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Sample records for antibody mediated cell

  1. Natural Killer Cell Mediated Antibody-Dependent Cellular Cytotoxicity in Tumor Immunotherapy with Therapeutic Antibodies

    PubMed Central

    Seidel, Ursula J. E.; Schlegel, Patrick; Lang, Peter

    2013-01-01

    In the last decade several therapeutic antibodies have been Federal Drug Administration (FDA) and European Medicines Agency (EMEA) approved. Although their mechanisms of action in vivo is not fully elucidated, antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is presumed to be a key effector function. A substantial role of ADCC has been demonstrated in vitro and in mouse tumor models. However, a direct in vivo effect of ADCC in tumor reactivity in humans remains to be shown. Several studies revealed a predictive value of FcγRIIIa-V158F polymorphism in monoclonal antibody treatment, indicating a potential effect of ADCC on outcome for certain indications. Furthermore, the use of therapeutic antibodies after allogeneic hematopoietic stem cell transplantation is an interesting option. Studying the role of the FcγRIIIa-V158F polymorphism and the influence of Killer-cell Immunoglobuline-like Receptor (KIR) receptor ligand incompatibility on ADCC in this approach may contribute to future transplantation strategies. Despite the success of approved second-generation antibodies in the treatment of several malignancies, efforts are made to further augment ADCC in vivo by antibody engineering. Here, we review currently used therapeutic antibodies for which ADCC has been suggested as effector function. PMID:23543707

  2. Role of complement and NK cells in antibody mediated rejection.

    PubMed

    Akiyoshi, Takurin; Hirohashi, Tsutomu; Alessandrini, Alessandro; Chase, Catherine M; Farkash, Evan A; Neal Smith, R; Madsen, Joren C; Russell, Paul S; Colvin, Robert B

    2012-12-01

    Despite extensive research on T cells and potent immunosuppressive regimens that target cellular mediated rejection, few regimens have been proved to be effective on antibody-mediated rejection (AMR), particularly in the chronic setting. C4d deposition in the graft has been proved to be a useful marker for AMR; however, there is an imperfect association between C4d and AMR. While complement has been considered as the main player in acute AMR, the effector mechanisms in chronic AMR are still debated. Recent studies support the role of NK cells and direct effects of antibody on endothelium cells in a mechanism suggesting the presence of a complement-independent pathway. Here, we review the history, currently available systems and progress in experimental animal research. Although there are consistent findings from human and animal research, transposing the experimental results from rodent to human has been hampered by the differences in endothelial functions between species. We briefly describe the findings from patients and compare them with results from animals, to propose a combined perspective.

  3. B cells mediate chronic allograft rejection independently of antibody production.

    PubMed

    Zeng, Qiang; Ng, Yue-Harn; Singh, Tripti; Jiang, Ke; Sheriff, Khaleefathullah A; Ippolito, Renee; Zahalka, Salwa; Li, Qi; Randhawa, Parmjeet; Hoffman, Rosemary A; Ramaswami, Balathiripurasundari; Lund, Frances E; Chalasani, Geetha

    2014-03-01

    Chronic rejection is the primary cause of long-term failure of transplanted organs and is often viewed as an antibody-dependent process. Chronic rejection, however, is also observed in mice and humans with no detectable circulating alloantibodies, suggesting that antibody-independent pathways may also contribute to pathogenesis of transplant rejection. Here, we have provided direct evidence that chronic rejection of vascularized heart allografts occurs in the complete absence of antibodies, but requires the presence of B cells. Mice that were deficient for antibodies but not B cells experienced the same chronic allograft vasculopathy (CAV), which is a pathognomonic feature of chronic rejection, as WT mice; however, mice that were deficient for both B cells and antibodies were protected from CAV. B cells contributed to CAV by supporting splenic lymphoid architecture, T cell cytokine production, and infiltration of T cells into graft vessels. In chimeric mice, in which B cells were present but could not present antigen, both T cell responses and CAV were markedly reduced. These findings establish that chronic rejection can occur in the complete absence of antibodies and that B cells contribute to this process by supporting T cell responses through antigen presentation and maintenance of lymphoid architecture.

  4. Antibody-dependent cell-mediated virus inhibition (ADCVI) antibody activity does not correlate with risk of HIV-1 superinfection

    PubMed Central

    FORTHAL, Donald N.; LANDUCCI, Gary; CHOHAN, Bhavna; RICHARDSON, Barbra A.; MCCLELLAND, R. Scott; JAOKO, Walter; BLISH, Catherine; OVERBAUGH, Julie

    2013-01-01

    Previous studies of HIV-infected women with high risk behavior have indicated that neither neutralizing antibody nor cellular immunity elicited by an initial HIV-1 infection is associated with protection against superinfection with a different HIV-1 strain. Here, we measured antibody-dependent cell-mediated virus inhibition (ADCVI) antibody activity in the plasma of 12 superinfected cases and 36 singly infected matched controls against 2 heterologous viruses. We found no association between plasma ADCVI activity and superinfection status. ADCVI antibody activity against heterologous virus elicited by the original infection may not contribute to preventing a superinfecting HIV-1. PMID:23344546

  5. Reactive oxygen species induced by therapeutic CD20 antibodies inhibit natural killer cell-mediated antibody-dependent cellular cytotoxicity against primary CLL cells.

    PubMed

    Werlenius, Olle; Aurelius, Johan; Hallner, Alexander; Akhiani, Ali A; Simpanen, Maria; Martner, Anna; Andersson, Per-Ola; Hellstrand, Kristoffer; Thorén, Fredrik B

    2016-05-31

    The antibody-dependent cellular cytotoxicity (ADCC) of natural killer (NK) cells is assumed to contribute to the clinical efficacy of monoclonal antibodies (mAbs) in chronic lymphocytic leukemia (CLL) and other hematopoietic malignancies of B cell origin. We sought to determine whether reactive oxygen species (ROS)-producing monocytes regulate the ADCC of NK cells against primary CLL cells using anti-CD20 as the linking antibody. The monoclonal CD20 antibodies rituximab and ofatumumab were found to trigger substantial release of ROS from monocytes. Antibody-exposed monocytes induced NK cell apoptosis and restricted NK cell-mediated ADCC against autologous CLL cells. The presence of inhibitors of ROS formation and scavengers of ROS preserved NK cell viability and restored NK cell-mediated ADCC against primary CLL cells. We propose that limiting the antibody-induced induction of immunosuppressive ROS may improve the anti-leukemic efficacy of anti-CD20 therapy in CLL. PMID:27097113

  6. Macrophage-Mediated Trogocytosis Leads to Death of Antibody-Opsonized Tumor Cells.

    PubMed

    Velmurugan, Ramraj; Challa, Dilip K; Ram, Sripad; Ober, Raimund J; Ward, E Sally

    2016-08-01

    Understanding the complex behavior of effector cells such as monocytes or macrophages in regulating cancerous growth is of central importance for cancer immunotherapy. Earlier studies using CD20-specific antibodies have demonstrated that the Fcγ receptor (FcγR)-mediated transfer of the targeted receptors from tumor cells to these effector cells through trogocytosis can enable escape from antibody therapy, leading to the viewpoint that this process is protumorigenic. In the current study, we demonstrate that persistent trogocytic attack results in the killing of HER2-overexpressing breast cancer cells. Further, antibody engineering to increase FcγR interactions enhances this tumoricidal activity. These studies extend the complex repertoire of activities of macrophages to trogocytic-mediated cell death of HER2-overexpressing target cells and have implications for the development of effective antibody-based therapies. Mol Cancer Ther; 15(8); 1879-89. ©2016 AACR. PMID:27226489

  7. Cloned transgenic farm animals produce a bispecific antibody for T cell-mediated tumor cell killing.

    PubMed

    Grosse-Hovest, Ludger; Müller, Sigrid; Minoia, Rosa; Wolf, Eckhard; Zakhartchenko, Valeri; Wenigerkind, Hendrik; Lassnig, Caroline; Besenfelder, Urban; Müller, Mathias; Lytton, Simon D; Jung, Gundram; Brem, Gottfried

    2004-05-01

    Complex recombinant antibody fragments for modulation of immune function such as tumor cell destruction have emerged at a rapid pace and diverse anticancer strategies are being developed to benefit patients. Despite improvements in molecule design and expression systems, the quantity and stability, e.g., of single-chain antibodies produced in cell culture, is often insufficient for treatment of human disease, and the costs of scale-up, labor, and fermentation facilities are prohibitive. The ability to yield mg/ml levels of recombinant antibodies and the scale-up flexibility make transgenic production in plants and livestock an attractive alternative to mammalian cell culture as a source of large quantities of biotherapeutics. Here, we report on the efficient production of a bispecific single-chain antibody in the serum of transgenic rabbits and a herd of nine cloned, transgenic cattle. The bispecific protein, designated r28M, is directed to a melanoma-associated proteoglycan and the human CD28 molecule on T cells. Purified from the serum of transgenic animals, the protein is stable and fully active in mediating target cell-restricted T cell stimulation and tumor cell killing.

  8. Specialized proresolving mediators enhance human B cell differentiation to antibody secreting cells1

    PubMed Central

    Ramon, Sesquile; Gao, Fei; Serhan, Charles N.; Phipps, Richard P.

    2012-01-01

    The resolution of inflammation is an active and dynamic process critical in maintaining homeostasis. Newly identified lipid mediators have been recognized as key players during the resolution phase. These specialized proresolving mediators (SPM) constitute separate families that include lipoxins, resolvins, protectins and maresins each derived from essential polyunsaturated fatty acids. New results demonstrate that SPM regulate aspects of the immune response, including reduction of neutrophil infiltration, decreased T cell cytokine production and stimulation of macrophage phagocytic activity. The actions of SPM on B lymphocytes remain unknown. Our study shows for the first time that the novel SPM 17-hydroxydosahexaenoic acid (17-HDHA), resolvin D1 (RvD1) and protectin D1 (PD1) are present in the spleen. Interestingly, 17-HDHA, RvD1 but not PD1, strongly increase activated human B cell IgM and IgG production. Furthermore, increased antibody production by 17-HDHA is due to augmented B cell differentiation towards a CD27+CD38+ antibody-secreting cell phenotype. 17-HDHA did not affect proliferation and was non-toxic to cells. Increase of plasma cell differentiation and antibody production supports the involvement of SPM during the late stages of inflammation and pathogen clearance. The present study provides new evidence for SPM activity in the humoral response. These new findings highlight the potential applications of SPM as endogenous and non-toxic adjuvants, and as anti-inflammatory therapeutic molecules. PMID:22711890

  9. Vaccination for invasive canine meningioma induces in situ production of antibodies capable of antibody-dependent cell-mediated cytotoxicity.

    PubMed

    Andersen, Brian M; Pluhar, G Elizabeth; Seiler, Charles E; Goulart, Michelle R; SantaCruz, Karen S; Schutten, Melissa M; Meints, Joyce P; O'Sullivan, M Gerard; Bentley, R Timothy; Packer, Rebecca A; Thomovsky, Stephanie A; Chen, Annie V; Faissler, Dominik; Chen, Wei; Hunt, Matthew A; Olin, Michael R; Ohlfest, John R

    2013-05-15

    Malignant and atypical meningiomas are resistant to standard therapies and associated with poor prognosis. Despite progress in the treatment of other tumors with therapeutic vaccines, this approach has not been tested preclinically or clinically in these tumors. Spontaneous canine meningioma is a clinically meaningful but underutilized model for preclinical testing of novel strategies for aggressive human meningioma. We treated 11 meningioma-bearing dogs with surgery and vaccine immunotherapy consisting of autologous tumor cell lysate combined with toll-like receptor ligands. Therapy was well tolerated, and only one dog had tumor growth that required intervention, with a mean follow up of 585 days. IFN-γ-elaborating T cells were detected in the peripheral blood of 2 cases, but vaccine-induced tumor-reactive antibody responses developed in all dogs. Antibody responses were polyclonal, recognizing both intracellular and cell surface antigens, and HSP60 was identified as one common antigen. Tumor-reactive antibodies bound allogeneic canine and human meningiomas, showing common antigens across breed and species. Histologic analysis revealed robust infiltration of antibody-secreting plasma cells into the brain around the tumor in posttreatment compared with pretreatment samples. Tumor-reactive antibodies were capable of inducing antibody-dependent cell-mediated cytotoxicity to autologous and allogeneic tumor cells. These data show the feasibility and immunologic efficacy of vaccine immunotherapy for a large animal model of human meningioma and warrant further development toward human trials.

  10. Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells.

    PubMed

    Romain, Gabrielle; Senyukov, Vladimir; Rey-Villamizar, Nicolas; Merouane, Amine; Kelton, William; Liadi, Ivan; Mahendra, Ankit; Charab, Wissam; Georgiou, George; Roysam, Badrinath; Lee, Dean A; Varadarajan, Navin

    2014-11-20

    The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effector-mediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cell-mediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia.

  11. Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells

    PubMed Central

    Romain, Gabrielle; Senyukov, Vladimir; Rey-Villamizar, Nicolas; Merouane, Amine; Kelton, William; Liadi, Ivan; Mahendra, Ankit; Charab, Wissam; Georgiou, George; Roysam, Badrinath; Lee, Dean A.

    2014-01-01

    The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effector-mediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cell-mediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia. PMID:25232058

  12. Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells.

    PubMed

    Romain, Gabrielle; Senyukov, Vladimir; Rey-Villamizar, Nicolas; Merouane, Amine; Kelton, William; Liadi, Ivan; Mahendra, Ankit; Charab, Wissam; Georgiou, George; Roysam, Badrinath; Lee, Dean A; Varadarajan, Navin

    2014-11-20

    The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effector-mediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cell-mediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia. PMID:25232058

  13. Antibody-mediated targeting of the Orai1 calcium channel inhibits T cell function.

    PubMed

    Cox, Jennifer H; Hussell, Scott; Søndergaard, Henrik; Roepstorff, Kirstine; Bui, John-Vu; Deer, Jen Running; Zhang, Jun; Li, Zhan-Guo; Lamberth, Kasper; Kvist, Peter Helding; Padkjær, Søren; Haase, Claus; Zahn, Stefan; Odegard, Valerie H

    2013-01-01

    Despite the attractiveness of ion channels as therapeutic targets, there are no examples of monoclonal antibodies directed against ion channels in clinical development. Antibody-mediated inhibition of ion channels could offer a directed, specific therapeutic approach. To investigate the potential of inhibiting ion channel function with an antibody, we focused on Orai1, the pore subunit of the calcium channel responsible for store-operated calcium entry (SOCE) in T cells. Effector T cells are key drivers of autoimmune disease pathogenesis and calcium signaling is essential for T cell activation, proliferation, and cytokine production. We show here the generation of a specific anti-human Orai1 monoclonal antibody (mAb) against an extracellular loop of the plasma membrane-spanning protein. The anti-Orai1 mAb binds native Orai1 on lymphocytes and leads to cellular internalization of the channel. As a result, T cell proliferation, and cytokine production is inhibited in vitro. In vivo, anti-Orai1 mAb is efficacious in a human T cell-mediated graft-versus host disease (GvHD) mouse model. This study demonstrates the feasibility of antibody-mediated inhibition of Orai1 function and, more broadly, reveals the possibility of targeting ion channels with biologics for the treatment of autoimmunity and other diseases. PMID:24376610

  14. Augmentation of antibody dependent cell mediated cytotoxicity following in vivo therapy with recombinant interleukin 2.

    PubMed

    Hank, J A; Robinson, R R; Surfus, J; Mueller, B M; Reisfeld, R A; Cheung, N K; Sondel, P M

    1990-09-01

    Monoclonal antibodies (mAB) with tumor specificity are able to enhance the immunological specificity of interleukin 2 (IL-2)-activated lymphokine activated killer (LAK) cells. Antibodies may also be used to broaden the range of tumor types susceptible to immune mediated cytotoxicity by the activated LAK cells. In these studies, mAB with relative tumor specificity were used to target immunologically activated effector cells in an in vitro antibody dependent cell mediated cytotoxicity (ADCC) assay. The mAB included: 3F8 and 14.G2a, which are both specific for neuroblastoma and melanoma and recognize ganglioside GD2, and mAB ING-1, a mouse-human chimeric antibody with constant regions from human IgG1 and kappa chains and variable regions from a mouse mAB that binds to a broad range of human adenocarcinomas. Each of these mAB was able to mediate ADCC with fresh effector cells and antibody binding targets. When peripheral blood mononuclear cells were obtained from cancer patients prior to and following in vivo therapy with interleukin 2, a significant increase was noted in ADCC activity by peripheral blood mononuclear cells obtained following IL-2 therapy. Inclusion of IL-2 in the medium during the cytotoxic assay with mAB further boosted ADCC. The total activity seen was often greater than the sum of the independent LAK activity and standard ADCC activity. The cells responsible for this ADCC had the CD16+ Fc receptor. Combining IL-2 with mAB in clinical tumor therapy may lead to a wider range of tumor types being responsive to immunotherapy and may also enhance the efficacy of therapy by specifically targeting activated effector cells to tumor cells recognized by mAB. Our results provide strong support for the testing of these hypotheses in clinical trials by combining in vivo treatment with IL-2 and mAB able to mediate ADCC.

  15. NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Cancer Immunotherapy.

    PubMed

    Wang, Wei; Erbe, Amy K; Hank, Jacquelyn A; Morris, Zachary S; Sondel, Paul M

    2015-01-01

    Natural killer (NK) cells play a major role in cancer immunotherapies that involve tumor-antigen targeting by monoclonal antibodies (mAbs). NK cells express a variety of activating and inhibitory receptors that serve to regulate the function and activity of the cells. In the context of targeting cells, NK cells can be "specifically activated" through certain Fc receptors that are expressed on their cell surface. NK cells can express FcγRIIIA and/or FcγRIIC, which can bind to the Fc portion of immunoglobulins, transmitting activating signals within NK cells. Once activated through Fc receptors by antibodies bound to target cells, NK cells are able to lyse target cells without priming, and secrete cytokines like interferon gamma to recruit adaptive immune cells. This antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells is utilized in the treatment of various cancers overexpressing unique antigens, such as neuroblastoma, breast cancer, B cell lymphoma, and others. NK cells also express a family of receptors called killer immunoglobulin-like receptors (KIRs), which regulate the function and response of NK cells toward target cells through their interaction with their cognate ligands that are expressed on tumor cells. Genetic polymorphisms in KIR and KIR-ligands, as well as FcγRs may influence NK cell responsiveness in conjunction with mAb immunotherapies. This review focuses on current therapeutic mAbs, different strategies to augment the anti-tumor efficacy of ADCC, and genotypic factors that may influence patient responses to antibody-dependent immunotherapies.

  16. Hormone Conjugated with Antibody to CD3 Mediates Cytotoxic T Cell Lysis of Human Melanoma Cells

    NASA Astrophysics Data System (ADS)

    Liu, Margaret Ann; Nussbaum, Samuel R.; Eisen, Herman N.

    1988-01-01

    Cytotoxic T lymphocytes can be activated by antibodies to their antigen-specific receptor complex (TCR-CD3) to destroy target cells, regardless of the specificity of the cytotoxic T cells. A novel hormone-antibody conjugate, consisting of an analog of melanocyte-stimulating hormone chemically coupled to a monoclonal antibody to CD3, the invariant component of the T cell receptor complex, was used to target human melanoma cells for destruction by human cytotoxic T lymphocytes that bear no specificity for the tumor cells. As targeting components of such anti-CD3 conjugates, hormones or growth factors are expected to prove more effective than antibodies to tumor-associated antigens in focusing the destructive activity of cytotoxic T cells on tumor target cells.

  17. Compromised NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Chronic SIV/SHIV Infection

    PubMed Central

    He, Xuan; Li, Dan; Luo, Zhenwu; Liang, Hua; Peng, Hong; Zhao, Yangyang; Wang, Nidan; Liu, Donghua; Qin, Chuan; Wei, Qiang; Yan, Huimin; Shao, Yiming

    2013-01-01

    Increasing evidence indicates that antibody-dependent cellular cytotoxicity (ADCC) contributes to the control of HIV/SIV infection. However, little is known about the ADCC function of natural killer (NK) cells in non-human primate model. Here we demonstrated that ADCC function of NK cells was significantly compromised in chronic SIV/SHIV infection, correlating closely with the expression of FcγRIIIa receptor (CD16) on NK cells. CD32, another class of IgG Fc receptors, was identified on NK cells with higher expression in the infected macaques and the blockade of CD32 impacted the ability of NK cells to respond to antibody-coated target cells. The inhibition of matrix metalloproteases (MMPs), a group of enzymes normally involved in tissue/receptor remodeling, could restore NK cell-mediated ADCC with increased CD16 expression on macaque NK cells. These data offer a clearer understanding of NK cell-mediated ADCC in rhesus macaques, which will allow us to evaluate the ADCC repertoire arising from preclinical vaccination studies in non-human primates and inform us in the future design of effective HIV vaccination strategies. PMID:23424655

  18. 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.

  19. Oncolytic and immunotherapeutic vaccinia induces antibody-mediated complement-dependent cancer cell lysis in humans.

    PubMed

    Kim, Mi Kyung; Breitbach, Caroline J; Moon, Anne; Heo, Jeong; Lee, Yu Kyoung; Cho, Mong; Lee, Jun Woo; Kim, Seong-Geun; Kang, Dae Hwan; Bell, John C; Park, Byeong Ho; Kirn, David H; Hwang, Tae-Ho

    2013-05-15

    Oncolytic viruses cause direct cytolysis and cancer-specific immunity in preclinical models. The goal of this study was to demonstrate induction of functional anticancer immunity that can lyse target cancer cells in humans. Pexa-Vec (pexastimogene devacirepvec; JX-594) is a targeted oncolytic and immunotherapeutic vaccinia virus engineered to express human granulocyte-macrophage colony-stimulating factor (GM-CSF). Pexa-Vec demonstrated replication, GM-CSF expression, and tumor responses in previous phase 1 trials. We now evaluated whether Pexa-Vec induced functional anticancer immunity both in the rabbit VX2 tumor model and in patients with diverse solid tumor types in phase 1. Antibody-mediated complement-dependent cancer cell cytotoxicity (CDC) was induced by intravenous Pexa-Vec in rabbits; transfer of serum from Pexa-Vec-treated animals to tumor-bearing animals resulted in tumor necrosis and improved survival. In patients with diverse tumor types treated on a phase 1 trial, CDC developed within 4 to 8 weeks in most patients; normal cells were resistant to the cytotoxic effects. T lymphocyte activation in patients was evidenced by antibody class switching. We determined that patients with the longest survival duration had the highest CDC activity, and identified candidate target tumor cell antigens. Thus, we demonstrated that Pexa-Vec induced polyclonal antibody-mediated CDC against multiple tumor antigens both in rabbits and in patients with diverse solid tumor types.

  20. An engineered substance P variant for receptor-mediated delivery of synthetic antibodies into tumor cells.

    PubMed

    Rizk, Shahir S; Luchniak, Anna; Uysal, Serdar; Brawley, Crista M; Rock, Ronald S; Kossiakoff, Anthony A

    2009-07-01

    We have developed and tested a robust delivery method for the transport of proteins to the cytoplasm of mammalian cells without compromising the integrity of the cell membrane. This receptor-mediated delivery (RMD) technology utilizes a variant of substance P (SP), a neuropeptide that is rapidly internalized upon interaction with the neurokinin-1 receptor (NK1R). Cargos in the form of synthetic antibody fragments (sABs) were conjugated to the engineered SP variant (SPv) and efficiently internalized by NK1R-expressing cells. The sABs used here were generated to bind specific conformational forms of actin. The internalized proteins appear to escape the endosome and retain their binding activity within the cells as demonstrated by co-localization with the actin cytoskeleton. Further, since the NK1R is over-expressed in many cancers, SPv-mediated delivery provides a highly specific method for therapeutic utilization of affinity reagents targeting intracellular processes in diseased tissue.

  1. Enhanced killing of chordoma cells by antibody-dependent cell-mediated cytotoxicity employing the novel anti-PD-L1 antibody avelumab.

    PubMed

    Fujii, Rika; Friedman, Eitan R; Richards, Jacob; Tsang, Kwong Y; Heery, Christopher R; Schlom, Jeffrey; Hodge, James W

    2016-06-01

    Chordoma, a rare bone tumor derived from the notochord, has been shown to be resistant to conventional therapies. Checkpoint inhibition has shown great promise in immune-mediated therapy of diverse cancers. The anti-PD-L1 mAb avelumab is unique among checkpoint inhibitors in that it is a fully human IgG1 capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) of PD-L1-expressing tumor cells. Here, we investigated avelumab as a potential therapy for chordoma. We examined 4 chordoma cell lines, first for expression of PD-L1, and in vitro for ADCC killing using NK cells and avelumab. PD-L1 expression was markedly upregulated by IFN-γ in all 4 chordoma cell lines, which significantly increased sensitivity to ADCC. Brachyury is a transcription factor that is uniformly expressed in chordoma. Clinical trials are ongoing in which chordoma patients are treated with brachyury-specific vaccines. Co-incubating chordoma cells with brachyury-specific CD8+ T cells resulted in significant upregulation of PD-L1 on the tumor cells, mediated by the CD8+ T cells' IFN-γ production, and increased sensitivity of chordoma cells to avelumab-mediated ADCC. Residential cancer stem cell subpopulations of chordoma cells were also killed by avelumab-mediated ADCC to the same degree as non-cancer stem cell populations. These findings suggest that as a monotherapy for chordoma, avelumab may enable endogenous NK cells, while in combination with T-cell immunotherapy, such as a vaccine, avelumab may enhance NK-cell killing of chordoma cells via ADCC.

  2. Enhanced killing of chordoma cells by antibody-dependent cell-mediated cytotoxicity employing the novel anti-PD-L1 antibody avelumab

    PubMed Central

    Fujii, Rika; Friedman, Eitan R.; Richards, Jacob; Tsang, Kwong Y.; Heery, Christopher R.; Schlom, Jeffrey; Hodge, James W.

    2016-01-01

    Chordoma, a rare bone tumor derived from the notochord, has been shown to be resistant to conventional therapies. Checkpoint inhibition has shown great promise in immune-mediated therapy of diverse cancers. The anti-PD-L1 mAb avelumab is unique among checkpoint inhibitors in that it is a fully human IgG1 capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) of PD-L1-expressing tumor cells. Here, we investigated avelumab as a potential therapy for chordoma. We examined 4 chordoma cell lines, first for expression of PD-L1, and in vitro for ADCC killing using NK cells and avelumab. PD-L1 expression was markedly upregulated by IFN-γ in all 4 chordoma cell lines, which significantly increased sensitivity to ADCC. Brachyury is a transcription factor that is uniformly expressed in chordoma. Clinical trials are ongoing in which chordoma patients are treated with brachyury-specific vaccines. Co-incubating chordoma cells with brachyury-specific CD8+ T cells resulted in significant upregulation of PD-L1 on the tumor cells, mediated by the CD8+ T cells' IFN-γ production, and increased sensitivity of chordoma cells to avelumab-mediated ADCC. Residential cancer stem cell subpopulations of chordoma cells were also killed by avelumab-mediated ADCC to the same degree as non-cancer stem cell populations. These findings suggest that as a monotherapy for chordoma, avelumab may enable endogenous NK cells, while in combination with T-cell immunotherapy, such as a vaccine, avelumab may enhance NK-cell killing of chordoma cells via ADCC. PMID:27172898

  3. Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy

    PubMed Central

    Roit, Fabio Da; Engelberts, Patrick J.; Taylor, Ronald P.; Breij, Esther C.W.; Gritti, Giuseppe; Rambaldi, Alessandro; Introna, Martino; Parren, Paul W.H.I.; Beurskens, Frank J.; Golay, Josée

    2015-01-01

    The novel Bruton tyrosine kinase inhibitor ibrutinib and phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib are promising drugs for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma, either alone or in combination with anti-CD20 antibodies. We investigated the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells, and antibody-dependent cellular cytotoxicity by these cells, as well as phagocytosis by macrophages or neutrophils were inhibited by ibrutinib with a half maximal effective concentration of 0.3–3 μM. Analysis of anti-CD20 mediated activation of natural killer cells isolated from patients on continued oral ibrutinib treatment suggested that repeated drug dosing inhibits these cells in vivo. Finally we show that the phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib similarly inhibited the immune cell-mediated mechanisms induced by anti-CD20 antibodies, although the effects of this drug at 10 μM were weaker than those observed with ibrutinib at the same concentration. We conclude that the design of combined treatment schedules of anti-CD20 antibodies with these kinase inhibitors should consider the multiple negative interactions between these two classes of drugs. PMID:25344523

  4. Antibody-mediated radiotherapy

    SciTech Connect

    Bloomer, W.D.; Lipsztein, R.; Dalton, J.F.

    1985-05-01

    Antibodies that react with antigens on the surface of tumor cells but not normal cells have great potential for cancer detection and therapy. If radiolabeled without loss of immunologic specificity, such antibodies may be able to deliver cytoxic amounts of radiation. Target- cell specificity and a high extraction coefficient are necessary with any radionuclide in order to minimize normal tissue irradiation. Tumor- cell-retention time and the rate of catabolized radionuclide will also influence ultimate applicability. Among the unanswered questions for choosing a radionuclide is the choice of particle emitter. Although classic beta emitters have been used in a number of clinical situations, they have not had a major impact on disease outcome except in diseases of the thyroid. Unfortunately, Auger emitters such as iodine 125 are cytotoxic only when localized within close proximity to the genome. On the other hand, alpha emitters such as astatine 211 eliminate the need for subcellular sequestration but not cell-specific localization. 34 references.

  5. The role of apoptosis in bispecific antibody-mediated T-cell cytotoxicity.

    PubMed Central

    Kroesen, B. J.; Wellenberg, G. J.; Bakker, A.; Helfrich, W.; The, T. H.; de Leij, L.

