and to self (autoantibodies).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunization of chickens with an agonistic monoclonal anti-chicken CD40 antibody-hapten complex: rapid and robust IgG response induced by a single subcutaneous injection.

PubMed

Chen, Chang-Hsin; Abi-Ghanem, Daad; Waghela, Suryakant D; Chou, Wen-Ko; Farnell, Morgan B; Mwangi, Waithaka; Berghman, Luc R

2012-04-30

Producing diagnostic antibodies in chicken egg yolk represents an alternate animal system that offers many advantages including high productivity at low cost. Despite being an excellent counterpart to mammalian antibodies, chicken IgG from yolk still represents an underused resource. The potential of agonistic monoclonal anti-CD40 antibodies (mAb) as a powerful immunological adjuvant has been demonstrated in mammals, but not in chickens. We recently reported an agonistic anti-chicken CD40 mAb (designated mAb 2C5) and showed that it may have potential as an immunological adjuvant. In this study, we examined the efficacy of targeting a short peptide to chicken CD40 [expressed by the antigen-presenting cells (APCs)] in enhancing an effective IgG response in chickens. For this purpose, an immune complex consisting of one streptavidin molecule, two directionally biotinylated mAb 2C5 molecules, and two biotinylated peptide molecules was produced. Chickens were immunized subcutaneously with doses of this complex ranging from 10 to 90 μg per injection once, and relative quantification of the peptide-specific IgG response showed that the mAb 2C5-based complex was able to elicit a strong IgG response as early as four days post-immunization. This demonstrates that CD40-targeting antigen to chicken APCs can significantly enhance antibody responses and induce immunoglobulin isotype-switching. This immunization strategy holds promise for rapid production of hapten-specific IgG in chickens. Copyright © 2012 Elsevier B.V. All rights reserved.

Generation and Characterization of Inhibitory Antibodies Specific to Guinea Pig CXCR1 and CXCR2.

PubMed

Tanaka, Kento; Yoshimura, Chigusa; Shiina, Tetsuo; Terauchi, Tomoko; Yoshitomi, Tomomi; Hirahara, Kazuki

2017-04-01

CXCR1 and CXCR2 are chemokine receptors that have different selectivity of chemokine ligands, but the distinct role of each receptor is not clearly understood. This is due to the absence of specific inhibitors in guinea pigs, which are the appropriate species for investigation of CXCR1 and CXCR2 because of their functional similarity to humans. In this study, we generated and evaluated monoclonal antibodies that specifically bound to guinea pig CXCR1 (gpCXCR1) and guinea pig CXCR2 (gpCXCR2) for acquisition of specific inhibitors. To assess the activity of antibodies, we established CHO-K1 cells stably expressing either gpCXCR1 or gpCXCR2 (CHO/gpCXCR1 or CHO/gpCXCR2). CHO/gpCXCR1 showed migration in response to guinea pig interleukin (IL)-8, and CHO/gpCXCR2 showed migration in response to both guinea pig IL-8 and guinea pig growth-regulated oncogene α. The receptor selectivities of the chemokines of guinea pigs were the same as the human orthologs. The inhibitory activities of the anti-gpCXCR1 and anti-gpCXCR2 monoclonal antibodies on cell migration were observed in a concentration-dependent manner. In conclusion, we successfully obtained inhibitory antibodies specific to gpCXCR1 and gpCXCR2. These inhibitory antibodies will be useful to clarify the physiological roles of CXCR1 and CXCR2 in guinea pigs.

Identification of a novel chemokine-dependent molecular mechanism underlying rheumatoid arthritis-associated autoantibody-mediated bone loss.

PubMed

Krishnamurthy, Akilan; Joshua, Vijay; Haj Hensvold, Aase; Jin, Tao; Sun, Meng; Vivar, Nancy; Ytterberg, A Jimmy; Engström, Marianne; Fernandes-Cerqueira, Cátia; Amara, Khaled; Magnusson, Malin; Wigerblad, Gustaf; Kato, Jungo; Jiménez-Andrade, Juan Miguel; Tyson, Kerry; Rapecki, Stephen; Lundberg, Karin; Catrina, Sergiu-Bogdan; Jakobsson, Per-Johan; Svensson, Camilla; Malmström, Vivianne; Klareskog, Lars; Wähämaa, Heidi; Catrina, Anca I

2016-04-01

Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) appear before disease onset and are associated with bone destruction. We aimed to dissect the role of ACPAs in osteoclast (OC) activation and to identify key cellular mediators in this process. Polyclonal ACPA were isolated from the synovial fluid (SF) and peripheral blood of patients with RA. Monoclonal ACPAs were isolated from single SF B-cells of patients with RA. OCs were developed from blood cell precursors with or without ACPAs. We analysed expression of citrullinated targets and peptidylarginine deiminases (PAD) enzymes by immunohistochemistry and cell supernatants by cytometric bead array. The effect of an anti-interleukin (IL)-8 neutralising antibody and a pan-PAD inhibitor was tested in the OC cultures. Monoclonal ACPAs were injected into mice and bone structure was analysed by micro-CT before and after CXCR1/2 blocking with reparixin. Protein citrullination by PADs is essential for OC differentiation. Polyclonal ACPAs enhance OC differentiation through a PAD-dependent IL-8-mediated autocrine loop that is completely abolished by IL-8 neutralisation. Some, but not all, human monoclonal ACPAs derived from single SF B-cells of patients with RA and exhibiting distinct epitope specificities promote OC differentiation in cell cultures. Transfer of the monoclonal ACPAs into mice induced bone loss that was completely reversed by the IL-8 antagonist reparixin. We provide novel insights into the key role of citrullination and PAD enzymes during OC differentiation and ACPA-induced OC activation. Our findings suggest that IL8-dependent OC activation may constitute an early event in the initiation of the joint specific inflammation in ACPA-positive RA. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

DOEpatents

Vanderlaan, M.; Watkins, B.E.; Stanker, L.H.

1991-10-01

Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described. 14 figures.

Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

DOEpatents

Vanderlaan, Martin; Watkins, Bruce E.; Stanker, Larry H.

1991-01-01

Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described.

Novel Anti-CRR9/CLPTM1L Antibodies with Antitumorigenic Activity Inhibit Cell Surface Accumulation, PI3K Interaction, and Survival Signaling

PubMed Central

Puskás, László G.; Mán, Imola; Szebeni, Gabor; Tiszlavicz, László; Tsai, Susan; James, Michael A.

2016-01-01

We and others have recently shown Cisplatin Resistance-Related Protein 9 (CRR9)/Cleft Lip and Palate Transmembrane 1-Like (CLPTM1L) to affect survival and proliferation in lung and pancreatic tumor cells. Our research has indicated that CLPTM1L affects multiple survival signaling pathways in tumor cells under oncogenic, genotoxic, and microenvironmental stress. We have confirmed the association of CLPTM1L with pancreatic cancer by demonstrating overexpression of CLPTM1L in pancreatic tumors and poor survival in patients with high tumor expression of CLPTM1L. Predicting a transmembrane structure, we determined that CLPTM1L could be targeted at the plasma membrane. Herein, we describe the development of monoclonal antibodies targeting CLPTM1L. Lead antibodies inhibited surface accumulation of CLPTM1L, Akt phosphorylation, anchorage-independent growth, and chemotherapeutic resistance in lung and pancreatic tumor cells. Gemcitabine promoted a physical interaction between CLPTM1L and p110α in pancreatic tumor cells, which was inhibited by anti-CLPTM1L. In-vivo treatment with anti-CLPTM1L robustly inhibited the growth of both lung and pancreatic adenocarcinoma xenografts. The efficacy of anti-CLPTM1L correlated with specific epitopes representing important targets in human cancers, particularly those driven by KRas, for which effective targeted therapies have been elusive. This study is the first to report cell-surface exposure of the tumor survival protein CLPTM1L and inhibition of the function of surface CLPTM1L with novel, systematically developed inhibitory monoclonal antibodies establishing proof of concept of clinically practical agents inhibiting this compelling new tumor survival target in cancer. PMID:26939707

Differential cross-reactivity of monoclonal antibody OPD4 (anti-CD45RO) in macaques.

PubMed

Wang, Xiaolei; Pahar, Bapi; Rasmussen, Terri; Alvarez, Xavier; Dufour, Jason; Rasmussen, Kelsi; Lackner, Andrew A; Veazey, Ronald S

2008-01-01

Immunologic research in nonhuman primates is occasionally limited by the availability of reagents that cross-react in nonhuman primates. One major limitation has been the lack of a monoclonal antibody to CD45RO. Although the monoclonal antibody UCHL-1 is used to detect CD45RO isoforms in humans, it does not react with nonhuman primates, mandating the use of alternative strategies to define "memory" T cell responses in nonhuman primates. The current study examined the reactivity and specificity of another antibody against CD45RO, clone OPD4, in macaques. Here we demonstrate that OPD4 specifically labels memory CD4+ T cells in approximately 44% of rhesus macaques (Macaca mulatta) of Indian but not Chinese origin. In contrast, tissues from pigtail macaques (Macaca nemestrina) react with this clone, indicating that OPD4 may be useful for examining memory CD4+ T cells in certain macaques, but its utility may be limited in other species or even among individual macaques.

Differential cross-reactivity of monoclonal antibody OPD4 (anti-CD45RO) in macaques

PubMed Central

Wang, Xiaolei; Pahar, Bapi; Rasmussen, Terri; Alvarez, Xavier; Dufour, Jason; Rasmussen, Kelsi; Lackner, Andrew A.; Veazey, Ronald S.

2008-01-01

Immunologic research in nonhuman primates is occasionally limited by the availability of reagents that cross react in nonhuman primates. One major limitation has been the lack of a monoclonal antibody to CD45RO. Although the monoclonal antibody UCHL-1 is used to detect CD45RO isoforms in humans, it does not react with nonhuman primates, mandating the use of alternative strategies to define “memory” T cell responses in nonhuman primates. The current study examined the reactivity and specificity of another antibody against CD45RO, clone OPD4, in macaques. Here we demonstrate that OPD4 specifically labels memory CD4+ T cells in ~44% of rhesus macaques (Macaca mulatta) of Indian, but not Chinese origin. In contrast, tissues from pigtail macaques (Macaca nemestrina) react with this clone, indicating that OPD4 may be useful for examining memory CD4+ T cells in certain macaques, but its utility may be limited in other species or even among individual macaques. PMID:18304631

Complement-mediated bactericidal activity of anti-factor H binding protein monoclonal antibodies against the meningococcus relies upon blocking factor H binding.

PubMed

Giuntini, Serena; Reason, Donald C; Granoff, Dan M

2011-09-01

Binding of the complement-downregulating protein factor H (fH) to the surface of the meningococcus is important for survival of the organism in human serum. The meningococcal vaccine candidate factor H binding protein (fHbp) is an important ligand for human fH. While some fHbp-specific monoclonal antibodies (MAbs) block binding of fH to fHbp, the stoichiometry of blocking in the presence of high serum concentrations of fH and its effect on complement-mediated bactericidal activity are unknown. To investigate this question, we constructed chimeric antibodies in which the human IgG1 constant region was paired with three murine fHbp-specific binding domains designated JAR 3, JAR 5, and MAb502. By surface plasmon resonance, the association rates for binding of all three MAbs to immobilized fHbp were >50-fold higher than that for binding of fH to fHbp, and the MAb dissociation rates were >500-fold lower than that for fH. While all three MAbs elicited similar C1q-dependent C4b deposition on live bacteria (classical complement pathway), only those antibodies that inhibited binding of fH to fHbp (JAR 3 and JAR 5) had bactericidal activity with human complement. MAb502, which did not inhibit fH binding, had complement-mediated bactericidal activity only when tested with fH-depleted human complement. When an IgG1 anti-fHbp MAb binds to sparsely exposed fHbp on the bacterial surface, there appears to be insufficient complement activation for bacteriolysis unless fH binding also is inhibited. The ability of fHbp vaccines to elicit protective antibodies, therefore, is likely to be enhanced if the antibody repertoire is of high avidity and includes fH-blocking activity.

Correlation between anti-PD-L1 tumor concentrations and tumor-specific and nonspecific biomarkers in a melanoma mouse model

PubMed Central

Contreras, Ana M.; Merino, María; Vasquez, Marcos; Trocóniz, Iñaki F.

2016-01-01

Blockade of PD-L1 with specific monoclonal antibodies (anti-PD-L1) represents a therapeutic strategy to increase the capability of the immune system to modulate the tumor immune-resistance. The relationship between anti-PD-L1 tumor exposition and anti-tumor effect represents a challenge that has been addressed in this work through the identification of certain biomarkers implicated in the antibody's mechanism of action, using a syngeneic melanoma mouse model. The development of an in-vitro/in-vivo platform has allowed us to investigate the PD-L1 behavior after its blockage with anti-PD-L1 at cellular level and in animals. In-vitro studies showed that the complex PD-L1/anti-PD-L1 was retained mainly at the cell surface. The antibody concentration and time exposure affected directly the recycling or ligand turnover. In-vivo studies showed that anti-PD-L1 was therapeutically active at all stage of the disease, with a rapid onset, a low but durable efficacy and non-relevant toxic effect. This efficacy measured as tumor shrinkage correlated with tumor-specific infiltrating lymphocytes (TILs), which increased as antibody tumor concentrations increased. Both, TILS and antibody concentrations followed similar kinetic patterns, justifying the observed anti-PD-L1 rapid onset. Interestingly, peripheral lymphocytes (PBLs) behave as infiltrating lymphocytes, suggesting that these PBLs might be considered as a possible biomarker for antibody activity. PMID:27764774

A novel monoclonal antibody-based enzyme-linked immunosorbent assay to determine luteinizing hormone in bovine plasma.