    1996-01-01

    In this report we describe the role of apoptosis in the process of tumour cell killing by bispecific monoclonal antibody (BsMAb)-redirected cytolytic T cells. The BsMAb used, BIS-1, has dual specificity for the CD3 complex on T cells and the pancarcinoma-associated 38 kDa transmembrane antigen EGP-2. BIS-1 allows activated T cells to specifically recognise and kill EGP-2-positive but not EGP-2-negative target cells. An assay was developed to quantify apoptosis in cells by separation of 3H-thymidine-labelled low-molecular, i.e. fragmented, from high-molecular, i.e. non-fragmented DNA. The presence of low molecular weight DNA was measured both within the target cells and in the cell-free supernatant. After exposure to BIS-1-redirected, -activated T cells, apoptosis was observed in EGP-2-positive target cells but not in EGP-2-negative target cells. Also no DNA fragmentation proved to be induced in the activated effector cells during assay. The degree of EGP-2-positive target DNA fragmentation depended on the concentration of BsMAb, the E/T ratio and the incubation time. Using a low E/T ratio (1/1), DNA fragmentation in and 51Cr release from target cells showed similar characteristics and kinetics. At higher E/T ratio (20/1), the 51Cr release from the target cells increased to a greater extent than the percentage fragmented target cell DNA. Inhibitors of DNA fragmentation added to the cytotoxicity assay inhibited not only DNA fragmentation, but also the release of chromium-51 from the target cells, suggesting that apoptosis and cell lysis are closely related in BsMAb-mediated cell killing. Images Figure 1 PMID:8611371

  6. HIV-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) -Mediating Antibodies Decline while NK Cell Function Increases during Antiretroviral Therapy (ART).

    PubMed

    Jensen, Sanne Skov; Fomsgaard, Anders; Borggren, Marie; Tingstedt, Jeanette Linnea; Gerstoft, Jan; Kronborg, Gitte; Rasmussen, Line Dahlerup; Pedersen, Court; Karlsson, Ingrid

    2015-01-01

    Understanding alterations in HIV-specific immune responses during antiretroviral therapy (ART), such as antibody-dependent cellular cytotoxicity (ADCC), is important in the development of novel strategies to control HIV-1 infection. This study included 53 HIV-1 positive individuals. We evaluated the ability of effector cells and antibodies to mediate ADCC separately and in combination using the ADCC-PanToxiLux assay. The ability of the peripheral blood mononuclear cells (PBMCs) to mediate ADCC was significantly higher in individuals who had been treated with ART before seroconversion, compared to the individuals initiating ART at a low CD4+ T cell count (<350 cells/μl blood) and the ART-naïve individuals. The frequency of CD16 expressing natural killer (NK) cells correlated with both the duration of ART and Granzyme B (GzB) activity. In contrast, the plasma titer of antibodies mediating ADCC declined during ART. These findings suggest improved cytotoxic function of the NK cells if initiating ART early during infection, while the levels of ADCC mediating antibodies declined during ART.

  7. HIV-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) -Mediating Antibodies Decline while NK Cell Function Increases during Antiretroviral Therapy (ART)

    PubMed Central

    Jensen, Sanne Skov; Fomsgaard, Anders; Borggren, Marie; Tingstedt, Jeanette Linnea; Gerstoft, Jan; Kronborg, Gitte; Rasmussen, Line Dahlerup; Pedersen, Court; Karlsson, Ingrid

    2015-01-01

    Understanding alterations in HIV-specific immune responses during antiretroviral therapy (ART), such as antibody-dependent cellular cytotoxicity (ADCC), is important in the development of novel strategies to control HIV-1 infection. This study included 53 HIV-1 positive individuals. We evaluated the ability of effector cells and antibodies to mediate ADCC separately and in combination using the ADCC-PanToxiLux assay. The ability of the peripheral blood mononuclear cells (PBMCs) to mediate ADCC was significantly higher in individuals who had been treated with ART before seroconversion, compared to the individuals initiating ART at a low CD4+ T cell count (<350 cells/μl blood) and the ART-naïve individuals. The frequency of CD16 expressing natural killer (NK) cells correlated with both the duration of ART and Granzyme B (GzB) activity. In contrast, the plasma titer of antibodies mediating ADCC declined during ART. These findings suggest improved cytotoxic function of the NK cells if initiating ART early during infection, while the levels of ADCC mediating antibodies declined during ART. PMID:26696395

  8. Small CD4 Mimetics Prevent HIV-1 Uninfected Bystander CD4 + T Cell Killing Mediated by Antibody-dependent Cell-mediated Cytotoxicity

    PubMed Central

    Richard, Jonathan; Veillette, Maxime; Ding, Shilei; Zoubchenok, Daria; Alsahafi, Nirmin; Coutu, Mathieu; Brassard, Nathalie; Park, Jongwoo; Courter, Joel R.; Melillo, Bruno; Smith, Amos B.; Shaw, George M.; Hahn, Beatrice H.; Sodroski, Joseph; Kaufmann, Daniel E.; Finzi, Andrés

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection causes a progressive depletion of CD4 + T cells. Despite its importance for HIV-1 pathogenesis, the precise mechanisms underlying CD4 + T-cell depletion remain incompletely understood. Here we make the surprising observation that antibody-dependent cell-mediated cytotoxicity (ADCC) mediates the death of uninfected bystander CD4 + T cells in cultures of HIV-1-infected cells. While HIV-1-infected cells are protected from ADCC by the action of the viral Vpu and Nef proteins, uninfected bystander CD4 + T cells bind gp120 shed from productively infected cells and are efficiently recognized by ADCC-mediating antibodies. Thus, gp120 shedding represents a viral mechanism to divert ADCC responses towards uninfected bystander CD4 + T cells. Importantly, CD4-mimetic molecules redirect ADCC responses from uninfected bystander cells to HIV-1-infected cells; therefore, CD4-mimetic compounds might have therapeutic utility in new strategies aimed at specifically eliminating HIV-1-infected cells. PMID:26870823

  9. Small CD4 Mimetics Prevent HIV-1 Uninfected Bystander CD4 + T Cell Killing Mediated by Antibody-dependent Cell-mediated Cytotoxicity.

    PubMed

    Richard, Jonathan; Veillette, Maxime; Ding, Shilei; Zoubchenok, Daria; Alsahafi, Nirmin; Coutu, Mathieu; Brassard, Nathalie; Park, Jongwoo; Courter, Joel R; Melillo, Bruno; Smith, Amos B; Shaw, George M; Hahn, Beatrice H; Sodroski, Joseph; Kaufmann, Daniel E; Finzi, Andrés

    2016-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection causes a progressive depletion of CD4 + T cells. Despite its importance for HIV-1 pathogenesis, the precise mechanisms underlying CD4 + T-cell depletion remain incompletely understood. Here we make the surprising observation that antibody-dependent cell-mediated cytotoxicity (ADCC) mediates the death of uninfected bystander CD4 + T cells in cultures of HIV-1-infected cells. While HIV-1-infected cells are protected from ADCC by the action of the viral Vpu and Nef proteins, uninfected bystander CD4 + T cells bind gp120 shed from productively infected cells and are efficiently recognized by ADCC-mediating antibodies. Thus, gp120 shedding represents a viral mechanism to divert ADCC responses towards uninfected bystander CD4 + T cells. Importantly, CD4-mimetic molecules redirect ADCC responses from uninfected bystander cells to HIV-1-infected cells; therefore, CD4-mimetic compounds might have therapeutic utility in new strategies aimed at specifically eliminating HIV-1-infected cells.

  10. Anti-Sp17 monoclonal antibody with antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity activities against human ovarian cancer cells.

    PubMed

    Song, Jia-xi; Cao, Wang-li; Li, Fang-qiu; Shi, Li-ning; Jia, Xuan

    2012-12-01

    Sperm protein 17 (Sp17) is a cancer testis antigen that has been shown to be overexpressed in a variety of gynecologic malignancies, in particular ovarian cancer. Emerging evidences indicate that Sp17 is involved in tumorigenesis and in the migration of malignant cells. It has been proposed as a useful target for tumor-vaccine strategies and a novel marker to define tumor subsets and predict drug response. However, the antitumor activity of anti-Sp17 monoclonal antibody (anti-Sp17 mAb) has not been investigated. In this study, the in vitro cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities of anti-Sp17 mAb were evaluated using Sp17-positive ovarian cancer cells as targets, Sp17-negative ovarian cancer cells as the control, and healthy human peripheral blood monocytes and healthy human serum as effectors. Our preliminary results indicate that the direct cytotoxicity of anti-Sp17 mAb against the investigated ovarian cancer cells was very weak. However, the cytotoxicity of anti-Sp17 mAb, mediated by peripheral blood mononuclear cells (PBMCs), as ADCC, or by human serum, as CDC, was relatively strong in the Sp17-positive ovarian cancer cells. This finding suggested that anti-Sp17 mAb could be a useful tool against ovarian cancer and may provide insight into the development of low side-effect targeting therapy for this malignant disease.

  11. Antiparietal cell antibody test

    MedlinePlus

    APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; Vitamin B12 - anti- ...

  12. Measurement of Anti-Erythropoiesis-Stimulating Agent IgG4 Antibody as an Indicator of Antibody-Mediated Pure Red Cell Aplasia

    PubMed Central

    Weeraratne, Dohan K.; Kuck, Andrew J.; Chirmule, Narendra

    2013-01-01

    Patients treated with erythropoietin-based erythropoiesis-stimulating agents (ESAs) can develop a rare but life-threatening condition called antibody-mediated pure red cell aplasia (amPRCA). The antibody characteristics in a nephrology patient with amPRCA include high antibody concentrations with neutralizing activity and a mixed IgG subclass including anti-ESA IgG4 antibodies. In contrast, anti-ESA IgG4 antibody is generally not detected in baseline samples and antibody-positive non-PRCA patients. Therefore, we validated a highly sensitive immunoassay on the ImmunoCAP 100 instrument to quantitate anti-ESA IgG4 antibodies using a human recombinant anti-epoetin alfa (EPO) IgG4 antibody as a calibrator. The biotinylated ESA was applied to a streptavidin ImmunoCAP, and bound anti-ESA IgG4 antibodies were detected using a β-galactosidase-conjugated mouse anti-human IgG4 antibody. The validated assay was used to detect anti-ESA IgG4 in amPRCA and non-PRCA patients. The immunoassay detected 15 ng/ml of human anti-EPO IgG4 antibody in the presence of a 200 M excess of human anti-ESA IgG1, IgG2, or IgM antibody and tolerated 2 μg/ml of soluble erythropoietin. All patient samples with confirmed amPRCA had measurable anti-ESA IgG4 antibodies. In addition, 94% (17/18) of non-PRCA patient samples were antibody negative or had below 15 ng/ml of anti-ESA IgG4 antibodies. This novel immunoassay can measure low-nanogram quantities of human anti-ESA IgG4 antibodies in the presence of other anti-ESA antibodies. An increased concentration of anti-ESA IgG4 antibody is associated with the development of amPRCA. We propose that the measurement of anti-ESA specific IgG4 antibodies may facilitate early detection of amPRCA in patients receiving all ESAs structurally related to human erythropoietin. PMID:23114696

  13. Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities.

    PubMed

    Chung, Shan; Quarmby, Valerie; Gao, Xiaoying; Ying, Yong; Lin, Linda; Reed, Chae; Fong, Chris; Lau, Wendy; Qiu, Zhihua J; Shen, Amy; Vanderlaan, Martin; Song, An

    2012-01-01

    The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its "regular" fucosylated counterpart and a series of mixtures containing varying proportions of "regular" and afucosylated materials. Compared with the "regular" fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa, moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC 50 values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells.

  14. Bispecific Antibodies that Mediate Killing of Cells Infected with Human Immunodeficiency Virus of Any Strain

    NASA Astrophysics Data System (ADS)

    Berg, Jorg; Lotscher, Erika; Steimer, Kathelyn S.; Capon, Daniel J.; Baenziger, Jurg; Jack, Hans-Martin; Wabl, Matthias

    1991-06-01

    Although AIDS patients lose human immunodeficiency virus (HIV)-specific cytotoxic T cells, their remaining CD8-positive T lymphocytes maintain cytotoxic function. To exploit this fact we have constructed bispecific antibodies that direct cytotoxic T lymphocytes of any specificity to cells that express gp120 of HIV. These bispecific antibodies comprise one heavy/light chain pair from an antibody to CD3, linked to a heavy chain whose variable region has been replaced with sequences from CD4 plus a second light chain. CD3 is part of the antigen receptor on T cells and is responsible for signal transduction. In the presence of these bispecific antibodies, T cells of irrelevant specificity effectively lyse HIV-infected cells in vitro.

  15. Incidence of erythropoietin antibody-mediated pure red cell aplasia: the Prospective Immunogenicity Surveillance Registry (PRIMS)

    PubMed Central

    Macdougall, Iain C.; Casadevall, Nicole; Locatelli, Francesco; Combe, Christian; London, Gerard M.; Di Paolo, Salvatore; Kribben, Andreas; Fliser, Danilo; Messner, Hans; McNeil, John; Stevens, Paul; Santoro, Antonio; De Francisco, Angel L.M.; Percheson, Paul; Potamianou, Anna; Foucher, Arnaud; Fife, Daniel; Mérit, Véronique; Vercammen, Els

    2015-01-01

    Background Subcutaneous administration of Eprex® (epoetin alfa) in patients with chronic kidney disease (CKD) was contraindicated in the European Union between 2002 and 2006 after increased reports of anti-erythropoietin antibody-mediated pure red cell aplasia (PRCA). The Prospective Immunogenicity Surveillance Registry (PRIMS) was conducted to estimate the incidence of antibody-mediated PRCA with subcutaneous administration of a new coated-stopper syringe presentation of Eprex® and to compare this with the PRCA incidence with subcutaneous NeoRecormon® (epoetin beta) and Aranesp® (darbepoetin alfa). Methods PRIMS was a multicentre, multinational, non-interventional, parallel-group, immunogenicity surveillance registry. Adults with CKD receiving or about to initiate subcutaneous Eprex®, NeoRecormon® or Aranesp® for anaemia were enrolled and followed for up to 3 years. Unexplained loss or lack of effect (LOE), including suspected PRCA, was reported, with antibody testing for confirmation of PRCA. Results Of the 15 333 patients enrolled, 5948 received Eprex® (8377 patient-years) and 9356 received NeoRecormon®/Aranesp® (14 286 patient-years). No treatment data were available for 29 patients. Among 23 patients with LOE, five cases of PRCA were confirmed (Eprex®, n = 3; NeoRecormon®, n = 1; Aranesp®, n = 1). Based on exposed time, PRCA incidence was 35.8/100 000 patient-years (95% CI 7.4–104.7) for Eprex® versus 14.0/100 000 patient-years (95% CI 1.7–50.6) for NeoRecormon®/Aranesp®. The incidence of PRCA with Eprex® was not significantly different versus comparator ESAs (rate ratio: 2.56; 95% CI 0.43–15.31). An analysis based on observed time produced similar findings. Conclusion This large, prospective registry demonstrates that PRCA is rare with subcutaneous administration of either the new coated-stopper syringe presentation of Eprex®, or NeoRecormon® or Aranesp®. PMID:25239637

  16. C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells.

    PubMed

    Charest-Morin, Xavier; Pépin, Rémy; Gagné-Henley, Angélique; Morissette, Guillaume; Lodge, Robert; Marceau, François

    2013-01-01

    The C-C chemokine receptor-7 (CCR7) is a G protein coupled receptor that has a role in leukocyte homing, but that is also expressed in aggressive tumor cells. Preclinical research supports that CCR7 is a valid target in oncology. In view of the increasing availability of therapeutic monoclonal antibodies that carry cytotoxic cargoes, we studied the feasibility of forcing intact cells to internalize known monoclonal antibodies by exploiting the cycle of endocytosis and recycling triggered by the CCR7 agonist CCL19. Firstly, an anti-CCR7 antibody (CD197; clone 150503) labeled surface recombinant CCR7 expressed in intact HEK 293a cells and the fluorescent antibody was internalized following CCL19 treatment. Secondly, a recombinant myc-tagged CCL19 construction was exploited along the anti-myc monoclonal antibody 4A6. The myc-tagged ligand was produced as a conditioned medium of transfected HEK 293a cells that contained the equivalent of 430 ng/ml of immunoreactive CCL19 (average value, ELISA determination). CCL19-myc, but not authentic CCL19, carried the fluorophore-labeled antibody 4A6 into other recipient cells that expressed recombinant CCR7 (microscopy, cytofluorometry). The immune complexes were apparent in endosomal structures, co-localized well with the small GTPase Rab5 and progressed toward Rab7-positive endosomes. A dominant negative form of Rab5 (GDP-locked) inhibited this endocytosis. Further, endosomes in CCL19-myc- or CCL19-stimulated cells were positive for β-arrestin2, but rarely for β-arrestin1. Following treatment with CCL19-myc and the 4A6 antibody, the melanoma cell line A375 that expresses endogenous CCR7 was specifically stained using a secondary peroxidase-conjugated antibody. Agonist-stimulated CCR7 can transport antibody-based cargoes, with possible therapeutic applications in oncology.

  17. Anti-β₂M monoclonal antibodies kill myeloma cells via cell- and complement-mediated cytotoxicity.

    PubMed

    Zhang, Mingjun; Qian, Jianfei; Lan, Yongsheng; Lu, Yong; Li, Haiyan; Hong, Bangxing; Zheng, Yuhuan; He, Jin; Yang, Jing; Yi, Qing

    2014-09-01

    Our previous studies showed that anti-β2M monoclonal antibodies (mAbs) at high doses have direct apoptotic effects on myeloma cells, suggesting that anti-β2M mAbs might be developed as a novel therapeutic agent. In this study, we investigated the ability of the mAbs at much lower concentrations to indirectly kill myeloma cells by utilizing immune effector cells or molecules. Our results showed that anti-β2M mAbs effectively lysed MM cells via antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which were correlated with and dependent on the surface expression of β2M on MM cells. The presence of MM bone marrow stromal cells or addition of IL-6 did not attenuate anti-β2M mAb-induced ADCC and CDC activities against MM cells. Furthermore, anti-β2M mAbs only showed limited cytotoxicity toward normal B cells and nontumorous mesenchymal stem cells, indicating that the ADCC and CDC activities of the anti-β2M mAbs were more prone to the tumor cells. Lenalidomide potentiated in vitro ADCC activity against MM cells and in vivo tumor inhibition capacity induced by the anti-β2M mAbs by enhancing the activity of NK cells. These results support clinical development of anti-β2M mAbs, both as a monotherapy and in combination with lenalidomide, to improve MM patient outcome.

  18. LGR5 expressing cells of hair follicle as potential targets for antibody mediated anti-cancer laser therapy

    NASA Astrophysics Data System (ADS)

    Popov, Boris V.

    2013-02-01

    Near infrared laser immunotherapy becomes now a new promising research field to cure the patients with cancers. One of the critical limitation in medical application of this treatment is availability of the specific markers for delivery of laser-sensitive nanoparticles. When coupled to antibodies to the cancer stem cells markers these nanoparticles may be delivered to the cancer tissue and mediate the laser induced thermolysis of the cancer stem cells that initiate and drive growth of cancer. This paper addresses the Lgr5 cell surface marker mediating the Wnt/β-catenin signal transduction as a potential target for anti-cancer laser immunotherapy of skin cancers.

  19. Disialoganglioside GD2 on human neuroblastoma cells: target antigen for monoclonal antibody-mediated cytolysis and suppression of tumor growth.

    PubMed

    Mujoo, K; Cheresh, D A; Yang, H M; Reisfeld, R A

    1987-02-15

    A murine monoclonal antibody 14.18 specifically recognizes disialoganglioside GD2, the major ganglioside expressed on the surface of human neuroblastoma cells. This monoclonal antibody (Mab) is of immunoglobulin G3 isotype, has an affinity constant (KA) of 3.5 X 10(8) M-1, and reacts preferentially with tumor cells and fresh frozen tumor tissues of neuroectodermal origin in enzyme-linked immunosorbent assay and immunoperoxidase assays, respectively. Mab 14.18 effectively lyses a number of human neuroblastoma cell lines by two distinct mechanisms, i.e., antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. There is a good correlation between the average number of antibody-binding sites per neuroblastoma cell and the amount of cell lysis observed in complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity. In addition, Mab 14.18 suppresses establishment as well as growth of progressively growing, established human neuroblastoma tumors in nude mice when injected 24 h and 9 days, respectively, after the initial s.c. inoculation of tumor cells. These data suggest that Mab 14.18 can mediate tumor cell killing in vivo and in vitro and may thereby prove useful for immunotherapy of human neuroblastoma.

  20. Antisperm antibody-mediated alterations in the cellular activity of human trophoblast cells in culture.

    PubMed

    Sinha, D; Chattopadhyay, S

    1994-04-01

    Immune recognition of the fetus is well documented, yet the immunological basis of pregnancy loss awaits elucidation. Identification of trophoblast membrane epitopes as non-self either by preformed immunoglobulins or by circulating immunocompetent cells would lead to immunological rejection of the tissue. Such an event may occur in cases of cross-reacting antibodies developed as a consequence of exposure of sperm surface antigens. This hypothesis was tested by developing specific antibodies in rabbits against intact sperm surface antigens. These were subjected to different schedules of IgG purification and characterization. By means of nuclide precursor incorporation, the effect of antisperm antibody on DNA, RNA and protein synthesis of trophoblast cells in culture were studied. The results showed that the antibody inhibits incorporation into cells but after a delay of 72 hours some cells gradually recover. The interaction also led to a reduced rate of hCG production. Lysosomal enzyme activity was inhibited in the spent medium of antibody-treated cells but lysosome rich fractions showed no effect. This indicated that the major effect of the antibody was growth inhibitory rather than cytolytic. PMID:7520885

  1. Tumor gene therapy by MVA-mediated expression of T-cell-stimulating antibodies.

    PubMed

    Paul, Stephane; Regulier, Etienne; Rooke, Ronald; Stoeckel, Fabienne; Geist, Michel; Homann, Horst; Balloul, Jean-Marc; Villeval, Dominique; Poitevin, Yves; Kieny, Marie-Paule; Acres, R Bruce

    2002-05-01

    Immune responses to tumor-associated antigens are often dampened by a tumor-induced state of immune anergy. Previous work has attempted to overcome tumor-induced T-cell anergy by the direct injection of vectors carrying the genes encoding one of a variety of cytokines. We hypothesised that the polyclonal stimulation of T cells, preferably through the TCR complex, would result in a cascade of cytokines associated with T-cell activation and would be best able to overcome T-cell anergy. Here we use the highly attenuated MVA poxvirus to express on tumor cells, in vitro and in vivo, either of three membrane-bound monoclonal antibodies specific for murine TCR complex. Using this system, we have expressed antibodies specific for the CD3 epsilon chain (KT3), TCR alpha/beta complex (H57-597), and V beta 7 chain (TR310). Tumor cells bristling with these antibodies are capable of inducing murine T-cell proliferation and cytokine production. When injected into growing tumors (P815, RenCa, and B16F10), these constructs induce the activation of immune effector cells and result in the rejection of the tumor. Histological and FACS analysis of tumor-infiltrating leukocytes reveal that the injection of recombinant virus-expressing antibodies specific for the TCR complex attracts and activates (CD25(+), CD69(+)) CD4 and CD8 lymphocytes. This approach represents a novel strategy to overcome T-cell anergy in tumors and allow the stimulation of tumor-specific T cells. PMID:11961670

  2. X-linked inhibitor of apoptosis protein mediates tumor cell resistance to antibody-dependent cellular cytotoxicity

    PubMed Central

    Evans, M K; Sauer, S J; Nath, S; Robinson, T J; Morse, M A; Devi, G R

    2016-01-01

    Inflammatory breast cancer (IBC) is the deadliest, distinct subtype of breast cancer. High expression of epidermal growth factor receptors [EGFR or human epidermal growth factor receptor 2 (HER2)] in IBC tumors has prompted trials of anti-EGFR/HER2 monoclonal antibodies to inhibit oncogenic signaling; however, de novo and acquired therapeutic resistance is common. Another critical function of these antibodies is to mediate antibody-dependent cellular cytotoxicity (ADCC), which enables immune effector cells to engage tumors and deliver granzymes, activating executioner caspases. We hypothesized that high expression of anti-apoptotic molecules in tumors would render them resistant to ADCC. Herein, we demonstrate that the most potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), overexpressed in IBC, drives resistance to ADCC mediated by cetuximab (anti-EGFR) and trastuzumab (anti-HER2). Overexpression of XIAP in parental IBC cell lines enhances resistance to ADCC; conversely, targeted downregulation of XIAP in ADCC-resistant IBC cells renders them sensitive. As hypothesized, this ADCC resistance is in part a result of the ability of XIAP to inhibit caspase activity; however, we also unexpectedly found that resistance was dependent on XIAP-mediated, caspase-independent suppression of reactive oxygen species (ROS) accumulation, which otherwise occurs during ADCC. Transcriptome analysis supported these observations by revealing modulation of genes involved in immunosuppression and oxidative stress response in XIAP-overexpressing, ADCC-resistant cells. We conclude that XIAP is a critical modulator of ADCC responsiveness, operating through both caspase-dependent and -independent mechanisms. These results suggest that strategies targeting the effects of XIAP on caspase activation and ROS suppression have the potential to enhance the activity of monoclonal antibody-based immunotherapy. PMID:26821068

  3. Antibody-Mediated Enhancement of Parvovirus B19 Uptake into Endothelial Cells Mediated by a Receptor for Complement Factor C1q

    PubMed Central

    von Kietzell, Kristina; Pozzuto, Tanja; Heilbronn, Regine; Grössl, Tobias; Fechner, Henry

    2014-01-01

    ABSTRACT Despite its strong host tropism for erythroid progenitor cells, human parvovirus B19 (B19V) can also infect a variety of additional cell types. Acute and chronic inflammatory cardiomyopathies have been associated with a high prevalence of B19V DNA in endothelial cells of the myocardium. To elucidate the mechanisms of B19V uptake into endothelium, we first analyzed the surface expression of the well-characterized primary B19V receptor P antigen and the putative coreceptors α5β1 integrins and Ku80 antigen on primary and permanent endothelial cells. The receptor expression pattern and also the primary attachment levels were similar to those in the UT7/Epo-S1 cell line regarded as functional for B19V entry, but internalization of the virus was strongly reduced. As an alternative B19V uptake mechanism in endothelial cells, we demonstrated antibody-dependent enhancement (ADE), with up to a 4,000-fold increase in B19V uptake in the presence of B19V-specific human antibodies. ADE was mediated almost exclusively at the level of virus internalization, with efficient B19V translocation to the nucleus. In contrast to monocytes, where ADE of B19V has been described previously, enhancement does not rely on interaction of the virus-antibody complexes with Fc receptors (FcRs), but rather, involves an alternative mechanism mediated by the heat-sensitive complement factor C1q and its receptor, CD93. Our results suggest that ADE represents the predominant mechanism of endothelial B19V infection, and it is tempting to speculate that it may play a role in the pathogenicity of cardiac B19V infection. IMPORTANCE Both efficient entry and productive infection of human parvovirus B19 (B19V) seem to be limited to erythroid progenitor cells. However, in vivo, the viral DNA can also be detected in additional cell types, such as endothelial cells of the myocardium, where its presence has been associated with acute and chronic inflammatory cardiomyopathies. In this study, we

  4. Antibody- and cell-mediated immune responses of Actinobacillus pleuropneumoniae-infected and bacterin-vaccinated pigs.

    PubMed Central

    Furesz, S E; Mallard, B A; Bossé, J T; Rosendal, S; Wilkie, B N; MacInnes, J I

    1997-01-01

    Current porcine pleuropneumonia bacterins afford only partial protection by decreasing mortality but not morbidity. In order to better understand the type(s) of immune response associated with protection, antibody- and cell-mediated immune responses (CMIR) were compared for piglets before and after administration of a commercial bacterin, which confers partial protection, or a low-dose (10(5) CFU/ml) aerosol challenge with Actinobacillus pleuropneumoniae CM5 (LD), which induces complete protection. Control groups received phosphate-buffered saline or adjuvant. Serum antibody response, antibody avidity, delayed-type hypersensitivity (DTH), and lymphocyte blastogenic responses were measured and compared among treatment groups to the lipopolysaccharide (LPS), capsular polysaccharide (CPS), hemolysin (HLY), and outer membrane proteins (OMP) of A. pleuropneumoniae. Peripheral blood lymphocytes and sera were collected prior to and following primary and secondary immunization-infection and high-dose A. pleuropneumoniae CM5 (10(7) CFU/ml) aerosol challenge. Serum antibody and DTH, particularly that to HLY, differed significantly between treatment groups, and increases were associated with protection. LD-infected piglets had higher antibody responses (P < or = 0.01) and antibody avidity (P < or = 0.10) than bacterin-vaccinated and control groups. Anti-HLY antibodies were consistently associated with protection, whereas anti-LPS and anti-CPS antibodies were not. LD-infected animals had higher DTH responses, particularly to HLY, than bacterin-vaccinated pigs (P < or = 0.03). The LD-infected group maintained consistent blastogenic responses to HLY, LPS, CPS, and OMP over the course of infection, unlike the bacterin-vaccinated and control animals. These data suggest that the immune responses induced by a commercial bacterin are very different from those induced by LD aerosol infection and that current bacterins may be modified, for instance, by addition of HLY, so as to

  5. Control of Toll-like receptor-mediated T cell-independent type 1 antibody responses by the inducible nuclear protein IκB-ζ.

    PubMed

    Hanihara-Tatsuzawa, Fumito; Miura, Hanae; Kobayashi, Shuhei; Isagawa, Takayuki; Okuma, Atsushi; Manabe, Ichiro; MaruYama, Takashi

    2014-11-01

    Antibody responses have been classified as being either T cell-dependent or T cell-independent (TI). TI antibody responses are further classified as being either type 1 (TI-1) or type 2 (TI-2), depending on their requirement for B cell-mediated antigen receptor signaling. Although the mechanistic basis of antibody responses has been studied extensively, it remains unclear whether different antibody responses share similarities in their transcriptional regulation. Here, we show that mice deficient in IκB-ζ, specifically in their B cells, have impaired TI-1 antibody responses but normal T cell-dependent and TI-2 antibody responses. The absence of IκB-ζ in B cells also impaired proliferation triggered by Toll-like receptor (TLR) activation, plasma cell differentiation, and class switch recombination (CSR). Mechanistically, IκB-ζ-deficient B cells could not induce TLR-mediated induction of activation-induced cytidine deaminase (AID), a class-switch DNA recombinase. Retroviral transduction of AID in IκB-ζ-deficient B cells restored CSR activity. Furthermore, acetylation of histone H3 in the vicinity of the transcription start site of the gene that encodes AID was reduced in IκB-ζ-deficient B cells relative to IκB-ζ-expressing B cells. These results indicate that IκB-ζ regulates TLR-mediated CSR by inducing AID. Moreover, IκB-ζ defines differences in the transcriptional regulation of different antibody responses.

  6. Clinically effective monoclonal antibody 3F8 mediates nonoxidative lysis of human neuroectodermal tumor cells by polymorphonuclear leukocytes.

    PubMed

    Kushner, B H; Cheung, N K

    1991-09-15

    Most studies of antibody-dependent cellular cytotoxicity (ADCC) by polymorphonuclear leukocytes (PMN) have supported oxidative lytic processes. This may be because the studies used nonhuman or nonneoplastic cells that were highly sensitive to reactive oxygen species or were small enough to be phagocytosed by PMN. We therefore investigated whether oxygen radicals participate in PMN cytotoxicity toward human neuroectodermal solid tumor cells sensitized by 3F8, which is an anti-ganglioside GD2 murine IgG3 monoclonal antibody with documented anticancer activity in humans. A 4-h 51Cr release assay was used to assess tumor cell lysis by hydrogen peroxide, superoxide, and hypochlorite. Nine of 11 GD2(+) human melanoma and neuroblastoma cell lines had equal or greater resistance to these oxidants as compared to a GD2(-) human carcinoma line (SKBr1-III) found by others (and confirmed by us) to be significantly more resistant to oxidative lysis than a murine cell line (P388D1) representative of those commonly used in cytotoxicity assays. To facilitate detection of oxidant-mediated lysis, subsequent studies of 3F8-mediated ADCC used GD2(+) targets that were relatively sensitive and others that were relatively resistant to oxygen radicals. Normal PMN and PMN obtained from children with chronic granulomatous disease, which do not generate reactive oxygen species, were equally effective in ADCC. Granulocyte-macrophage colony-stimulating factor, which primes oxidative responses of normal but not of chronic granulomatous disease PMN, enhanced ADCC by both kinds of PMN. During ADCC of 3F8-sensitized targets, with or without granulocyte-macrophage colony-stimulating factor, GD2(-) "innocent bystander" tumor cells (including P388D1) were not lysed, a finding consistent with unimportant extracellular release of cytotoxic mediators. Finally, antioxidant and antimyeloperoxidase moieties did not block ADCC. We conclude that oxidants are not key factors in 3F8-mediated lysis by PMN of

  7. Evidence of T-cell mediated neuronal injury in stiff-person syndrome with anti-amphiphysin antibodies.

    PubMed

    Poh, Mervyn Q W; Simon, Neil G; Buckland, Michael E; Salisbury, Elizabeth; Watson, Shaun

    2014-02-15

    Paraneoplastic stiff-person syndrome (SPS) has been associated with antibodies against amphiphysin. Current evidence supports a pathogenic role for anti-amphiphysin antibodies. A 74-year-old female was diagnosed with amphiphysin-associated paraneoplastic stiff-person syndrome and associated encephalomyelitis. She had initial response to IVIG, however her symptoms worsened after two months and were resistant to further treatment. Subsequently the patient died and a post-mortem was performed. Neuropathology revealed perivascular and parenchymal lymphocytic infiltrates, with neuronophagia mediated by CD8+ T cells and microglia in brainstem, spinal cord, and mesial temporal lobe structures. These findings suggest a pathogenic role of cytotoxic CD8+ T-cells, with potential implication for therapy of future patients.

  8. Lapatinib enhances trastuzumab-mediated antibody-dependent cellular cytotoxicity via upregulation of HER2 in malignant mesothelioma cells

    PubMed Central

    OKITA, RIKI; SHIMIZU, KATSUHIKO; NOJIMA, YUJI; YUKAWA, TAKURO; MAEDA, AI; SAISHO, SHINSUKE; NAKATA, MASAO

    2015-01-01

    EGFR/HER2 are frequently expressed in MPM tissues, however, no studies have shown the clinical benefit of using EGFR/HER2-targeting drugs in patients with malignant pleural mesothelioma (MPM). It was reported that the tyrosine kinase inhibitor (TKI) lapatinib enhanced trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC) in HER2-positive breast cancer, suggesting that this combination is a promising strategy for MPM treatment. The aim of the present study was to explore the possibility of a TKI combined with trastuzumab to enhance ADCC in MPM cells. Five MPM cell lines were used to test the effects of TKIs targeting EGFR (gefitinib, afatinib and lapatinib) on cell proliferation and the expression of the HER family receptor. The combined effects of TKI with trastuzumab on ADCC were evaluated using the LDH release assay. Additionally, MPM cells were isolated from patients and evaluated for lapatinib-induced upregulation of HER family receptors and trastuzumab- or cetuximab-mediated ADCC. In MPM cell lines, HER2 expression was upregulated by lapatinib, downregulated by afatinib and unaffected by gefitinib. As expected, more trastuzumab bound to MPM cells pretreated with lapatinib than untreated cells, resulting in the enhancement of trastuzumab-mediated ADCC in MPM cells. In patient-derived MPM cells, both HER2 and EGFR were upregulated by lapatinib, resulting in the enhancement of both trastuzumab- and cetuximab-mediated ADCC. Of the three TKIs, only lapatinib enhanced trastuzumab-mediated ADCC via the upregulation of HER2 expression in MPM cells, suggesting that sequential combination of lapatinib and trastuzumab may be a promising strategy for MPM treatment. PMID:26503698

  9. Lapatinib enhances trastuzumab-mediated antibody-dependent cellular cytotoxicity via upregulation of HER2 in malignant mesothelioma cells.

    PubMed

    Okita, Riki; Shimizu, Katsuhiko; Nojima, Yuji; Yukawa, Takuro; Maeda, Ai; Saisho, Shinsuke; Nakata, Masao

    2015-12-01

    EGFR/HER2 are frequently expressed in MPM tissues, however, no studies have shown the clinical benefit of using EGFR/HER2-targeting drugs in patients with malignant pleural mesothelioma (MPM). It was reported that the tyrosine kinase inhibitor (TKI) lapatinib enhanced trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC) in HER2-positive breast cancer, suggesting that this combination is a promising strategy for MPM treatment. The aim of the present study was to explore the possibility of a TKI combined with trastuzumab to enhance ADCC in MPM cells. Five MPM cell lines were used to test the effects of TKIs targeting EGFR (gefitinib, afatinib and lapatinib) on cell proliferation and the expression of the HER family receptor. The combined effects of TKI with trastuzumab on ADCC were evaluated using the LDH release assay. Additionally, MPM cells were isolated from patients and evaluated for lapatinib-induced upregulation of HER family receptors and trastuzumab- or cetuximab‑mediated ADCC. In MPM cell lines, HER2 expression was upregulated by lapatinib, downregulated by afatinib and unaffected by gefitinib. As expected, more trastuzumab bound to MPM cells pretreated with lapatinib than untreated cells, resulting in the enhancement of trastuzumab-mediated ADCC in MPM cells. In patient-derived MPM cells, both HER2 and EGFR were upregulated by lapatinib, resulting in the enhancement of both trastuzumab- and cetuximab-mediated ADCC. Of the three TKIs, only lapatinib enhanced trastuzumab-mediated ADCC via the upregulation of HER2 expression in MPM cells, suggesting that sequential combination of lapatinib and trastuzumab may be a promising strategy for MPM treatment. PMID:26503698

  10. Antibody-Dependent Cell-Mediated Cytotoxicity to Hemagglutinin of Influenza A Viruses After Influenza Vaccination in Humans.

    PubMed

    Zhong, Weimin; Liu, Feng; Wilson, Jason R; Holiday, Crystal; Li, Zhu-Nan; Bai, Yaohui; Tzeng, Wen-Pin; Stevens, James; York, Ian A; Levine, Min Z

    2016-04-01

    Background.  Detection of neutralizing antibodies (nAbs) to influenza A virus hemagglutinin (HA) antigens by conventional serological assays is currently the main immune correlate of protection for influenza vaccines However, current prepandemic avian influenza vaccines are poorly immunogenic in inducing nAbs despite considerable protection conferred. Recent studies show that Ab-dependent cell-mediated cytotoxicity (ADCC) to HA antigens are readily detectable in the sera of healthy individuals and patients with influenza infection. Methods.  Virus neutralization and ADCC activities of serum samples from individuals who received either seasonal or a stock-piled H5N1 avian influenza vaccine were evaluated by hemagglutination inhibition assay, microneutralization assay, and an improved ADCC natural killer (NK) cell activation assay. Results.  Immunization with inactivated seasonal influenza vaccine led to strong expansion of both nAbs and ADCC-mediating antibodies (adccAbs) to H3 antigen of the vaccine virus in 24 postvaccination human sera. In sharp contrast, 18 individuals vaccinated with the adjuvanted H5N1 avian influenza vaccine mounted H5-specific antibodies with strong ADCC activities despite moderate virus neutralization capacity. Strength of HA-specific ADCC activities is largely associated with the titers of HA-binding antibodies and not with the fine antigenic specificity of anti-HA nAbs. Conclusions.  Detection of both nAbs and adccAbs may better reflect protective capacity of HA-specific antibodies induced by avian influenza vaccines. PMID:27419174

  11. Antibody Targeting of “Steady-State” Dendritic Cells Induces Tolerance Mediated by Regulatory T Cells

    PubMed Central

    Mahnke, Karsten; Ring, Sabine; Enk, Alexander H.

    2016-01-01

    Dendritic cells (DCs) are often defined as pivotal inducers of immunity, but these proinflammatory properties only develop after stimulation or ex vivo manipulation of DCs. Under non-inflammatory conditions in vivo, DCs are embedded into a tissue environment and encounter a plethora of self-antigens derived from apoptotic material. This material is transported to secondary lymphoid organs. As DCs maintain their non-activated phenotype in a sterile tissue environment, interaction with T cells will induce rather regulatory T cells than effector T cells. Thus, DCs are not only inducers of immunity but are also critical for maintenance of peripheral tolerance. Therapeutically, intervention for the induction of long-lasting tolerance in several autoimmune conditions may therefore be possible by manipulating DC activation and/or targeting of DCs in their “natural” tissue environment. PMID:26941742

  12. Antibody-mediated pure red cell aplasia (PRCA) on switching from darbepoetin alfa to epoetin beta: what are the implications?

    PubMed Central

    Assunção, José; Vinhas, José

    2008-01-01

    We report the development of antibody-mediated pure red cell aplasia (PRCA) in a 63-year-old man with end-stage renal disease following a switch from darbepoetin alfa to epoetin beta. Haemoglobin levels began to decrease 6 months after the switch. Increasing the epoetin beta dose produced no response and regular blood transfusions were required; PRCA was confirmed and epoetin beta was discontinued. The patient responded positively to immunosuppression; after 2 months on prednisone and cyclophosphamide, haemoglobin levels stabilized and no further transfusions were required. This case highlights the difficulty in establishing a cause-effect relationship where more than one erythropoiesis-stimulating agent is involved. PMID:25983889

  13. Everolimus inhibits anti-HLA I antibody-mediated endothelial cell signaling, migration and proliferation more potently than sirolimus.

    PubMed

    Jin, Y-P; Valenzuela, N M; Ziegler, M E; Rozengurt, E; Reed, E F

    2014-04-01

    Antibody (Ab) crosslinking of HLA I molecules on the surface of endothelial cells triggers proliferative and pro-survival intracellular signaling, which is implicated in the process of chronic allograft rejection, also known as transplant vasculopathy (TV). The purpose of this study was to investigate the role of mammalian target of rapamycin (mTOR) in HLA I Ab-induced signaling cascades. Everolimus provides a tool to establish how the mTOR signal network regulates HLA I-mediated migration, proliferation and survival. We found that everolimus inhibits mTOR complex 1 (mTORC1) by disassociating Raptor from mTOR, thereby preventing class I-induced phosphorylation of mTOR, p70S6K, S6RP and 4E-BP1, and resultant class I-stimulated cell migration and proliferation. Furthermore, we found that everolimus inhibits class I-mediated mTORC2 activation (1) by disassociating Rictor and Sin1 from mTOR; (2) by preventing class I-stimulated Akt phosphorylation and (3) by preventing class I-mediated ERK phosphorylation. These results suggest that everolimus is more effective than sirolimus at antagonizing both mTORC1 and mTORC2, the latter of which is critical in endothelial cell functional changes leading to TV in solid organ transplantation after HLA I crosslinking. Our findings point to a potential therapeutic effect of everolimus in prevention of chronic Ab-mediated rejection. PMID:24580843

  14. An anti–PR1/HLA-A2 T-cell receptor–like antibody mediates complement-dependent cytotoxicity against acute myeloid leukemia progenitor cells

    PubMed Central

    Sergeeva, Anna; Alatrash, Gheath; He, Hong; Ruisaard, Kathryn; Lu, Sijie; Wygant, James; McIntyre, Bradley W.; Ma, Qing; Li, Dan; St John, Lisa; Clise-Dwyer, Karen

    2011-01-01

    PR1 (VLQELNVTV) is a human leukocyte antigen-A2 (HLA-A2)–restricted leukemia-associated peptide from proteinase 3 (P3) and neutrophil elastase (NE) that is recognized by PR1-specific cytotoxic T lymphocytes that contribute to cytogenetic remission of acute myeloid leukemia (AML). We report a novel T-cell receptor (TCR)–like immunoglobulin G2a (IgG2a) antibody (8F4) with high specific binding affinity (dissociation constant [KD] = 9.9nM) for a combined epitope of the PR1/HLA-A2 complex. Flow cytometry and confocal microscopy of 8F4-labeled cells showed significantly higher PR1/HLA-A2 expression on AML blasts compared with normal leukocytes (P = .046). 8F4 mediated complement-dependent cytolysis of AML blasts and Lin−CD34+CD38− leukemia stem cells (LSCs) but not normal leukocytes (P < .005). Although PR1 expression was similar on LSCs and hematopoietic stem cells, 8F4 inhibited AML progenitor cell growth, but not normal colony-forming units from healthy donors (P < .05). This study shows that 8F4, a novel TCR-like antibody, binds to a conformational epitope of the PR1/HLA-A2 complex on the cell surface and mediates specific lysis of AML, including LSCs. Therefore, this antibody warrants further study as a novel approach to targeting leukemia-initiating cells in patients with AML. PMID:21296998

  15. Supraagonistic, bispecific single-chain antibody purified from the serum of cloned, transgenic cows induces T-cell-mediated killing of glioblastoma cells in vitro and in vivo.

    PubMed

    Grosse-Hovest, Ludger; Wick, Wolfgang; Minoia, Rosa; Weller, Michael; Rammensee, Hans-Georg; Brem, Gottfried; Jung, Gundram

    2005-12-20

    Here we characterize the antitumor activity of a recombinant bispecific single-chain antibody isolated from the serum of cloned transgenic cows. The antibody, termed r28M, is directed to a melanoma-associated proteoglycan, also expressed on glioblastoma cells, and to human CD28. Bound to tumor cells, r28M induced exceedingly efficient supraagonistic T-cell activation via the CD28 molecule without an additional stimulus via the TCR/CD3 complex. In vitro, T cells and NK cells contributed to tumor cell killing after r28M-mediated activation of peripheral blood mononuclear cells. However, NK activity depended on T-cell-derived cytokines. In vivo, r28M markedly inhibited the growth of human glioblastoma cells in nude mice. The serum half-life of the protein after i.v. injection was approximately 6 hr. Thus, r28M is unique not only in inducing supraagonistic CD28-mediated T-cell activation against tumor cells in vitro and in vivo, it also meets 2 additional requirements that are critical for clinical application: a relatively long serum half-life and the possibility of obtaining large amounts of active material from cloned transgenic livestock.

  16. Interleukin 1 and tumour necrosis factor alpha may be responsible for the lytic mechanism during anti-tumour antibody-dependent cell-mediated cytotoxicity.

    PubMed Central

    Pullyblank, A. M.; Guillou, P. J.; Monson, J. R.

    1995-01-01

    Antibodies are thought to bring about tumour cell lysis by antibody-dependent cell-mediated cytotoxicity (ADCC), but the exact mechanism is not well elucidated. Monocytes are known to be important mediators of anti-tumour ADCC and are also known to secrete the cytokines tumour necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta), both of which have been shown to bring about tumour cell lysis. We examined the release of these cytokines during ADCC and attempted to elucidate which components of the ADCC reaction were necessary for cytokine production. We measured TNF-alpha and IL-1 beta in supernatants collected from a standard ADCC assay using each of the anti-colorectal antibodies m17-1A, c17-1A and cSF25. We found that there was significant TNF-alpha and IL-1 beta release during ADCC mediated by each of these three antibodies and that the magnitude of cytokine release seemed to reflect the degree of tumour cell lysis produced by each antibody. Furthermore, we found that effector cells, target cells and a specific anti-tumour antibody were necessary for this to occur. The presence of only some of the components of the reaction or of an irrelevant antibody produced little or no TNF-alpha or IL-1 beta. We conclude that TNF-alpha and IL-1 beta are released when an effector and tumour target cell are united by a specific tumour antibody and that these cytokines may be important in bringing about tumour cell lysis during the ADCC reaction. PMID:7669568

  17. Antibody-mediated opsonization of red blood cells in parvovirus B19 infection.

    PubMed

    Chehadeh, Wassim; Halim, Medhat A; Al-Nakib, Widad

    2009-07-20

    Red blood cells (RBCs) express abundantly parvovirus B19 receptor, and their role in the dissemination or clearance of B19 infection is unknown. In this study, we report that in early, acute or persistent infection, B19 viremia is mostly associated with RBCs. The capacity of different patients' plasma or IgG to opsonize RBCs collected from patients with early B19 infection, was investigated. The highest opsonization activity was observed with plasma or IgG fractions from patients with past B19 infection. In contrast, IgG samples from patients with acute or persistent infection showed no or little opsonization activity. The depletion of antibodies specific to B19 VP1, but not VP2, from IgG samples, resulted in a significant suppression of opsonization. Furthermore, IgG samples preincubated with heated B19 particles exposing VP1-unique (VP1u) region were unable to opsonize RBCs. These observations clearly suggest a role for anti-VP1u IgG in the opsonization of RBC-bound B19 particles.

  18. IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines.

    PubMed

    Ho, Steven C L; Bardor, Muriel; Feng, Huatao; Mariati; Tong, Yen Wah; Song, Zhiwei; Yap, Miranda G S; Yang, Yuansheng

    2012-01-01

    A Tricistronic vector utilizing internal ribosome entry site (IRES) elements to express the light chain (LC), heavy chain (HC), and a neomycin phosphotransferase (NPT) selection marker from one transcript is designed for generation of mAb expressing CHO cell lines. As compared to the commonly used vectors, benefits of this design include: (1) minimized non-expressing clones, (2) enhanced stable mAb productivity without gene amplification, (3) control of LC and HC expression at defined ratios, and (4) consistent product quality. After optimization of the LC and HC arrangement and increasing selection stringency by weakening the NPT selection marker, this Tricistronic vector is able to generate stably transfected pools with specific productivity (qmAb) greater than 5pg/cell/day (pcd) and titers over 150mg/L. 5% of clones from these pools have qmAb greater than 20pcd and titers ranging from 300 to more than 500mg/L under non-optimized shake flask batch cultures using commercially available protein-free medium. The mAb produced by these clones have low aggregation and consistent glycosylation profiles. The entire process of transfection to high-expressing clones requires only 6 months. The IRES-mediated Tricistronic vector provides an attractive alternative to commonly used vectors for fast generation of mAb CHO cell lines with high productivity. PMID:22024589

  19. Mediation of cytotoxic functions by classes and subclasses of sheep antibody reactive with cell surface immunoglobulin idiotypic and constant region determinants.

    PubMed Central

    Stevenson, F K; Elliott, E V

    1978-01-01

    Sheep antibodies, reactive with either the idiotypic or constant region antigenic determinants of the immunoglobulin light chain on guinea-pig L2C leukaemic cells, were separated into IgM and into the two subclasses of IgG, IgG1 and IgG2. Antibody of both IgG subclasses inhibited the migration of L2C cells along plastic surfaces; IgM was only weakly inhibitory. Antibody of class IgM and of subclass IgG1 mediated complement cytotoxicity against the L2C cells whereas only that of subclass IgG2 mediated K-cell cytotoxicity; the effector arms were rabbit complement and sheep peripheral leucocytes, respectively. PMID:75184

  20. Evaluation of B cell maturation antigen as a target for antibody drug conjugate mediated cytotoxicity in multiple myeloma.

    PubMed

    Lee, Lydia; Bounds, Danton; Paterson, Jennifer; Herledan, Gaelle; Sully, Katherine; Seestaller-Wehr, Laura M; Fieles, William E; Tunstead, James; McCahon, Lee; Germaschewski, Fiona M; Mayes, Patrick A; Craigen, Jenny L; Rodriguez-Justo, Manuel; Yong, Kwee L

    2016-09-01

    B-cell maturation antigen (BCMA, also termed TNFRSF17) is an attractive therapeutic target due to its restricted expression on normal and malignant plasma cells (PC). GSK2857916 (or J6M0-MMAF) is a BCMA-specific antibody conjugated to the microtubule-disrupting agent monomethyl auristatin F (MMAF) via a protease-resistant linker. To evaluate the clinical potential of this agent, tumour cells from seventy multiple myeloma (MM) patients were assessed for BCMA expression by immunohistochemistry and flow cytometry. All patients tested expressed BCMA, at varying levels, and both surface and intracellular expression were observed. BCMA expression is maintained through relapse, extramedullary spread and in residual disease post therapy. BCMA levels may also be prognostically useful as higher levels of BCMA were associated with poorer outcomes, even taking into account genetic risk. We observed rapid internalization of surface BCMA and newly expressed protein by 1 h, suggesting a mechanism for J6M0-MMAF activity even with low surface antigen. J6M0-MMAF mediated cytotoxicity of MM cells varied with dose and antigen levels, with clonogenic progenitors killed at lower doses than mature cells. In comparison, J6M0-MMAF killing of primary CD138(+) myeloma cells occurred with slower kinetics. Our observations support BCMA to be a promising therapeutic target in MM for novel therapies such as J6M0-MMAF. PMID:27313079

  1. Removal of terminal alpha-galactosyl residues from xenogeneic porcine endothelial cells. Decrease in complement-mediated cytotoxicity but persistence of IgG1-mediated antibody-dependent cell-mediated cytotoxicity.

    PubMed

    Watier, H; Guillaumin, J M; Piller, F; Lacord, M; Thibault, G; Lebranchu, Y; Monsigny, M; Bardos, P

    1996-07-15

    To determine the role of the terminal alpha-galactosyl residue in the endothelial damage mediated by human xenoreactive natural antibodies (IgM and IgG), we treated porcine endothelial cells in culture with green coffee bean alpha-galactosidase. A practically complete removal of terminal alpha-Gal residues (as evaluated by flow cytometry with Bandeiraea simplicifolia isolectin B4) and concomitant exposure of N-acetyllactosamine were obtained without altering cell viability. A dramatic decrease in IgM and IgG binding (from a pool of human sera) was observed, confirming the key role of the alpha-galactosyl residues. The enzyme treatment did not induce any nonspecific immunoglobulin binding sites, but led to the exposure of new epitopes for a minor fraction of IgM. The main residual IgM and IgG binding could be due to xenoantigens other than the alpha-galactosyl residues. When alpha-galactosidase-treated endothelial cells were used as targets in cytotoxicity experiments, they were less susceptible than untreated cells to complement-mediated cytotoxicity induced by fresh human serum. In contrast, they did not acquire resistance to human IgG-dependent cellular cytotoxicity, despite the decrease in IgG binding. Because it is known that antibody-dependent cytotoxicity mediated by CD16+ NK cells is dependent on IgG1 and IgG3, and not on IgG2 or IgG4, which was confirmed by blocking experiments, we studied the binding of all four subclasses to intact and alpha-galactosidase-treated endothelial cells. Two major subclasses, IgG1 and IgG2, bound to untreated endothelial cells, whereas IgG3 binding was low and IgG4 binding was negligible. A decrease in IgG1, IgG2, and IgG3 binding was observed upon alpha-galactosidase treatment, indicating that antibodies belonging to these three subclasses recognize alpha-galactosyl residues. The decrease in IgG2 binding was more pronounced than the decrease in IgG1 binding. Collectively, these data indicate that IgG1 xenoreactive natural

  2. Fenretinide sensitizes multidrug-resistant human neuroblastoma cells to antibody-independent and ch14.18-mediated NK cell cytotoxicity.

    PubMed

    Shibina, Anastasia; Seidel, Diana; Somanchi, Srinivas S; Lee, Dean A; Stermann, Alexander; Maurer, Barry J; Lode, Holger N; Reynolds, C Patrick; Huebener, Nicole

    2013-04-01

    Neuroblastoma (NB) is the most common extracranial solid tumor in children. Combining passive immunotherapy with an antibody to the disialoganglioside GD2 (ch14.18/SP2/0) and cytokines with 13-cis-retinoic acid for post-myeloablative maintenance therapy increased survival in high-risk NB, but the overall prognosis for these children is still in need of improvement. Fenretinide (4-HPR) is a synthetic retinoid that has shown clinical activity in recurrent NB and is cytotoxic to a variety of cancer cells, in part via the accumulation of dihydroceramides, which are precursors of GD2. We investigated the effect of 4-HPR on CHO-derived, ch14.18-mediated anti-NB effector functions, complement-dependent cytotoxicity (CDC), and antibody-dependent and antibody-independent cellular cytotoxicity (ADCC and AICC, respectively). Here, we demonstrate for the first time that pretreatment of fenretinide-resistant NB cells with 4-HPR significantly enhanced ch14.18/CHO-mediated CDC and ADCC and AICC by both human natural killer cells and peripheral blood mononuclear cells. Treatment with 4-HPR increased GD2 and death receptor (DR) expression in resistant NB cells and induced an enhanced granzyme B and perforin production by effector cells. Blocking of ganglioside synthesis with a glucosylceramide synthase inhibitor abrogated the increased ADCC response but had no effect on the AICC, indicating that GD2 induced by 4-HPR mediates the sensitization of NB cells for ADCC. We also showed that 4-HPR induced increased GD2 and DR expression in a resistant NB xenograft model that was associated with an increased ADCC and AICC response using explanted tumor target cells from 4-HPR-treated mice. In summary, these findings provide an important baseline for the combination of 4-HPR and passive immunotherapy with ch14.18/CHO in future clinical trials for high-risk NB patients.

  3. Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding

    PubMed Central

    Borrok, M Jack; Luheshi, Nadia M; Beyaz, Nurten; Davies, Gareth C; Legg, James W; Wu, Herren; Dall'Acqua, William F; Tsui, Ping

    2015-01-01

    Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs. PMID:25970007

  4. The CD25-binding antibody Daclizumab High-Yield Process has a distinct glycosylation pattern and reduced antibody-dependent cell-mediated cytotoxicity in comparison to Zenapax®

    PubMed Central

    Ganguly, Bishu; Balasa, Balaji; Efros, Lyubov; Hinton, Paul R.; Hartman, Stephen; Thakur, Archana; Xiong, Joanna M.; Schmidt, Brian; Robinson, Randy R.; Sornasse, Thierry; Vexler, Vladimir; Sheridan, James P.

    2016-01-01

    ABSTRACT The CD25-binding antibody daclizumab high-yield process (DAC HYP) is an interleukin (IL)-2 signal modulating antibody that shares primary amino acid sequence and CD25 binding affinity with Zenapax®, a distinct form of daclizumab, which was approved for the prevention of acute organ rejection in patients receiving renal transplants as part of an immunosuppressive regimen that includes cyclosporine and corticosteroids. Comparison of the physicochemical properties of the two antibody forms revealed the glycosylation profile of DAC HYP differs from Zenapax in both glycan distribution and the types of oligosaccharides, most notably high-mannose, galactosylated and galactose-α-1,3-galactose (α-Gal) oligosaccharides, resulting in a DAC HYP antibody material that is structurally distinct from Zenapax. Although neither antibody elicited complement-dependent cytotoxicity in vitro, DAC HYP antibody had significantly reduced levels of antibody-dependent cell-mediated cytotoxicity (ADCC). The ADCC activity required natural killer (NK) cells, but not monocytes, suggesting the effects were mediated through binding to Fc-gamma RIII (CD16). Incubation of each antibody with peripheral blood mononuclear cells also caused the down-modulation of CD16 expression on NK cells and the CD16 down-modulation was greater for Zenapax in comparison to that observed for DAC HYP. The substantive glycosylation differences between the two antibody forms and corresponding greater Fc-mediated effector activities by Zenapax, including cell killing activity, manifest as a difference in the biological function and pharmacology between DAC HYP and Zenapax. PMID:27367933

  5. Cutting edge: An antibody recognizing ancestral endogenous virus glycoproteins mediates antibody-dependent cellular cytotoxicity on HIV-1-infected cells.

    PubMed

    Michaud, Henri-Alexandre; SenGupta, Devi; de Mulder, Miguel; Deeks, Steven G; Martin, Jeffrey N; Kobie, James J; Sacha, Jonah B; Nixon, Douglas F

    2014-08-15

    The failure of antiviral vaccines is often associated with rapid viral escape from specific immune responses. In the past, conserved epitope or algorithmic epitope selections, such as mosaic vaccines, have been designed to diversify immunity and to circumvent potential viral escape. An alternative approach is to identify conserved stable non-HIV-1 self-epitopes present exclusively in HIV-1-infected cells. We showed previously that human endogenous retroviral (HERV) mRNA transcripts and protein are found in cells of HIV-1-infected patients and that HERV-K (HML-2)-specific T cells can eliminate HIV-1-infected cells in vitro. In this article, we demonstrate that a human anti-HERV-K (HML-2) transmembrane protein Ab binds specifically to HIV-1-infected cells and eliminates them through an Ab-dependent cellular cytotoxicity mechanism in vitro. Thus, Abs directed against epitopes other than HIV-1 proteins may have a role in eliminating HIV-1-infected cells and could be targeted in novel vaccine approaches or immunotherapeutic modalities. PMID:25024383

  6. Anti-neuroblastoma effect of ch14.18 antibody produced in CHO cells is mediated by NK-cells in mice.

    PubMed

    Zeng, Yan; Fest, Stefan; Kunert, Renate; Katinger, Hermann; Pistoia, Vito; Michon, Jean; Lewis, Gillan; Ladenstein, Ruth; Lode, Holger N

    2005-07-01

    Successful treatment of stage 4 neuroblastoma remains a major challenge in pediatric oncology. In order to improve the outcome, passive immunotherapy using human-mouse chimeric monoclonal anti-disialoganglioside GD2 antibody ch14.18 has been evaluated in early phase clinical trials with promising results in progressing stage 4 neuroblastoma patients. In preparation of European phase III clinical trial (HR-NBL-1/ESIOP), the cell line used for production of ch14.18 was changed. Specifically, the plasmid encoding for ch14.18 antibody was recloned into CHO cells. Here, we report the in vitro and in vivo anti-neuroblastoma activity of antibody ch14.18 produced in CHO cells (ch14.18/CHO) compared to that of ch14.18 manufactured from SP2/0 (ch14.18/SP2/0) and NS0 cells (ch14.18/NS0). First, we demonstrate identical binding of ch14.18/CHO to the nominal antigen disialoganglioside GD2 in vitro compared to ch14.18/SP2/0 and ch14.18/NS0. Binding was GD2-specific, since all precursor- and metabolite-gangliosides of GD2 tested were not recognized by ch14.18/CHO. Second, the functional properties of ch14.18/CHO were determined in complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) reactions against GD2 positive neuroectodermal tumor cell lines in vitro. There was no difference in CDC mediated specific tumor cell lysis among the three different ch14.18 antibody preparations. Interestingly, ch14.18/CHO showed superior ADCC activity at low antibody concentrations. Third, the efficacy of ch14.18/CHO was evaluated in the NXS2 neuroblastoma model in vivo. Importantly, the ch14.18/CHO preparation was effective in suppression of experimental liver metastasis in this model. In vivo depletion of NK-cells completely abrogated this effect, suggesting that the mechanism involved in the ch14.18/CHO induced anti-neuroblastoma effect is mediated by NK-dependent ADCC.