PubMed

Borromeo, V; Berrini, A; De Grandi, F; Cremonesi, F; Fiandanese, N; Pocar, P; Secchi, C

2014-07-01

The development of a novel enzyme-linked immunosorbent assay (ELISA) for determining luteinizing hormone (LH) in bovine plasma is described. Anti-bovine LH (bLH) monoclonal antibodies (mAbs) were produced and characterized. One mAb recognizing the bLH β subunit was used for immunoaffinity purification of substantial amounts of biologically active bLH from pituitary glands. The purified bLH in combination with 2 anti-bLH β subunit mAbs was used to develop a sandwich ELISA, which satisfied all the criteria required to investigate LH secretory patterns in the bovine species. The ELISA standard curve was linear over the range 0.05 to 2.5 ng/mL, and the assay proved suitable for measuring bLH in plasma without any prior treatment of samples. Cross-reactivity and recovery tests confirmed the specificity of the method. The intra- and inter-assay coefficients of variation ranged between 3.41% and 9.40%, and 9.29% and 15.84%, respectively. The analytical specificity of the method was validated in vivo by provocative tests for LH in heifers, using the LH releasing peptide gonadotropin-releasing hormone. In conclusion, the adoption of mAbs for this ELISA for coating the wells and labeling, combined with the easy one-step production of reference bLH, ensures long-term continuity in large-scale measurements of LH in the bovine species. Copyright © 2014 Elsevier Inc. All rights reserved.

Antibody-mediated immunotherapy of macaques chronically infected with SHIV suppresses viraemia

NASA Astrophysics Data System (ADS)

Shingai, Masashi; Nishimura, Yoshiaki; Klein, Florian; Mouquet, Hugo; Donau, Olivia K.; Plishka, Ronald; Buckler-White, Alicia; Seaman, Michael; Piatak, Michael; Lifson, Jeffrey D.; Dimitrov, Dimiter; Nussenzweig, Michel C.; Martin, Malcolm A.

2013-11-01

Neutralizing antibodies can confer immunity to primate lentiviruses by blocking infection in macaque models of AIDS. However, earlier studies of anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies administered to infected individuals or humanized mice reported poor control of virus replication and the rapid emergence of resistant variants. A new generation of anti-HIV-1 monoclonal antibodies, possessing extraordinary potency and breadth of neutralizing activity, has recently been isolated from infected individuals. These neutralizing antibodies target different regions of the HIV-1 envelope glycoprotein including the CD4-binding site, glycans located in the V1/V2, V3 and V4 regions, and the membrane proximal external region of gp41 (refs 9, 10, 11, 12, 13, 14). Here we have examined two of the new antibodies, directed to the CD4-binding site and the V3 region (3BNC117 and 10-1074, respectively), for their ability to block infection and suppress viraemia in macaques infected with the R5 tropic simian-human immunodeficiency virus (SHIV)-AD8, which emulates many of the pathogenic and immunogenic properties of HIV-1 during infections of rhesus macaques. Either antibody alone can potently block virus acquisition. When administered individually to recently infected macaques, the 10-1074 antibody caused a rapid decline in virus load to undetectable levels for 4-7days, followed by virus rebound during which neutralization-resistant variants became detectable. When administered together, a single treatment rapidly suppressed plasma viraemia for 3-5weeks in some long-term chronically SHIV-infected animals with low CD4+ T-cell levels. A second cycle of anti-HIV-1 monoclonal antibody therapy, administered to two previously treated animals, successfully controlled virus rebound. These results indicate that immunotherapy or a combination of immunotherapy plus conventional antiretroviral drugs might be useful as a treatment for chronically HIV-1-infected individuals experiencing immune dysfunction.

Immunoblotting validation of research antibodies generated against HS1-associated protein X-1 in the human neutrophil model cell line PLB-985.

PubMed

Cavnar, Peter; Inman, Kristina

2015-01-01

HS1-associated protein X-1 (Hax1) is a 35 kDa protein that is ubiquitously expressed. Hax1 is an anti-apoptotic protein with additional roles in cell motility, and autosomal recessive loss of Hax1 results in Kostmann syndrome, a form of severe congenital neutropenia. Because of the important role of Hax1 in neutrophils we demonstrate here validation of two commercially available research antibodies directed against human Hax1 in the human myeloid leukemia cell line PLB-985 cells. We show that both the mouse anti-Hax1 monoclonal IgG directed against amino acids 10-148 of Hax1 and a rabbit anti-Hax1 polyclonal IgG antibody directed against full-length Hax1 reliably and consistently detect Hax1 during immunoblotting of three different PLB-985 cell densities. Using shRNA mediated Hax1 knockdown, we demonstrate the specificity of both Hax1 antibodies. In addition, our results suggest that the rabbit anti-Hax1 polyclonal antibody provides a stronger intensity in detecting Hax1 protein, with detection in as few as 0.1 x 10 (6) cells in 6 total replicates we have performed.

Generation of a Canine Anti-EGFR (ErbB-1) Antibody for Passive Immunotherapy in Dog Cancer Patients

PubMed Central

Wang, Wei; Weichselbaumer, Marlene; Matz, Miroslawa; Mader, Alexander; Steinfellner, Willibald; Meitz, Sarah; Mechtcheriakova, Diana; Sobanov, Yuri; Willmann, Michael; Stockner, Thomas; Spillner, Edzard; Kunert, Renate; Jensen-Jarolim, Erika

2014-01-01

Passive immunotherapy with monoclonal antibodies represents a cornerstone of human anticancer therapies, but has not been established in veterinary medicine yet. As the tumor-associated antigen EGFR (ErbB-1) is highly conserved between humans and dogs, and considering the effectiveness of the anti-EGFR antibody cetuximab in human clinical oncology, we present here a “caninized” version of this antibody, can225IgG, for comparative oncology studies. Variable region genes of 225, the murine precursor of cetuximab, were fused with canine constant heavy gamma and kappa chain genes, respectively, and transfected into Chinese hamster ovary (CHO) DUKX-B11 cells. Of note, 480 clones were screened and the best clones were selected according to productivity and highest specificity in EGFR-coated ELISA. Upon purification with Protein G, the recombinant cetuximab-like canine IgG was tested for integrity, correct assembly, and functionality. Specific binding to the surface of EGFR-overexpressing cells was assessed by flow cytometry and immunofluorescence; moreover, binding to canine mammary tissue was demonstrated by immunohistochemistry. In cell viability and proliferation assays, incubation with can225IgG led to significant tumor cell growth inhibition. Moreover, this antibody mediated significant tumor cell killing via phagocytosis in vitro. We thus present here, for the first time, the generation of a canine IgG antibody and its hypothetical structure. On the basis of its cetuximab-like binding site, on the one hand, and the expression of a 91% homologous EGFR molecule in canine cancer, on the other hand, this antibody may be a promising research compound to establish passive immunotherapy in dog patients with cancer. PMID:24755200

Polyclonal and monoclonal antibodies in clinic.

PubMed

Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

2014-01-01

Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic.

Anti-PD-L1/TGFβR2 (M7824) fusion protein induces immunogenic modulation of human urothelial carcinoma cell lines, rendering them more susceptible to immune-mediated recognition and lysis.

PubMed

Grenga, Italia; Donahue, Renee N; Gargulak, Morgan L; Lepone, Lauren M; Roselli, Mario; Bilusic, Marijo; Schlom, Jeffrey

2018-03-01

Avelumab has recently been approved by the Food and Drug Administration for the therapy of Merkel cell carcinoma and urothelial carcinoma. M7824 is a novel first-in-class bifunctional fusion protein comprising a monoclonal antibody against programmed death-ligand 1 (PD-L1, avelumab), fused to the extracellular domain of human transforming growth factor beta (TGFβ) receptor 2, which functions as a TGFβ "trap." Advanced urothelial tumors have been shown to express TGFβ, which possesses immunosuppressive properties that promote cancer progression and metastasis. The rationale for a combined molecule is to block the PD-1/PD-L1 interaction between tumor cells and immune cell infiltrate and simultaneously reduce or eliminate TGFβ from the tumor microenvironment. In this study, we explored the effect of M7824 on invasive urothelial carcinoma cell lines. Human urothelial (transitional cell) carcinoma cell lines HTB-4, HTB-1, and HTB-5 were treated with M7824, M7824mut (M7824 that is mutated in the anti-PD-L1 portion of the molecule and thus does not bind PD-L1), anti-PD-L1 (avelumab), or IgG1 isotype control monoclonal antibody, and were assessed for gene expression, cell-surface phenotype, and sensitivity to lysis by TRAIL, antigen-specific cytotoxic T lymphocytes and natural killer cells. M7824 retains the ability to mediate antibody-dependent cellular cytotoxicity of tumor cells, although in some cases to a lesser extent than anti-PD-L1. However, compared to anti-PD-L1, M7824 increases (A) gene expression of molecules involved in T-cell trafficking in the tumor (e.g., CXCL11), (B) TRAIL-mediated tumor cell lysis, and (C) antigen-specific CD8 + T-cell-mediated lysis of tumor cells. These studies demonstrate the immunomodulatory properties of M7824 on both tumor cell phenotype and immune-mediated lysis. Compared to anti-PD-L1 or M7824mut, M7824 induces immunogenic modulation of urothelial carcinoma cell lines, rendering them more susceptible to immune-mediated recognition and lysis. These findings show the relevance of the dual blockade of PD-L1 and TGFβ in urothelial carcinoma cell lines and thus support the rationale for future clinical studies of M7824 in patients with urothelial cancer. Published by Elsevier Inc.

Fiber Optic Immunochemical Sensors For Continuous Monitoring Of Hapten Concentrations

NASA Astrophysics Data System (ADS)

Miller, W. Greg; Anderson, F. Philip

1989-06-01

We describe a fiber optic sensor based on a homogeneous fluorescence energy transfer immunoassay which operates in a continuous, reversible manner to quantitate the anticonvulsant drug phenytoin. B-phycoerythrin-phenytoin and Texas Red labeled anti-phenytoin antibody were sealed inside a short length of cellulose dialysis tubing which was cemented to the distal end of an optical fiber. When the sensor was placed into a solution of phenytoin, the drug crossed the dialysis membrane, displaced a fraction of the B-phycoerythrin-phenytoin from the antibody, and produced a change in fluorescence signal which was measured with a fiber optic fluorometer. The sensor had a concentration response of 5 to 500μmo1/L phenytoin with a response time of 5 to 15 min and precision of <2.5% CV. The chemical kinetics of the antibody-hapten indicator reaction were modeled mathematically and simulation showed that response time in the minutes range can be achieved when the dissociation rate constant is greater than approximately 10-3 sec-1. The dissociation rate constant influences the time to reach equilibrium and the unbound P* concentration range available for instrumental measurement. The ratio of the labeled and unlabeled hapten dissociation rate constants influences the analyte concentration range to which the sensor will respond.

Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer.

PubMed

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Chang, Yao-Wen; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2017-12-01

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal, lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-119 (IgG 1 , kappa), and characterized its efficacy against pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. H 2 Mab-119 reacted with pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 in flow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for MIA PaCa-2, although H 2 Mab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical analyses with H 2 Mab-119 revealed sensitive and specific reactions against breast and colon cancers but did not react with pancreatic cancers, indicating that H 2 Mab-119 is useful for detecting HER2 overexpression in pancreatic cancers using flow cytometry and Western blot analyses.

Idiotypic analysis of antibodies to hen egg-white lysozyme (HEL). II. Distribution and frequency of occurrence of idiotypes shared by A/J anti-HEL antibody.

PubMed Central

Semma, M; Sakato, N; Fujio, H; Amano, T

1981-01-01

Anti-HEL Ab from one A/J mouse (No. a-4),Ida-4(HEL), was s.c. inoculated into rabbits to induce anti-idiotypic (anti-Id) sera. After absorption with normal A/J Ig, two anti-Id serra (R101 and R102) were obtained. The interaction between 125I-Id(a-4)(HEL) and anti-Id sera was completely inhibited by unlabelled Ida-4(HEL), but not by non-specific Ig. The anti-Id sera were used to investigate the distribution of A/J (No. a-4) anti-HEL idiotypes in sera obtained from HEL-immunized animals. Idiotypes shared by Ida-4(HEL) were also detected in the sera of five examined mouse strains, but not in any of the examined rat, guinea-pig, goat, and sheep sear. Our experiments suggest the presence of inter- as well as intrastrain idiotype in mice. However, cross-reactivity appeared to be weak. When R101 and R102, respectively, were the anti-Id sera used, the frequency of occurrence of Ida-4(HEL) in stain A wa 1/106 and 1/53; in other mouse strains it was 1/277 and 1/85. PMID:7461724

The two novel DLL4-targeting antibody-drug conjugates MvM03 and MGD03 show potent anti-tumour activity in breast cancer xenograft models.

PubMed

Wang, Shijing; Zhou, Rihong; Sun, Fumou; Li, Renjie; Wang, Min; Wu, Min

2017-11-28

The anti-human Delta-like 4 (DLL4) monoclonal antibody MMGZ01 has a high affinity to hrDLL4 and arrests the DLL4-mediated human umbilical vein endothelial cell (HUVEC) phenotype, promotes immature vessels, and effectively reduces breast cancer cell growth in vivo. To develop a much more effective therapy, we conjugated MMGZ01 with two small-molecule cytotoxic agents, i.e., monomethyl auristatin E (MMAE) and doxorubicin (DOX), with different linkers to generate antibody-drug conjugates (ADCs), i.e., MMGZ01-vc-MMAE (named MvM03) and MMGZ01-GMBS-DOX (named MGD03), that are more potent therapeutic agents than naked antibody therapeutic agents. The produced anti-DLL4 ADCs can be effectively directed against DLL4 and internalized. Then, the release of MMAE or DOX into the cytosol can induce G2/M or G0/G1 phase growth arrest and cell death through the induction of apoptosis. In vitro, MvM03 was highly potent and selective against DLL4 cell lines. The anti-DLL4 ADCs, particularly MvM03, showed more potent anti-tumour activity than Docetaxel, which is an inhibitor of the depolymerisation of microtubules, in two xenograft breast cancer tumour models. Our findings indicate that anti-DLL4 ADCs have promising potential as an effective therapy for breast cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

Anti-PD-1/PD-L1 antibodies in non-small cell lung cancer: the era of immunotherapy.