  7. Antibodies to P-selectin glycoprotein ligand-1 block dendritic cell-mediated enterovirus 71 transmission and prevent virus-induced cells death.

    PubMed

    Ren, Xiao-Xin; Li, Chuan; Xiong, Si-Dong; Huang, Zhong; Wang, Jian-Hua; Wang, Hai-Bo

    2015-01-01

    P-selectin glycoprotein ligand-1 (PSGL-1) has been proved to serve as the functional receptor for enterovirus 71 (EV71). We found the abundant expression of PSGL-1 on monocyte-derived dendritic cells (MDDCs). However, we have previously demonstrated that MDDCs did not support efficient replication of EV71. Dendritic cells (DCs) have been described to be subverted by various viruses including EV71 for viral dissemination, we thus explore the potential contribution of PSGL-1 on DC-mediated EV71 transmission. We found that the cell-surface-expressing PSGL-1 on MDDCs mediated EV71 binding, and intriguingly, these loaded-viruses on MDDCs could be transferred to encountered target cells; Prior-treatment with PSGL-1 antibodies or interference with PSGL-1 expression diminished MDDC-mediated EV71 transfer and rescued virus-induced cell death. Our data uncover a novel role of PSGL-1 in DC-mediated EV71 spread, and provide an insight into blocking primary EV71 infection.

  8. DNA-based vaccine against La Crosse virus: protective immune response mediated by neutralizing antibodies and CD4+ T cells.

    PubMed

    Schuh, T; Schultz, J; Moelling, K; Pavlovic, J

    1999-07-01

    La Crosse virus (LACV)-mediated encephalitis is the most frequently reported arboviral disease in the United States, but to date no vaccine against this virus is available. We have established a new animal model, genetically targeted mice lacking a functional interferon type I receptor (IFNAR-1). These mice show an age-independent susceptibility to LACV and develop an acute encephalitis within 6 days of infection, thereby allowing the evaluation of vaccines against LACV. Taking advantage of this knockout mouse model, we have assessed the feasibility of DNA vaccination against this viral disease. Plasmid DNAs, encoding either the virus surface glycoproteins G1 and G2 or the internal nucleocapsid protein N, were used to immunize IFNAR-1-deficient mice. Mice vaccinated with DNA encoding the glycoproteins G1 and G2 produced neutralizing antibodies and exhibited a high degree of protection against challenge with high doses of LACV. Depletion of CD4+ T cells in mice vaccinated with DNA encoding G1/G2 reduced their capacity to control the infection. Virus titration and immunohistological analysis revealed that the protected mice showed no evidence of LACV particles in the brain. This indicates that the vaccine-induced immune response efficiently blocked viral spreading from the primary replication site to the brain. In contrast, immunization with DNA encoding protein N yielded only a partial protective effect that can be attributed to the cellular immune response. Taken together, this study shows that DNA vaccines can be designed to efficiently induce a protective immune response based on neutralizing antibodies and CD4+ T cells.

  9. The combination of trastuzumab and pertuzumab administered at approved doses may delay development of trastuzumab resistance by additively enhancing antibody-dependent cell-mediated cytotoxicity

    PubMed Central

    Tóth, Gábor; Szöőr, Árpád; Simon, László; Yarden, Yosef; Szöllősi, János; Vereb, György

    2016-01-01

    ABSTRACT Although the recently concluded CLEOPATRA trial showed clinical benefits of combining trastuzumab and pertuzumab for treating HER2-positive metastatic breast cancer, trastuzumab monotherapy is still the mainstay in adjuvant settings. Since trastuzumab resistance occurs in over half of these cancers, we examined the mechanisms by which treatment of intrinsically trastuzumab-resistant and -sensitive tumors can benefit from the combination of these antibodies. F(ab′)2 of both trastuzumab and pertuzumab were generated and validated in order to separately analyze antibody-dependent cell-mediated cytotoxicity (ADCC)-based and direct biological effects of the antibodies. Compared to monotherapy, combination of the two antibodies at clinically permitted doses enhanced the recruitment of natural killer cells responsible for ADCC, and significantly delayed the outgrowth of xenografts from intrinsically trastuzumab-resistant JIMT-1 cells. Antibody dose-response curves of in vitro ADCC showed that antibody-mediated killing can be saturated, and the two antibodies exert an additive effect at sub-saturation doses. Thus, the additive effect in vivo indicates that therapeutic tissue levels likely do not saturate ADCC. Additionally, isobole studies with the in vitro trastuzumab-sensitive BT-474 cells showed that the direct biological effect of combined treatment is additive, and surpasses the maximum effect of either monotherapy. Our results suggest the combined therapy is expected to give results that are superior to monotherapy, whatever the type of HER2-positive tumor may be. The combination of both antibodies at maximum clinically approved doses should thus be administered to patients to recruit maximum ADCC and cause maximum direct biological growth inhibition. PMID:27380003

  10. Antibody induced CD4 down-modulation of T cells is site-specifically mediated by CD64(+) cells.

    PubMed

    Vogel, Stephanie; Grabski, Elena; Buschjäger, Daniela; Klawonn, Frank; Döring, Marius; Wang, Junxi; Fletcher, Erika; Bechmann, Ingo; Witte, Torsten; Durisin, Martin; Schraven, Burkhart; Mangsbo, Sara M; Schönfeld, Kurt; Czeloth, Niklas; Kalinke, Ulrich

    2015-12-16

    Treatment of PBMC with the CD4-specific mAb BT-061 induces CD4 down-modulation of T cells. Here we report that addition of BT-061 to purified T cells did not confer this effect, whereas incubation of T cells in BT-061 coated wells restored CD4 down-modulation. These results implied that Fcγ receptor mediated cell-cell interactions played a role. In consistence with this hypothesis PBMC depleted of CD64(+) monocytes did not confer CD4 down-modulation of BT-061 decorated T cells. Strikingly, CD4 down-modulation was observed in BT-061 treated synovial fluid punctuated from patients' inflamed joints that comprised enhanced numbers of CD64(+) cells. In contrast, in a circulating whole blood system injection of BT-061 did not induce CD4 down-modulation, due to CD64 saturation by serum IgG. Similarly, tonsil derived mononuclear cells devoid of CD64(+) cells did not show CD4 down-modulation, whereas addition of blood derived monocytes restored the effect. Thus, the interaction of BT-061 decorated T cells with CD64(+) cells is needed for CD4 down-modulation, implying that in patients BT-061 would primarily induce CD4 down-modulation at inflammatory sites. These results highlight the need not only to examine the interaction of a given mAb with single FcγR, but also the immunological environment that is appropriate to support such interactions.

  11. Antibody induced CD4 down-modulation of T cells is site-specifically mediated by CD64(+) cells.

    PubMed

    Vogel, Stephanie; Grabski, Elena; Buschjäger, Daniela; Klawonn, Frank; Döring, Marius; Wang, Junxi; Fletcher, Erika; Bechmann, Ingo; Witte, Torsten; Durisin, Martin; Schraven, Burkhart; Mangsbo, Sara M; Schönfeld, Kurt; Czeloth, Niklas; Kalinke, Ulrich

    2015-01-01

    Treatment of PBMC with the CD4-specific mAb BT-061 induces CD4 down-modulation of T cells. Here we report that addition of BT-061 to purified T cells did not confer this effect, whereas incubation of T cells in BT-061 coated wells restored CD4 down-modulation. These results implied that Fcγ receptor mediated cell-cell interactions played a role. In consistence with this hypothesis PBMC depleted of CD64(+) monocytes did not confer CD4 down-modulation of BT-061 decorated T cells. Strikingly, CD4 down-modulation was observed in BT-061 treated synovial fluid punctuated from patients' inflamed joints that comprised enhanced numbers of CD64(+) cells. In contrast, in a circulating whole blood system injection of BT-061 did not induce CD4 down-modulation, due to CD64 saturation by serum IgG. Similarly, tonsil derived mononuclear cells devoid of CD64(+) cells did not show CD4 down-modulation, whereas addition of blood derived monocytes restored the effect. Thus, the interaction of BT-061 decorated T cells with CD64(+) cells is needed for CD4 down-modulation, implying that in patients BT-061 would primarily induce CD4 down-modulation at inflammatory sites. These results highlight the need not only to examine the interaction of a given mAb with single FcγR, but also the immunological environment that is appropriate to support such interactions. PMID:26670584

  12. Antibody induced CD4 down-modulation of T cells is site-specifically mediated by CD64+ cells

    PubMed Central

    Vogel, Stephanie; Grabski, Elena; Buschjäger, Daniela; Klawonn, Frank; Döring, Marius; Wang, Junxi; Fletcher, Erika; Bechmann, Ingo; Witte, Torsten; Durisin, Martin; Schraven, Burkhart; Mangsbo, Sara M.; Schönfeld, Kurt; Czeloth, Niklas; Kalinke, Ulrich

    2015-01-01

    Treatment of PBMC with the CD4-specific mAb BT-061 induces CD4 down-modulation of T cells. Here we report that addition of BT-061 to purified T cells did not confer this effect, whereas incubation of T cells in BT-061 coated wells restored CD4 down-modulation. These results implied that Fcγ receptor mediated cell-cell interactions played a role. In consistence with this hypothesis PBMC depleted of CD64+ monocytes did not confer CD4 down-modulation of BT-061 decorated T cells. Strikingly, CD4 down-modulation was observed in BT-061 treated synovial fluid punctuated from patients’ inflamed joints that comprised enhanced numbers of CD64+ cells. In contrast, in a circulating whole blood system injection of BT-061 did not induce CD4 down-modulation, due to CD64 saturation by serum IgG. Similarly, tonsil derived mononuclear cells devoid of CD64+ cells did not show CD4 down-modulation, whereas addition of blood derived monocytes restored the effect. Thus, the interaction of BT-061 decorated T cells with CD64+ cells is needed for CD4 down-modulation, implying that in patients BT-061 would primarily induce CD4 down-modulation at inflammatory sites. These results highlight the need not only to examine the interaction of a given mAb with single FcγR, but also the immunological environment that is appropriate to support such interactions. PMID:26670584

  13. Immobilized surfactant-nanotube complexes support selectin-mediated capture of viable circulating tumor cells in the absence of capture antibodies.

    PubMed

    Mitchell, Michael J; Castellanos, Carlos A; King, Michael R

    2015-10-01

    The metastatic spread of tumor cells from the primary site to anatomically distant organs leads to a poor patient prognosis. Increasing evidence has linked adhesive interactions between circulating tumor cells (CTCs) and endothelial cells to metastatic dissemination. Microscale biomimetic flow devices hold promise as a diagnostic tool to isolate CTCs and develop metastatic therapies, utilizing E-selectin (ES) to trigger the initial rolling adhesion of tumor cells under flow. To trigger firm adhesion and capture under flow, such devices also typically require antibodies against biomarkers thought to be expressed on CTCs. This approach is challenged by the fact that CTCs are now known to exhibit heterogeneous expression of conventional biomarkers. Here, we describe surfactant-nanotube complexes to enhance ES-mediated capture and isolation of tumor cells without the use of capture antibodies. While the majority of tumor cells exhibited weaker rolling adhesion on halloysite nanotubes (HNT) coated with ES, HNT functionalization with the sodium dodecanoate (NaL) surfactant induced a switch to firm cellular adhesion under flow. Conversely, surfactant-nanotube complexes significantly reduced the number of primary human leukocytes captured via ES-mediated adhesion under flow. The switch in tumor cell adhesion was exploited to capture and isolate tumor cells in the absence of EpCAM antibodies, commonly utilized as the gold standard for CTC isolation. Additionally, HNT-NaL complexes were shown to capture tumor cells with low to negligible EpCAM expression, that are not efficiently captured using conventional approaches.

  14. Immobilized Surfactant-Nanotube Complexes Support Selectin-Mediated Capture of Viable Circulating Tumor Cells in the Absence of Capture Antibodies

    PubMed Central

    Mitchell, Michael J.; Castellanos, Carlos A.; King, Michael R.

    2015-01-01

    The metastatic spread of tumor cells from the primary site to anatomically distant organs leads to a poor patient prognosis. Increasing evidence has linked adhesive interactions between circulating tumor cells (CTCs) and endothelial cells to metastatic dissemination. Microscale biomimetic flow devices hold promise as a diagnostic tool to isolate CTCs and develop metastatic therapies, utilizing E-selectin (ES) to trigger the initial rolling adhesion of tumor cells under flow. To trigger firm adhesion and capture under flow, such devices also typically require antibodies against biomarkers thought to be expressed on CTCs. This approach is challenged by the fact that CTCs are now known to exhibit heterogeneous expression of conventional biomarkers. Here, we describe surfactant-nanotube complexes to enhance ES-mediated capture and isolation of tumor cells without the use of capture antibodies. While the majority of tumor cells exhibited weaker rolling adhesion on halloysite nanotubes (HNT) coated with ES, HNT functionalization with the sodium dodecanoate (NaL) surfactant induced a switch to firm cellular adhesion under flow. Conversely, surfactant-nanotube complexes significantly reduced the number of primary human leukocytes captured via ES-mediated adhesion under flow. The switch in tumor cell adhesion was exploited to capture and isolate tumor cells in the absence of EpCAM antibodies, commonly utilized as the gold standard for CTC isolation. Additionally, HNT-NaL complexes were shown to capture tumor cells with low to negligible EpCAM expression, that are not efficiently captured using conventional approaches. PMID:25761664

  15. Chediak-Higashi gene in humans. II. The selectivity of the defect in natural- killer and antibody-dependent cell-mediated cytotoxicity function

    PubMed Central

    Klein, M; Roder, J; Haliotis, T; Korec, S; Jett; Herberman, RB; Katz, P; Fauci, AS

    1980-01-01

    Antibody-dependent cell-mediated cytolysis (ADCC) of human tumor cells by FcR(+) nonadherent effector lymphocytes as well as natural killer (NK) activity was markedly impaired in Chediak-Steinbrinck-Higashi Syndrome (C-HS) patients. Compared to a more than 400-fold defect in NK activity in terms of lytic units, the abnormal ADCC response in C-HS donors was 24-fold below normal suggesting a partial but not complete overlap of lymphocytes or lytic mechanisms responsible for ADCC and NK. The ADCC mechanism against erythrocyte targets, however, was normal, thereby suggesting a qualitative difference in these two forms of ADCC. Other effector-cell functions against tumor-cell targets were normal as measured by (a) spontaneous cytolysis mediated by monocytes, (b) spontaneous cytostasis mediated by neutrophils, and (c) lectin-dependent cytolysis mediated by neutrophils. Although one C-HS patient was low in lectin-dependent cytolysis mediated by lymphocytes, the other C-HS patient was normal, thereby suggesting that cytolytic T function was not linked to the NK-ADCC defect. In addition, the proliferative response to T-dependent mitogens was also relatively normal. These results, combined with other studies showing normal cell-mediated and humoral immunity in these same patients, suggest that patients with C-HS have an immunodeficiency which is selective for NK and ADCC activity. PMID:6966316

  16. Survival of residual neutrophils and accelerated myelopoiesis limit the efficacy of antibody-mediated depletion of Ly-6G+ cells in tumor-bearing mice.

    PubMed

    Moses, Katrin; Klein, Johanna C; Männ, Linda; Klingberg, Anika; Gunzer, Matthias; Brandau, Sven

    2016-06-01

    Expansion of Ly-6G(+) myeloid cells has been reported in most murine cancer models. However, divergent findings exist regarding the role and effect of these cells on host immunity and tumor progression. Antibody-mediated depletion of Ly-6G(+) cells is a common technique to assess the in vivo relevance of these cells. Interpretation of results crucially depends on the efficacy and course of depletion. We established murine head and neck cancer models and analyzed the efficacy of antibody-mediated depletion by flow cytometry, conventional histology, and intravital imaging with a novel Ly-6G-transgenic mouse model. The first phase of depletion was characterized by effective elimination of Ly-6G(+) cells from the peripheral blood. Nevertheless, viable, resistant cells were found to reside in the tumor tissue and spleen. This peripheral depletion phase was associated with high systemic levels of granulocyte colony-stimulating factor and KC and enhanced splenic production of Ly-6G(+) cells. Even under sustained treatment with either αGr-1 or αLy-6G antibodies, peripheral blood depletion ended after approximately 1 wk and was followed by reappearance of immature Ly-6G(+) cells with an immunoregulatory phenotype. Reappearance of these depletion-resistant immature cells was enhanced in tumor-bearing, compared with naïve, control mice. Collectively, our data suggest that depletion of Ly-6G(+) myeloid cells in tumor-bearing mice is counteracted by the persistence of intratumoral cells, enhanced extramedullary granulopoiesis, and accelerated reappearance of immature cells. Hence, extensive monitoring of in vivo kinetics and tissue distribution of Ly-6G(+) cells is required in depletion studies.

  17. Intravital imaging reveals improved Kupffer cell-mediated phagocytosis as a mode of action of glycoengineered anti-CD20 antibodies

    PubMed Central

    Grandjean, Capucine L.; Montalvao, Fabricio; Celli, Susanna; Michonneau, David; Breart, Beatrice; Garcia, Zacarias; Perro, Mario; Freytag, Olivier; Gerdes, Christian A.; Bousso, Philippe

    2016-01-01

    Anti-CD20 monoclonal antibodies (mAbs) represent an effective treatment for a number of B cell malignancies and autoimmune disorders. Glycoengineering of anti-CD20mAb may contribute to increased anti-tumor efficacy through enhanced antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP) as reported by in vitro studies. However, where and how glycoengineered Ab may potentiate therapeutic responses in vivo is yet to be elucidated. Here, we have performed mouse liver transplants to demonstrate that the liver is sufficient to mediate systemic B cells depletion after anti-CD20 treatment. Relying on intravital two-photon imaging of human CD20-expressing mice, we provide evidence that ADP by Kupffer cells (KC) is a major mechanism for rituximab-mediated B cell depletion. Notably, a glycoengineered anti-mouse CD20 Ab but not its wild-type counterpart triggered potent KC-mediated B cell depletion at low doses. Finally, distinct thresholds for KC phagocytosis were also observed for GA101 (obinutuzumab), a humanized glycoengineered type II anti-CD20 Ab and rituximab. Thus, we propose that enhanced phagocytosis of circulating B cells by KC represents an important in vivo mechanism underlying the improved activity of glycoengineered anti-CD20 mAbs. PMID:27698437

  18. Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses

    PubMed Central

    McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T.; Dennison, S. Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S. Munir; Haynes, Barton F.; Tomaras, Georgia D.

    2016-01-01

    Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine

  19. Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses.

    PubMed

    Tay, Matthew Zirui; Liu, Pinghuang; Williams, LaTonya D; McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T; Dennison, S Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S Munir; Moody, M Anthony; Hope, Thomas J; Haynes, Barton F; Tomaras, Georgia D

    2016-08-01

    Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine

  20. Increased infectivity in human cells and resistance to antibody-mediated neutralization by truncation of the SIV gp41 cytoplasmic tail.

    PubMed

    Kuwata, Takeo; Kaori, Takaki; Enomoto, Ikumi; Yoshimura, Kazuhisa; Matsushita, Shuzo

    2013-01-01

    The role of antibodies in protecting the host from human immunodeficiency virus type 1 (HIV-1) infection is of considerable interest, particularly because the RV144 trial results suggest that antibodies contribute to protection. Although infection of non-human primates with simian immunodeficiency virus (SIV) is commonly used as an animal model of HIV-1 infection, the viral epitopes that elicit potent and broad neutralizing antibodies to SIV have not been identified. We isolated a monoclonal antibody (MAb) B404 that potently and broadly neutralizes various SIV strains. B404 targets a conformational epitope comprising the V3 and V4 loops of Env that intensely exposed when Env binds CD4. B404-resistant variants were obtained by passaging viruses in the presence of increasing concentration of B404 in PM1/CCR5 cells. Genetic analysis revealed that the Q733stop mutation, which truncates the cytoplasmic tail of gp41, was the first major substitution in Env during passage. The maximal inhibition by B404 and other MAbs were significantly decreased against a recombinant virus with a gp41 truncation compared with the parental SIVmac316. This indicates that the gp41 truncation was associated with resistance to antibody-mediated neutralization. The infectivities of the recombinant virus with the gp41 truncation were 7,900-, 1,000-, and 140-fold higher than those of SIVmac316 in PM1, PM1/CCR5, and TZM-bl cells, respectively. Immunoblotting analysis revealed that the gp41 truncation enhanced the incorporation of Env into virions. The effect of the gp41 truncation on infectivity was not obvious in the HSC-F macaque cell line, although the resistance of viruses harboring the gp41 truncation to neutralization was maintained. These results suggest that viruses with a truncated gp41 cytoplasmic tail were selected by increased infectivity in human cells and by acquiring resistance to neutralizing antibody. PMID:23717307

  1. Complement mediates human immunodeficiency virus type 1 infection of a human T cell line in a CD4- and antibody-independent fashion.

    PubMed

    Boyer, V; Desgranges, C; Trabaud, M A; Fischer, E; Kazatchkine, M D

    1991-05-01

    Incubation of the human T cell lymphotropic virus (HTLV)-IIIB and HTLV-RF strains of human immunodeficiency virus type 1 (HIV-1) with normal seronegative human serum under conditions that allow complement activation resulted in enhancement of infection of the MT2 human T cell line cultured in the presence of low amounts of virus. Infection of MT2 cells was assessed by measuring reverse transcriptase activity in supernatants at day 9 of culture. Complement activation by viral suspensions occurred through the alternative pathway. Opsonization of HTLV-RF viral particles with complement was sufficient to allow a productive infection to occur in cells exposed to suboptimal amounts of virus. Infection of MT2 cells with suboptimal amounts of serum-opsonized HIV-1 was suppressed by blocking the C3dg receptor (CR2, CD21) on MT2 cells with monoclonal anti-CR2 antibody and rabbit F(ab')2 anti-mouse immunoglobulin antibodies. Blocking of the gp120-binding site on CD4 under similar experimental conditions had no inhibitory effect on infection of MT2 cells with opsonized virus. Opsonization of HIV-1 with seronegative serum also resulted in a CR2-mediated enhancement of the infection of normal peripheral blood mononuclear cells and T lymphocytes. These results indicate that complement in the absence of antibody may enhance infection of C3 receptor-bearing T cells with HIV-1, and that the interaction of opsonized virus with the CR2 receptor may result by itself in the infection of target T cells in a CD4- and antibody-independent fashion. PMID:1827139

  2. Principles of antibody-mediated TNF receptor activation

    PubMed Central

    Wajant, H

    2015-01-01

    From the beginning of research on receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF), agonistic antibodies have been used to stimulate TNFRSF receptors in vitro and in vivo. Indeed, CD95, one of the first cloned TNFRSF receptors, was solely identified as the target of cell death-inducing antibodies. Early on, it became evident from in vitro studies that valency and Fcγ receptor (FcγR) binding of antibodies targeting TNFRSF receptors can be of crucial relevance for agonistic activity. TNFRSF receptor-specific antibodies of the IgM subclass and secondary cross-linked or aggregation prone dimeric antibodies typically display superior agonistic activity compared with dimeric antibodies. Likewise, anchoring of antibodies to cell surface-expressed FcγRs potentiate their ability to trigger TNFRSF receptor signaling. However, only recently has the relevance of oligomerization and FcγR binding for the in vivo activity of antibody-induced TNFRSF receptor activation been straightforwardly demonstrated in vivo. This review discusses the crucial role of oligomerization and/or FcγR binding for antibody-mediated TNFRSF receptor stimulation in light of current models of TNFRSF receptor activation and especially the overwhelming relevance of these issues for the rational development of therapeutic TNFRSF receptor-targeting antibodies. PMID:26292758

  3. Rational clinical trial design for antibody mediated renal allograft injury

    PubMed Central

    Sandal, Shaifali; Zand, Martin S.

    2015-01-01

    Antibody mediated renal allograft rejection is a significant cause of acute and chronic graft loss. Recent work has revealed that AMR is a complex processes, involving B and plasma cells, donor-specific antibodies, complement, vascular endothelial cells, NK cells, Fc receptors, cytokines and chemokines. These insights have led to the development of numerous new therapies, and adaptation of others originally developed for treatment of hemetologic malignancies, autoimmune and complement mediated conditions. Here we review emerging insights into the pathophysiology of AMR as well as current and emerging therapies for both acute and chronic AMR. Finally, we discuss rational clinical trial design in light of antibody and B cell immunobiology, as well as appropriate efficacy metrics to identify robust protocols and therapeutic agents. PMID:25553476

  4. Adoptive cell transfer of contact sensitivity-initiation mediated by nonimmune cells sensitized with monoclonal IgE antibodies. Dependence on host skin mast cells.

    PubMed

    Matsuda, H; Ushio, H; Paliwal, V; Ptak, W; Askenase, P W

    1995-05-15

    A role for mast cell release of serotonin (5-HT), via Ag-specific factors derived from Thy-1+ B220+ lymphoid cells in the initiation of murine contact sensitivity (CS) has been suggested. However, because CS in mast cell-deficient mice was intact, a role for mast cells in CS initiation was unclear. Therefore, we examined whether CS could be initiated by i.v. injection of nonimmune mixed lymphoid cells that were sensitized in vitro with IgE. When naive mice received IgE-sensitized nonimmune spleen or lymph node cells, or IgE-sensitized purified mast cells, together with immune CS-effector B220- T cells, which therefore were depleted of CS-initiating, Thy-1+, B220+ cells, which could not transfer CS, then reconstitution of CS occurred. Mast cell-deficient W/Wv mice could not elicit this IgE-dependent CS ear swelling, but when mast cell deficiency was reversed by ear injection of normal bone marrow-derived cultured mast cells, then CS was restored. In vitro pretreatment with irrelevant monoclonal anti-OVA IgE prevented CS initiation mediated by Ag-specific, IgE mAb-sensitized cells, presumably by blocking sensitization with IgE. Thus Fc epsilon R on the normal lymphoid cells were involved. When ketanserin, a 5-HT2 receptor antagonist, was injected i.v. before cell transfer, CS initiation via IgE-sensitized cells and CS were no longer elicited. Thus, in this system, IgE Abs bound to circulating IgE Fc epsilon R bearing lymphoid cells sensitized in vitro (most likely basophils), probably mediated early activation of these circulating basophils to release mediators, causing 5-HT release from cutaneous mast cells, to mediate CS initiation. PMID:7730614

  5. Antibody-dependent cellular cytotoxicity-mediated serotherapy against murine neuroblastoma. II. In vitro and in vivo treatment using effector cells from normal and X-irradiated humans.

    PubMed

    Byfield, J E; Zerubavel, R; Fonkalsrud, E W

    1983-01-01

    Human peripheral lymphocytes (HLc) have been studied in vitro as possible effector cells in an antibody-dependent cellular cytotoxicity (ADCC) reaction. HLc were found to be active against murine neuroblastoma cells (MNB) inoculated into the flank of syngeneic mice. Both the time of onset of tumor appearance and the mean survival time of tumor-bearing host mice were beneficially influenced. Occasional animals could be cured of up to 10(5) tumor cells (1--10 cells of MNB are lethal). This level of tumor cytotoxicity approaches that of tolerance-dose chemotherapy and is without demonstrable side-effects. HLc from patients who had just received = 3,000 rads fractionated therapeutic X-irradiation were equally effective as HLc from control non-irradiated donors when assayed at equivalent HLc : tumor cell ratios. HLc could also inhibit MNB tumor cell growth in the ascitic form, confirming in vivo activity. Overall, HLc appeared almost as active as rat spleen cells in mediating a useful anti-tumor ADCC. This approach may ultimately prove useful in man, especially in the peritoneal cavity, and is currently limited only by the need to develop appropriate antisera. It is proposed and emphasized that such antisera need not necessarily be directed at tumor-specific antigens. Organ-specific antibodies such are already known to develop spontaneously in some human auto-immune diseases might be equally useful and are a naturally occurring potential source of appropriately expressed genetic material.

  6. Red Blood Cell Antibody Identification

    MedlinePlus

    ... be limited. Home Visit Global Sites Search Help? RBC Antibody Identification Share this page: Was this page helpful? Also known as: Alloantibody Identification; Antibody ID, RBC; RBC Ab ID Formal name: Red Blood Cell ...

  7. Provirus activation plus CD59 blockage triggers antibody-dependent complement-mediated lysis of latently HIV-1-infected cells.

    PubMed

    Lan, Jie; Yang, Kai; Byrd, Daniel; Hu, Ningjie; Amet, Tohti; Shepherd, Nicole; Desai, Mona; Gao, Jimin; Gupta, Samir; Sun, Yongtao; Yu, Qigui

    2014-10-01

    Latently HIV-1-infected cells are recognized as the last barrier toward viral eradication and cure. To purge these cells, we combined a provirus stimulant with a blocker of human CD59, a key member of the regulators of complement activation, to trigger Ab-dependent complement-mediated lysis. Provirus stimulants including prostratin and histone deacetylase inhibitors such as romidepsin and suberoylanilide hydroxamic acid activated proviruses in the latently HIV-1-infected T cell line ACH-2 as virion production and viral protein expression on the cell surface were induced. Romidepsin was the most attractive provirus stimulant as it effectively activated proviruses at nanomolar concentrations that can be achieved clinically. Antiretroviral drugs including two protease inhibitors (atazanavir and darunavir) and an RT inhibitor (emtricitabine) did not affect the activity of provirus stimulants in the activation of proviruses. However, saquinavir (a protease inhibitor) markedly suppressed virus production, although it did not affect the percentage of cells expressing viral Env on the cell surface. Provirus-activated ACH-2 cells expressed HIV-1 Env that colocalized with CD59 in lipid rafts on the cell surface, facilitating direct interaction between them. Blockage of CD59 rendered provirus-activated ACH-2 cells and primary human CD4(+) T cells that were latently infected with HIV-1 sensitive to Ab-dependent complement-mediated lysis by anti-HIV-1 polyclonal Abs or plasma from HIV-1-infected patients. Therefore, a combination of provirus stimulants with regulators of complement activation blockers represents a novel approach to eliminate HIV-1.