PubMed

Valecha, Gautam Kishore; Vennepureddy, Adarsh; Ibrahim, Uroosa; Safa, Firas; Samra, Bachar; Atallah, Jean Paul

2017-01-01

Advanced non-small cell lung cancer (NSCLC) has been conventionally treated with cytotoxic chemotherapy with short-lived responses and significant toxicities. Monoclonal antibodies to programmed death-1 receptor (PD-1) and programmed death ligand 1 (PD-L1) have shown tremendous promise in the treatment of advanced NSCLC in various clinical trials. Areas covered: In this article, we will review the outcomes of various trials of anti-PD-1/anti-PD-L1 antibodies in the treatment of NSCLC. We will also discuss their mechanism of action and toxicities. Expert commentary: Anti-PD-1/PD-L1 antibodies offer several advantages including significant antitumor activity, induction of long lasting responses, and favorable safety profile. Several trials are now being conducted to evaluate their efficacy as first line agents as well as in combination with other agents. More research is also needed to identify other biomarkers, in addition to PD-L1 expression, that could more reliably predict response to these drugs, and aid in better patient selection.

A survey of manufacturing and handling practices for monoclonal antibodies by pharmacy, nursing and medical personnel.

PubMed

Alexander, M; King, J; Lingaratnam, S; Byrne, J; MacMillan, K; Mollo, A; Kirsa, S; Green, M

2016-04-01

There is a paucity of data available to assess the occupational health and safety risk associated with exposure to monoclonal antibodies. Industry standards and published guidelines are conflicting or outdated. Guidelines offer contrary recommendations based on an array of methodological approaches. This survey aimed to describe current practices, beliefs and attitudes relating to the handling of monoclonal antibodies by Australian medical, nursing and pharmacy clinicians. An electronic survey was distributed between June and September 2013. Respondents were surveyed on three focus areas: institutional guideline availability and content, current practices and attitudes. Demographic data relating to respondent and primary place of practice were also collected. A total of 222 clinicians completed the survey, with representation from all targeted professional groups and from a variety of geographic locations. 92% of respondents reported that their institution prepared or administered monoclonal antibodies, with 87% specifically handling anti-cancer monoclonal antibodies. Monoclonal antibodies were mostly prepared onsite (84-90%) and mostly within pharmacy clean-rooms (75%) and using cytotoxic cabinets (61%). 43% of respondents reported access to institutional monoclonal antibody handling guidelines with risk reduction strategies including training and education (71%), spill and waste management (71%), procedures for transportation (57%) and restricted handling (50%). Nurses had a stronger preference towards pharmacy manufacturing than both doctors and pharmacists for a range of clinical scenarios. 95% of all respondents identified that professional or regulatory body guidelines are an important resource when considering handling practices. Monoclonal antibodies are most commonly handled according to cytotoxic drug standards and often in the absence of formal guidelines. © The Author(s) 2014.

Sensitivity of Breast Cancer Stem Cells to TRA-8 Anti-DR5 Monoclonal Antibody

DTIC Science & Technology

2013-02-01

1 AD_________________ Award Number: W81XWH-11-1-0151 TITLE: Sensitivity of Breast Cancer Stem Cells to TRA-8 Anti-DR5...Annual Summary 3. DATES COVERED 15-January-2012 – 14-January-2013 4. TITLE AND SUBTITLE Sensitivity of Breast Cancer Stem Cells to TRA-8 Anti-DR5...release; distribution unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Basal-like breast cancers (BLBCs) generally become resistant to

Generation of monoclonal pan-hemagglutinin antibodies for the quantification of multiple strains of influenza

PubMed Central

Zou, Wei; Marcil, Anne; Paquet, Eric; Gadoury, Christine; Jaentschke, Bozena; Li, Xuguang; Petiot, Emma; Durocher, Yves; Baardsnes, Jason; Rosa-Calatrava, Manuel; Ansorge, Sven; Kamen, Amine A.

2017-01-01

Vaccination is the most effective course of action to prevent influenza. About 150 million doses of influenza vaccines were distributed for the 2015–2016 season in the USA alone according to the Centers for Disease Control and Prevention. Vaccine dosage is calculated based on the concentration of hemagglutinin (HA), the main surface glycoprotein expressed by influenza which varies from strain to strain. Therefore yearly-updated strain-specific antibodies and calibrating antigens are required. Preparing these quantification reagents can take up to three months and significantly slows down the release of new vaccine lots. Therefore, to circumvent the need for strain-specific sera, two anti-HA monoclonal antibodies (mAbs) against a highly conserved sequence have been produced by immunizing mice with a novel peptide-conjugate. Immunoblots demonstrate that 40 strains of influenza encompassing HA subtypes H1 to H13, as well as B strains from the Yamagata and Victoria lineage were detected when the two mAbs are combined to from a pan-HA mAb cocktail. Quantification using this pan-HA mAbs cocktail was achieved in a dot blot assay and results correlated with concentrations measured in a hemagglutination assay with a coefficient of correlation of 0.80. A competitive ELISA was also optimised with purified viral-like particles. Regardless of the quantification method used, pan-HA antibodies can be employed to accelerate process development when strain-specific antibodies are not available, and represent a valuable tool in case of pandemics. These antibodies were also expressed in CHO cells to facilitate large-scale production using bioreactor technologies which might be required to meet industrial needs for quantification reagents. Finally, a simulation model was created to predict the binding affinity of the two anti-HA antibodies to the amino acids composing the highly conserved epitope; different probabilities of interaction between a given amino acid and the antibodies might explain the affinity of each antibody against different influenza strains. PMID:28662134

Safety and immunogenicity of inactivated varicella-zoster virus vaccine in adults with hematologic malignancies receiving treatment with anti-CD20 monoclonal antibodies.

PubMed

Parrino, Janie; McNeil, Shelly A; Lawrence, Steven J; Kimby, Eva; Pagnoni, Marco F; Stek, Jon E; Zhao, Yanli; Chan, Ivan S F; Kaplan, Susan S

2017-03-27

Immunocompromised patients can experience significant morbidity and occasional mortality from complications associated with herpes zoster (HZ), but live attenuated HZ vaccine is contraindicated for these patients. Inactivated zoster vaccine (ZV IN ) is in development for prevention of HZ in immunocompromised patients. However, there are limited data in the literature regarding the effect of anti-CD20 monoclonal antibodies on vaccine-related cell-mediated immune response. This study evaluated safety and immunogenicity of ZV IN in patients with hematologic malignancies (HM) receiving anti-CD20 monoclonal antibodies (alone or in combination chemotherapy regimens) and not likely to undergo hematopoietic cell transplant (HCT) (n=80). This was an open-label, single-arm, multicenter Phase I study (NCT01460719) of a 4-dose ZV IN regimen (∼30days between doses) in patients ⩾18years old. Blood samples were collected prior to dose 1 and 28days Postdose 4 to measure varicella zoster virus (VZV)-specific T-cell responses using interferon-γ enzyme-linked immunospot (IFN-γ ELISPOT). The primary hypothesis was that ZV IN would elicit significant VZV-specific immune responses at ∼28days Postdose 4, with a geometric fold rise (GMFR) >1.0. All vaccinated patients were evaluated for adverse events (AE) through 28days Postdose 4. ZV IN elicited a statistically significant VZV-specific immune response measured by IFN-γ ELISPOT at 28days Postdose 4 (GMFR=4.34 [90% CI:3.01, 6.24], p-value<0.001), meeting the pre-specified success criterion. Overall, 85% (68/80) of patients reported ⩾1 AE, 44% (35/80) reported ⩾1 injection-site AE, and 74% (59/80) reported ⩾1 systemic AE. The majority of systemic AEs were non-serious and considered unrelated to vaccination by the investigator. Frequencies of AEs did not increase with subsequent doses of vaccine. No recipient of ZV IN had rash polymerase chain reaction (PCR) positive for VZV vaccine strain. In adults with HM receiving anti-CD20 monoclonal antibodies, ZV IN was well-tolerated and elicited statistically significant VZV-specific T-cell responses ∼28days Postdose 4. CLINICALTRIALS.GOV identifier: NCT01460719. Copyright © 2017 Elsevier Ltd. All rights reserved.

A human monoclonal antibody drug and target discovery platform for B-cell chronic lymphocytic leukemia based on allogeneic hematopoietic stem cell transplantation and phage display.

PubMed

Baskar, Sivasubramanian; Suschak, Jessica M; Samija, Ivan; Srinivasan, Ramaprasad; Childs, Richard W; Pavletic, Steven Z; Bishop, Michael R; Rader, Christoph

2009-11-12

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only potentially curative treatment available for patients with B-cell chronic lymphocytic leukemia (B-CLL). Here, we show that post-alloHSCT antibody repertoires can be mined for the discovery of fully human monoclonal antibodies to B-CLL cell-surface antigens. Sera collected from B-CLL patients at defined times after alloHSCT showed selective binding to primary B-CLL cells. Pre-alloHSCT sera, donor sera, and control sera were negative. To identify post-alloHSCT serum antibodies and subsequently B-CLL cell-surface antigens they recognize, we generated a human antibody-binding fragment (Fab) library from post-alloHSCT peripheral blood mononuclear cells and selected it on primary B-CLL cells by phage display. A panel of Fab with B-CLL cell-surface reactivity was strongly enriched. Selection was dominated by highly homologous Fab predicted to bind the same antigen. One Fab was converted to immunoglobulin G1 and analyzed for reactivity with peripheral blood mononuclear cells from B-CLL patients and healthy volunteers. Cell-surface antigen expression was restricted to primary B cells and up-regulated in primary B-CLL cells. Mining post-alloHSCT antibody repertoires offers a novel route to discover fully human monoclonal antibodies and identify antigens of potential therapeutic relevance to B-CLL and possibly other cancers. Trials described herein were registered at www.clinicaltrials.gov as nos. NCT00055744 and NCT00003838.

Development and evaluation of an anti-rabies virus phosphoprotein-specific monoclonal antibody for detection of rabies neutralizing antibodies using RFFIT

PubMed Central

Chun, Byung Chul; Lee, Yeong Seon; Hwang, Kyu Jam; Yang, Dong-Kun

2017-01-01

Background Rabies is a major public health problem with a fatality rate close to 100%; however, complete prevention can be achieved through pre- or post-exposure prophylaxis. The rapid fluorescent focus inhibition test (RFFIT) is one of the recommended testing methods to determine the production of neutralizing antibodies after vaccination. Here, we report the development of a new monoclonal antibody (mAb) designed to react specifically with Rabies virus (RABV) phosphoprotein (P protein), and the evaluation of its applicability to the RFFIT and its effectiveness as a diagnostic reagent for human rabies. Methodology/principal findings The mAb KGH P 16B8 was produced to target the P protein of the Korean KGH RABV strain. An indirect immunofluorescence assay (IFA) was conducted to detect various strains of RABV in various cell lines. Alexa-conjugated KGH P 16B8 (16B8-Alexa) was developed for the RFFIT. The IFA test could detect RABV up to a 1:2,500 dilution, with a detection limit comparable to that of a commercial diagnostic reagent. The sensitivity, specificity, positive predictive value, and negative predictive value of the RFFIT using 16B8-Alexa in 414 clinical specimens were 98.67%, 99.47%, 99.55%, and 98.42%, respectively. The results of the RFFIT with 16B8-Alexa were strongly correlated with those obtained using an existing commercial diagnostic reagent (r = 0.995, p<0.001). Conclusions/significance The mAb developed in this study shows high sensitivity and specificity, confirming its clinical utility with the RFFIT to measure the rabies neutralizing antibody titer and establish a diagnosis in human. Thus, 16B8-Alexa is expected to serve as an alternative diagnostic reagent that is widely accessible, with potentially broad applications beyond those of the RFFIT in Korea. Further studies with 16B8-Alexa should provide insight into the immunological mechanism of the P protein of Korean RABV. PMID:29267277

Development and evaluation of an anti-rabies virus phosphoprotein-specific monoclonal antibody for detection of rabies neutralizing antibodies using RFFIT.

PubMed

Um, Jihye; Chun, Byung Chul; Lee, Yeong Seon; Hwang, Kyu Jam; Yang, Dong-Kun; Park, Jun-Sun; Kim, Su Yeon

2017-12-01

Rabies is a major public health problem with a fatality rate close to 100%; however, complete prevention can be achieved through pre- or post-exposure prophylaxis. The rapid fluorescent focus inhibition test (RFFIT) is one of the recommended testing methods to determine the production of neutralizing antibodies after vaccination. Here, we report the development of a new monoclonal antibody (mAb) designed to react specifically with Rabies virus (RABV) phosphoprotein (P protein), and the evaluation of its applicability to the RFFIT and its effectiveness as a diagnostic reagent for human rabies. The mAb KGH P 16B8 was produced to target the P protein of the Korean KGH RABV strain. An indirect immunofluorescence assay (IFA) was conducted to detect various strains of RABV in various cell lines. Alexa-conjugated KGH P 16B8 (16B8-Alexa) was developed for the RFFIT. The IFA test could detect RABV up to a 1:2,500 dilution, with a detection limit comparable to that of a commercial diagnostic reagent. The sensitivity, specificity, positive predictive value, and negative predictive value of the RFFIT using 16B8-Alexa in 414 clinical specimens were 98.67%, 99.47%, 99.55%, and 98.42%, respectively. The results of the RFFIT with 16B8-Alexa were strongly correlated with those obtained using an existing commercial diagnostic reagent (r = 0.995, p<0.001). The mAb developed in this study shows high sensitivity and specificity, confirming its clinical utility with the RFFIT to measure the rabies neutralizing antibody titer and establish a diagnosis in human. Thus, 16B8-Alexa is expected to serve as an alternative diagnostic reagent that is widely accessible, with potentially broad applications beyond those of the RFFIT in Korea. Further studies with 16B8-Alexa should provide insight into the immunological mechanism of the P protein of Korean RABV.

Nuclear Chk1 prevents premature mitotic entry.