  8. Nanoparticle mediated drug delivery of rolipram to tyrosine kinase B positive cells in the inner ear with targeting peptides and agonistic antibodies

    PubMed Central

    Glueckert, Rudolf; Pritz, Christian O.; Roy, Soumen; Dudas, Jozsef; Schrott-Fischer, Anneliese

    2015-01-01

    Aim: Systemic pharmacotherapies have limitation due to blood-labyrinth barrier, so local delivery via the round window membrane opens a path for effective treatment. Multifunctional nanoparticle (NP)-mediated cell specific drug delivery may enhance efficacy and reduce side effects. Different NPs with ligands to target TrkB receptor were tested. Distribution, uptake mechanisms, trafficking, and bioefficacy of drug release of rolipram loaded NPs were evaluated. Methods: We tested lipid based nanocapsules (LNCs), Quantum Dot, silica NPs with surface modification by peptides mimicking TrkB or TrkB activating antibodies. Bioefficacy of drug release was tested with rolipram loaded LNCs to prevent cisplatin-induced apoptosis. We established different cell culture models with SH-SY-5Y and inner ear derived cell lines and used neonatal and adult mouse explants. Uptake and trafficking was evaluated with FACS and confocal as well as transmission electron microscopy. Results: Plain NPs show some selectivity in uptake related to the in vitro system properties, carrier material, and NP size. Some peptide ligands provide enhanced targeted uptake to neuronal cells but failed to show this in cell cultures. Agonistic antibodies linked to silica NPs showed TrkB activation and enhanced binding to inner ear derived cells. Rolipram loaded LNCs proved as effective carriers to prevent cisplatin-induced apoptosis. Discussion: Most NPs with targeting ligands showed limited effects to enhance uptake. NP aggregation and unspecific binding may change uptake mechanisms and impair endocytosis by an overload of NPs. This may affect survival signaling. NPs with antibodies activate survival signaling and show effective binding to TrkB positive cells but needs further optimization for specific internalization. Bioefficiacy of rolipram release confirms LNCs as encouraging vectors for drug delivery of lipophilic agents to the inner ear with ideal release characteristics independent of endocytosis

  9. Humanizing murine IgG3 anti-GD2 antibody m3F8 substantially improves antibody-dependent cell-mediated cytotoxicity while retaining targeting in vivo

    PubMed Central

    Cheung, Nai-Kong V.; Guo, Hongfen; Hu, Jian; Tassev, Dimiter V.; Cheung, Irene Y.

    2012-01-01

    Murine IgG3 anti-GD2 antibody m3F8 has shown anti-neuroblastoma activity in Phase I/II studies, where antibody-dependent cell-mediated cytotoxicity (ADCC) played a key role. Humanization of m3F8 should circumvent human anti-mouse antibody (HAMA) response and enhance its ADCC properties to reduce dosing and pain side effect. Chimeric 3F8 (ch3F8) and humanized 3F8 (hu3F8-IgG1 and hu3F8-IgG4) were produced and purified by protein A affinity chromatography. In vitro comparison was made with m3F8 and other anti-GD2 antibodies in binding, cytotoxicity, and cross-reactivity assays. In GD2 binding studies by SPR, ch3F8 and hu3F8 maintained KD comparable to m3F8. Unlike other anti-GD2 antibodies, m3F8, ch3F8 and hu3F8 had substantially slower koff.. Similar to m3F8, both ch3F8 and hu3F8 inhibited tumor cell growth in vitro, while cross-reactivity with other gangliosides was comparable to that of m3F8. Both peripheral blood mononuclear cell (PBMC)-ADCC and polymorphonuclear leukocytes (PMN)-ADCC of ch3F8 and hu3F8-IgG1 were more potent than m3F8. This superiority was consistently observed in ADCC assays, irrespective of donors or NK-92MI-transfected human CD16 or CD32, whereas complement mediated cytotoxicity (CMC) was reduced. As expected, hu3F8-IgG4 had near absent PBMC-ADCC and CMC. Hu3F8 and m3F8 had similar tumor-to-non tumor ratios in biodistribution studies. Anti-tumor effect against neuroblastoma xenografts was better with hu3F8-IgG1 than m3F8. In conclusion, humanizing m3F8 produced next generation anti-GD2 antibodies with substantially more potent ADCC in vitro and anti-tumor activity in vivo. By leveraging ADCC over CMC, they may be clinically more effective, while minimizing pain and HAMA side effects. A Phase I trial using hu3F8-IgG1 is ongoing. PMID:22754766

  10. Pharmacological intervention in antibody mediated disease.

    PubMed

    Coutts, S M; Plunkett, M L; Iverson, G M; Barstad, P A; Berner, C M

    1996-04-01

    The use of single signal anergy to inactive pathological B cells in an antigen-specific manner is discussed. Cross-linking surface immunoglobulin, with a construct which contains oligovalent B cell epitopes on a non-immunogenic molecular framework can be used to inactivate the target B cells if the construct lacks T cells epitopes. An example of such a B cell toleragen is LJP 394, which inactivates anti-dsDNA-specific B cells in vivo in murine immunized and spontaneous disease models. The drug enhances survival and lowers renal pathology in BXSB mice. Appropriate definition of epitopes of pathological (auto) antibodies thus offers an opportunity for pharmacological intervention.

  11. Analysis of leukocyte populations in Canadian Holsteins classified as high or low immune responders for antibody- or cell-mediated immune response

    PubMed Central

    Hine, Brad C.; Cartwright, Shannon L.; Mallard, Bonnie A.

    2012-01-01

    Selection of dairy cattle for increased milk production with little or no emphasis on health traits leads to an increased prevalence of disease. A possible genetic solution to this problem is to combine production and immune response traits in a weighted selection index. In the current study, leukocyte populations in heifers identified as having a high antibody-mediated immune response (HiAMIR) or high cell-mediated immune response (HiCMIR) phenotype were compared before and after immunization in order to identify leukocyte population profiles associated with these phenotypes. The results demonstrated that the HiCMIR-phenotype animals had a higher baseline proportion of gamma-delta T-cells in peripheral blood. Also, the observed increase in the proportion of B-cells in peripheral blood in response to immunization was greater in the HiAMIR-phenotype animals. It is expected that identifying leukocyte population profiles associated with immune response phenotypes will improve our ability to identify animals with enhanced overall immune responsiveness. PMID:23024458

  12. CD147 monoclonal antibody mediated by chitosan nanoparticles loaded with α-hederin enhances antineoplastic activity and cellular uptake in liver cancer cells

    PubMed Central

    Zhu, Rong; Zhang, Chun-ge; Liu, Yang; Yuan, Zhi-qiang; Chen, Wei-liang; Yang, Shu-di; Li, Ji-zhao; Zhu, Wen-jing; Zhou, Xiao-feng; You, Ben-gang; Zhang, Xue-nong

    2015-01-01

    An antibody that specifically interacts with an antigen could be applied to an active targeting delivery system. In this study, CD147 antibody was coupled with α-hed chitosan nanoparticles (α-Hed-CS-NPs). α-Hed-CS-CD147-NPs were round and spherical in shape, with an average particle size of 148.23 ± 1.75 nm. The half-maximum inhibiting concentration (IC50) of α-Hed-CS-CD147-NPs in human liver cancer cell lines HepG2 and SMMC-7721 was lower than that of free α-Hed and α-Hed-CS-NPs. α-Hed-induced cell death was mainly triggered by apoptosis. The increase in intracellular accumulation of α-Hed-CS-CD147-NPs was also related to CD147-mediated internalization through the Caveolae-dependent pathway and lysosomal escape. The higher targeting antitumor efficacy of α-Hed-CS-CD147-NPs than that α-Hed-CS-NPs was attributed to its stronger fluorescence intensity in the tumor site in nude mice. PMID:26639052

  13. Silica vesicles as nanocarriers and adjuvants for generating both antibody and T-cell mediated immune resposes to Bovine Viral Diarrhoea Virus E2 protein.

    PubMed

    Mody, Karishma T; Mahony, Donna; Zhang, Jun; Cavallaro, Antonino S; Zhang, Bing; Popat, Amirali; Mahony, Timothy J; Yu, Chengzhong; Mitter, Neena

    2014-12-01

    Bovine Viral Diarrhoea Virus (BVDV) is widely distributed in cattle industries and causes significant economic losses worldwide annually. A limiting factor in the development of subunit vaccines for BVDV is the need to elicit both antibody and T-cell-mediated immunity as well as addressing the toxicity of adjuvants. In this study, we have prepared novel silica vesicles (SV) as the new generation antigen carriers and adjuvants. With small particle size of 50 nm, thin wall (~6 nm), large cavity (~40 nm) and large entrance size (5.9 nm for SV-100 and 16 nm for SV-140), the SV showed high loading capacity (∼ 250 μg/mg) and controlled release of codon-optimised E2 (oE2) protein, a major immunogenic determinant of BVDV. The in vivo functionality of the system was validated in mice immunisation trials comparing oE2 plus Quil A (50 μg of oE2 plus 10 μg of Quil A, a conventional adjuvant) to the oE2/SV-140 (50 μg of oE2 adsorbed to 250 μg of SV-140) or oE2/SV-140 together with 10 μg of Quil A. Compared to the oE2 plus Quil A, which generated BVDV specific antibody responses at a titre of 10(4), the oE2/SV-140 group induced a 10 times higher antibody response. In addition, the cell-mediated response, which is essential to recognise and eliminate the invading pathogens, was also found to be higher [1954-2628 spot forming units (SFU)/million cells] in mice immunised with oE2/SV-140 in comparison to oE2 plus Quil A (512-1369 SFU/million cells). Our study has demonstrated that SV can be used as the next-generation nanocarriers and adjuvants for enhanced veterinary vaccine delivery. PMID:25239045

  14. Antibody-Mediated Pathogen Resistance in Plants.

    PubMed

    Peschen, Dieter; Schillberg, Stefan; Fischer, Rainer

    2016-01-01

    The methods described in this chapter were developed in order to produce transgenic plants expressing pathogen-specific single-chain variable fragment (scFv) antibodies fused to antifungal peptides (AFPs), conferring resistance against fungal pathogens. We describe the selection from a phage display library of avian scFv antibodies that recognize cell surface proteins on fungi from the genus Fusarium, and the construction of scFv-AFP fusion protein constructs followed by their transient expression in tobacco (Nicotiana spp.) plants and stable expression in Arabidopsis thaliana plants. Using these techniques, the antibody fusion with the most promising in vitro activity can be used to generate transgenic plants that are resistant to pathogens such as Fusarium oxysporum f. sp. matthiolae.

  15. Strong Hepatitis C Virus (HCV)–specific Cell-mediated Immune Responses in the Absence of Viremia or Antibodies Among Uninfected Siblings of HCV Chronically Infected Children

    PubMed Central

    El-Karaksy, Hanaa; Shata, Mohamed T.; Sobhy, Maha; Helmy, Heba; El-Naghi, Suzan; Galal, Gehan; Ali, Zainab Z.; Esmat, Gamal; Abdelwahab, Sayed F.; Strickland, G. Thomas; El-Kamary, Samer S.

    2011-01-01

    Background. Cell-mediated immune (CMI) responses to hepatitis C virus (HCV) antigens in adults without seroconversion or viremia are biomarkers for prior transient infection. We investigated HCV-specific CMI responses in seronegative children living with HCV-infected siblings. Methods. Children 3–18 years of age living with HCV-infected siblings were screened for HCV antibodies and HCV RNA. Peripheral blood mononuclear cells (PBMCs) were evaluated for HCV-specific CMI responses by interferon γ (IFN-γ) enzyme-linked immunospot assay using 3 recombinant HCV protein antigens. Flow cytometry phenotypically characterized IFN-γ-secreting cells. Results. Forty-five seronegative children and 5 seropositive viremic siblings had functionally viable PBMCs. Among the 45 seronegative siblings, 15 (33.3%) had positive HCV-specific IFN-γ responses, and subsequent RNA testing revealed that 3 were viremic. Compared with the 5 seropositive viremic children, the median number of HCV-specific spot-forming units was significantly higher in the 12 seronegative aviremic children (P = .002) and in the 3 seronegative viremic children (P = .025). Flow cytometric analysis revealed that IFN-γ was synthesized mainly by CD4+ T cells. Conclusion. Strong HCV-specific CD4+ T cell responses were detectable in higher frequency among seronegative, aviremic children compared with persistently infected siblings. Further studies are needed to determine whether these immune responses are protective against HCV infection. PMID:21257736

  16. Donor antibodies to HNA-3a implicated in TRALI reactions prime neutrophils and cause PMN-mediated damage to human pulmonary microvascular endothelial cells in a two-event in vitro model.

    PubMed

    Silliman, Christopher C; Curtis, Brian R; Kopko, Patricia M; Khan, Samina Y; Kelher, Marguerite R; Schuller, Randy M; Sannoh, Baindu; Ambruso, Daniel R

    2007-02-15

    Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality. Antibodies to HNA-3a are commonly implicated in TRALI. We hypothesized that HNA-3a antibodies prime neutrophils (PMNs) and cause PMN-mediated cytotoxicity through a two-event pathogenesis. Isolated HNA-3a+ or HNA-3a- PMNs were incubated with plasma containing HNA-3a antibodies implicated in TRALI, and their ability to prime the oxidase was measured. Human pulmonary microvascular endothelial cells (HMVECs) were activated with endotoxin or buffer, HNA-3a+ or HNA-3a- PMNs were added, and the coculture was incubated with plasma+/-antibodies to HNA-3a. PMN-mediated damage was measured by counting viable HMVECs/mm2. Plasma containing HNA-3a antibodies primed the fMLP-activated respiratory burst of HNA-3a+, but not HNA-3a-, PMNs and elicited PMN-mediated damage of LPS-activated HMVECs when HNA-3a+, but not HNA-3a-, PMNs were used. Thus, antibodies to HNA-3a primed PMNs and caused PMN-mediated HMVEC cytotoxicity in a two-event model identical to biologic response modifiers implicated in TRALI. PMID:17038531

  17. Neutralization capacity and antibody dependent cell-mediated cytotoxicity of separated IgG subclasses 1, 3 and 4 against herpes simplex virus.

    PubMed Central

    Mathiesen, T; Persson, M A; Sundqvist, V A; Wahren, B

    1988-01-01

    IgG subclasses 1, 3 and 4 in sera from herpes simplex virus type 1 (HSV-1) seropositive donors were separated and their functions assayed. The main neutralizing activity to HSV-1 was found in the IgG1 fractions. Both IgG3 and IgG4 possessed higher neutralizing titres than IgG1 in relation to the respective HSV IgG subclass enzyme-linked immunosorbent assay (ELISA) titre. Addition of complement resulted in a strong enhancement of IgG3 neutralizing activity. HSV neutralizations by IgG1 and, surprisingly, IgG4 were also somewhat enhanced by complement. With the addition of complement, the contribution to neutralizing activity of IgG3 was calculated to increase from 31 to 40% of total IgG in HSV neutralization in native sera. The avidities of the IgG fractions to HSV glycoprotein C (gC) were estimated in a few sera but could not be correlated to neutralization results. Antibody dependent cell-mediated cytotoxicity (ADCC) was detectable mainly in IgG1 and 3 fractions of sera with high anti-HSV antibody titres. PMID:2842096

  18. CHO-S antibody titers >1 gram/liter using flow electroporation-mediated transient gene expression followed by rapid migration to high-yield stable cell lines.

    PubMed

    Steger, Krista; Brady, James; Wang, Weili; Duskin, Meg; Donato, Karen; Peshwa, Madhusudan

    2015-04-01

    In recent years, researchers have turned to transient gene expression (TGE) as an alternative to CHO stable cell line generation for early-stage antibody development. Despite advances in transfection methods and culture optimization, the majority of CHO-based TGE systems produce insufficient antibody titers for extensive use within biotherapeutic development pipelines. Flow electroporation using the MaxCyte STX Scalable Transfection System is a highly efficient, scalable means of CHO-based TGE for gram-level production of antibodies without the need for specialized expression vectors or genetically engineered CHO cell lines. CHO cell flow electroporation is easily scaled from milligram to multigram quantities without protocol reoptimization while maintaining transfection performance and antibody productivity. In this article, data are presented that demonstrate the reproducibility, scalability, and antibody production capabilities of CHO-based TGE using the MaxCyte STX. Data show optimization of posttransfection parameters such as cell density, media composition, and feed strategy that result in secreted antibody titers >1 g/L and production of multiple grams of antibody within 2 weeks of a single CHO-S cell transfection. In addition, data are presented to demonstrate the application of scalable electroporation for the rapid generation of high-yield stable CHO cell lines to bridge the gap between early- and late-stage antibody development activities.

  19. Gamma Interferon Is Required for Optimal Antibody-Mediated Immunity against Genital Chlamydia Infection

    PubMed Central

    Naglak, Elizabeth K.; Morrison, Sandra G.

    2016-01-01

    Defining the mechanisms of immunity conferred by the combination of antibody and CD4+ T cells is fundamental to designing an efficacious chlamydial vaccine. Using the Chlamydia muridarum genital infection model of mice, which replicates many features of human C. trachomatis infection and avoids the characteristic low virulence of C. trachomatis in the mouse, we previously demonstrated a significant role for antibody in immunity to chlamydial infection. We found that antibody alone was not protective. Instead, protection appeared to be conferred through an undefined antibody-cell interaction. Using gene knockout mice and in vivo cellular depletion methods, our data suggest that antibody-mediated protection is dependent on the activation of an effector cell population in genital tract tissues by CD4+ T cells. Furthermore, the CD4+ T cell-secreted cytokine gamma interferon (IFN-γ) was found to be a key component of the protective antibody response. The protective function of IFN-γ was not related to the immunoglobulin class or to the magnitude of the Chlamydia-specific antibody response or to recruitment of an effector cell population to genital tract tissue. Rather, IFN-γ appears to be necessary for activation of the effector cell population that functions in antibody-mediated chlamydial immunity. Our results confirm the central role of antibody in immunity to chlamydia reinfection and demonstrate a key function for IFN-γ in antibody-mediated protection. PMID:27600502

  20. Activation of cellular cytotoxicity and complement-mediated lysis of melanoma and neuroblastoma cells in vitro by murine antiganglioside antibodies MB 3.6 and 14.G2a.

    PubMed

    Mayer, P; Handgretinger, R; Bruchelt, G; Schaber, B; Rassner, G; Fierlbeck, G

    1994-04-01

    Mouse monoclonal antibodies against tumour-associated gangliosides GD2 (14.G2a) and GD3 (MB 3.6) were tested to mediate antibody-dependent cellular cytotoxicity (ADCC) with various effector cells or complement-dependent cytolysis (CDC). We also evaluated the immunomodulating potential of interferons in combination with cellular cytotoxicity. Using effector:target (E/T) ratios of 40:1, ADCC with effector cells such as granulocytes or mononuclear blood cells was not detectable against melanoma cell lines GR, SK-MEL-28 and G-361 which preferentially express GD3 and bind antibody MB 3.6. Neuroblastoma cell line SK-N-LO, which was used for comparative purposes, mainly expressed GD2 and the tumour cells were killed effectively after labelling with antibody 14.G2a. Granulocytes did not show significant killing of melanoma cells by ADCC, but neuroblastoma cells were killed very efficiently. Peripheral blood mononuclear cells (PBMC) also failed to kill melanoma cells. Interferon-beta slightly stimulated PBMC and increased killing of neuroblastoma cells, but no additive effects with ADCC were detectable. Incubation of target cells with interferons produced no significant differences in susceptibility of the target cells to interferon-activated PBMC cytotoxicity. Despite the lack of effectiveness in mediating cellular cytotoxicity, GD3 antibody MB 3.6 showed strong complement-dependent cytolysis in the presence of human plasma. There were remarkable differences in individual activity and different susceptibility of the melanoma cell lines. We assume that CDC may have more activity against melanoma cells than cytotoxicity associated with various effector cells.

  1. Suppression of natural killer cell activity by rabbit antibody to human beta 2-microglobulin (beta 2m) is an Fc-mediated phenomenon and is not beta 2m specific.

    PubMed

    Jones, R A; Richards, S J; Patel, D; Scott, C S

    1991-07-01

    Natural killer (NK) cell cytotoxicity constitutes an important component of the host immune defence system. The NK effector cell has been relatively well defined in terms of immunophenotypic characteristics, but in contrast to the functional T-cell receptor molecule associated with major histocompatibility complex (MHC)-restricted cytotoxic activity, the NK cell receptor has not to date been defined. However, several studies have suggested that the beta 2-microglobulin (beta 2m) molecule is functionally associated with NK cell activity. Using various heterospecific and monoclonal antibodies, this study has shown that intact rabbit IgG antibody bound either directly or indirectly to peripheral mononuclear cell (PMNC) effector populations significantly reduced their lytic activity against K562 targets. Substitution of F(ab)2 fragments for rabbit IgG antibodies, or the use of monoclonal antibodies alone, failed to reduce peripheral blood mononuclear cell (PMNC) lytic activity. Addition of non-NK cell components labelled with rabbit anti-beta 2m to purified NK-enriched effector cell populations also suppressed K562 lysis. In contrast, pre-treatment of a NK-enriched PMNC fraction with rabbit anti-beta 2m enhanced target lysis. These results strongly suggest that antibody-induced suppression of PMNC NK activity is mediated via rabbit Fc attached to co-existing non-NK cells in the mononuclear fraction, and are inconsistent with the previously suggested functional association between NK activity and membrane beta 2m.

  2. Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors.

    PubMed

    Devarapu, Satish Kumar; Mamidi, Srinivas; Plöger, Frank; Dill, Othmar; Blixt, Ola; Kirschfink, Michael; Schwartz-Albiez, Reinhard

    2016-06-15

    A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies. PMID:26830059

  3. Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors.

    PubMed

    Devarapu, Satish Kumar; Mamidi, Srinivas; Plöger, Frank; Dill, Othmar; Blixt, Ola; Kirschfink, Michael; Schwartz-Albiez, Reinhard

    2016-06-15

    A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies.

  4. Cationic liposomes enhance targeted delivery and expression of exogenous DNA mediated by N-terminal modified poly(L-lysine)-antibody conjugate in mouse lung endothelial cells.

    PubMed

    Trubetskoy, V S; Torchilin, V P; Kennel, S; Huang, L

    1992-07-15

    A new and improved system for targeted gene delivery and expression is described. Transfection efficiency of N-terminal modified poly(L-lysine) (NPLL) conjugated with anti-thrombomodulin antibody 34A can be improved by adding to the system a lipophilic component, cationic liposomes. DNA, antibody conjugate and cationic liposomes form a ternary electrostatic complex which preserves the ability to bind specifically to the target cells. At the same time the addition of liposomes enhance the specific transfection efficiency of antibody-polylysine/DNA binary complex by 10 to 20-fold in mouse lung endothelial cells in culture.

  5. Antibody-mediated targeting of iron oxide nanoparticles to the folate receptor alpha increases tumor cell association in vitro and in vivo

    PubMed Central

    Ndong, Christian; Toraya-Brown, Seiko; Kekalo, Katsiaryna; Baker, Ian; Gerngross, Tillman U; Fiering, Steven N; Griswold, Karl E

    2015-01-01

    Active molecular targeting has become an important aspect of nanoparticle development for oncology indications. Here, we describe molecular targeting of iron oxide nanoparticles (IONPs) to the folate receptor alpha (FOLRα) using an engineered antibody fragment (Ffab). Compared to control nanoparticles targeting the non-relevant botulinum toxin, the Ffab-IONP constructs selectively accumulated on FOLRα-overexpressing cancer cells in vitro, where they exhibited the capacity to internalize into intracellular vesicles. Similarly, Ffab-IONPs homed to FOLRα-positive tumors upon intraperitoneal administration in an orthotopic murine xenograft model of ovarian cancer, whereas negative control particles showed no detectable tumor accumulation. Interestingly, Ffab-IONPs built with custom 120 nm nanoparticles exhibited lower in vitro targeting efficiency when compared to those built with commercially sourced 180 nm nanoparticles. In vivo, however, the two Ffab-IONP platforms achieved equivalent tumor homing, although the smaller 120 nm IONPs were more prone to liver sequestration. Overall, the results show that Ffab-mediated targeting of IONPs yields specific, high-level accumulation within cancer cells, and this fact suggests that Ffab-IONPs could have future utility in ovarian cancer diagnostics and therapy. PMID:25878495

  6. Antibody-mediated targeting of iron oxide nanoparticles to the folate receptor alpha increases tumor cell association in vitro and in vivo.

    PubMed

    Ndong, Christian; Toraya-Brown, Seiko; Kekalo, Katsiaryna; Baker, Ian; Gerngross, Tillman U; Fiering, Steven N; Griswold, Karl E

    2015-01-01

    Active molecular targeting has become an important aspect of nanoparticle development for oncology indications. Here, we describe molecular targeting of iron oxide nanoparticles (IONPs) to the folate receptor alpha (FOLRα) using an engineered antibody fragment (Ffab). Compared to control nanoparticles targeting the non-relevant botulinum toxin, the Ffab-IONP constructs selectively accumulated on FOLRα-overexpressing cancer cells in vitro, where they exhibited the capacity to internalize into intracellular vesicles. Similarly, Ffab-IONPs homed to FOLRα-positive tumors upon intraperitoneal administration in an orthotopic murine xenograft model of ovarian cancer, whereas negative control particles showed no detectable tumor accumulation. Interestingly, Ffab-IONPs built with custom 120 nm nanoparticles exhibited lower in vitro targeting efficiency when compared to those built with commercially sourced 180 nm nanoparticles. In vivo, however, the two Ffab-IONP platforms achieved equivalent tumor homing, although the smaller 120 nm IONPs were more prone to liver sequestration. Overall, the results show that Ffab-mediated targeting of IONPs yields specific, high-level accumulation within cancer cells, and this fact suggests that Ffab-IONPs could have future utility in ovarian cancer diagnostics and therapy.

  7. Antibody-Mediated Autoimmune Encephalopathies and Immunotherapies.

    PubMed

    Gastaldi, Matteo; Thouin, Anaïs; Vincent, Angela

    2016-01-01

    Over the last 15 years it has become clear that rare but highly recognizable diseases of the central nervous system (CNS), including newly identified forms of limbic encephalitis and other encephalopathies, are likely to be mediated by antibodies (Abs) to CNS proteins. The Abs are directed against membrane receptors and ion channel-associated proteins that are expressed on the surface of neurons in the CNS, such as N-methyl D-aspartate receptors and leucine-rich, glioma inactivated 1 protein and contactin-associated protein like 2, that are associated with voltage-gated potassium channels. The diseases are not invariably cancer-related and are therefore different from the classical paraneoplastic neurological diseases that are associated with, but not caused by, Abs to intracellular proteins. Most importantly, the new antibody-associated diseases almost invariably respond to immunotherapies with considerable and sometimes complete recovery, and there is convincing evidence of their pathogenicity in the relatively limited studies performed so far. Treatments include first-line steroids, intravenous immunoglobulins, and plasma exchange, and second-line rituximab and cyclophosphamide, followed in many cases by steroid-sparing agents in the long-term. This review focuses mainly on N-methyl D-aspartate receptor- and voltage-gated potassium channel complex-related Abs in adults, the clinical phenotypes, and treatment responses. Pediatric cases are referred to but not reviewed in detail. As there have been very few prospective studies, the conclusions regarding immunotherapies are based on retrospective studies. PMID:26692392

  8. Evaluation of antibody-dependent cell-mediated cytotoxicity activity and cetuximab response in KRAS wild-type metastatic colorectal cancer patients

    PubMed Central

    Lo Nigro, Cristiana; Ricci, Vincenzo; Vivenza, Daniela; Monteverde, Martino; Strola, Giuliana; Lucio, Francesco; Tonissi, Federica; Miraglio, Emanuela; Granetto, Cristina; Fortunato, Mirella; Merlano, Marco Carlo

    2016-01-01

    AIM: To investigate the prognostic role of invariant natural killer T (iNKT) cells and antibody-dependent cell-mediated cytotoxicity (ADCC) in wild type KRAS metastatic colorectal cancer (mCRC) patients treated with cetuximab. METHODS: Forty-one KRAS wt mCRC patients, treated with cetuximab and irinotecan-based chemotherapy in II and III lines were analyzed. Genotyping of single nucleotide polymorphism (SNP)s in the FCGR2A, FCGR3A and in the 3’ untranslated regions of KRAS and mutational analysis for KRAS, BRAF and NRAS genes was determined either by sequencing or allelic discrimination assays. Enriched NK cells were obtained from lymphoprep-peripheral blood mononuclear cell and iNKT cells were defined by co-expression of CD3, TCRVα24, TCRVβ11. ADCC was evaluated as ex vivo NK-dependent activity, measuring lactate dehydrogenase release. RESULTS: At basal, mCRC patients performing ADCC activity above the median level (71%) showed an improved overall survival (OS) compared to patients with ADCC below (median 16 vs 8 mo; P = 0.026). We did not find any significant correlation of iNKT cells with OS (P = 0.19), albeit we observed a trend to a longer survival after 10 mo in patients with iNKT above median basal level (0.382 cells/microliter). Correlation of OS and progression-free survival (PFS) with interesting SNPs involved in ADCC ability revealed not to be significant. Patients carrying alleles both with A in FCGR2A and TT in FCGR3A presented a trend of longer PFS (median 9 vs 5 mo; P = 0.064). Chemotherapy impacted both iNKT cells and ADCC activity. Their prognostic values get lost when we analysed them after 2 and 4 mo of treatment. CONCLUSION: Our results suggest a link between iNKT cells, basal ADCC activity, genotypes in FCGR2A and FCGR3A, and efficacy of cetuximab in KRAS wt mCRC patients. PMID:26909137

  9. gp49B-mediated negative regulation of antibody production by memory and marginal zone B cells.