PubMed

Matsuyama, Makoto; Goto, Hidemasa; Kasahara, Kousuke; Kawakami, Yoshitaka; Nakanishi, Makoto; Kiyono, Tohru; Goshima, Naoki; Inagaki, Masaki

2011-07-01

Chk1 inhibits the premature activation of the cyclin-B1-Cdk1. However, it remains controversial whether Chk1 inhibits Cdk1 in the centrosome or in the nucleus before the G2-M transition. In this study, we examined the specificity of the mouse monoclonal anti-Chk1 antibody DCS-310, with which the centrosome was stained. Conditional Chk1 knockout in mouse embryonic fibroblasts reduced nuclear but not centrosomal staining with DCS-310. In Chk1(+/myc) human colon adenocarcinoma (DLD-1) cells, Chk1 was detected in the nucleus but not in the centrosome using an anti-Myc antibody. Through the combination of protein array and RNAi technologies, we identified Ccdc-151 as a protein that crossreacted with DCS-310 on the centrosome. Mitotic entry was delayed by expression of the Chk1 mutant that localized in the nucleus, although forced immobilization of Chk1 to the centrosome had little impact on the timing of mitotic entry. These results suggest that nuclear but not centrosomal Chk1 contributes to correct timing of mitotic entry.

Induction of neutralizing antibodies by a tobacco chloroplast-derived vaccine based on a B cell epitope from canine parvovirus.

PubMed

Molina, Andrea; Veramendi, Jon; Hervás-Stubbs, Sandra

2005-11-25

The 2L21 epitope of the VP2 protein from the canine parvovirus (CPV), fused to the cholera toxin B subunit (CTB-2L21), was expressed in transgenic tobacco chloroplasts. Mice and rabbits that received protein-enriched leaf extracts by parenteral route produced high titers of anti-2L21 antibodies able to recognize the VP2 protein. Rabbit sera were able to neutralize CPV in an in vitro infection assay with an efficacy similar to the anti-2L21 neutralizing monoclonal antibody 3C9. Anti-2L21 IgG and seric IgA antibodies were elicited when mice were gavaged with a suspension of pulverized tissues from CTB-2L21 transformed plants. Combined immunization (a single parenteral injection followed by oral boosters) shows that oral boosters help to maintain the anti-2L21 IgG response induced after a single injection, whereas parenteral administration of the antigen primes the subsequent oral boosters by promoting the induction of anti-2L21 seric IgA antibodies. Despite the induced humoral response, antibodies elicited by oral delivery did not show neutralizing capacity in the in vitro assay. The high yield of the fusion protein permits the preparation of a high number of vaccine doses from a single plant and makes feasible the oral vaccination using a small amount of crude plant material. However, a big effort has still to be done to enhance the protective efficacy of subunit vaccines by the oral route.

Durable antitumor responses to CD47 blockade require adaptive immune stimulation

PubMed Central

Sockolosky, Jonathan T.; Dougan, Michael; Ingram, Jessica R.; Ho, Chia Chi M.; Kauke, Monique J.; Almo, Steven C.; Ploegh, Hidde L.; Garcia, K. Christopher

2016-01-01

Therapeutic antitumor antibodies treat cancer by mobilizing both innate and adaptive immunity. CD47 is an antiphagocytic ligand exploited by tumor cells to blunt antibody effector functions by transmitting an inhibitory signal through its receptor signal regulatory protein alpha (SIRPα). Interference with the CD47–SIRPα interaction synergizes with tumor-specific monoclonal antibodies to eliminate human tumor xenografts by enhancing macrophage-mediated antibody-dependent cellular phagocytosis (ADCP), but synergy between CD47 blockade and ADCP has yet to be demonstrated in immunocompetent hosts. Here, we show that CD47 blockade alone or in combination with a tumor-specific antibody fails to generate antitumor immunity against syngeneic B16F10 tumors in mice. Durable tumor immunity required programmed death-ligand 1 (PD-L1) blockade in combination with an antitumor antibody, with incorporation of CD47 antagonism substantially improving response rates. Our results highlight an underappreciated contribution of the adaptive immune system to anti-CD47 adjuvant therapy and suggest that targeting both innate and adaptive immune checkpoints can potentiate the vaccinal effect of antitumor antibody therapy. PMID:27091975

LC-MS/MS strategies for therapeutic antibodies and investigation into the quantitative impact of antidrug-antibodies.

PubMed

Ewles, Matthew; Mannu, Ranbir; Fox, Chris; Stanta, Johannes; Evans, Graeme; Goodwin, Lee; Duffy, James; Bell, Len; Estdale, Sian; Firth, David

2016-12-01

We aimed to establish novel, high-throughput LC-MS/MS strategies for quantification of monoclonal antibodies in human serum and examine the potential impact of antidrug antibodies. We present two strategies using a thermally stable immobilized trypsin. The first strategy uses whole serum digestion and the second introduces Protein G enrichment to improve the selectivity. The impact of anti-trastuzumab antibodies on the methods was tested. Whole serum digestion has been validated for trastuzumab (LLOQ 0.25 µg/ml). Protein G enrichment has been validated for trastuzumab (LLOQ 0.1 µg/ml), bevacizumab (LLOQ 0.1 µg/ml) and adalimumab (LLOQ 0.25 µg/ml). We have shown the potential for anti-drug antibodies to impact on the quantification and we have subsequently established a strategy to overcome this impact where total quantification is desired.

Antibody treatment of human tumor xenografts elicits active anti-tumor immunity in nude mice

PubMed Central

Liebman, Meredith A.; Roche, Marly I.; Williams, Brent R.; Kim, Jae; Pageau, Steven C.; Sharon, Jacqueline

2007-01-01

Athymic nude mice bearing subcutaneous tumor xenografts of the human anti-colorectal cancer cell line SW480 were used as a preclinical model to explore anti-tumor immunotherapies. Intratumor or systemic treatment of the mice with murine anti-SW480 serum, recombinant anti-SW480 polyclonal antibodies, or the anti-colorectal cancer monoclonal antibody CO17-1A, caused retardation or regression of SW480 tumor xenografts. Interestingly, when mice that had regressed their tumors were re-challenged with SW480 cells, these mice regressed the new tumors without further antibody treatment. Adoptive transfer of spleen cells from mice that had regressed their tumors conferred anti-tumor immunity to naïve nude mice. Pilot experiments suggest that the transferred anti-tumor immunity is mediated by T cells of both γδ and αβ lineages. These results demonstrate that passive anti-tumor immunotherapy can elicit active immunity and support a role for extra-thymic γδ and αβ T cells in tumor rejection. Implications for potential immunotherapies include injection of tumor nodules in cancer patients with anti-tumor antibodies to induce anti-tumor T cell immunity. PMID:17920694

Humanization of the anti-CD18 antibody 6.7: an unexpected effect of a framework residue in binding to antigen.

PubMed

Caldas, Cristina; Coelho, Verônica; Kalil, Jorge; Moro, Ana Maria; Maranhão, Andrea Q; Brígido, Marcelo M

2003-05-01

Humanization of monoclonal antibodies by complementary determinant region (CDR)-grafting has become a standard procedure to improve the clinical usage of animal antibodies. However, antibody humanization may result in loss of activity that has been attributed to structural constraints in the framework structure. In this paper, we report the complete humanization of the 6.7 anti-human CD18 monoclonal antibody in a scFv form. We used a germline-based approach to design a humanized VL gene fragment and expressed it together with a previously described humanized VH. The designed humanized VL has only 14 mutations compared to the closest human germline sequence. The resulting humanized scFv maintained the binding capacity and specificity to human CD18 expressed on the cell surface of peripheral blood mononuclear cells (PBMC), and showed the same pattern of staining T-lymphocytes sub-populations, in comparison to the original monoclonal antibody. We observed an unexpected effect of a conserved mouse-human framework position (L37) that hinders the binding of the humanized scFv to antigen. This paper reveals a new framework residue that interferes with paratope and antigen binding and also reinforces the germline approach as a successful strategy to humanize antibodies.

A SPR-based immunosensor for the detection of isoproturon.

PubMed

Gouzy, Marie-Françoise; Kess, Melanie; Krämer, Petra M

2009-02-15

The proof of principle of a reusable surface plasmon resonance (SPR)-based immunosensor for the monitoring of isoproturon (IPU), a selective and systemic herbicide, is presented. The detecting rat monoclonal anti-isoproturon antibody (mAb IOC 7E1) was reversibly immobilized through the use of a capture mouse anti-rat (kappa-chain) monoclonal antibody (mAb TIB 172), which was covalently immobilized on the sensor chip surface. Such strategy features a controlled binding of the captured detecting antibody as well as facilitates the surface regeneration. The capture of the anti-IPU mAb by the antibody (TIB 172) coated sensor surface could be carried out up to 120 times (immobilization/regeneration cycles) without any evidence of activity loss. With a high test midpoint and a low associated SPR signal, the direct detection format was shown to be unsuitable for the routine analysis of isoproturon. However, the limit of detection (LOD) could be easily enhanced by using a strategy based on a surface competition assay, which improved all immunosensor parameters. Moreover, the sensitivity and working range of the indirect format were found to be dependent on the surface density of the anti-IPU mAb IOC 7E1. As expected for competitive formats, the lowest surface coverage (0.5 ng/mm(2)) allowed a lower detection of the herbicide isoproturon with a calculated LOD of 0.1 microg/l, an IC(50) (50% inhibition) of 5.3+/-0.6 microg/l, and a working range (20-80% inhibition) of 1.3-16.3 microg/l.

Antibody neutralization of cell-surface gC1qR/HABP1/SF2-p32 prevents lamellipodia formation and tumorigenesis

PubMed Central

Kim, Beom-Chan; Hwang, Hyun-Jung; An, Hyoung-Tae; Lee, Hyun; Park, Jun-Sub; Hong, Jin; Ko, Jesang; Kim, Chungho; Lee, Jae-Seon; Ko, Young-Gyu

2016-01-01

We previously demonstrated that cell-surface gC1qR is a key regulator of lamellipodia formation and cancer metastasis. Here, we screened a monoclonal mouse antibody against gC1qR to prevent cell migration by neutralizing cell-surface gC1qR. The anti-gC1qR antibody prevented growth factor-stimulated lamellipodia formation, cell migration and focal adhesion kinase activation by inactivating receptor tyrosine kinases (RTKs) in various cancer cells such as A549, MDA-MB-231, MCF7 and HeLa cells. The antibody neutralization of cell-surface gC1qR also inhibited angiogenesis because the anti-gC1qR antibody prevented growth factor-stimulated RTK activation, lamellipodia formation, cell migration and tube formation in HUVEC. In addition, we found that A549 tumorigenesis was reduced in a xenograft mouse model by following the administration of the anti-gC1qR antibody. With these data, we can conclude that the antibody neutralization of cell-surface gC1qR could be a good therapeutic strategy for cancer treatment. PMID:27363031

Novel mechanism of antibodies to hepatitis B virus in blocking viral particle release from cells.

PubMed

Neumann, Avidan U; Phillips, Sandra; Levine, Idit; Ijaz, Samreen; Dahari, Harel; Eren, Rachel; Dagan, Shlomo; Naoumov, Nikolai V

2010-09-01

Antibodies are thought to exert antiviral activities by blocking viral entry into cells and/or accelerating viral clearance from circulation. In particular, antibodies to hepatitis B virus (HBV) surface antigen (HBsAg) confer protection, by binding circulating virus. Here, we used mathematical modeling to gain information about viral dynamics during and after single or multiple infusions of a combination of two human monoclonal anti-HBs (HepeX-B) antibodies in patients with chronic hepatitis B. The antibody HBV-17 recognizes a conformational epitope, whereas antibody HBV-19 recognizes a linear epitope on the HBsAg. The kinetic profiles of the decline of serum HBV DNA and HBsAg revealed partial blocking of virion release from infected cells as a new antiviral mechanism, in addition to acceleration of HBV clearance from the circulation. We then replicated this approach in vitro, using cells secreting HBsAg, and compared the prediction of the mathematical modeling obtained from the in vivo kinetics. In vitro, HepeX-B treatment of HBsAg-producing cells showed cellular uptake of antibodies, resulting in intracellular accumulation of viral particles. Blocking of HBsAg secretion also continued after HepeX-B was removed from the cell culture supernatants. These results identify a novel antiviral mechanism of antibodies to HBsAg (anti-HBs) involving prolonged blocking of the HBV and HBsAg subviral particles release from infected cells. This may have implications in designing new therapies for patients with chronic HBV infection and may also be relevant in other viral infections.

Population pharmacokinetic and pharmacodynamic analysis of BIIB023, an anti-TNF-like weak inducer of apoptosis (anti-TWEAK) monoclonal antibody.

PubMed

Galluppi, Gerald R; Wisniacki, Nicolas; Stebbins, Chris

2016-07-01

Tumour necrosis factor-like weak inducer of apoptosis (TWEAK) is implicated in the pathogenesis of lupus nephritis. This study evaluated the pharmacokinetics, using the population approach, and pharmacodynamics of BIIB023, an anti-TWEAK monoclonal antibody, in healthy Chinese, Japanese and Caucasian volunteers. In this single-dose, randomized, double-blind, phase 1 study of BIIB023 in healthy volunteers, BIIB023 was administered by intravenous infusion (3 or 20 mg kg(-1) ) on Day 1; follow-up occurred through Day 71. BIIB023 serum concentration was measured using a validated enzyme-linked immunosorbent assay; BIIB023 concentration-time data were subjected to noncompartmental analysis. Population pharmacokinetic analysis was performed using data from this study and a prior phase 1 study of BIIB023 in subjects with rheumatoid arthritis. Soluble TWEAK and BIIB023 complex were evaluated. There were no differences in BIIB023 pharmacokinetics requiring dose adjustment among the three ethnic groups or between healthy volunteers and arthritis patients. BIIB023 central compartment volume (3050 ml) and clearance (7.42 ml h(-1) ) were comparable to those observed for other monoclonal antibody drugs. BIIB023 serum exposure increased in a dose-dependent manner in all groups, but not in direct proportion to dose level; at concentrations below ~10 μg ml(-1) , nonlinear clearance was observed. Soluble TWEAK levels decreased to below the level of quantitation after BIIB023 treatment, with concomitant changes in BIIB023 complex levels. No clinically meaningful differences were observed in BIIB023 pharmacokinetic and pharmacodynamic properties in healthy Chinese, Japanese and Caucasian volunteers; pharmacodynamic measures suggested target engagement. TWEAK may be an attractive therapeutic target for lupus nephritis treatment. © 2016 The British Pharmacological Society.

Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

NASA Astrophysics Data System (ADS)

Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

1995-07-01

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

[Anti-M3 muscarinic acetylcholine receptor antibodies and Sjögren's syndrome].

PubMed

Tsuboi, Hiroto; Iizuka, Mana; Asashima, Hiromitsu; Sumida, Takayuki

2013-01-01

Sjögren's syndrome (SS) is an autoimmune disease that affects exocrine glands including salivary and lacrimal glands. It is characterized by lymphocytic infiltration into exocrine glands, leading to dry mouth and eyes. A number of auto-antibodies are detected in patients with SS. However, no SS-specific pathologic auto-antibodies have yet been found in this condition. M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva. It is reported that some patients with SS carried inhibitory auto-antibodies against M3R. To clarify the epitopes and function of anti-M3R antibodies in SS, we examined antibodies to the extracellular domains (N terminal region, the first, second, and third extracellular loop) of M3R by ELISA using synthesized peptide antigens encoding these domains in 42 SS and 42 healthy controls (HC). Titers and positivity of anti-M3R antibodies to every extracellular domain of M3R were significantly higher in SS than in HC. Our results indicated the presence of several B cell epitopes on M3R in SS. Moreover, we analyzed the functions of anti-M3R antibodies by Ca(2+)-influx assays using a human salivary gland (HSG) cell line. The functional analysis indicated that the influence of such anti-M3R antibodies on Ca(2+)-influx in HSG cells might differ based on the epitopes to which they bind. Interestingly, both IgG from anti-M3R antibodies to the second extracellular loop positive SS and anti-M3R monoclonal antibodies against the second extracellular loop of M3R, which we generated, suppressed Ca(2+)-influx in the HSG cells induced by cevimeline stimulation. These observations suggested that auto-antibodies against the second extracellular loop of M3R could be involved in salivary dysfunction in patients with SS. These results indicated the presence of several B cell epitopes on M3R in SS and the influence of anti-M3R antibodies on salivary secretion might differ based on these epitopes. Thus, anti-M3R antibodies could be not only potential pathologic auto-antibodies, but also new diagnostic makers and therapeutic targets for SS.

Humanized Antibodies for Antiviral Therapy

NASA Astrophysics Data System (ADS)

Co, Man Sung; Deschamps, Marguerite; Whitley, Richard J.; Queen, Cary

1991-04-01

Antibody therapy holds great promise for the treatment of cancer, autoimmune disorders, and viral infections. Murine monoclonal antibodies are relatively easy to produce but are severely restricted for therapeutic use by their immunogenicity in humans. Production of human monoclonal antibodies has been problematic. Humanized antibodies can be generated by introducing the six hypervariable regions from the heavy and light chains of a murine antibody into a human framework sequence and combining it with human constant regions. We humanized, with the aid of computer modeling, two murine monoclonal antibodies against herpes simplex virus gB and gD glycoproteins. The binding, virus neutralization, and cell protection results all indicate that both humanized antibodies have retained the binding activities and the biological properties of the murine monoclonal antibodies.

A human recombinant monoclonal antibody to cocaine: Preparation, characterization and behavioral studies

PubMed Central

Eubanks, Lisa M.; Ellis, Beverly A.; Cai, Xiaoqing; Schlosburg, Joel E.; Janda, Kim D.

2014-01-01

Cocaine abuse remains prevalent worldwide and continues to be a major health concern; nonetheless, there is no effective therapy. Immunopharmacothery has emerged as a promising treatment strategy by which anti-cocaine antibodies bind to the drug blunting its effects. Previous passive immunization studies using our human monoclonal antibody, GNCgzk, resulted in protection against cocaine overdose and acute toxicity. To further realize the clinical potential of this antibody, a recombinant IgG form of the antibody has been produced in mammalian cells. This antibody displayed a high binding affinity for cocaine (low nanomolar) in line with the superior attributes of the GNCgzk antibody and reduced cocaine-induced ataxia in a cocaine overdose model. PMID:25205191

Effect of monoclonal antibodies against lipoteichoic acid from the oral bacterium Streptococcus mutans on its adhesion and plaque-accumulation in vitro.

PubMed

Stashenko, P; Peros, W J; Gibbons, R J; Dearborn, S M

1986-01-01

Five monoclonal antibodies directed against Streptococcus mutans strain JBP lipoteichoic acid (LTA) were characterized. They were all similarly reactive with the immunizing LTA-containing extract, with intact Strep. mutans JBP cells and with LTA purified from Lactobacillus casei. Immobilized anti-LTA antibodies removes LTA from LTA-containing extracts. The binding of antibodies to LTA was inhibited by the aqueous extract but not by the organic extract of de-acylated LTA, indicating reactivity with the polyglycerol-phosphate portion of the molecule. Antibodies were reactive with all serotypes of Strep. mutans, as well as with strains of Streptococcus salivarius, Streptococcus sanguis and L. casei, but not with LTA-negative species Streptococcus mitis or Actinomyces viscosus. Anti-LTA antibodies at doses of 0.3 or 3.0 micrograms/ml, had no effect on the adherence of Strep. mutans JBP to experimental salivary pellicles formed on hydroxyapatite, but enhanced adherence 150-300 per cent at 30 micrograms/ml. There was no effect of anti-LTA antibodies in a chemostat model which measured sucrose-dependent plaque accumulation by Strep. mutans. The results argue against a major role for LTA in Strep. mutans adherence or plaque accumulation in vitro.

From rabbit antibody repertoires to rabbit monoclonal antibodies.

PubMed

Weber, Justus; Peng, Haiyong; Rader, Christoph

2017-03-24

In this review, we explain why and how rabbit monoclonal antibodies have become outstanding reagents for laboratory research and increasingly for diagnostic and therapeutic applications. Starting with the unique ontogeny of rabbit B cells that affords highly distinctive antibody repertoires rich in in vivo pruned binders of high diversity, affinity and specificity, we describe the generation of rabbit monoclonal antibodies by hybridoma technology, phage display and alternative methods, along with an account of successful humanization strategies.

Generation of anti-idiotype scFv for pharmacokinetic measurement in lymphoma patients treated with chimera anti-CD22 antibody SM03.

PubMed

Zhao, Qi; Wong, Pui-Fan; Lee, Susanna S T; Leung, Shui-On; Cheung, Wing-Tai; Wang, Jun-Zhi

2014-01-01

Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.

Generation of Anti-Idiotype scFv for Pharmacokinetic Measurement in Lymphoma Patients Treated with Chimera Anti-CD22 Antibody SM03

PubMed Central

Zhao, Qi; Wong, Pui-Fan; Lee, Susanna S. T.; Leung, Shui-On; Cheung, Wing-Tai; Wang, Jun-Zhi

2014-01-01

Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen. PMID:24816427

Determination of trace amount of cyanobacterial toxin in water by microchip based enzyme-linked immunosorbent assay.

PubMed

Pyo, Dongjin; Hahn, Jong Hoon

2009-01-01

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is usually lower than the detection limits. As a more sensitive detection method for microcystin, we developed a microchip based enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies. New monoclonal antibodies against the microcystin leucine-arginine variant (MCLR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. We used keyhole limpet hemocyanin(KLH)-conjugated MCLR as an immunogen for the production of mouse monoclonal antibody. The immunization, cell fusion, and screening of hybridoma cells producing anti-MCLR antibody were conducted. Since the ELISA test was highly sensitive, the newly developed microchip based ELISA can be suitable for the trace analysis of cyanobacterial hepatotoxins, microcystins in water. The linear responses of monoclonal antibodies with different concentrations of microcystin LR were established between 0.025 and 0.3 ng/mL.

Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by XMetA, an allosteric partial agonist antibody

USDA-ARS?s Scientific Manuscript database

XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by xmeta, an allosteric partial agonist antibody

USDA-ARS?s Scientific Manuscript database

XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

B cell epitopes on infliximab identified by oligopeptide microarray with unprocessed patient sera.

PubMed

Homann, Arne; Röckendorf, Niels; Kromminga, Arno; Frey, Andreas; Jappe, Uta

2015-10-29

Autoimmune diseases like rheumatoid arthritis and inflammatory bowel disease are treated with TNF-alpha-blocking antibodies such as infliximab and adalimumab. A common side effect of therapeutic antibodies is the induction of anti-drug antibodies, which may reduce therapeutic efficacy. In order to reveal immunogenic epitopes on infliximab which are responsible for the adverse effects, sera from patients treated with infliximab were screened by ELISA for anti-infliximab antibodies. Sera containing high levels of anti-drug-antibodies (>1.25 µg/ml) were analyzed in an oligopeptide microarray system containing immobilized 15-meric oligopeptides from the infliximab amino acid sequence. Immunogenic infliximab IgG-epitopes were identified by infrared fluorescence scanning and comparison of infliximab-treated patients versus untreated controls. Six relevant epitopes on infliximab were recognized by the majority of all patient sera: 4 in the variable and 2 in the constant region. Three of the epitopes in the variable region are located in the TNF-alpha binding region of infliximab. The fourth epitope of the variable part of infliximab is located close to the TNF-alpha binding region and contains an N-glycosylation sequon. The sera positive for anti-infliximab antibodies do not contain antibodies against adalimumab as determined by ELISA. Thus, there is no infliximab-adalimumab cross-reactivity as determined by these systems. Our data shall contribute to a knowledge-based recommendation for a potentially necessary therapy switch from infliximab to another type of TNF-alpha-blocker. The characterization of immunogenic epitopes on therapeutic monoclonal antibodies using unprocessed patient sera shall lead to direct translational aspects for the development of less immunogenic therapeutic antibodies. Patients benefit from less adverse events and longer lasting drug effects.

Antigenic Properties of the HIV Envelope on Virions in Solution

PubMed Central

Mengistu, Meron; Lewis, George K.; Lakowicz, Joseph R.

2014-01-01

The structural flexibility found in human immunodeficiency virus (HIV) envelope glycoproteins creates a complex relationship between antigenicity and sensitivity to antiviral antibodies. The study of this issue in the context of viral particles is particularly problematic as conventional virus capture approaches can perturb antigenicity profiles. Here, we employed a unique analytical system based on fluorescence correlation spectroscopy (FCS), which measures antibody-virion binding with all reactants continuously in solution. Panels of nine anti-envelope monoclonal antibodies (MAbs) and five virus types were used to connect antibody binding profiles with neutralizing activities. Anti-gp120 MAbs against the 2G12 or b12 epitope, which marks functional envelope structures, neutralized viruses expressing CCR5-tropic envelopes and exhibited efficient virion binding in solution. MAbs against CD4-induced (CD4i) epitopes considered hidden on functional envelope structures poorly bound these viruses and were not neutralizing. Anti-gp41 MAb 2F5 was neutralizing despite limited virion binding. Similar antigenicity patterns occurred on CXCR4-tropic viruses, except that anti-CD4i MAbs 17b and 19e were neutralizing despite little or no virion binding. Notably, anti-gp120 MAb PG9 and anti-gp41 MAb F240 bound to both CCR5-tropic and CXCR4-tropic viruses without exerting neutralizing activity. Differences in the virus production system altered the binding efficiencies of some antibodies but did not enhance antigenicity of aberrant gp120 structures. Of all viruses tested, only JRFL pseudoviruses showed a direct relationship between MAb binding efficiency and neutralizing potency. Collectively, these data indicate that the antigenic profiles of free HIV particles generally favor the exposure of functional over aberrant gp120 structures. However, the efficiency of virion-antibody interactions in solution inconsistently predicts neutralizing activity in vitro. PMID:24284318

Evaluation of oriented lysozyme immobilized with monoclonal antibody

NASA Astrophysics Data System (ADS)

Aoyagi, Satoka; Okada, Keigo; Shigyo, Ayako; Man, Naoki; Karen, Akiya

2008-12-01

The orientation of a lysozyme immobilized with a monoclonal antibody was evaluated based on determination of the uppermost surface structure using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Specific peaks of the oriented lysozyme immobilized with monoclonal anti-lysozyme antibody were obtained in comparison with reference samples, non-oriented immobilized lysozyme and immobilized anti-lysozyme antibody. All samples were freeze-dried before TOF-SIMS measurement, and then each sample was measured using TOF-SIMS with a bismuth cluster ion source. TOF-SIMS spectra were analyzed to select peaks specific to the oriented immobilized lysozyme as well as to identify their chemical formula and ensemble of amino acids. The possible chemical formulae of the lysozyme fragments were then investigated with an element matching program and a residue matching program. The results from TOF-SIMS spectra analysis were compared to the amino acid sequence of the lysozyme and its three-dimensional structure registered in the protein data bank. Finally, the fragment-ion-generating regions of the oriented immobilized lysozyme were determined based on the suggested residues and the three-dimensional structure.

A 1H NMR method for the analysis of antigen-antibody interactions: binding of a peptide fragment of lysozyme to anti-lysozyme monoclonal antibody.

PubMed

Ito, W; Nishimura, M; Sakato, N; Fujio, H; Arata, Y

1987-09-01

A proton nuclear magnetic resonance (NMR) study is reported of the molecular structural basis of antigen-antibody interactions. An immunologically reactive proteolytic fragment corresponding to one of the antigenic regions on hen egg-white lysozyme (HEL) was used in combination with a monoclonal antibody that recognizes this site. Using spin diffusion, we prepared an antibody in which the magnetization of the antigen binding site was saturated by non-specific nuclear Overhauser effect. Under these conditions the effect of the saturation of the antibody was observed to spread over the peptide fragment through the antigen binding site. On the basis of the results obtained for the intermolecular nuclear Overhauser effect, we discuss how the peptide fragment interacts with the antibody. The side chains of aromatic residues, Trp, Tyr, and His, and of ionic residues, especially Arg, Lys, and Glu, are suggested to be important in the antigen-antibody interaction.

The Fc and not CD4 Receptor Mediates Antibody Enhancement of HIV Infection in Human Cells

NASA Astrophysics Data System (ADS)

Homsy, Jacques; Meyer, Mia; Tateno, Masatoshi; Clarkson, Sarah; Levy, Jay A.