    PubMed

    Fukao, Saori; Haniuda, Kei; Nojima, Takuya; Takai, Toshiyuki; Kitamura, Daisuke

    2014-07-15

    The rapid Ab responses observed after primary and secondary immunizations are mainly derived from marginal zone (MZ) and memory B cells, respectively, but it is largely unknown how these responses are negatively regulated. Several inhibitory receptors have been identified and their roles have been studied, but mainly on follicular B cells and much less so on MZ B, and never on memory B cells. gp49B is an Ig superfamily member that contains two ITIMs in its cytoplasmic tail, and it has been shown to negatively regulate mast cell, macrophage, and NK cell responses. In this study, we demonstrate that gp49B is preferentially expressed on memory and MZ B cells. We show that gp49B(-/-) mice produce more IgM after a primary immunization and more IgM and IgG1 after a secondary immunization than gp49B(+/+) mice in T cell-dependent immune responses. Memory and MZ B cells from gp49B(-/-) mice also produce more Abs upon in vitro stimulation with CD40 than those from gp49B(+/+) mice. The in vitro IgM production by MZ B cells from gp49B(+/+), but not gp49B(-/-), mice is suppressed by interaction with a putative gp49B ligand, the integrin αvβ3 heterodimer. In addition, gp49B(-/-) mice exhibited exaggerated IgE production in the memory recall response. These results suggest that plasma cell development from memory and MZ B cells, as well as subsequent Ab production, are suppressed via gp49B. In memory B cells, this suppression also prevents excessive IgE production, thus curtailing allergic diseases.

  10. Generation of new peptide-Fc fusion proteins that mediate antibody-dependent cellular cytotoxicity against different types of cancer cells

    PubMed Central

    Sioud, Mouldy; Westby, Phuong; Olsen, Julie Kristine E.; Mobergslien, Anne

    2015-01-01

    Antibody-dependent cellular cytotoxicity (ADCC), a key effector function for the clinical effectiveness of monoclonal antibodies, is triggered by the engagement of the antibody Fc domain with the Fcγ receptors expressed by innate immune cells such as natural killer (NK) cells and macrophages. Here, we fused cancer cell-binding peptides to the Fc domain of human IgG1 to engineer novel peptide-Fc fusion proteins with ADCC activity. The designed fusion proteins were expressed in human embryonic kidney 293T cells, followed by purification and characterization by western blots. One of the engineered variants (WN-Fc), bound with high affinity to a wide range of solid tumor cell lines (e.g., colon, lung, prostate, skin, ovarian, and mammary tumors). Treatment of cancer cells with the engineered peptide-Fc fusions in the presence of effector NK cells potentially enhanced cytotoxicity, degranulation, and interferon-γ production by NK cells when compared to cells treated with the Fc control. The presence of competing peptides inhibited NK cell activation. Furthermore, a bispecific peptide-Fc fusion protein activated NK cells against HER-1- and/or HER-2-expressing cancer cells. Collectively, the engineered peptide-Fc fusions constitute a new promising strategy to recruit and activate NK cells against tumor cells, a primary goal of cancer immunotherapy. PMID:26605373

  11. Impact of viral attachment factor expression on antibody-mediated neutralization of flaviviruses.

    PubMed

    Obara, Christopher J; Dowd, Kimberly A; Ledgerwood, Julie E; Pierson, Theodore C

    2013-03-01

    Neutralization of flaviviruses requires engagement of the virion by antibodies with a stoichiometry that exceeds a required threshold. Factors that modulate the number of antibodies bound to an individual virion when it contacts target cells impact neutralization potency. However, the contribution of cellular factors to the potency of neutralizing antibodies has not been explored systematically. Here we investigate the relationship between expression level of a viral attachment factor on cells and the neutralizing potency of antibodies. Analysis of the attachment factor DC-SIGNR on cells in neutralization studies failed to identify a correlation between DC-SIGNR expression and antibody-mediated protection. Furthermore, neutralization potency was equivalent on a novel Jurkat cell line induced to express DC-SIGNR at varying levels. Finally, blocking virus-attachment factor interactions had no impact on neutralization activity. Altogether, our studies suggest that cellular attachment factor expression is not a significant contributor to the potency of neutralizing antibodies to flaviviruses.

  12. Updates on antibody-mediated rejection in intestinal transplantation

    PubMed Central

    Wu, Guo-Sheng

    2016-01-01

    Antibody-mediated rejection (ABMR) has increasingly emerged as an important cause of allograft loss after intestinal transplantation (ITx). Compelling evidence indicates that donor-specific antibodies can mediate and promote acute and chronic rejection after ITx. However, diagnostic criteria for ABMR after ITx have not been established yet and the mechanisms of antibody-mediated graft injury are not well-known. Effective approaches to prevent and treat ABMR are required to improve long-term outcomes of intestine recipients. Clearly, ABMR after ITx has become an important area for research and clinical investigation. PMID:27683635

  13. Updates on antibody-mediated rejection in intestinal transplantation.

    PubMed

    Wu, Guo-Sheng

    2016-09-24

    Antibody-mediated rejection (ABMR) has increasingly emerged as an important cause of allograft loss after intestinal transplantation (ITx). Compelling evidence indicates that donor-specific antibodies can mediate and promote acute and chronic rejection after ITx. However, diagnostic criteria for ABMR after ITx have not been established yet and the mechanisms of antibody-mediated graft injury are not well-known. Effective approaches to prevent and treat ABMR are required to improve long-term outcomes of intestine recipients. Clearly, ABMR after ITx has become an important area for research and clinical investigation. PMID:27683635

  14. Updates on antibody-mediated rejection in intestinal transplantation

    PubMed Central

    Wu, Guo-Sheng

    2016-01-01

    Antibody-mediated rejection (ABMR) has increasingly emerged as an important cause of allograft loss after intestinal transplantation (ITx). Compelling evidence indicates that donor-specific antibodies can mediate and promote acute and chronic rejection after ITx. However, diagnostic criteria for ABMR after ITx have not been established yet and the mechanisms of antibody-mediated graft injury are not well-known. Effective approaches to prevent and treat ABMR are required to improve long-term outcomes of intestine recipients. Clearly, ABMR after ITx has become an important area for research and clinical investigation.

  15. Targeting Tumor Cells with Anti-CD44 Antibody Triggers Macrophage-Mediated Immune Modulatory Effects in a Cancer Xenograft Model

    PubMed Central

    Maisel, Daniela; Birzele, Fabian; Voss, Edgar; Nopora, Adam; Bader, Sabine; Friess, Thomas; Goller, Bernhard; Laifenfeld, Daphna; Weigand, Stefan; Runza, Valeria

    2016-01-01

    CD44, a transmembrane receptor reported to be involved in various cellular functions, is overexpressed in several cancer types and supposed to be involved in the initiation, progression and prognosis of these cancers. Since the sequence of events following the blockage of the CD44-HA interaction has not yet been studied in detail, we profiled xenograft tumors by RNA Sequencing to elucidate the mode of action of the anti-CD44 antibody RG7356. Analysis of tumor and host gene-expression profiles led us to the hypothesis that treatment with RG7356 antibody leads to an activation of the immune system. Using cytokine measurements we further show that this activation involves the secretion of chemo-attractants necessary for the recruitment of immune cells (i.e. macrophages) to the tumor site. We finally provide evidence for antibody-dependent cellular phagocytosis (ADCP) of the malignant cells by macrophages. PMID:27463372

  16. Neuropeptide-mediated regulation of hapten-specific IgE responses in mice. II. Mechanisms of substance P-mediated isotype-specific suppression of BPO-specific IgE antibody-forming cell responses induced in vitro.

    PubMed

    Carucci, J A; Herrick, C A; Durkin, H G

    1994-01-01

    Previous studies in our laboratory have shown that substance P (SP), injected into benzylpenicilloyl-keyhole limpet hemocyanin (BPO-KLH) sensitized mice at the peak of the benzylpenicilloyl (BPO)-specific IgE response, suppressed these responses in isotype-specific fashion within 48 h. These studies also showed that SP, but not neurotensin (NT), serotonin (5-HT), somatostatin (SOM) or gastrin, suppressed BPO-specific memory IgE antibody-forming cell (AFC) responses induced in vitro, also in isotype-specific fashion. To investigate the mechanisms by which SP suppressed BPO-specific IgE AFC responses were induced in vitro, these responses were induced by culturing spleen cells from BPO-KLH sensitized mice for 5 days with BPO-KLH with or without whole SP, amino terminal SP (SP 1-4: Arg-Lys-Pro-Lys), or carboxy terminal SP (SP 8-11: Phe-Gly-Leu-Met). In some experiments, the SP receptor antagonist (D-Pro2, D-Phe7, D-Trp9)-SP (D-SP) was included in culture. In other experiments anti-interferon monoclonal antibody (anti-IFN gamma mAb) was in culture. Whole SP and SP 8-11, but not SP 1-4, suppressed BPO-specific IgE AFC responses induced in vitro. The suppression obtained was IgE isotype-specific and dose-dependent. Inclusion of SP receptor antagonist (D-Pro2, D-Phe7, D-Trp9)-SP inhibited suppression of BPO-specific memory IgE AFC responses by SP or SP 8-11. The SP-mediated suppression of BPO-specific memory IgE responses appeared to involve interferon gamma (IFN gamma).

  17. Cell-targeting antibodies in immunity to Ebola.

    PubMed

    Schmaljohn, Alan; Lewis, George K

    2016-06-01

    As the 2014-15 Ebola virus epidemic in West Africa evolved from emergency to lesson, developers of both vaccines and therapeutic antibodies were left with the puzzlement of what kinds of anti-Ebola antibodies are predictably desirable in treating the afflicted, and what antibodies might account for the specific and lasting protection elicited by the more effective vaccines. The facile answer in virology is that neutralizing antibody (NAb) is desired and required. However, with Ebola and other filoviruses (as with many prior viral examples), there are multiple discordances in which neutralizing antibodies fail to protect animals, and others in which antibody-mediated protection is observed in the absence of measured virus neutralization. Explanation presumably resides in the protective role of antibodies that bind and functionally 'target' virus-infected cells, here called 'cell-targeting antibody', or CTAb. To be clear, many NAbs are also CTAbs, and in the case of Ebola the great majority of NAbs are likely CTAbs. Isotype, glycosylation, and other features of CTAbs are likely crucial in their capacity to mediate protection. Overall, results and analysis invite an increasingly complex view of antibody-mediated immunity to enveloped viruses. PMID:27005312

  18. Antibody-mediated Xenograft Injury: Mechanisms and Protective Strategies

    PubMed Central

    Pierson, Richard N.

    2009-01-01

    The use of porcine organs for clinical transplantation is a promising potential solution to the shortage of human organs. Preformed anti-pig antibody is the primary cause of hyperacute rejection, while elicited antibody can contribute to subsequent “delayed” xenograft rejection. This article will review recent progress to overcome antibody mediated xenograft rejection, through modification of the host immunity and use of genetically engineered pig organs. PMID:19376229

  19. Characterization of rabbit cells by monoclonal and polyclonal antibodies.

    PubMed Central

    Ponsard, D C; Cinader, B; Chou, C T; Dubiski, S

    1986-01-01

    Reagents for the identification of rabbit cell markers have been developed at a relatively slow rate. In this paper, rabbit cells are being characterized by polyclonal antibodies against a T-cell antigen (RTLA), a B-cell antigen (RABELA) and an analogue of murine Ia antigen. A number of monoclonal antibodies, specific for lymphocytes and/or bone marrow and/or polymorphonuclear leucocytes, have been used for the analysis of cells with identifiable membrane antigens. Populations that have cells with two of the above antigens in the membranes were identified. To these ends, complement-mediated cell kill by antisera alone and in mixtures was employed. PMID:3489667

  20. B Cells, Antibodies, and More

    PubMed Central

    Hoffman, William; Lakkis, Fadi G.

    2016-01-01

    B cells play a central role in the immunopathogenesis of glomerulonephritides and transplant rejection. B cells secrete antibodies that contribute to tissue injury via multiple mechanisms. In addition, B cells contribute to disease pathogenesis in autoimmunity and alloimmunity by presenting antigens as well as providing costimulation and cytokines to T cells. B cells also play an immunomodulatory role in regulating the immune response by secreting cytokines that inhibit disease onset and/or progression. B cell–targeted approaches for treating immune diseases of the kidney and other organs have gained significant momentum. However, much remains to be understood about B-cell biology in order to determine the timing, duration, and context of optimal therapeutic response to B cell–targeted approaches. In this review, we discuss the multifaceted roles of B cells as enhancers and regulators of immunity with relevance to kidney disease and transplantation. PMID:26700440

  1. Postoperative rebound of antiblood type antibodies and antibody-mediated rejection after ABO-incompatible living-related kidney transplantation.

    PubMed

    Ishida, Hideki; Kondo, Tsunenori; Shimizu, Tomokazu; Nozaki, Taiji; Tanabe, Kazunari

    2015-03-01

    The purpose of this study is to examine whether postoperative antiblood type antibody rebound is attributed to kidney allograft rejection in ABO blood type-incompatible (ABO-I) living-related kidney transplantation (KTx). A total of 191 ABO-I recipients who received ABO-I living-related KTx between 2001 and 2013 were divided into two groups: Group 1 consisted of low rebound [(≦1:32), N = 170] and Group 2 consisted of high rebound [(≧1:64), N = 21], according to the levels of the rebounded antiblood type antibodies within 1 year after transplantation. No prophylactic treatment for rejection was administered for elevated antiblood type antibodies, regardless of the levels of the rebounded antibodies. Within 1 year after transplantation, T-cell-mediated rejection was observed in 13 of 170 recipients (13/170, 8%) in Group 1 and in 2 of 21 recipients (2/21, 10%) in Group 2 (Groups 1 vs. 2, P = 0.432). Antibody-mediated rejection was observed in 15 of 170 recipients (15/170, 9%) and 2 of 21 recipients (2/21, 10%) in Groups 1 and 2, respectively (P = 0.898). In this study, we found no correlation between the postoperative antiblood type antibody rebound and the incidence of acute rejection. We concluded that no treatment is necessary for rebounded antiblood type antibodies.

  2. Infection of CD4{sup +} T lymphocytes by the human T cell leukemia virus type 1 is mediated by the glucose transporter GLUT-1: Evidence using antibodies specific to the receptor's large extracellular domain

    SciTech Connect

    Jin, Qingwen; Agrawal, Lokesh; VanHorn-Ali, Zainab; Alkhatib, Ghalib . E-mail: galkhati@iupui.edu

    2006-05-25

    To analyze HTLV-1 cytotropism, we developed a highly sensitive vaccinia virus-based assay measuring activation of a reporter gene upon fusion of two distinct cell populations. We used this system in a functional cDNA screening to isolate and confirm that the glucose transporter protein 1 (GLUT-1) is a receptor for HTLV-1. GLUT-1 is a ubiquitously expressed plasma membrane glycoprotein with 12 transmembrane domains and 6 extracellular loops (ECL). We demonstrate for the first time that peptide antibodies (GLUT-IgY) raised in chicken to the large extracellular loop (ECL1) detect GLUT-1 at the cell surface and inhibit envelope (Env)-mediated fusion and infection. Efficient GLUT-IgY staining was detected with peripheral blood CD4{sup +} lymphocytes purified by positive selection. Further, GLUT-IgY caused efficient inhibition of Env-mediated fusion and infection of CD4{sup +} T and significantly lower inhibition of CD8{sup +} T lymphocytes. The specificity of GLUT-IgY antibodies to GLUT-1 was demonstrated by ECL1 peptide competition studies. Grafting ECL1 of GLUT-1 onto the receptor-negative GLUT-3 conferred significant receptor activity. In contrast, grafting ECL1 of GLUT-3 onto GLUT-1 resulted in a significant loss of the receptor activity. The ECL1-mediated receptor activity was efficiently blocked with four different human monoclonal antibody (HMab) to HTLV-1 Env. The ECL1-derived peptide blocked HTLV-1 Env-mediated fusion with several nonhuman mammalian cell lines. The results demonstrate the utilization of cell surface GLUT-1 in HTLV-1 infection of CD4{sup +} T lymphocytes and implicate a critical role for the ECL1 region in viral tropism.

  3. HIV-1 Tat Promotes Integrin-Mediated HIV Transmission to Dendritic Cells by Binding Env Spikes and Competes Neutralization by Anti-HIV Antibodies

    PubMed Central

    Monini, Paolo; Cafaro, Aurelio; Srivastava, Indresh K.; Moretti, Sonia; Sharma, Victoria A.; Andreini, Claudia; Chiozzini, Chiara; Ferrantelli, Flavia; Cossut, Maria R. Pavone.; Tripiciano, Antonella; Nappi, Filomena; Longo, Olimpia; Bellino, Stefania; Picconi, Orietta; Fanales-Belasio, Emanuele; Borsetti, Alessandra; Toschi, Elena; Schiavoni, Ilaria; Bacigalupo, Ilaria; Kan, Elaine; Sernicola, Leonardo; Maggiorella, Maria T.; Montin, Katy; Porcu, Marco; Leone, Patrizia; Leone, Pasqualina; Collacchi, Barbara; Palladino, Clelia; Ridolfi, Barbara; Falchi, Mario; Macchia, Iole; Ulmer, Jeffrey B.; Buttò, Stefano; Sgadari, Cecilia; Magnani, Mauro; Federico, Maurizio P. M.; Titti, Fausto; Banci, Lucia; Dallocchio, Franco; Rappuoli, Rino; Ensoli, Fabrizio; Barnett, Susan W.; Garaci, Enrico; Ensoli, Barbara

    2012-01-01

    Use of Env in HIV vaccine development has been disappointing. Here we show that, in the presence of a biologically active Tat subunit vaccine, a trimeric Env protein prevents in monkeys virus spread from the portal of entry to regional lymph nodes. This appears to be due to specific interactions between Tat and Env spikes that form a novel virus entry complex favoring R5 or X4 virus entry and productive infection of dendritic cells (DCs) via an integrin-mediated pathway. These Tat effects do not require Tat-transactivation activity and are blocked by anti-integrin antibodies (Abs). Productive DC infection promoted by Tat is associated with a highly efficient virus transmission to T cells. In the Tat/Env complex the cysteine-rich region of Tat engages the Env V3 loop, whereas the Tat RGD sequence remains free and directs the virus to integrins present on DCs. V2 loop deletion, which unshields the CCR5 binding region of Env, increases Tat/Env complex stability. Of note, binding of Tat to Env abolishes neutralization of Env entry or infection of DCs by anti-HIV sera lacking anti-Tat Abs, which are seldom present in natural infection. This is reversed, and neutralization further enhanced, by HIV sera containing anti-Tat Abs such as those from asymptomatic or Tat-vaccinated patients, or by sera from the Tat/Env vaccinated monkeys. Thus, both anti-Tat and anti-Env Abs are required for efficient HIV neutralization. These data suggest that the Tat/Env interaction increases HIV acquisition and spreading, as a mechanism evolved by the virus to escape anti-Env neutralizing Abs. This may explain the low effectiveness of Env-based vaccines, which are also unlikely to elicit Abs against new Env epitopes exposed by the Tat/Env interaction. As Tat also binds Envs from different clades, new vaccine strategies should exploit the Tat/Env interaction for both preventative and therapeutic interventions. PMID:23152803

  4. Cell-targeting antibodies in immunity to Ebola

    PubMed Central

    Schmaljohn, Alan; Lewis, George K.

    2016-01-01

    As the 2014–15 Ebola virus epidemic in West Africa evolved from emergency to lesson, developers of both vaccines and therapeutic antibodies were left with the puzzlement of what kinds of anti-Ebola antibodies are predictably desirable in treating the afflicted, and what antibodies might account for the specific and lasting protection elicited by the more effective vaccines. The facile answer in virology is that neutralizing antibody (NAb) is desired and required. However, with Ebola and other filoviruses (as with many prior viral examples), there are multiple discordances in which neutralizing antibodies fail to protect animals, and others in which antibody-mediated protection is observed in the absence of measured virus neutralization. Explanation presumably resides in the protective role of antibodies that bind and functionally ‘target’ virus-infected cells, here called ‘cell-targeting antibody’, or CTAb. To be clear, many NAbs are also CTAbs, and in the case of Ebola the great majority of NAbs are likely CTAbs. Isotype, glycosylation, and other features of CTAbs are likely crucial in their capacity to mediate protection. Overall, results and analysis invite an increasingly complex view of antibody-mediated immunity to enveloped viruses. PMID:27005312

  5. Vector-mediated antibody gene transfer for infectious diseases.

    PubMed

    Schnepp, Bruce C; Johnson, Philip R

    2015-01-01

    This chapter discusses the emerging field of vector-mediated antibody gene transfer as an alternative vaccine for infectious disease, with a specific focus on HIV. However, this methodology need not be confined to HIV-1; the general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets like hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis. This approach is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. With vector-mediated gene transfer, the antibody gene is delivered to the host, via a recombinant adeno-associated virus (rAAV) vector; this in turn results in long-term endogenous antibody expression from the injected muscle that confers protective immunity. Vector-mediated antibody gene transfer can rapidly move existing, potent broadly cross-neutralizing HIV-1-specific antibodies into the clinic. The gene transfer products demonstrate a potency and breadth identical to the original product. This strategy eliminates the need for immunogen design and interaction with the adaptive immune system to generate protection, a strategy that so far has shown limited promise.

  6. Antibodies as Mediators of Brain Pathology.

    PubMed

    Brimberg, Lior; Mader, Simone; Fujieda, Yuichiro; Arinuma, Yoshiyuki; Kowal, Czeslawa; Volpe, Bruce T; Diamond, Betty

    2015-11-01

    The brain is normally sequestered from antibody exposure by the blood brain barrier. However, antibodies can access the brain during fetal development before the barrier achieves full integrity, and in disease states when barrier integrity is compromised. Recent studies suggest that antibodies contribute to brain pathology associated with autoimmune diseases such as systemic lupus erythematosus and neuromyelitis optica, and can lead to transient or permanent behavioral or cognitive abnormalities. We review these findings here and examine the circumstances associated with antibody entry into the brain, the routes of access and the mechanisms that then effect pathology. Understanding these processes and the nature and specificity of neuronal autoantibodies may reveal therapeutic strategies toward alleviating or preventing the neurological pathologies and behavioral abnormalities associated with autoimmune disease.

  7. Antibodies as Mediators of Brain Pathology.

    PubMed

    Brimberg, Lior; Mader, Simone; Fujieda, Yuichiro; Arinuma, Yoshiyuki; Kowal, Czeslawa; Volpe, Bruce T; Diamond, Betty

    2015-11-01

    The brain is normally sequestered from antibody exposure by the blood brain barrier. However, antibodies can access the brain during fetal development before the barrier achieves full integrity, and in disease states when barrier integrity is compromised. Recent studies suggest that antibodies contribute to brain pathology associated with autoimmune diseases such as systemic lupus erythematosus and neuromyelitis optica, and can lead to transient or permanent behavioral or cognitive abnormalities. We review these findings here and examine the circumstances associated with antibody entry into the brain, the routes of access and the mechanisms that then effect pathology. Understanding these processes and the nature and specificity of neuronal autoantibodies may reveal therapeutic strategies toward alleviating or preventing the neurological pathologies and behavioral abnormalities associated with autoimmune disease. PMID:26494046

  8. Antibodies as Mediators of Brain Pathology

    PubMed Central

    Brimberg, Lior; Mader, Simone; Fujieda, Yuichiro; Arinuma, Yoshiyuki; Kowal, Czeslawa; Volpe, Bruce T.; Diamond, Betty

    2016-01-01

    The brain is normally sequestered from antibody exposure by the blood brain barrier. However, antibodies can access the brain during fetal development before the barrier achieves full integrity, and in disease states when barrier integrity is compromised. Recent studies suggest that antibodies contribute to brain pathology associated with autoimmune diseases such as systemic lupus erythematosus and neuromyelitis optica, and can lead to transient or permanent behavioral or cognitive abnormalities. We review these findings here and examine the circumstances associated with antibody entry into the brain, the routes of access and the mechanisms that then effect pathology. Understanding these processes and the nature and specificity of neuronal autoantibodies may reveal therapeutic strategies toward alleviating or preventing the neurological pathologies and behavioral abnormalities associated with autoimmune disease. PMID:26494046

  9. Decreased complement mediated binding of antibody//sup 3/-dsDNA immune complexes to the red blood cells of patients with systemic lupus erythematosus, rheumatoid arthritis, and hematologic malignancies

    SciTech Connect

    Taylor, R.P.; Horgan, C.; Buschbacher, R.; Brunner, C.M.; Hess, C.E.; O'Brien, W.M.; Wanebo, H.J.

    1983-06-01

    The complement mediated binding of prepared antibody//sup 3/H-dsDNA immune complexes to the red blood cells obtained from a number of patient populations has been investigated. Patients with solid tumors have binding activity similar to that seen in a normal group of individuals. However, a significant fraction of patients with systemic lupus erythematosus, rheumatoid arthritis, and hematologic malignancies have lowered binding activity compared with normal subjects. Quantitative studies indicate the lowered activity probably arises due to a decrease in complement receptors on the respective red blood cells. The potential importance and implications of these findings are briefly discussed.

  10. Impaired Clearance of Early Apoptotic Cells Mediated by Inhibitory IgG Antibodies in Patients with Primary Sjögren's Syndrome

    PubMed Central

    Manoussakis, Menelaos N.; Fragoulis, George E.; Vakrakou, Aigli G.; Moutsopoulos, Haralampos M.

    2014-01-01

    Objectives Deficient efferocytosis (i.e. phagocytic clearance of apoptotic cells) has been frequently reported in systemic lupus erythematosus (SLE). Todate, patients with primary Sjögren's syndrome (SS) have not been assessed for phagocytosis of apoptotic cells (ApoCell-phagocytosis) and of particulate targets (microbeads, MB-phagocytosis). Design ApoCell-phagocytosis and MB-phagocytosis were comparatively assessed by flow cytometry in peripheral blood specimens and monocyte-derived macrophage (MDM) preparations from healthy blood donors (HBD) and consecutive SS, SLE and rheumatoid arthritis (RA) patients. Cross-admixture ApoCell-phagocytosis experiments were also performed using phagocytes from HBD or patients, and apoptotic cells pretreated with whole sera or purified serum IgG derived from patients or HBD. Results Compared to HBD, approximately half of SS and SLE patients studied (but not RA) manifested significantly reduced ApoCell-phagocytosis (p<0.001) and MB-phagocytosis (p<0.003) by blood-borne phagocytes that correlated inversely with disease activity (p≤0.004). In cross-admixture assays, healthy monocytes showed significantly reduced ApoCell-phagocytosis when fed with apoptotic cells that were pretreated with sera or purified serum IgG preparations from SS and SLE patients (p<0.0001, compared to those from HBD or RA). Such aberrant effect of the SS and SLE sera and IgG preparations correlated linearly with their content of IgG antibodies against apoptotic cells (p≤0.0001). Phagocytic dysfunction maybe also present in certain SS and SLE patients, as supported by deficient capacity of MDM for ApoCell-phagocytosis and MB-phagocytosis under patients' serum-free conditions. Conclusion Similarly to SLE, efferocytosis is frequently impaired in SS and is primarily due to the presence of inhibitory IgG anti-ApoCell antibodies and secondarily to phagocytes' dysfunction. PMID:25396412

  11. Selection of Antibodies Interfering with Cell Surface Receptor Signaling Using Embryonic Stem Cell Differentiation.

    PubMed

    Melidoni, Anna N; Dyson, Michael R; McCafferty, John

    2016-01-01

    Antibodies able to bind and modify the function of cell surface signaling components in vivo are increasingly being used as therapeutic drugs. The identification of such "functional" antibodies from within large antibody pools is, therefore, the subject of intense research. Here we describe a novel cell-based expression and reporting system for the identification of functional antibodies from antigen-binding populations preselected with phage display. The system involves inducible expression of the antibody gene population from the Rosa-26 locus of embryonic stem (ES) cells, followed by secretion of the antibodies during ES cell differentiation. Target antigens are cell-surface signaling components (receptors or ligands) with a known effect on the direction of cell differentiation (FGFR1 mediating ES cell exit from self renewal in this particular protocol). Therefore, inhibition or activation of these components by functional antibodies in a few elite clones causes a shift in the differentiation outcomes of these clones, leading to their phenotypic selection. Functional antibody genes are then recovered from positive clones and used to produce the purified antibodies, which can be tested for their ability to affect cell fates exogenously. Identified functional antibody genes can be further introduced in different stem cell types. Inducible expression of functional antibodies has a temporally controlled protein-knockdown capability, which can be used to study the unknown role of the signaling pathway in different developmental contexts. Moreover, it provides a means for control of stem cell differentiation with potential in vivo applications.

  12. Antibody-mediated modulation of arthritis induced by Chlamydia.

    PubMed Central

    Rank, R. G.; Ramsey, K. H.; Hough, A. J.

    1988-01-01

    The purpose of this investigation was to determine the role of the humoral immune response in the production of arthritis in mice immunized with the chlamydial agent of mouse pneumonitis (MoPn) (Chlamydia trachomatis biovar). Mice were made B cell deficient (BCD) by treatment with rabbit antiserum to murine IgM. Control mice included animals treated similarly with normal rabbit serum or phosphate-buffered saline. Male mice were immunized with MoPn inactivated with ultraviolet irradiation while female mice were immunized by genital tract infection with viable chlamydiae. Arthritis was elicited in all mice by intra-articular inoculation of inactivated MoPn. When knee joints were examined for pathologic changes at varying times after challenge, a marked enhancement of the arthritis was observed in both male and female BCD mice when compared with controls at all time points. These data indicated that the humoral immune response is not essential for the production of arthritic disease in this model but may have some role in the modulation of the process in immunologically intact animals. Persistence of chlamydial antigen in joint tissue of BCD mice suggested that antibody may play a role in the elimination of antigen, thus decreasing the stimulus for the development of cell-mediated immunologic injury. Regulatory role for T suppressor cells cannot be ruled out however, because B cell deficient mice have been shown to lack certain T suppressor cell subsets. Images Figure 3 Figure 4 Figure 6 Figure 7 Figure 9 PMID:3400779

  13. A Nanoparticle Platform To Evaluate Bioconjugation and Receptor-Mediated Cell Uptake Using Cross-Linked Polyion Complex Micelles Bearing Antibody Fragments.