1989-06-01

Antibodies that enhance human immunodeficiency virus (HIV) infectivity have been found in the blood of infected individuals and in infected or immunized animals. These findings raise serious concern for the development of a safe vaccine against acquired immunodeficiency syndrome. To address the in vivo relevance and mechanism of this phenomenon, antibody-dependent enhancement of HIV infectivity in peripheral blood macrophages, lymphocytes, and human fibroblastoid cells was studied. Neither Leu3a, a monoclonal antibody directed against the CD4 receptor, nor soluble recombinant CD4 even at high concentrations prevented this enhancement. The addition of monoclonal antibody to the Fc receptor III (anti-FcRIII), but not of antibodies that react with FcRI or FcRII, inhibited HIV type 1 and HIV type 2 enhancement in peripheral blood macrophages. Although enhancement of HIV infection in CD4+ lymphocytes could not be blocked by anti-FcRIII, it was inhibited by the addition of human immunoglobulin G aggregates. The results indicate that the FcRIII receptor on human macrophages and possibly another Fc receptor on human CD4+ lymphocytes mediate antibody-dependent enhancement of HIV infectivity and that this phenomenon proceeds through a mechanism independent of the CD4 protein.

Construction of Rabbit Immune Antibody Libraries.

PubMed

Nguyen, Thi Thu Ha; Lee, Jong Seo; Shim, Hyunbo

2018-01-01

Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice, and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage-display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from nonmurine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.

Thymic hormone-containing cells. Characterization and localization of serum thymic factor in young mouse thymus studied by monoclonal antibodies

PubMed Central

1982-01-01

The characterization and distribution of cells containing the serum thymic factor (FTS) in the thymus of young mice was studied by immunofluorescence using monoclonal anti-FTS antibodies. FTS+ cells were distributed throughout the thymic parenchyma but were more frequent in the medullary region than in the cortex. FTS-containing cells presented a stellate or globular aspect, and some of them exhibited fluorescent cytoplasmic granules. The epithelial nature of FTS+ cells was confirmed by double-labeling experiments using an anti- keratin antiserum (as an epithelial cell marker). Nevertheless, only a minority of keratin-positive epithelial reticular cells contained FTS. All controls, including the incubation of sections from nonthymic tissues with the anti-FTS antibodies, were negative. Taken together, these results confirm the exclusive localization of FTS-containing cells within the mouse thymus. PMID:7047671

Characterization of two anti-dengue human monoclonal antibodies prepared from PBMCs of patients with dengue illness in Thailand.

PubMed

Li, Z-Y; Yamashita, A; Kawashita, N; Sasaki, T; Pan, Y; Ono, K-I; Ikuta, K; Li, Y-G

2016-06-01

The global spread of the four dengue virus (DENV) serotypes (dengue-1 to -4) has made this virus a major and growing public health concern. Generally, pre-existing neutralizing antibodies derived from primary infection play a significant role in protecting against subsequent infection with the same serotype. By contrast, these pre-existing antibodies are believed to mediate a non-protective response to subsequent heterotypic DENV infections, leading to the onset of dengue illness. In this study, two monoclonal antibodies prepared by using peripheral blood mononuclear cells (PBMCs) from patients with dengue fever were characterized. Epitope mapping revealed that amino acid residues 254-278 in domain II of the viral envelope protein E were the target region of these antibodies. A database search revealed that certain sequences in this epitope region showed high conservation among the four serotypes of DENV. These two human monoclonal antibodies could neutralize DENV-2,-4 more effectively than DENV-1,-3. The amino acid sequences could not explain this difference in neutralizing activity. However, the 3D structure results showed that amino acid 274 could be the critical residue for the difference in neutralization. These results may provide basic information for the development of a dengue vaccine.

Generation of Murine Monoclonal Antibodies by Hybridoma Technology.

PubMed

Holzlöhner, Pamela; Hanack, Katja

2017-01-02

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

Protective effects of murine monoclonal antibodies in experimental septicemia: E. coli antibodies protect against different serotypes of E. coli.

PubMed

Salles, M F; Mandine, E; Zalisz, R; Guenounou, M; Smets, P

1989-04-01

Murine monoclonal antibodies that bind outer membrane antigens of the J5 mutant of Escherichia coli O111:B4 were derived from spleen cells of BALB/c mice immunized with killed whole cells and boosted with lipopolysaccharide (LPS) and LPS-associated proteins. Seven hybridomas were selected for their reactivity against the J5 LPS; they cross-reacted with O111, O55, O127, and O128 E. coli LPS. One (B7B3) also reacted with the Serratia marcescens LPS and Klebsiella pneumoniae lipid A. A protective effect was obtained with D6B4 antibody in a lethal endotoxemia model induced by LPS from O111, O127, and O128 E. coli serotypes in D-galactosamine-sensitized mice. D6B4 and D6B3 antibodies protected mice infected with E. coli O111:B4, when administered before infection. The D6B4 antibody was also protective when administered after infection. The antibodies D6B3 and D4B5 were protective in heterologous infection induced by E. coli O2:K1.

Detection of Circulating Tumor Cells in Hepatocellular Carcinoma Using Antibodies against Asialoglycoprotein Receptor, Carbamoyl Phosphate Synthetase 1 and Pan-Cytokeratin

PubMed Central

Zhang, Yu; Liu, Huiying; Sun, Bin; Zhao, Linlin; Ge, Naijian; Qian, Haihua; Yang, Yefa; Wu, Mengchao; Yin, Zhengfeng

2014-01-01

Background Asialoglycoprotein receptor (ASGPR)-ligand-based separation combined with identification with Hep Par 1 or pan-cytokeratin (P-CK) antibody have been demonstrated to detect circulating tumor cells (CTCs) in hepatocellular carcinoma (HCC). The aim of this study was to develop an improved enrichment and identification system that allows the detection of all types of HCC CTCs. Methods The specificity of the prepared anti-ASGPR monoclonal antibody was characterized. HCC cells were bound by ASGPR antibody and subsequently magnetically isolated by second antibody-coated magnetic beads. Isolated HCC cells were identified by immunofluorescence staining using a combination of anti-P-CK and anti-carbamoyl phosphate synthetase 1 (CPS1) antibodies. Blood samples spiked with HepG2 cells were used to determine recovery and sensitivity. CTCs were detected in blood samples from HCC patients and other patients. Results ASGPR was exclusively expressed in human hepatoma cell line, normal hepatocytes and HCC cells in tissue specimens detected by the ASGPR antibody staining. More HCC cells could be identified by the antibody cocktail for CPS1 and P-CK compared with a single antibody. The current approach obtained a higher recovery rate of HepG2 cells and more CTC detection from HCC patients than the previous method. Using the current method CTCs were detected in 89% of HCC patients and no CTCs were found in the other test subjects. Conclusions Our anti-ASGPR antibody could be used for specific and efficient HCC CTC enrichment, and anti-P-CK combined with anti-CPS1 antibodies is superior to identification with one antibody alone in the sensitivity for HCC CTC detection. PMID:24763545

B7-1 (CD80) as target for immunotoxin therapy for Hodgkin's disease.

PubMed Central

Vooijs, W. C.; Otten, H. G.; van Vliet, M.; van Dijk, A. J.; de Weger, R. A.; de Boer, M.; Bohlen, H.; Bolognesi, A.; Polito, L.; de Gast, G. C.

1997-01-01

In this preclinical study, the potential applicability of an anti-B7-1 immunotoxin (IT) for the treatment of Hodgkin's disease (HD) was investigated. Immunohistochemical analysis demonstrated strong expression of B7-1 on Hodgkin and Reed-Sternberg (R-S) cells and clear expression on dendritic cells, macrophages and some B-cells in tissues, but not on other tissue cells. Flow cytometric analysis demonstrated that B7-1 was expressed on a few monocytes, but not on CD34+ cells from bone marrow, resting T- or B-cells from peripheral blood or epithelial and endothelial cell lines. An anti-B7-1 immunotoxin containing the anti-B7-1 monoclonal antibody (MAb) B7-24 and saporin as toxin moiety was constructed and showed an affinity similar to that shown by the native MAb. It exhibited strong cytotoxicity against the B7-1+ B-cell line Raji (IC50 10(-11) M), R-S cell lines HDLM2, KM/H2 and L428 and also against a B7-1-transfected epithelial cell line, A431, whose parental line lacks expression of B7-1. In clonogenic assays with Raji cells or KM/H2 cells, a 3- or 4-log kill, respectively, was observed. No cytotoxicity was found against the B7-1- epithelial and endothelial cell lines or against haematopoietic progenitor cells. In conclusion, an anti-B7-1 immunotoxin was developed that had good cytotoxicity against R-S cell lines and that may be used in the elimination of R-S cells in vivo. A concomitant elimination of activated antigen-presenting cells may avoid development of antitoxin and anti-mouse Ig responses and allow repeated administration. Images Figure 1 PMID:9365164

A novel micro-emulsion and micelle assembling method to prepare DEC205 monoclonal antibody coupled cationic nanoliposomes for simulating exosomes to target dendritic cells.

PubMed

Li, Kexin; Chang, Shasha; Wang, Zhongyan; Zhao, Xiuli; Chen, Dawei

2015-08-01

Cationic biomimetic exosomes were prepared using a novel micro-emulsion and micelle assembling method by introducing DEC205 monoclonal antibody as specific ligand to target dendritic cells (DCs). The Box-Behnken experimental design was applied for optimization of nanoliposomes (NLip) and DEC205 monoclonal antibody was then conjugated on the surface of NLip (DEC205-NLip). NLip and DEC205-NLip respectively had an average size of 62.7 ± 6.33 nm and 81.64 ± 4.25 nm, zeta potential of +30.5 ± 2.3 mV and +19.8 ± 1.8 mV and encapsulation efficiency of 91.02 ± 3.1% and 93.10 ± 2.2%. In addition, the toxicity studies confirmed DEC205 monoclonal antibody could significantly reduce the cytotoxicity of the cationic lipid against DCs. And the cellular uptake experiment evaluated the significant targeting effect of the DEC205 monoclonal antibody on DC cells. In conclusion, the novel method presented here to prepare biomimetic exosomes was an efficient approach to develop antigen carriers for specific DCs targeting. Copyright © 2015 Elsevier B.V. All rights reserved.

Production of Autoantibodies by Murine B-1a Cells Stimulated with Helicobacter pylori Urease through Toll-Like Receptor 2 Signaling ▿ †

PubMed Central

Kobayashi, Fumiko; Watanabe, Eri; Nakagawa, Yohko; Yamanishi, Shingo; Norose, Yoshihiko; Fukunaga, Yoshitaka; Takahashi, Hidemi

2011-01-01

Helicobacter pylori infection is associated with several autoimmune diseases, in which autoantibody-producing B cells must be activated. Among these B cells, CD5-positive B-1a cells from BALB/c mice were confirmed to secrete autoantibodies when cocultured with purified H. pylori urease in the absence of T cells. To determine the mechanisms for autoantibody production, CD5-positive B-1a cells were sorted from murine spleen cells and stimulated with either purified H. pylori urease or H. pylori coated onto plates (referred to hereafter as plate-coated H. pylori), and autoantibody production was measured by enzyme-linked immunosorbent assay (ELISA). Complete urease was not secreted from H. pylori but was visually expressed over the bacterium-like endotoxin. Urease-positive plated-coated H. pylori stimulated B-1a cells to produce autoantibodies, although urease-deficient isotype-matched H. pylori did not. Autoantibody secretion by B-1a cells was inhibited when bacteria were pretreated with anti-H. pylori urease-specific antibody having neutralizing ability against urease enzymatic activity but not with anti-H. pylori urease-specific antibody without neutralizing capacity. The B-1a cells externally express various Toll-like receptors (TLRs): TLR1, TLR2, TLR4, and TLR6. Among the TLRs, blocking of TLR2 on B-1a cells with a specific monoclonal antibody (MAb), T2.5, inhibited autoantibody secretion when B-1a cells were stimulated with plate-coated H. pylori or H. pylori urease. Moreover, B-1a cells from TLR2-knockout mice did not produce those autoantibodies. The present study provides evidence that functional urease expressed on the surface of H. pylori will directly stimulate B-1a cells via innate TLR2 to produce various autoantibodies and may induce autoimmune disorders. PMID:21947775

In Vitro and In Vivo Characterization of a Fully Felinized Therapeutic Anti-Nerve Growth Factor Monoclonal Antibody for the Treatment of Pain in Cats.

PubMed

Gearing, D P; Huebner, M; Virtue, E R; Knight, K; Hansen, P; Lascelles, B D X; Gearing, R P; Drew, A C

2016-07-01

Limited options are available for the treatment of pain in cats. Monoclonal antibodies (mAbs) that neutralize nerve growth factor (NGF) have demonstrated analgesic capacity in rodent models, people with osteoarthritis, and dogs with degenerative joint disease. This study describes the design and characterization of a fully felinized anti-NGF monoclonal antibody. In vitro potency, pharmacokinetics, and the ability of the antibody to treat pain in a self-resolving, acute inflammation model were investigated in cats. Thirty-eight cats at a research colony at Charles River Laboratories, Ireland. Felinized anti-NGF mAb, NV-02, was produced using a complementary DNA (cDNA)-based method (PETization). Purified NV-02 was tested for affinity, potency, and immunoreactivity in vitro, then for safety and plasma pharmacokinetic distribution in vivo, and analgesic efficacy in a model of kaolin-induced inflammatory pain. Anti-NGF mAb, NV-02 neutralized NGF with high affinity and potency and did not bind complement. NV-02-administered SC had a plasma half-life of 7-15 days and was well tolerated at dosages up to 28 mg/kg. A dosage of 2 mg/kg NV-02 SC significantly decreased signs of lameness on day 2 (P = .0027), day 3 (P = .016), day 4, (P = .0063), day 5 (P = .0085), day 6 (P = .0014), and day 7 (P = .0034) after induction of inflammation. The high affinity, long plasma half-life, safety, and analgesic efficacy of felinized anti-NGF mAb (NV-02) support further investigation of the analgesic potential of this antibody in the cat. Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Rituximab used in three cases with relapsed non-Hodgkin’s lymphoma

PubMed Central

ELLI, MURAT; YILMAZ, SEMA; AYDIN, RAMAZAN; MURAT, SADRIYE; BILGICI, MELTEM CEYHAN; DAGDEMIR, AYHAN

2013-01-01

Relapsed or refractory B-cell non-Hodgkin’s lymphoma (B-NHL) patients have a poor prognosis. New treatment modalities have been used to improve survival rates in children with relapsed or refractory B-NHL. CD20 is expressed in >98% of childhood B-NHL and a chimeric anti-CD20 monoclonal antibody, rituximab, is increasingly being used at relapse. The aim of the present study was to determine the efficacy of rituximab on relapsed B-NHL. Three B-NHL cases were treated successfully with a combination of intensive chemotherapy protocol plus rituximab. PMID:24649209

Therapeutic preparations of IVIg contain naturally occurring anti-HLA-E antibodies that react with HLA-Ia (HLA-A/-B/-Cw) alleles.