    PubMed

    Florinas, Stelios; Liu, Marc; Fleming, Ryan; Van Vlerken-Ysla, Lilian; Ayriss, Joanne; Gilbreth, Ryan; Dimasi, Nazzareno; Gao, Changshou; Wu, Herren; Xu, Ze-Qi; Chen, Shaoyi; Dirisala, Anjaneyulu; Kataoka, Kazunori; Cabral, Horacio; Christie, R James

    2016-05-01

    Targeted nanomedicines are a promising technology for treatment of disease; however, preparation and characterization of well-defined protein-nanoparticle systems remain challenging. Here, we describe a platform technology to prepare antibody binding fragment (Fab)-bearing nanoparticles and an accompanying real-time cell-based assay to determine their cellular uptake compared to monoclonal antibodies (mAbs) and Fabs. The nanoparticle platform was composed of core-cross-linked polyion complex (PIC) micelles prepared from azide-functionalized PEG-b-poly(amino acids), that is, azido-PEG-b-poly(l-lysine) [N3-PEG-b-PLL] and azido-PEG-b-poly(aspartic acid) [N3-PEG-b-PAsp]. These PIC micelles were 30 nm in size and contained approximately 10 polymers per construct. Fabs were derived from an antibody binding the EphA2 receptor expressed on cancer cells and further engineered to contain a reactive cysteine for site-specific attachment and a cleavable His tag for purification from cell culture expression systems. Azide-functionalized micelles and thiol-containing Fab were linked using a heterobifunctional cross-linker (FPM-PEG4-DBCO) that contained a fluorophenyl-maleimide for stable conjugation to Fabs thiols and a strained alkyne (DBCO) group for coupling to micelle azide groups. Analysis of Fab-PIC micelle conjugates by fluorescence correlation spectroscopy, size exclusion chromatography, and UV-vis absorbance determined that each nanoparticle contained 2-3 Fabs. Evaluation of cellular uptake in receptor positive cancer cells by real-time fluorescence microscopy revealed that targeted Fab-PIC micelles achieved higher cell uptake than mAbs and Fabs, demonstrating the utility of this approach to identify targeted nanoparticle constructs with unique cellular internalization properties. PMID:27007881

  14. T Cell Mediated Antibody lnvariance in an Immune Response Against A Bacterial Carbohydrate Antigen Requires CD28/B7–1 Costimulation

    PubMed Central

    Kölsch, Eckehart; Specht, Christoph

    2001-01-01

    The humoral immune response against α(1→3) dextran (Dex) in BALB/c mice is characterized by the formation of predominantly IgM antibodies bearing the J558 idiotype. IgG antibodies do not appear in euthymic mice. In athymic animals however, the response proceeds to a vigorous IgG production. In euthymic mice formation of IgG is suppressed by J558 idiotype- specific regulatory T cells recognizing in association with I-Ed and in cognate T/B interaction the VH CDR3 derived peptide of the J558 idiotpye. Only B-2 lymphocytes produce IgG whereas B-1 cells do not participate in the production of this Ig class. Using a novel synthetic all α(1→3)-D-gluco configurated tetrasaccharide the Dex-specific B cells can for the first time be analyzed in FACS. In experiments using this newly designed low molecular Dex no signs of B cell apoptosis can be found. This demonstrates a true silencing of persisting Bγ memory cells and supports previous by adoptive transfer experiments. In this suppression an involvement of CD28/B7–1 interaction can be demonstrated which is a necessary costimulatory suppression signal in addition to the cognate TCR/peptide-I-Ed interaction between J558 Id-specific T cells and J558 idiotype beating B cells. This results in an activation of 178–4 Ts cells, leading to an overall suppression of the Dex-specific IgG isotype production on the one hand and on the other hand provides a signal for the survival and clonal expansion of J558 Id-positive B cells. PMID:11785674

  15. The effects of environmental enrichment and transport stress on the weights of lymphoid organs, cell-mediated immune response, heterophil functions and antibody production in laying hens.

    PubMed

    Matur, Erdal; Akyazi, İbrahim; Eraslan, Evren; Ergul Ekiz, Elif; Eseceli, Hüseyin; Keten, Mehmet; Metiner, Kemal; Aktaran Bala, Deniz

    2016-02-01

    The effects of environmental enrichment and transport stress on the immune system were investigated in laying hens. A total of 48 1-day-old chickens were used, half of the chickens were reared in conventional cages (RCC) and the rest in enriched cages (REC). Transport stress was applied in the 17th week. Liver weight decreased, spleen and bursa of Fabricius weights, white blood cell count, CD4+ and CD8+ cell proportions increased due to the transport. Environmental enrichment significantly increased antibody production and tended to increase monocyte percentage and CD8+ cell proportion. The effect of transport on, heterophil (H) and lymphocyte (L) percentages was not significant in RCC chickens. While heterophil percentage and H:L ratio increased, lymphocyte percentage decreased in REC chickens subjected to transport. Transport stress increased heterophil functions both in REC and RCC chickens, but the increase was higher in REC hens than in RCC hens. In conclusion, although environmental enrichment did not neutralize the effect of transport on lymphoid organs, it activated the non-specific immune system, cellular and the humoral branches of the specific immune system by increasing heterophil functions, CD8+ cells and antibody production, respectively. Therefore, environmental enrichment suggested for improving animal welfare may also be beneficial to improve the immune system of birds exposed to stress.

  16. Lung injury mediated by antibodies to endothelium. II. Study of the effect of repeated antigen-antibody interactions in rabbits tolerant to heterologous antibody.

    PubMed Central

    Camussi, G.; Caldwell, P. R.; Andres, G.; Brentjens, J. R.

    1987-01-01

    The effect of repeated interactions of antibodies with cell surface antigens have been examined in in vitro, but not in in vivo systems. In this study are described the results of multiple antibody-cell surface antigen interactions in vivo. Rabbits were given repeated intravenous injections of goat antibodies to angiotensin converting enzyme (ACE), an antigen expressed on the surface of lung endothelial cells. For prevention of anaphylactic reactions, which would have been induced by multiple injections of heterologous immune or nonimmune IgG, the rabbits were made neonatally tolerant to goat IgG. Divalent immune IgG given daily for 21 days induced chronic antigenic modulation (antigen disappearance) with resistance to antibody-mediated inflammatory lesions. The rabbits, however, developed degenerative changes of alveolar endothelial and epithelial cells. Administration of immune IgG every other day for 43 days allowed partial reexpression of ACE and was associated with intravascular, but not interstitial, inflammatory changes. In contrast, repeated administration of monovalent immune Fab did not induce antigenic modulation but caused severe, lethal, interstitial pneumonitis. Thus, in this experimental model the development of acute interstitial inflammatory changes correlates with persistence of antigen and is abrogated by disappearance of antigen induced by divalent antibodies. Further, repeated endothelial antigen antibody interactions fail to induce chronic inflammatory or sclerosing lung lesions. Images Figure 5 Figure 6 Figure 11 Figure 12 Figure 13 Figure 1 Figure 2 Figure 3 Figure 4 Figure 7 Figure 7 Figure 9 Figure 10 PMID:3034065

  17. Antibody-Mediated Clearance of Alphavirus Infection from Neurons

    NASA Astrophysics Data System (ADS)

    Levine, Beth; Hardwick, J. Marie; Trapp, Bruce D.; Crawford, Thomas O.; Bollinger, Robert C.; Griffin, Diane E.

    1991-11-01

    Humoral immunity is important for protection against viral infection and neutralization of extracellular virus, but clearance of virus from infected tissues is thought to be mediated solely by cellular immunity. However, in a SCID mouse model of persistent alphavirus encephalomyelitis, adoptive transfer of hyperimmune serum resulted in clearance of infectious virus and viral RNA from the nervous system, whereas adoptive transfer of sensitized T lymphocytes had no effect on viral replication. Three monoclonal antibodies to two different epitopes on the E2 envelope glycoprotein mediated viral clearance. Treatment of alphavirus-infected primary cultured rat neurons with these monoclonal antibodies to E2 resulted in decreased viral protein synthesis, followed by gradual termination of mature infectious virion production. Thus, antibody can mediate clearance of alphavirus infection from neurons by restricting viral gene expression.

  18. Antibody-mediated cofactor-driven reactions

    DOEpatents

    Schultz, Peter G.

    1993-01-01

    Chemical reactions capable of being rate-enhanced by auxiliary species which interact with the reactants but do not become chemically bound to them in the formation of the final product are performed in the presence of antibodies which promote the reactions. The antibodies contain regions within their antigen binding sites which recognize the auxiliary species in a conformation which promotes the reaction. The antigen binding site frequently recognizes a particular transition state complex or other high energy complex along the reaction coordinate, thereby promoting the progress of the reaction along the desired route as opposed to other less favorable routes. Various classes of reaction together with appropriate antigen binding site specificities tailored for each are disclosed.

  19. Varicella-zoster virus-specific, cell-mediated immunity with interferon-gamma release assay after vaccination of college students with no or intermediate IgG antibody response.

    PubMed

    Terada, Kihei; Itoh, Yuri; Fujii, Akihide; Kitagawa, Seiko; Ogita, Satoko; Ouchi, Kazunobu

    2015-02-01

    This study measured Varicella-zoster virus (VZV) specific cell-mediated immunity (CMI) and antibodies to clarify immune response after vaccination in 68 college students with negative or intermediate IgG antibody status. The enrolled numbers of negative, intermediate, and positive VZV-IgG antibody were 27, 41, and 28 students, respectively. The positive rates of CMI were 3.7% (1/27), 41.5% (17/41), and 96.4% (27/28) before vaccination, respectively. After vaccination, the IgG antibody titers became significantly higher in the intermediate IgG group compared to those in the negative IgG group (P < 0.01), but CMI did not differ significantly between the two groups. Ninety-three percent (38/41) of the intermediate IgG antibody group and 41% (11/27) of the negative IgG antibody group became positive for the IgG antibody after vaccination (P < 0.0001). When subjects were divided into negative, intermediate, and positive CMI by interferon-gamma values before vaccination, the IgG antibody and interferon-gamma values increased significantly in the positive CMI group compared to the negative CMI group after vaccination (P < 0.01 and P < 0.01, respectively). All (17/17) of positive CMI group and 61% (27/44) of negative CMI group became positive for the IgG antibody after vaccination (P < 0.01). Ninety-four percent (16/17) of positive CMI group and 59% (28/44) of negative CMI group became positive for CMI after vaccination (P < 0.05). Ninety-six percent (22/23) of the subjects with a history of vaccination became IgG seropositive after a second dose of vaccination, but 22% (5/23) of them remained negative for CMI. CMI is valuable information to identify potential non-responders to vaccination and to predict risk of clinical VZV infection.

  20. Diversification of the Primary Antibody Repertoire by AID-Mediated Gene Conversion.

    PubMed

    Lanning, Dennis K; Knight, Katherine L

    2015-01-01

    Gene conversion, mediated by activation-induced cytidine deaminase (AID), has been found to contribute to generation of the primary antibody repertoire in several vertebrate species. Generation of the primary antibody repertoire by gene conversion of immunoglobulin (Ig) genes occurs primarily in gut-associated lymphoid tissues (GALT) and is best described in chicken and rabbit. Here, we discuss current knowledge of the mechanism of gene conversion as well as the contribution of the microbiota in promoting gene conversion of Ig genes. Finally, we propose that the antibody diversification strategy used in GALT species, such as chicken and rabbit, is conserved in a subset of human and mouse B cells.

  1. HIV-1 Replication in Langerhans and Interstitial Dendritic Cells Is Inhibited by Neutralizing and Fc-Mediated Inhibitory Antibodies ▿ †

    PubMed Central

    Peressin, M.; Holl, V.; Schmidt, S.; Decoville, T.; Mirisky, D.; Lederle, A.; Delaporte, M.; Xu, K.; Aubertin, A. M.; Moog, C.

    2011-01-01

    Langerhans cells (LCs) and interstitial dendritic cells (IDCs) may be among the first human immunodeficiency virus type 1 (HIV-1) targets after sexual transmission. We generated cells of these types by differentiation of purified CD34+ cord blood cells. After in vitro infection with R5-tropic strains, we obtained similar percentages of infected cells for both dendritic cell (DC) subsets. Moreover, LC infection was not increased by blockage of langerin by antilangerin. These results indicate that, under our experimental conditions, there was no evidence of any preference of HIV replication in LCs versus IDCs. The inhibitory activity of HIV-1-specific IgAs and IgGs against HIV-1 replication in LCs and IDCs was analyzed. We found that neutralizing antibodies inhibit HIV-1 infection of both DC subsets. Interestingly, HIV-1 was inhibited more efficiently by the IgGs than the corresponding IgA, due to an Fcγ receptor-dependent mechanism. Moreover, nonneutralizing inhibitory IgGs were able to inhibit infection of both LCs and IDCs. These results underline the importance of HIV-1 inhibition by the binding of the Fc part of IgGs to Fcγ receptors and suggest that the induction of neutralizing and nonneutralizing inhibitory IgGs in addition to neutralizing IgAs at mucosal sites may contribute to protection against sexual transmission of HIV-1. PMID:21084491

  2. Antibody-dependent cellular cytotoxicity-mediating antibodies from an HIV-1 vaccine efficacy trial target multiple epitopes and preferentially use the VH1 gene family.

    PubMed

    Bonsignori, Mattia; Pollara, Justin; Moody, M Anthony; Alpert, Michael D; Chen, Xi; Hwang, Kwan-Ki; Gilbert, Peter B; Huang, Ying; Gurley, Thaddeus C; Kozink, Daniel M; Marshall, Dawn J; Whitesides, John F; Tsao, Chun-Yen; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Rerks-Ngarm, Supachai; Kim, Jerome H; Michael, Nelson L; Tomaras, Georgia D; Montefiori, David C; Lewis, George K; DeVico, Anthony; Evans, David T; Ferrari, Guido; Liao, Hua-Xin; Haynes, Barton F

    2012-11-01

    The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-dependent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vaccinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment. Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes (n = 19), a non-A32-blockable conformational epitope (n = 1), and the gp120 Env variable loops (n = 3). Fourteen antibodies mediated cross-clade target cell killing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also displayed a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The polyclonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation of these ADCC-mediating responses compared to VH1 bNAbs. PMID:22896626

  3. Oral administration of Aloe vera gel, anti-microbial and anti-inflammatory herbal remedy, stimulates cell-mediated immunity and antibody production in a mouse model

    PubMed Central

    Bałan, Barbara Joanna; Niemcewicz, Marcin; Kocik, Janusz; Jung, Leszek; Skopiński, Piotr

    2014-01-01

    Introduction Aloe vera (L.) Burm. f. (Aloe barbadensis Mill) Liliaceae, succulent plant native to northern Africa, is presently cultivated in many regions of the world. Traditionally, its inner part of parenchyma, which contains aloe gel, was used for the treatment of minor wounds, inflammatory skin disorders, thermal and radiation burns and to alleviate chronic osteoarthritis pain. It also possesses some antimicrobial activity. Now, aloe gel is also increasingly consumed as a dietary supplement. Some data suggest its immunomodulatory properties. The aim of the study The aim of the study was to evaluate the influence of orally administered aloe gel on some parameters of cellular and humoral immunity viz. mitogen-induced proliferation of splenic lymphocytes and their chemokinetic activity, and anti-sheep red blood cells (SRBC) antibody production in Balb/c mice. Results Daily treatment of mice for 14 and 21 days with 50 µl or 150 µl of aloe gel dose resulted in enhanced chemokinetic activity and stronger response of their splenic lymphocytes to mitogen PHA and enhancement of anti-SRBC antibody production. PMID:26155113

  4. Fc-glycosylation influences Fcγ receptor binding and cell-mediated anti-HIV activity of monoclonal antibody 2G12.

    PubMed

    Forthal, Donald N; Gach, Johannes S; Landucci, Gary; Jez, Jakub; Strasser, Richard; Kunert, Renate; Steinkellner, Herta

    2010-12-01

    Interactions between the Fc segment of IgG and FcγRs on a variety of cells are likely to play an important role in the anti-HIV activity of Abs. Because the nature of the glycan structure on the Fc domain is a critical determinant of Fc-FcγR binding, proper Fc glycosylation may contribute to Ab-mediated protection. We have generated five different glycoforms of the broadly HIV-1-neutralizing mAb 2G12 in wild-type and glycoengineered plants and Chinese hamster ovary cells. Plant-derived 2G12 exhibited highly homogeneous glycosylation profiles with a single dominant N-glycan species. Using flow cytometry with FcγR-expressing cell lines, all 2G12 glycoforms demonstrated similar binding to FcγRI, FcγRIIa, and FcγRIIb. In contrast, two glycoforms derived from glycoengineered plants that lack plant-specific xylose and core α1,3-fucose, and instead carry human-like glycosylation with great uniformity, showed significantly enhanced binding to FcγRIIIa compared with Chinese hamster ovary or wild-type plant-derived 2G12. Using surface plasmon resonance, we show that binding of 2G12 to FcγRIIIa is markedly affected by core fucose, irrespective of its plant-specific α1,3 or mammalian-type α1,6 linkage. Consistent with this finding, 2G12 glycoforms lacking core fucose (and xylose) mediated higher antiviral activity against HIV-1 or simian immunodeficiency virus as measured by Ab-dependent cell-mediated virus inhibition. This is, to our knowledge, the first demonstration that specific alterations of Fc glycosylation can improve antiviral activity. Such alterations may result in better immunotherapeutic reagents. Moreover, biasing vaccine-induced immune responses toward optimal Fc glycosylation patterns could result in improved vaccine efficacy. PMID:21041724

  5. Mediation of macrophage cytolytic and phagocytic activities by antibodies of different classes and class-specific Fc-receptors.

    PubMed

    Walker, W S

    1977-08-01

    The classes of antibodies that mediate the phagocytosis and cytolysis of 51Cr-labeled chicken erythrocytes by IC-21 macrophages, an established line of mouse peritoneal macrophages, were identified. The phagocytic activity of IC-21 macrophages, as determined by a functional inhibition assay with mouse myeloma proteins, depended mainly on IgM and IgG2a antibodies and to a lesser extent on IgG2b antibodies. Extracellular cytolysis of target cells was mediated solely by IgG2b antibodies. These results correlate with the previously documented specificities of discrete Fc-receptors for IgG2a and IgG2b immunoglobulins on IC-21 cells. Thus, phagocytosis and cytolysis appear to be mediated by antibodies of different classes operating through separate and distinct sites on the surface of IC-21 macrophages. PMID:886183

  6. Patient-Derived Antibody Targets Tumor Cells

    Cancer.gov

    An NCI Cancer Currents blog on an antibody derived from patients that killed tumor cells in cell lines of several cancer types and slowed tumor growth in mouse models of brain and lung cancer without evidence of side effects.

  7. Effective protein inhibition in intact mouse oocytes through peptide nanoparticle-mediated antibody transfection

    PubMed Central

    Li, Ruichao; Jin, Zhen; Gao, Leilei; Liu, Peng

    2016-01-01

    Female meiosis is a fundamental area of study in reproductive medicine, and the mouse oocyte model of in vitro maturation (IVM) is most widely used to study female meiosis. To investigate the probable role(s) of an unknown protein in female meiosis, the method traditionally used involves microinjecting a specific antibody into mouse oocytes. Recently, in studies on somatic cells, peptide nanoparticle-mediated antibody transfection has become a popular tool because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, untill now no researchers have tried using this technique on mouse oocytes because the zona pellucida surrounding the oocyte membrane (vitelline membrane) is usually thought or proved to be a tough barrier to macromolecules such as antibodies and proteins. Therefore, we attempted to introduce an antibody into mouse oocytes using a peptide nanoparticle. Here we show for the first time that with our optimized method, an antibody can be effectively delivered into mouse oocytes and inhibit its target protein with high specificity. We obtained significant results using small GTPase Arl2 as a test subject protein. We propose peptide nanoparticle-mediated antibody transfection to be a superior alternative to antibody microinjection for preliminary functional studies of unknown proteins in mouse oocytes. PMID:27114861

  8. Vaccine induced antibodies to the first variable loop of human immunodeficiency virus type 1 gp120, mediate antibody-dependent virus inhibition in macaques.

    PubMed

    Bialuk, Izabela; Whitney, Stephen; Andresen, Vibeke; Florese, Ruth H; Nacsa, Janos; Cecchinato, Valentina; Valeri, Valerio W; Heraud, Jean-Michel; Gordon, Shari; Parks, Robyn Washington; Montefiori, David C; Venzon, David; Demberg, Thorsten; Guroff, Marjorie Robert-; Landucci, Gary; Forthal, Donald N; Franchini, Genoveffa

    2011-12-01

    The role of antibodies directed against the hyper variable envelope region V1 of human immunodeficiency virus type 1 (HIV-1), has not been thoroughly studied. We show that a vaccine able to elicit strain-specific non-neutralizing antibodies to this region of gp120 is associated with control of highly pathogenic chimeric SHIV(89.6P) replication in rhesus macaques. The vaccinated animal that had the highest titers of antibodies to the amino terminus portion of V1, prior to challenge, had secondary antibody responses that mediated cell killing by antibody-dependent cellular cytotoxicity (ADCC), as early as 2 weeks after infection and inhibited viral replication by antibody-dependent cell-mediated virus inhibition (ADCVI), by 4 weeks after infection. There was a significant inverse correlation between virus level and binding antibody titers to the envelope protein, (R=-0.83, p=0.015), and ADCVI (R=-0.84 p=0.044). Genotyping of plasma virus demonstrated in vivo selection of three SHIV(89.6P) variants with changes in potential N-linked glycosylation sites in V1. We found a significant inverse correlation between virus levels and titers of antibodies that mediated ADCVI against all the identified V1 virus variants. A significant inverse correlation was also found between neutralizing antibody titers to SHIV(89.6) and virus levels (R=-0.72 p=0.0050). However, passive inoculation of purified immunoglobulin from animal M316, the macaque that best controlled virus, to a naïve macaque, resulted in a low serum neutralizing antibodies and low ADCVI activity that failed to protect from SHIV(89.6P) challenge. Collectively, while our data suggest that anti-envelope antibodies with neutralizing and non-neutralizing Fc(R-dependent activities may be important in the control of SHIV replication, they also demonstrate that low levels of these antibodies alone are not sufficient to protect from infection.

  9. An integrative structure-based framework for predicting biological effects mediated by antipeptide antibodies.

    PubMed

    Caoili, Salvador Eugenio C

    2015-12-01

    A general framework is presented for predicting quantitative biological effects mediated by antipeptide antibodies, primarily on the basis of antigen structure (possibly featuring intrinsic disorder) analyzed to estimate epitope-paratope binding affinities, which in turn is considered within the context of dose-response relationships as regards antibody concentration. This is illustrated mainly using an approach based on protein structural energetics, whereby expected amounts of solvent-accessible surface area buried upon epitope-paratope binding are related to the corresponding binding affinity, which is estimated from putative B-cell epitope structure with implicit treatment of paratope structure, for antipeptide antibodies either reacting with peptides or cross-reacting with cognate protein antigens. Key methods described are implemented in SAPPHIRE/SUITE (Structural-energetic Analysis Program for Predicting Humoral Immune Response Epitopes/SAPPHIRE User Interface Tool Ensemble; publicly accessible via http://freeshell.de/~badong/suite.htm). Representative results thus obtained are compared with published experimental data on binding affinities and quantitative biological effects, with special attention to loss of paratope sidechain conformational entropy (neglected in previous analyses) and in light of key in-vivo constraints on antigen-antibody binding affinity and antibody-mediated effects. Implications for further refinement of B-cell epitope prediction methods are discussed as regards envisioned biomedical applications including the development of prophylactic and therapeutic antibodies, peptide-based vaccines and immunodiagnostics. PMID:26410103

  10. Quantitative model of antibody- and soluble CD4-mediated neutralization of primary isolates and T-cell line-adapted strains of human immunodeficiency virus type 1.

    PubMed Central

    Klasse, P J; Moore, J P

    1996-01-01

    Primary isolates (PI) of human immunodeficiency virus type 1 (HIV-1) are considerably less sensitive than T-cell line-adapted strains to neutralization by soluble CD4 and by most cross-reactive monoclonal antibodies to the viral envelope (Env) glycoprotein, as well as by postinfection and postvaccination sera (J. P. Moore and D. D. Ho, AIDS 9 [suppl. A]:5117-5136, 1995). We developed a quantitative model to explain the neutralization resistance of PI. The factors incorporated into the model are the dissociation constants for the binding of the neutralizing agent to native Env oligomers, the number of outer Env molecules on the viral surface (which decreases by shedding), and the minimum number of Env molecules required for attachment and fusion. We conclude that modest differences in all these factors can, when combined, explain a relative neutralization resistance of PI versus T-cell line-adapted strains that sometimes amounts to several orders of magnitude. The hypothesis that neutralization of HIV is due to the reduction below a minimum number of the Env molecules on a virion available for attachment and fusion is at odds with single- and few-hit neutralization theories. Our analysis of these ideas favors the hypothesis that neutralization of HIV is instead a competitive blocking of interactions with cellular factors, including adsorption receptors. PMID:8648701

  11. Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on antibody-mediated immune response and gene expression of pattern recognition receptors and cytokines in broiler chickens immunized with T-cell dependent antigens.

    PubMed

    Alizadeh, M; Rodriguez-Lecompte, J C; Echeverry, H; Crow, G H; Slominski, B A

    2016-04-01

    This study evaluated the effect of yeast-derived products on innate and antibody mediated immune response in broiler chickens following immunization with sheep red blood cells (SRBC) and bovine serum albumin (BSA). One-day-old male broiler chickens (Ross-308) were randomly assigned to 6 dietary treatments of 9 replicate cages of 5 birds each per treatment. Dietary treatments consisted of a Control diet without antibiotic, and diets containing 11 mg/kg of virginiamycin, 0.25% of yeast cell wall (YCW), 0.2% of a commercial product Maxi-Gen Plus containing processed yeast and nucleotides, 0.05% of nucleotides, or a diet containing 10% of DDGS. On days 21 and 28 post-hatching, 5 birds per treatment were immunized intramuscularly with both SRBC and BSA. One week after each immunization, blood samples were collected. Serum samples were analyzed by hemagglutination test for antibody response to SRBC, and by ELISA for serum IgM and IgG response to BSA. On d 35, 5 birds per treatment were euthanized and the tissue samples from the cecal tonsils were collected to assess the gene expression of toll-like receptors TLR2b, TLR4, and TLR21, monocyte mannose receptor (MMR), and cytokines IL-10, IL-13, IL-4, IL-12p35, and IFN-γ. The results for gene expression analysis demonstrated that the diet supplemented with YCW increased the expression of TLR2b and T-helper type 2 cytokines IL-10, IL-4, and IL-13 relative to the Control; and the expression of TLR4 and IL-13 was upregulated in the nucleotide-containing diet. However, the diets containing antibiotics or Maxi-Gen Plus downregulated the expression of IFN-γ compared to the control. The primary antibody response to SRBC was not affected by diets. However, the diet containing YCW increased the secondary antibody response to SRBC compared to the antibiotic treatment. Neither primary nor secondary IgG and IgM response against BSA were affected by diets. In conclusion, supplementation of the diet with YCW stimulated Th2 cell-mediated

  12. B cell Rab7 mediates induction of activation-induced cytidine deaminase expression and class-switching in T-dependent and T-independent antibody responses.

    PubMed

    Pone, Egest J; Lam, Tonika; Lou, Zheng; Wang, Rui; Chen, Yuhui; Liu, Dongfang; Edinger, Aimee L; Xu, Zhenming; Casali, Paolo

    2015-04-01

    Class switch DNA recombination (CSR) is central to the maturation of the Ab response because it diversifies Ab effector functions. Like somatic hypermutation, CSR requires activation-induced cytidine deaminase (AID), whose expression is restricted to B cells, as induced by CD40 engagement or dual TLR-BCR engagement (primary CSR-inducing stimuli). By constructing conditional knockout Igh(+/C)γ(1-cre)Rab7(fl/fl) mice, we identified a B cell-intrinsic role for Rab7, a small GTPase involved in intracellular membrane functions, in mediating AID induction and CSR. Igh(+/C)γ(1-cre)Rab7(fl/fl) mice displayed normal B and T cell development and were deficient in Rab7 only in B cells undergoing Igh(C)γ(1-cre) Iγ1-Sγ1-Cγ1-cre transcription, as induced--like Igh germline Iγ1-Sγ1-Cγ1 and Iε-Sε-Cε transcription--by IL-4 in conjunction with a primary CSR-inducing stimulus. These mice could not mount T-independent or T-dependent class-switched IgG1 or IgE responses while maintaining normal IgM levels. Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells showed, in vivo and in vitro, normal proliferation and survival, normal Blimp-1 expression and plasma cell differentiation, as well as intact activation of the noncanonical NF-κB, p38 kinase, and ERK1/2 kinase pathways. They, however, were defective in AID expression and CSR in vivo and in vitro, as induced by CD40 engagement or dual TLR1/2-, TLR4-, TLR7-, or TLR9-BCR engagement. In Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells, CSR was rescued by enforced AID expression. These findings, together with our demonstration that Rab7-mediated canonical NF-κB activation, as critical to AID induction, outline a novel role of Rab7 in signaling pathways that lead to AID expression and CSR, likely by promoting assembly of signaling complexes along intracellular membranes.

  13. Interactions between natural killer cells and antibody Fc result in enhanced antibody neutralization of human immunodeficiency virus type 1.

    PubMed

    Forthal, Donald N; Landucci, Gary; Phan, Tran B; Becerra, Juan

    2005-02-01

    Antibodies can prevent lentivirus infections in animals and may play a role in controlling viral burden in established infection. In preventing and particularly in controlling infection, antibodies likely function in the presence of large quantities of virus. In this study, we explored the mechanisms by which antibodies neutralize large inocula of human immunodeficiency virus type 1 (HIV-1) on different target cells. Immunoglobulin G (IgG) from HIV-infected patients was tested for neutralizing activity against primary R5 strains of HIV-1 at inocula ranging from 100 to 20,000 50% tissue culture infective doses. At all virus inocula, inhibition by antibody was enhanced when target cells for virus growth were monocyte-depleted, peripheral blood mononuclear cells (PBMCs) rather than CD4(+) lymphocytes. However, enhanced inhibition on PBMCs was greatest with larger amounts of virus. Depleting PBMCs of natural killer (NK) cells, which express Fc receptors for IgG (FcgammaRs), abrogated the enhanced antibody inhibition, whereas adding NK cells to CD4(+) lymphocytes restored inhibition. There was no enhanced inhibition on PBMCs when F(ab')(2) was used. Further experiments demonstrated that the release of beta-chemokines, most likely through FcgammaR triggering of NK cells, contributed modestly to the antiviral activity of antibody on PBMCs and that antibody-coated virus adsorbed to uninfected cells provided a target for NK cell-mediated inhibition of HIV-1. These results indicate that Fc-FcgammaR interactions enhance the ability of antibody to neutralize HIV-1. Since FcgammaR-bearing cells are always present in vivo, FcgammaR-mediated antibody function may play a role in the ability of antibody to control lentivirus infection.