PubMed

Ravindranath, Mepur H; Terasaki, Paul I; Pham, Tho; Jucaud, Vadim; Kawakita, Satoru

2013-03-14

The US Food and Drug Administration approved intravenous immunoglobulin (IVIg), extracted from the plasma of thousands of blood donors, for removing HLA antibodies (Abs) in highly sensitized patients awaiting organ transplants. Since the blood of healthy individuals has HLA Abs, we tested different IVIg preparations for reactivity to HLA single antigen Luminex beads. All preparations showed high levels of HLA-Ia and -Ib reactivity. Since normal nonalloimmunized males have natural antibodies to the heavy chains (HCs) of HLA antigens, the preparations were then tested against iBeads coated only with intact HLA antigens. All IVIg preparations varied in level of antibody reactivity to intact HLA antigens. We raised monoclonal Abs against HLA-E that mimicked IVIg's HLA-Ia and HLA-Ib reactivity but reacted only to HLA-I HCs. Inhibition experiments with synthetic peptides showed that HLA-E shares epitopes with HLA-Ia alleles. Importantly, depleting anti-HLA-E Abs from IVIg totally eliminated the HLA-Ia reactivity of IVIg. Since anti-HLA-E mAbs react with HLA-Ia, they might be useful in suppressing HLA antibody production, similar to the way anti-RhD Abs suppress production. At the same time, anti-HLA-E mAb, which reacts only to HLA-I HCs, is unlikely to produce transfusion-related acute lung injury, in contrast to antibodies reacting to intact-HLA.

Impact of Depleting Therapeutic Monoclonal Antibodies on the Host Adaptive Immunity: A Bonus or a Malus?

PubMed Central

Deligne, Claire; Milcent, Benoît; Josseaume, Nathalie; Teillaud, Jean-Luc; Sibéril, Sophie

2017-01-01

Clinical responses to anti-tumor monoclonal antibody (mAb) treatment have been regarded for many years only as a consequence of the ability of mAbs to destroy tumor cells by innate immune effector mechanisms. More recently, it has also been shown that anti-tumor antibodies can induce a long-lasting anti-tumor adaptive immunity, likely responsible for durable clinical responses, a phenomenon that has been termed the vaccinal effect of antibodies. However, some of these anti-tumor antibodies are directed against molecules expressed both by tumor cells and normal immune cells, in particular lymphocytes, and, hence, can also strongly affect the host adaptive immunity. In addition to a delayed recovery of target cells, lymphocyte depleting-mAb treatments can have dramatic consequences on the adaptive immune cell network, its rebound, and its functional capacities. Thus, in this review, we will not only discuss the mAb-induced vaccinal effect that has emerged from experimental preclinical studies and clinical trials but also the multifaceted impact of lymphocytes-depleting therapeutic antibodies on the host adaptive immunity. We will also discuss some of the molecular and cellular mechanisms of action whereby therapeutic mAbs induce a long-term protective anti-tumor effect and the relationship between the mAb-induced vaccinal effect and the immune response against self-antigens. PMID:28855903

Long-term Outcomes and Risk Factors for Reoperation After Surgical Treatment for Gastrointestinal Crohn Disease According to Anti-tumor Necrosis Factor-α Antibody Use: 35 Years of Experience at a Single Institute in Korea.

PubMed

Lee, Sang Mok; Han, Eon Chul; Ryoo, Seung-Bum; Oh, Heung-Kwon; Choe, Eun Kyung; Moon, Sang Hui; Kim, Joo Sung; Jung, Hyun Chae; Park, Kyu Joo

2015-08-01

Crohn disease is characterized by high rates of recurrence and reoperations. However, few studies have investigated long-term surgical outcomes in Asian populations. We investigated risk factors for reoperation, particularly those associated with anti-tumor necrosis factor-α (anti-TNF-α) antibody use, and long-term follow-up results. We reviewed the records of 148 patients (100 males and 48 females) who underwent surgery for gastrointestinal Crohn disease and retrospectively analyzed long-term outcomes and risk factors. The mean age at diagnosis was 28.8 years. Thirty-eight patients (25.7%) received monoclonal antibody treatment before reoperation. A small bowel and colon resection was most commonly performed (83 patients, 56.1%). The median follow-up was 149 months, during which 47 patients underwent reoperation. The median interval between the primary and the secondary surgeries was 65 months, with accumulated reoperation rates of 16.5%, 31.8%, and 57.2% after 5, 10, and 15 years, respectively. Obstruction was the most common indication for reoperation (37 patients, 25.0%). In a multivariable analysis, age <17 years at diagnosis (A1) (odds ratio [OR], 2.20; P = 0.023), penetrating behavior (B3) (OR, 4.39; P < 0.001), and no azathioprine use (OR, 2.87; P = 0.003) were associated with reoperation. Anti-TNF-α antibody use did not affect the reoperation rate (P = 0.767). We showed a high reoperation rate regardless of treatment with anti-TNF-α antibody, which indicates that recurrent surgery is still needed to cure patients with gastrointestinal Crohn diseases. Younger age at primary operation, penetrating behavior, and no azathioprine use were significant factors associated with reoperation for gastrointestinal Crohn disease.

Binding and orientation of fibronectin on polystyrene surfaces using immobilized bacterial adhesin-related peptides.

PubMed

Klueh, U; Bryers, J D; Kreutzer, D L

2003-10-01

Fibronectin (FN) is known to bind to bacteria via high affinity receptors on bacterial surfaces known as adhesins. The binding of bacteria to FN is thought to have a key role in foreign device associated infections. For example, previous studies have indicated that Staphylococcus aureus adhesins bind to the 29 kDa NH(3) terminus end of FN, and thereby promote bacteria adherence to surfaces. Recently, the peptide sequences within the S. aureus adhesin molecule that are responsible for FN binding have been identified. Based on these observations, we hypothesize that functional FN can be bound and specifically oriented on polystyrene surfaces using bacterial adhesin-related (BRP-A) peptide. We further hypothesize that monoclonal antibodies that react with specific epitopes on the FN can be used to quantify both FN binding and orientation on these surfaces. Based on this hypothesis, we initiated a systematic investigation of the binding and orientation of FN on polystyrene surfaces using BRP-A peptide. To test this hypothesis, the binding and orientation of the FN to immobilized BRP-A was quantified using (125)I-FN, and monoclonal antibodies. (125)I-FN was used to quantitate FN binding to peptide-coated polystyrene surfaces. The orientation of bound FN was demonstrated by the use of monoclonal antibodies, which are reactive with the amine (N) or carboxyl (C) termini of the FN. The results of our studies demonstrated that when the BRP-A peptide was used to bind FN to surfaces that: 1. functional FN was bound to the peptide; 2. anti-C terminus antibodies bound to the peptide FN; and 3. only limited binding of anti-N terminus antibodies to peptide-bound FN occurred. We believe that the data that indicate an enhanced binding of anti-C antibodies reactive to anti-N antibodies are a result of the FN binding in an oriented manner with the N termini of FN bound tightly to the BRP-A on the polystyrene surface. Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 36-43, 2003

Detection of the Argonaute Protein Ago2 and microRNAs in the RNA Induced Silencing Complex (RISC) Using a Monoclonal Antibody

PubMed Central

Ikeda, Keigo; Satoh, Minoru; Pauley, Kaleb M.; Fritzler, Marvin J.; Reeves, Westley H.; Chan, Edward K.L.

2007-01-01

MicroRNAs (miRNAs) are short RNA molecules responsible for post-transcriptional gene silencing by the degradation or translational inhibition of their target messenger RNAs (mRNAs). This process of gene silencing, known as RNA interference (RNAi), is mediated by highly conserved Argonaute (Ago) proteins which are the key components of the RNA induced silencing complex (RISC). In humans, Ago2 is responsible for the endonuclease cleavage of targeted mRNA and it interacts with the mRNA-binding protein GW182, which is a marker for cytoplasmic foci referred to as GW bodies (GWBs). We demonstrated that the anti-Ago2 monoclonal antibody 4F9 recognized GWBs in a cell cycle dependent manner and was capable of capturing miRNAs associated with Ago2. Since Ago2 protein is the effector protein of RNAi, anti-Ago2 monoclonal antibody may be useful in capturing functional miRNAs. PMID:17054975

Detection of the argonaute protein Ago2 and microRNAs in the RNA induced silencing complex (RISC) using a monoclonal antibody.

PubMed

Ikeda, Keigo; Satoh, Minoru; Pauley, Kaleb M; Fritzler, Marvin J; Reeves, Westley H; Chan, Edward K L

2006-12-20

MicroRNAs (miRNAs) are short RNA molecules responsible for post-transcriptional gene silencing by the degradation or translational inhibition of their target messenger RNAs (mRNAs). This process of gene silencing, known as RNA interference (RNAi), is mediated by highly conserved Argonaute (Ago) proteins which are the key components of the RNA induced silencing complex (RISC). In humans, Ago2 is responsible for the endonuclease cleavage of targeted mRNA and it interacts with the mRNA-binding protein GW182, which is a marker for cytoplasmic foci referred to as GW bodies (GWBs). We demonstrated that the anti-Ago2 monoclonal antibody 4F9 recognized GWBs in a cell cycle dependent manner and was capable of capturing miRNAs associated with Ago2. Since Ago2 protein is the effector protein of RNAi, anti-Ago2 monoclonal antibody may be useful in capturing functional miRNAs.

Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134.

PubMed

Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Kato, Yukinari

2018-07-01

The epidermal growth factor receptor (EGFR) is a type-1 transmembrane receptor tyrosine kinase, which activates the downstream signaling cascades in many tumors, such as oral and lung cancers. We previously developed EMab-134, a novel anti-EGFR monoclonal antibody (mAb), which reacts with endogenous EGFR-expressing cancer cell lines and normal cells independent of glycosylation in Western blotting, flow cytometry, and immunohistochemical analysis. EMab-134 showed very high sensitivity (94.7%) to oral squamous cell carcinomas in immunohistochemical analysis. In this study, we performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of EMab-134. A blocking peptide (375-394 amino acids of EGFR) neutralized the EMab-134 reaction against oral cancer cells in flow cytometry and immunohistochemistry. The minimum epitope of EMab-134 was found to be the 377- RGDSFTHTPP -386 sequence. Our findings can be applied for the production of more functional anti-EGFR mAbs that in turn can be used for antitumor treatments.

Monoclonal Antibodies against Small Molecule Natural Products and Their Applications, Eastern Blotting and Knockout Extract

PubMed Central

Shoyama, Yukihiro

2011-01-01

To determine the hapten number in hapten-carrier protein conjugate matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry was applied. Highly specific anti-ginsenoside Rb1 and Rg1 monoclonal antibodies (MAbs) were prepared. Ginsenosides were developed on thin layer chromatography (TLC) plates which were covered by a polyvinylidene difluoride (PVDF) membrane resulting in blotting. The membrane was treated with NaIO4 solution to release the aldehyde group on the sugar moiety of the ginsenosides. By treatment of the membrane with a protein solution the ginsenoside-protein conjugation as a Schiff-base occurred, which can function to fix it to the PVDF membrane. A part of the ginsenoside aglycone was reacted with anti-ginsenoside Rb1 MAb, secondary MAb conjugated with enzyme and finally a substrate was added, resulting in a specific and highly sensitive staining that we named Eastern blotting. Furthermore, it makes one-step isolation of ginsenoside Rb1 possible using an immuno-affinity column conjugated with anti-ginsenoside Rb1 MAb. Furthermore, immunoaffinity concentration was carried out allowing high sensitivity analysis of lower concentrations of ginsenoside Rb1 so that several unknown bands could be structurally determined.

Anti-PD-L1 Treatment Induced Central Diabetes Insipidus.

PubMed

Zhao, Chen; Tella, Sri Harsha; Del Rivero, Jaydira; Kommalapati, Anuhya; Ebenuwa, Ifechukwude; Gulley, James; Strauss, Julius; Brownell, Isaac

2018-02-01

Immune checkpoint inhibitors, including anti-programmed cell death protein 1 (PD-1), anti-programmed cell death protein ligand 1 (PD-L1), and anti-cytotoxic T-lymphocyte antigen 4 (anti-CTLA4) monoclonal antibodies, have been widely used in cancer treatment. They are known to cause immune-related adverse events (irAEs), which resemble autoimmune diseases. Anterior pituitary hypophysitis with secondary hypopituitarism is a frequently reported irAE, especially in patients receiving anti-CTLA4 treatment. In contrast, posterior pituitary involvement, such as central diabetes insipidus (DI), is relatively rare and is unreported in patients undergoing PD-1/PD-L1 blockade. We describe a case of a 73-year-old man with Merkel cell carcinoma who received the anti-PD-L1 monoclonal antibody avelumab and achieved partial response. The patient developed nocturia, polydipsia, and polyuria 3 months after starting avelumab. Further laboratory testing revealed central DI. Avelumab was held and he received desmopressin for the management of central DI. Within 6 weeks after discontinuation of avelumab, the patient's symptoms resolved and he was eventually taken off desmopressin. The patient remained off avelumab and there were no signs or symptoms of DI 2 months after the discontinuation of desmopressin. To our knowledge, this is the first report of central DI associated with anti-PD-L1 immunotherapy. The patient's endocrinopathy was successfully managed by holding treatment with the immune checkpoint inhibitor. This case highlights the importance of early screening and appropriate management of hormonal irAEs in subjects undergoing treatment with immune checkpoint inhibitors to minimize morbidity and mortality. Copyright © 2017 Endocrine Society

B Lymphocyte intestinal homing in inflammatory bowel disease

PubMed Central

2011-01-01

Background Inflammatory bowel disease (IBD) is thought to be due to an abnormal interaction between the host immune system and commensal microflora. Within the intestinal immune system, B cells produce physiologically natural antibodies but pathologically atypical anti-neutrophil antibodies (xANCAs) are frequently observed in patients with IBD. The objective is to investigate the localisation of immunoglobulin-producing cells (IPCs) in samples of inflamed intestinal tissue taken from patients with IBD, and their possible relationship with clinical features. Methods The IPCs in small intestinal, colonic and rectal biopsy specimens of patients with IBD were analysed by means of immunofluorescence using polyclonal rabbit anti-human Ig and goat anti-human IgM. The B cell phenotype of the IPC-positive samples was assessed using monoclonal antibodies specific for CD79, CD20, CD23, CD21, CD5, λ and κ chains. Statistical correlations were sought between the histological findings and clinical expression. Results The study involved 96 patients (64 with ulcerative colitis and 32 with Crohn's disease). Two different patterns of B lymphocyte infiltrates were found in the intestinal tissue: one was characterised by a strong to moderate stromal localisation of small IgM+/CD79+/CD20-/CD21-/CD23-/CD5± IPCs (42.7% of cases); in the other (57.3%) no such small IPCs were detected in stromal or epithelial tissues. IPCs were significantly less frequent in the patients with Crohn's disease than in those with ulcerative colitis (p = 0.004). Conclusion Our findings suggest that different immunopathogenetic pathways underlie chronic intestinal inflammation with different clinical expressions. The presence of small B lymphocytes resembling B-1 cells also seemed to be negatively associated with Crohn's disease. It can therefore be inferred that the gut contains an alternative population of B cells that have a regulatory function. PMID:22208453

Cloning and molecular characterization of the cDNAs encoding the variable regions of an anti-CD20 monoclonal antibody.

PubMed

Shanehbandi, Dariush; Majidi, Jafar; Kazemi, Tohid; Baradaran, Behzad; Aghebati-Maleki, Leili

2017-01-01

CD20-based targeting of B-cells in hematologic malignancies and autoimmune disorders is associated with outstanding clinical outcomes. Isolation and characterization of VH and VL cDNAs encoding the variable regions of the heavy and light chains of monoclonal antibodies (MAb) is necessary to produce next generation MAbs and their derivatives such as bispecific antibodies (bsAb) and single-chain variable fragments (scFv). This study was aimed at cloning and characterization of the VH and VL cDNAs from a hybridoma cell line producing an anti-CD20 MAb. VH and VL fragments were amplified, cloned and characterized. Furthermore, amino acid sequences of VH, VL and corresponding complementarity-determining regions (CDR) were determined and compared with those of four approved MAbs including Rituximab (RTX), Ibritumomab tiuxetan, Ofatumumab and GA101. The cloned VH and VL cDNAs were found to be functional and follow a consensus pattern. Amino acid sequences corresponding to the VH and VL fragments also indicated noticeable homologies to those of RTX and Ibritumomab. Furthermore, amino acid sequences of the relating CDRs had remarkable similarities to their counterparts in RTX and Ibritumomab. Successful recovery of VH and VL fragments encourages the development of novel CD20 targeting bsAbs, scFvs, antibody conjugates and T-cells armed with chimeric antigen receptors.

Structural basis of clade-specific HIV-1 neutralization by humanized anti-V3 monoclonal antibody KD-247.

PubMed

Kirby, Karen A; Ong, Yee Tsuey; Hachiya, Atsuko; Laughlin, Thomas G; Chiang, Leslie A; Pan, Yun; Moran, Jennifer L; Marchand, Bruno; Singh, Kamalendra; Gallazzi, Fabio; Quinn, Thomas P; Yoshimura, Kazuhisa; Murakami, Toshio; Matsushita, Shuzo; Sarafianos, Stefan G

2015-01-01

Humanized monoclonal antibody KD-247 targets the Gly(312)-Pro(313)-Gly(314)-Arg(315) arch of the third hypervariable (V3) loop of the HIV-1 surface glycoprotein. It potently neutralizes many HIV-1 clade B isolates, but not of other clades. To understand the molecular basis of this specificity, we solved a high-resolution (1.55 Å) crystal structure of the KD-247 antigen binding fragment and examined the potential interactions with various V3 loop targets. Unlike most antibodies, KD-247 appears to interact with its target primarily through light chain residues. Several of these interactions involve Arg(315) of the V3 loop. To evaluate the role of light chain residues in the recognition of the V3 loop, we generated 20 variants of KD-247 single-chain variable fragments with mutations in the antigen-binding site. Purified proteins were assessed for V3 loop binding using AlphaScreen technology and for HIV-1 neutralization. Our data revealed that recognition of the clade-specificity defining residue Arg(315) of the V3 loop is based on a network of interactions that involve Tyr(L32), Tyr(L92), and Asn(L27d) that directly interact with Arg(315), thus elucidating the molecular interactions of KD-247 with its V3 loop target. © FASEB.

Clinical impact of occult hepatitis B virus infection in immunosuppressed patients

PubMed Central

Sagnelli, Evangelista; Pisaturo, Mariantonietta; Martini, Salvatore; Filippini, Pietro; Sagnelli, Caterina; Coppola, Nicola

2014-01-01

Occult hepatitis B infection (OBI), is characterized by low level hepatitis B virus (HBV) DNA in circulating blood and/or liver tissue. In clinical practice the presence of antibody to hepatitis B core antigen in hepatitis B surface antigen (HBsAg)-/anti-HBs-negative subjects is considered indicative of OBI. OBI is mostly observed in the window period of acute HBV infection in blood donors and in recipients of blood and blood products, in hepatitis C virus chronic carriers, in patients under pharmacological immunosuppression, and in those with immunodepression due to HIV infection or cancer. Reactivation of OBI mostly occurs in anti-HIV-positive subjects, in patients treated with immunosuppressive therapy in onco-hematological settings, in patients who undergo hematopoietic stem cell transplantation, in those treated with anti-CD20 or anti-CD52 monoclonal antibody, or anti-tumor necrosis factors antibody for rheumatological diseases, or chemotherapy for solid tumors. Under these conditions the mortality rate for hepatic failure or progression of the underlying disease due to discontinuation of specific treatment can reach 20%. For patients with OBI, prophylaxis with nucleot(s)ide analogues should be based on the HBV serological markers, the underlying diseases and the type of immunosuppressive treatment. Lamivudine prophylaxis is indicated in hemopoietic stem cell transplantation and in onco-hematological diseases when high dose corticosteroids and rituximab are used; monitoring may be indicated when rituximab-sparing schedules are used, but early treatment should be applied as soon as HBsAg becomes detectable. This review article presents an up-to-date evaluation of the current knowledge on OBI. PMID:25018849

Computational Prediction of Neutralization Epitopes Targeted by Human Anti-V3 HIV Monoclonal Antibodies

PubMed Central

Shmelkov, Evgeny; Krachmarov, Chavdar; Grigoryan, Arsen V.; Pinter, Abraham; Statnikov, Alexander; Cardozo, Timothy

2014-01-01

The extreme diversity of HIV-1 strains presents a formidable challenge for HIV-1 vaccine design. Although antibodies (Abs) can neutralize HIV-1 and potentially protect against infection, antibodies that target the immunogenic viral surface protein gp120 have widely variable and poorly predictable cross-strain reactivity. Here, we developed a novel computational approach, the Method of Dynamic Epitopes, for identification of neutralization epitopes targeted by anti-HIV-1 monoclonal antibodies (mAbs). Our data demonstrate that this approach, based purely on calculated energetics and 3D structural information, accurately predicts the presence of neutralization epitopes targeted by V3-specific mAbs 2219 and 447-52D in any HIV-1 strain. The method was used to calculate the range of conservation of these specific epitopes across all circulating HIV-1 viruses. Accurately identifying an Ab-targeted neutralization epitope in a virus by computational means enables easy prediction of the breadth of reactivity of specific mAbs across the diversity of thousands of different circulating HIV-1 variants and facilitates rational design and selection of immunogens mimicking specific mAb-targeted epitopes in a multivalent HIV-1 vaccine. The defined epitopes can also be used for the purpose of epitope-specific analyses of breakthrough sequences recorded in vaccine clinical trials. Thus, our study is a prototype for a valuable tool for rational HIV-1 vaccine design. PMID:24587168

Risk of fatigue in cancer patients receiving anti-EGFR monoclonal antibodies: results from a systematic review and meta-analysis of randomized controlled trial.

PubMed

Zhu, Jianhong; Zhao, Wenxia; Liang, Dan; Li, Guocheng; Qiu, Kaifeng; Wu, Junyan; Li, Jianfang

2018-04-01

To evaluate the association between fatigue and anti-epidermal growth factor receptor monoclonal antibodies (anti-EGFR MAbs), we conducted the first meta-analysis to access the incidence and risk of fatigue associated with anti-EGFR MAbs. Electronic databases were searched for randomized controlled trials (RCTs) published up to February 2017. Eligible studies were selected according to PRISMA statement. Incidence rates, risk ratio (RRs), and 95% confidence intervals (CIs) were calculated using fixed-effects or random-effects models. Outcomes of quality were summarized in accordance with the GRADE (Grading of Recommendations Assessment, Development, and Evaluation) methodology. Thirty-five RCTs (including 15,622 patients) were included; median follow-up ranged from 8.1 to 71.4 months, and the fatigue events were recorded and graded according to the Common Toxicity Criteria for Adverse Events version 2.0 or 3.0 in most of the included trials. For patients receiving anti-EGFR MAbs, the overall incidence of all-grade and high-grade fatigue was 54.1% and 10.5%, respectively. Compared with control, anti-EGFR MAbs significantly increased the risk of all-grade fatigue (RR 1.10, 95% CI, 1.05-1.14, moderate-quality evidence) and high-grade fatigue (RR 1.31, 95% CI, 1.19-1.45, moderate-quality evidence). No significant differences among subgroup analyses (anti-EGFR MAbs, tumor type, and median follow-up) on high-grade fatigue were observed. No evidence of publication bias was observed. The present study suggested that anti-EGFR MAbs may increase the risk of fatigue in cancer patients.

Early short-term treatment with neutralizing human monoclonal antibodies halts SHIV infection in newborn macaques

PubMed Central

Hessell, Ann J.; Jaworski, J. Pablo; Epson, Erin; Matsuda, Kenta; Pandey, Shilpi; Kahl, Christoph; Reed, Jason; Sutton, William F.; Hammond, Katherine B.; Cheever, Tracy A.; Barnette, Philip T.; Legasse, Alfred W.; Planer, Shannon; Stanton, Jeffrey J.; Pegu, Amarendra; Chen, Xuejun; Wang, Keyun; Siess, Don; Burke, David; Park, Byung S.; Axthelm, Michael K.; Lewis, Anne; Hirsch, Vanessa M.; Graham, Barney S.; Mascola, John R.; Sacha, Jonah B.; Haigwood, Nancy L.

2016-01-01

Prevention of mother to child transmission (MTCT) of HIV remains a major objective where antenatal care is not readily accessible. We tested anti-HIV-1 human neutralizing monoclonal antibodies (NmAb) as post-exposure therapy in an infant macaque model for intrapartum MTCT. One-month-old rhesus macaques were inoculated orally with SHIVSF162P3. On days 1, 4, 7, and 10 after virus exposure, we injected animals subcutaneously with NmAbs and quantified systemic distribution of NmAbs in multiple tissues within 24 h following administration. Replicating virus was found in multiple tissues by day 1 in animals without treatment. All NmAb-treated macaques were free of virus in blood and tissues at 6 months post-exposure. We detected no anti-SHIV T cell responses in blood or tissues at necropsy, and no virus emerged following CD8+ T cell depletion. These results suggest early passive immunotherapy can eliminate early viral foci and thereby prevent the establishment of viral reservoirs. PMID:26998834

Production of Murine Monoclonal Antibodies using Traditional and Novel Technology

DTIC Science & Technology

2010-03-01

1 17. LIMITATION OF ABSTRACT UL 18. NUMBER OF PAGES 17 19a. NAME OF RESPONSIBLE PERSON Sandra J . Johnson 19b. TELEPHONE NUMBER (include...users should direct such requests to the National Technical Information Service. Acknowledgments The author would like to acknowledge Dr. Bonnie J ...Monoclonal Antibodies: Principles and Practice; Academic Press: London, 1996. Goyache, Joaquin; Orden, Jose A.; Blanco , Jose L.; Hernandez , Javier

Rapid preparative separation of monoclonal antibody charge variants using laterally-fed membrane chromatography.

PubMed

Sadavarte, Rahul; Madadkar, Pedram; Filipe, Carlos Dm; Ghosh, Raja

2018-01-15

Monoclonal antibodies undergo various forms of chemical transformation which have been shown to cause loss in efficacy and alteration in pharmacokinetic properties of these molecules. Such modified antibody molecules are known as variants. They also display physical properties such as charge that are different from intact antibody molecules. However, the difference in charge is very subtle and separation based on it is quite challenging. Charge variants are usually separated using ion-exchange column chromatography or isoelectric focusing. In this paper, we report a rapid and scalable method for fractionating monoclonal antibody charge variants, based on the use of cation exchange laterally-fed membrane chromatography (LFMC). Starting with a sample of monoclonal antibody hIgG1-CD4, three well-resolved fractions were obtained using either pH or salt gradient. These fractions were identified as acidic, neutral and basic variants. Each of these fractions contained intact heavy and light chains and so antibody fragmentation had no role in variant generation. The separation was comparable to that using column chromatography but was an order of magnitude faster. Copyright © 2017 Elsevier B.V. All rights reserved.