  14. Interactions between Natural Killer Cells and Antibody Fc Result in Enhanced Antibody Neutralization of Human Immunodeficiency Virus Type 1

    PubMed Central

    Forthal, Donald N.; Landucci, Gary; Phan, Tran B.; Becerra, Juan

    2005-01-01

    Antibodies can prevent lentivirus infections in animals and may play a role in controlling viral burden in established infection. In preventing and particularly in controlling infection, antibodies likely function in the presence of large quantities of virus. In this study, we explored the mechanisms by which antibodies neutralize large inocula of human immunodeficiency virus type 1 (HIV-1) on different target cells. Immunoglobulin G (IgG) from HIV-infected patients was tested for neutralizing activity against primary R5 strains of HIV-1 at inocula ranging from 100 to 20,000 50% tissue culture infective doses. At all virus inocula, inhibition by antibody was enhanced when target cells for virus growth were monocyte-depleted, peripheral blood mononuclear cells (PBMCs) rather than CD4+ lymphocytes. However, enhanced inhibition on PBMCs was greatest with larger amounts of virus. Depleting PBMCs of natural killer (NK) cells, which express Fc receptors for IgG (FcγRs), abrogated the enhanced antibody inhibition, whereas adding NK cells to CD4+ lymphocytes restored inhibition. There was no enhanced inhibition on PBMCs when F(ab′)2 was used. Further experiments demonstrated that the release of β-chemokines, most likely through FcγR triggering of NK cells, contributed modestly to the antiviral activity of antibody on PBMCs and that antibody-coated virus adsorbed to uninfected cells provided a target for NK cell-mediated inhibition of HIV-1. These results indicate that Fc-FcγR interactions enhance the ability of antibody to neutralize HIV-1. Since FcγR-bearing cells are always present in vivo, FcγR-mediated antibody function may play a role in the ability of antibody to control lentivirus infection. PMID:15681406

  15. How antibodies alter the cell entry pathway of dengue virus particles in macrophages

    PubMed Central

    Ayala-Nunez, Nilda V.; Hoornweg, Tabitha E.; van de Pol, Denise P.I.; Sjollema, Klaas A.; Flipse, Jacky; van der Schaar, Hilde M.; Smit, Jolanda M.

    2016-01-01

    Antibody-dependent enhancement of dengue virus (DENV) infection plays an important role in the exacerbation of DENV-induced disease. To understand how antibodies influence the fate of DENV particles, we explored the cell entry pathway of DENV in the absence and presence of antibodies in macrophage-like P388D1 cells. Recent studies unraveled that both mature and immature DENV particles contribute to ADE, hence, both particles were studied. We observed that antibody-opsonized DENV enters P388D1 cells through a different pathway than non-opsonized DENV. Antibody-mediated DENV entry was dependent on FcγRs, pH, Eps15, dynamin, actin, PI3K, Rab5, and Rab7. In the absence of antibodies, DENV cell entry was FcγR, PI3K, and Rab5-independent. Live-cell imaging of fluorescently-labeled particles revealed that actin-mediated membrane protrusions facilitate virus uptake. In fact, actin protrusions were found to actively search and capture antibody-bound virus particles distantly located from the cell body, a phenomenon that is not observed in the absence of antibodies. Overall, similar results were seen for antibody-opsonized standard and antibody-bound immature DENV preparations, indicating that the maturation status of the virus does not control the entry pathway. Collectively, our findings suggest that antibodies alter the cell entry pathway of DENV and trigger a novel mechanism of initial virus-cell contact. PMID:27385443

  16. The antibody approach of labeling blood cells

    SciTech Connect

    Srivastava, S.C.

    1991-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  17. The antibody approach of labeling blood cells

    SciTech Connect

    Srivastava, S.C.

    1991-01-01

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  18. The antibody approach of labeling blood cells

    SciTech Connect

    Srivastava, S.C.

    1992-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are critically assessed and evaluated.

  19. Antibody-Mediated Rejection of Single Class I MHC-Disparate Cardiac Allografts

    PubMed Central

    Hattori, Yusuke; Bucy, R. Pat; Kubota, Yoshinobu; Baldwin, William M.; Fairchild, Robert L.

    2012-01-01

    Murine CCR5−/− recipients produce high titers of antibody to complete MHC-mismatched heart and renal allografts. To study mechanisms of class I MHC antibody-mediated allograft injury, we tested the rejection of heart allografts transgenically expressing a single class I MHC disparity in wild-type C57BL/6 (H-2b) and B6.CCR5−/− recipients. Donor-specific antibody titers in CCR5−/− recipients were 30-fold higher than in wild-type recipients. B6.Kd allografts survived longer than 60 days in wild-type recipients whereas CCR5−/− recipients rejected all allografts within 14 days. Rejection was accompanied by infiltration of CD8 T cells, neutrophils, and macrophages and C4d deposition in the graft capillaries. B6.Kd allografts were rejected by CD8−/−/CCR5−/−, but not μMT−/−/CCR5−/−, recipients indicating the need for antibody but not CD8 T cells. Grafts retrieved at day 10 from CCR5−/− and CD8−/−/CCR5−/− recipients and from RAG-1−/− allograft recipients injected with anti-Kd antibodies expressed high levels of perforin, myeloperoxidase and CCL5 mRNA. These studies indicate that the continual production of anti-donor class I MHC antibody can mediate allograft rejection, that donor-reactive CD8 T cells synergize with the antibody to contribute to rejection, and that expression of three biomarkers during rejection can occur in the absence of this CD8 T cell activity. PMID:22578247

  20. HIV-specific CD4-induced Antibodies Mediate Broad and Potent Antibody-dependent Cellular Cytotoxicity Activity and are Commonly Detected in Plasma from HIV-infected Humans

    PubMed Central

    Williams, Katherine L.; Cortez, Valerie; Dingens, Adam S.; Gach, Johannes S.; Rainwater, Stephanie; Weis, Julie F.; Chen, Xuemin; Spearman, Paul; Forthal, Donald N.; Overbaugh, Julie

    2015-01-01

    HIV-specific antibodies (Abs) can reduce viral burden by blocking new rounds of infection or by destroying infected cells via activation of effector cells through Fc–FcR interaction. This latter process, referred to as antibody-dependent cellular cytotoxicity (ADCC), has been associated with viral control and improved clinical outcome following both HIV and SIV infections. Here we describe an HIV viral-like particle (VLP)-based sorting strategy that led to identification of HIV-specific memory B cells encoding Abs that mediate ADCC from a subtype A-infected Kenyan woman at 914 days post-infection. Using this strategy, 12 HIV-envelope-specific monoclonal antibodies (mAbs) were isolated and three mediated potent ADCC activity when compared to well-characterized ADCC mAbs. The ADCC-mediating Abs also mediated antibody-dependent cell-mediated virus inhibition (ADCVI), which provides a net measure of Fc receptor-triggered effects against replicating virus. Two of the three ADCC-mediating Abs targeted a CD4-induced (CD4i) epitope also bound by the mAb C11; the third antibody targeted the N-terminus of V3. Both CD4i Abs identified here demonstrated strong cross-clade breadth with activity against 10 of 11 envelopes tested, including those from clades A, B, C, A/D and C/D, whereas the V3-specific antibody showed more limited breadth. Variants of these CD4i, C11-like mAbs engineered to interrupt binding to FcγRs inhibited a measurable percentage of the donor's ADCC activity starting as early as 189 days post-infection. C11-like antibodies also accounted for between 18–78% of ADCC activity in 9 chronically infected individuals from the same cohort study. Further, the two CD4i Abs originated from unique B cells, suggesting that antibodies targeting this epitope can be commonly produced. Taken together, these data provide strong evidence that CD4i, C11-like antibodies develop within the first 6 months of infection and they can arise from unique B-cell lineages in the

  1. Novel antimalarial antibodies highlight the importance of the antibody Fc region in mediating protection.

    PubMed

    Pleass, Richard J; Ogun, Solabomi A; McGuinness, David H; van de Winkel, Jan G J; Holder, Anthony A; Woof, Jenny M

    2003-12-15

    Parasite drug resistance and difficulties in developing effective vaccines have precipitated the search for alternative therapies for malaria. The success of passive immunization suggests that immunoglobulin (Ig)-based therapies are effective. To further explore the mechanism(s) by which antibody mediates its protective effect, we generated human chimeric IgG1 and IgA1 and a single-chain diabody specific for the C-terminal 19-kDa region of Plasmodium yoelii merozoite surface protein 1 (MSP119), a major target of protective immune responses. These novel human reagents triggered in vitro phagocytosis of merozoites but, unlike their parental mouse IgG2b, failed to protect against parasite challenge in vivo. Therefore, the Fc region appears critical for mediating protection in vivo, at least for this MSP119 epitope. Such antibodies may serve as prototype therapeutic agents, and as useful tools in the development of in vitro neutralization assays with Plasmodium parasites.

  2. Broadly neutralizing antibodies that inhibit HIV-1 cell to cell transmission

    PubMed Central

    Malbec, Marine; Porrot, Françoise; Rua, Rejane; Horwitz, Joshua; Klein, Florian; Halper-Stromberg, Ari; Scheid, Johannes F.; Eden, Caroline; Mouquet, Hugo; Nussenzweig, Michel C.

    2013-01-01

    The neutralizing activity of anti–HIV-1 antibodies is typically measured in assays where cell-free virions enter reporter cell lines. However, HIV-1 cell to cell transmission is a major mechanism of viral spread, and the effect of the recently described broadly neutralizing antibodies (bNAbs) on this mode of transmission remains unknown. Here we identify a subset of bNAbs that inhibit both cell-free and cell-mediated infection in primary CD4+ lymphocytes. These antibodies target either the CD4-binding site (NIH45-46 and 3BNC60) or the glycan/V3 loop (10-1074 and PGT121) on HIV-1 gp120 and act at low concentrations by inhibiting multiple steps of viral cell to cell transmission. These antibodies accumulate at virological synapses and impair the clustering and fusion of infected and target cells and the transfer of viral material to uninfected T cells. In addition, they block viral cell to cell transmission to plasmacytoid DCs and thereby interfere with type-I IFN production. Thus, only a subset of bNAbs can efficiently prevent HIV-1 cell to cell transmission, and this property should be considered an important characteristic defining antibody potency for therapeutic or prophylactic antiviral strategies. PMID:24277152

  3. Monoclonal antibodies specific for sickle cell hemoglobin

    SciTech Connect

    Jensen, R.H.; Vanderlaan, M.; Grabske, R.J.; Branscomb, E.W.; Bigbee, W.L.; Stanker, L.H.

    1985-01-01

    Two mouse hybridoma cell lines were isolated which produce monoclonal antibodies that bind hemoglobin S. The mice were immunized with peptide-protein conjugates to stimulate a response to the amino terminal peptide of the beta chain of hemoglobin S, where the single amino acid difference between A and S occurs. Immunocharacterization of the antibodies shows that they bind specifically to the immunogen peptide and to hemoglobin S. The specificity for S is high enough that one AS cell in a mixture with a million AA cells is labeled by antibody, and such cells can be analyzed by flow cytometry. Immunoblotting of electrophoretic gels allows definitive identification of hemoglobin S as compared with other hemoglobins with similar electrophoretic mobility. 12 references, 4 figures.

  4. Role of group 3 innate lymphoid cells in antibody production

    PubMed Central

    Magri, Giuliana; Cerutti, Andrea

    2015-01-01

    Innate lymphoid cells (ILCs) constitute a heterogeneous family of effector lymphocytes of the innate immune system that mediate lymphoid organogenesis, tissue repair, immunity and inflammation. The initial view that ILCs exert their protective functions solely during the innate phase of an immune response has been recently challenged by evidence indicating that ILCs shape adaptive immunity by establishing both contact-dependent and contact-independent interactions with multiple hematopoietic and non-hematopoietic cells, including B cells. Some of these interactions enhance antibody responses both systemically and at mucosal sites of entry. PMID:25621842

  5. Antibody-mediated immunity in CFW mice infected with Mycobacterium lepraemurium. Humoral immune response in murine leprosy.

    PubMed Central

    Rojas-Espinosa, O; Casoluengo-Méndez, M; Díaz, G V

    1976-01-01

    A depression in antibody-mediated immunity (AMI) measured both in terms of circulating antibody and plaque-forming cells in the spleen was observed in CFW mice infected with M. lepraemurium when sheep red blood cells (SRBC) and human gammaglobulin (HGG) were used as antigens. The impairment in AMI was evident only after 75 days of infection thereafter the antibody response to SRBC antigen progressively decreased until the last day of experimentation (135 days). Within the first 60 days of infection no alteration in AMI was observed with the HGG antigen while the response to the SRBC antigen was significantly higher in the infected animals than in uninfected controls. PMID:795574

  6. Fully Human Antagonistic Antibodies against CCR4 Potently Inhibit Cell Signaling and Chemotaxis

    PubMed Central

    Géraudie, Solène; Scheffler, Ulrike; Griep, Remko A.; Reiersen, Herald; Duncan, Alexander R.; Kiprijanov, Sergej M.

    2014-01-01

    Background CC chemokine receptor 4 (CCR4) represents a potentially important target for cancer immunotherapy due to its expression on tumor infiltrating immune cells including regulatory T cells (Tregs) and on tumor cells in several cancer types and its role in metastasis. Methodology Using phage display, human antibody library, affinity maturation and a cell-based antibody selection strategy, the antibody variants against human CCR4 were generated. These antibodies effectively competed with ligand binding, were able to block ligand-induced signaling and cell migration, and demonstrated efficient killing of CCR4-positive tumor cells via ADCC and phagocytosis. In a mouse model of human T-cell lymphoma, significant survival benefit was demonstrated for animals treated with the newly selected anti-CCR4 antibodies. Significance For the first time, successful generation of anti- G-protein coupled chemokine receptor (GPCR) antibodies using human non-immune library and phage display on GPCR-expressing cells was demonstrated. The generated anti-CCR4 antibodies possess a dual mode of action (inhibition of ligand-induced signaling and antibody-directed tumor cell killing). The data demonstrate that the anti-tumor activity in vivo is mediated, at least in part, through Fc-receptor dependent effector mechanisms, such as ADCC and phagocytosis. Anti-CC chemokine receptor 4 antibodies inhibiting receptor signaling have potential as immunomodulatory antibodies for cancer. PMID:25080123

  7. The influence of the swine major histocompatibility genes on antibody and cell-mediated immune responses to immunization with an aromatic-dependent mutant of Salmonella typhimurium.

    PubMed Central

    Lumsden, J S; Kennedy, B W; Mallard, B A; Wilkie, B N

    1993-01-01

    Eighty-two major histocompatibility complex (MHC) swine leukocyte antigen (SLA) defined miniature pigs from 16 litters were examined for serum agglutinating antibody titer and O-polysaccharide (O-ps) specific peripheral blood lymphocyte blastogenesis following two parenteral vaccinations with 1 x 10(8) aromatic-dependent (aroA) Salmonella typhimurium and following oral challenge with 1 x 10(12) virulent parent S. typhimurium. Least mean squares analysis allowed separate determinations of the effects of MHC genotype, dam, sire and litter. In most cases only litter significantly influenced both lymphocyte blastogenesis and antibody titer before and after vaccination and following challenge. However, pig SLA haplotype significantly influenced the degree of O-ps specific lymphocyte proliferation six days after the second vaccination (p < 0.004). Lymphocyte proliferation and serum agglutinating antibody response six days after primary vaccination were negatively correlated (r2 = -0.68, p < 0.001). In most cases, "dd" and "gg" homozygous and "dg" heterozygous pigs, having the same MHC class II region, behaved immunologically as a group distinct from the other genotypes. PMID:8431799

  8. Induction of CD4 suppressor T cells with anti-Leu-8 antibody

    SciTech Connect

    Kanof, M.E.; Strober, W.; James, S.P.

    1987-07-01

    To characterize the conditions under which CD4 T cells suppress polyclonal immunoglobulin synthesis, we investigated the capacity of CD4 T cells that coexpress the surface antigen recognized by the monoclonal antibody anti-Leu-8 to mediate suppression. In an in vitro system devoid of CD8 T cells, CD4, Leu-8+ T cells suppressed pokeweed mitogen-induced immunoglobulin synthesis. Similarly, suppressor function was induced in unfractionated CD4 T cell populations after incubation with anti-Leu-8 antibody under cross-linking conditions. This induction of suppressor function by anti-Leu-8 antibody was not due to expansion of the CD4, Leu-8+ T cell population because CD4 T cells did not proliferate in response to anti-Leu-8 antibody. However, CD4, Leu-8+ T cell-mediated suppression was radiosensitive. Finally, CD4, Leu-8+ T cells do not inhibit immunoglobulin synthesis when T cell lymphokines were used in place of helper CD4 T cells (CD4, Leu-8- T cells), suggesting that CD4 T cell-mediated suppression occurs at the T cell level. We conclude that CD4 T cells can be induced to suppress immunoglobulin synthesis by modulation of the membrane antigen recognized by anti-Leu-8 antibody.

  9. Immunological effects of the anti-programmed death-1 antibody on human peripheral blood mononuclear cells.

    PubMed

    Akiyama, Yasuto; Nonomura, Chizu; Kondou, Ryota; Miyata, Haruo; Ashizawa, Tadashi; Maeda, Chie; Mitsuya, Koichi; Hayashi, Nakamasa; Nakasu, Yoko; Yamaguchi, Ken

    2016-09-01

    Immune checkpoint antibody-mediated blockade has gained attention as a new cancer immunotherapy strategy. Accumulating evidence suggests that this therapy imparts a survival benefit to metastatic melanoma and non-small cell lung cancer patients. A substantial amount of data on immune checkpoint antibodies has been collected from clinical trials; however, the direct effect of the antibodies on human peripheral blood mononuclear cells (PBMCs) has not been exclusively investigated. In this study, we developed an anti-programmed death-1 (PD-1) antibody (with biosimilarity to nivolumab) and examined the effects of the antibody on PBMCs derived from cancer patients. Specifically, we investigated the effects of the anti-PD-1 antibody on proliferation, cytokine production, cytotoxic T lymphocytes (CTL) and regulatory T cells. These investigations yielded several important results. First, the anti-PD-1 antibody had no obvious effect on resting PBMCs; however, high levels of the anti-PD-1 antibody partly stimulated PBMC proliferation when accompanied by an anti-CD3 antibody. Second, the anti-PD-1 antibody restored the growth inhibition of anti-CD3 Ab-stimulated PBMCs mediated by PD-L1. Third, the anti-PD-1 antibody exhibited a moderate inhibitory effect on the induction of myeloid-derived suppressor cells (MDSCs) by anti-CD3 antibody stimulation. Additionally, the presence of the anti-PD-1 antibody promoted antigen-specific CTL induction, which suggests that combining anti-PD-1 antibody and conventional immunotherapy treatments may have beneficial effects. These results indicate that specific cellular immunological mechanisms are partly responsible for the antitumor effect exhibited by the anti-PD-1 antibody against advanced cancers in clinical trials. PMID:27573705

  10. Antibody-directed complement-mediated cytotoxicity to hepatocytes from patients with chronic hepatitis B.

    PubMed Central

    Michalak, T I; Lau, J Y; McFarlane, B M; Alexander, G J; Eddleston, A L; Williams, R

    1995-01-01

    The susceptibility of hepatocytes from patients with chronic hepatitis B to complement-dependent cytotoxicity mediated by heterologous antibodies to hepatitis B virus core (anti-HBc) and surface (anti-HBs) antigens and to hepatic asialoglycoprotein receptor was examined using a microcytotoxicity assay. The anti-HBc-induced cytotoxicity was found to be markedly enhanced against hepatocytes isolated from patients with chronic active hepatitis (72.6 +/- 9.5% (mean +/- s.e.m.); n = 6) over that against hepatocytes from individuals with chronic persistent hepatitis or inactive liver cirrhosis (40.6 +/- 18.6%; n = 4) (P = 0.019). Overall, values of the anti-HBc-directed cytotoxicity were higher in patients positive for HBcAg in hepatocytes and seropositive for hepatitis B virus e antigen (HBeAg). Hepatocytotoxicity was also exerted by anti-HBs and anti-asialoglycoprotein receptor antibodies in the presence of complement, but it was not seemingly related to disease activity. These results indicate that hepatitis B virus core and surface antigens and asialoglycoprotein receptor at the hepatocyte surface can be recognized by antibodies, and raise the possibility that complement-dependent cytolysis may contribute to the injury of hepatitis B virus-infected hepatocytes. The data also suggest that liver cells of patients with severe chronic hepatitis might be more susceptible to anti-HBc antibody-directed complement-mediated cytotoxicity than those with inactive liver histology. PMID:7743660

  11. Targeting breast cancer stem cells with HER2-specific antibodies and natural killer cells.

    PubMed

    Diessner, Joachim; Bruttel, Valentin; Becker, Kathrin; Pawlik, Miriam; Stein, Roland; Häusler, Sebastian; Dietl, Johannes; Wischhusen, Jörg; Hönig, Arnd

    2013-01-01

    Breast cancer is the most common cancer among women worldwide. Every year, nearly 1.4 million new cases of breast cancer are diagnosed, and about 450.000 women die of the disease. Approximately 15-25% of breast cancer cases exhibit increased quantities of the trans-membrane receptor tyrosine kinase human epidermal growth factor receptor 2 (HER2) on the tumor cell surface. Previous studies showed that blockade of this HER2 proto-oncogene with the antibody trastuzumab substantially improved the overall survival of patients with this aggressive type of breast cancer. Recruitment of natural killer (NK) cells and subsequent induction of antibody-dependent cell-mediated cytotoxicity (ADCC) contributed to this beneficial effect. We hypothesized that antibody binding to HER2-positive breast cancer cells and thus ADCC might be further improved by synergistically applying two different HER2-specific antibodies, trastuzumab and pertuzumab. We found that tumor cell killing via ADCC was increased when the combination of trastuzumab, pertuzumab, and NK cells was applied to HER2-positive breast cancer cells, as compared to the extent of ADCC induced by a single antibody. Furthermore, a subset of CD44(high)CD24(low)HER2(low) cells, which possessed characteristics of cancer stem cells, could be targeted more efficiently by the combination of two HER2-specific antibodies compared to the efficiency of one antibody. These in vitro results demonstrated the immunotherapeutic benefit achieved by the combined application of trastuzumab and pertuzumab. These findings are consistent with the positive results of the clinical studies, CLEOPATRA and NEOSPHERE, conducted with patients that had HER2-positive breast cancer. Compared to a single antibody treatment, the combined application of trastuzumab and pertuzumab showed a stronger ADCC effect and improved the targeting of breast cancer stem cells.

  12. Targeting breast cancer stem cells with HER2-specific antibodies and natural killer cells

    PubMed Central

    Diessner, Joachim; Bruttel, Valentin; Becker, Kathrin; Pawlik, Miriam; Stein, Roland; Häusler, Sebastian; Dietl, Johannes; Wischhusen, Jörg; Hönig, Arnd

    2013-01-01

    Breast cancer is the most common cancer among women worldwide. Every year, nearly 1.4 million new cases of breast cancer are diagnosed, and about 450.000 women die of the disease. Approximately 15-25% of breast cancer cases exhibit increased quantities of the trans-membrane receptor tyrosine kinase human epidermal growth factor receptor 2 (HER2) on the tumor cell surface. Previous studies showed that blockade of this HER2 proto-oncogene with the antibody trastuzumab substantially improved the overall survival of patients with this aggressive type of breast cancer. Recruitment of natural killer (NK) cells and subsequent induction of antibody-dependent cell-mediated cytotoxicity (ADCC) contributed to this beneficial effect. We hypothesized that antibody binding to HER2-positive breast cancer cells and thus ADCC might be further improved by synergistically applying two different HER2-specific antibodies, trastuzumab and pertuzumab. We found that tumor cell killing via ADCC was increased when the combination of trastuzumab, pertuzumab, and NK cells was applied to HER2-positive breast cancer cells, as compared to the extent of ADCC induced by a single antibody. Furthermore, a subset of CD44highCD24lowHER2low cells, which possessed characteristics of cancer stem cells, could be targeted more efficiently by the combination of two HER2-specific antibodies compared to the efficiency of one antibody. These in vitro results demonstrated the immunotherapeutic benefit achieved by the combined application of trastuzumab and pertuzumab. These findings are consistent with the positive results of the clinical studies, CLEOPATRA and NEOSPHERE, conducted with patients that had HER2-positive breast cancer. Compared to a single antibody treatment, the combined application of trastuzumab and pertuzumab showed a stronger ADCC effect and improved the targeting of breast cancer stem cells. PMID:23593542

  13. Antibody-Dependent Cellular Cytotoxicity against Reactivated HIV-1-Infected Cells

    PubMed Central

    Lee, Wen Shi; Richard, Jonathan; Lichtfuss, Marit; Smith, Amos B.; Park, Jongwoo; Courter, Joel R.; Melillo, Bruno N.; Sodroski, Joseph G.; Kaufmann, Daniel E.; Parsons, Matthew S.

    2015-01-01

    ABSTRACT Lifelong antiretroviral therapy (ART) for HIV-1 does not diminish the established latent reservoir. A possible cure approach is to reactivate the quiescent genome from latency and utilize immune responses to eliminate cells harboring reactivated HIV-1. It is not known whether antibodies within HIV-1-infected individuals can recognize and eliminate cells reactivated from latency through antibody-dependent cellular cytotoxicity (ADCC). We found that reactivation of HIV-1 expression in the latently infected ACH-2 cell line elicited antibody-mediated NK cell activation but did not result in antibody-mediated killing. The lack of CD4 expression on these HIV-1 envelope (Env)-expressing cells likely resulted in poor recognition of CD4-induced antibody epitopes on Env. To examine this further, cultured primary CD4+ T cells from HIV-1+ subjects were used as targets for ADCC. These ex vivo-expanded primary cells were modestly susceptible to ADCC mediated by autologous or heterologous HIV-1+ serum antibodies. Importantly, ADCC mediated against these primary cells could be enhanced following incubation with a CD4-mimetic compound (JP-III-48) that exposes CD4-induced antibody epitopes on Env. Our studies suggest that with sufficient reactivation and expression of appropriate Env epitopes, primary HIV-1-infected cells can be targets for ADCC mediated by autologous serum antibodies and innate effector cells. The results of this study suggest that further investigation into the potential of ADCC to eliminate reactivated latently infected cells is warranted. IMPORTANCE An HIV-1 cure remains elusive due to the persistence of long-lived latently infected cells. An HIV-1 cure strategy, termed “shock and kill,” aims to reactivate HIV-1 expression in latently infected cells and subsequently eliminate the reactivated cells through immune-mediated killing. While recent research efforts have focused on reversing HIV-1 latency, it remains unclear whether preexisting immune

  14. Molecular basis of antibody-mediated neutralization and protection against flavivirus.

    PubMed

    Dai, Lianpan; Wang, Qihui; Qi, Jianxun; Shi, Yi; Yan, Jinghua; Gao, George F

    2016-10-01

    Antibody-mediated humoral immunity plays a pivotal role in flavivirus control. Neutralizing antibodies targeting viral envelope (E) protein, provide protection against flaviviruses in vivo but can also promote virus infection by antibody-dependent enhancement when antibodies are weakly neutralizing or in subneutralizing concentrations. The molecular basis for antibody-mediated virus neutralization can be revealed by structural studies of monoclonal antibodies complexed with the E protein or virion. In addition, the flavivirus non-structural protein NS1 can also induce host antibody production, and some of these antibodies can provide protection against virus challenge. In this review, we summarize the known structures of flavivirus neutralizing or protective antibodies bound to their epitopes and describe the underlying molecular mechanisms. © 2016 IUBMB Life, 68(10):783-791, 2016.

  15. Molecular basis of antibody-mediated neutralization and protection against flavivirus.

    PubMed

    Dai, Lianpan; Wang, Qihui; Qi, Jianxun; Shi, Yi; Yan, Jinghua; Gao, George F

    2016-10-01

    Antibody-mediated humoral immunity plays a pivotal role in flavivirus control. Neutralizing antibodies targeting viral envelope (E) protein, provide protection against flaviviruses in vivo but can also promote virus infection by antibody-dependent enhancement when antibodies are weakly neutralizing or in subneutralizing concentrations. The molecular basis for antibody-mediated virus neutralization can be revealed by structural studies of monoclonal antibodies complexed with the E protein or virion. In addition, the flavivirus non-structural protein NS1 can also induce host antibody production, and some of these antibodies can provide protection against virus challenge. In this review, we summarize the known structures of flavivirus neutralizing or protective antibodies bound to their epitopes and describe the underlying molecular mechanisms. © 2016 IUBMB Life, 68(10):783-791, 2016. PMID:27604155

  16. Magnetic antibody-linked nanomatchmakers for therapeutic cell targeting.

    PubMed

    Cheng, Ke; Shen, Deliang; Hensley, M Taylor; Middleton, Ryan; Sun, Baiming; Liu, Weixin; De Couto, Geoffrey; Marbán, Eduardo

    2014-01-01

    Stem cell transplantation is a promising strategy for therapeutic cardiac regeneration, but current therapies are limited by inefficient interaction between potentially beneficial cells (either exogenously transplanted or endogenously recruited) and the injured tissue. Here we apply targeted nanomedicine to achieve in vivo cell-mediated tissue repair, imaging and localized enrichment without cellular transplantation. Iron nanoparticles are conjugated with two types of antibodies (one against antigens on therapeutic cells and the other directed at injured cells) to produce magnetic bifunctional cell engager (MagBICE). The antibodies link the therapeutic cells to the injured cells, whereas the iron core of MagBICE enables physical enrichment and imaging. We treat acute myocardial infarction by targeting exogenous bone marrow-derived stem cells (expressing CD45) or endogenous CD34-positive cells to injured cardiomyocytes (expressing myosin light chain. Targeting can be further enhanced by magnetic attraction, leading to augmented functional benefits. MagBICE represents a generalizable platform technology for regenerative medicine. PMID:25205020

  17. Diagnostic criteria of antibody-mediated rejection in kidney transplants.

    PubMed

    Mosquera Reboredo, J M; Vázquez Martul, E

    2011-01-01

    The diagnosis and treatment of anti-donor antibody-mediated rejection or humoral rejection (ABMR) is one of the main discussions at the moment in kidney transplantation. The search for histopathological markers that help us to diagnose ABMR has been more problematic, in contrast to the histological expression of cellular or tubulointerstitial rejection. Although the relationship between post-transplant anti-donor antibodies and the allograft's prognosis has been a topic of discussion for a long time, led in the main by P.Terasaki, it was not until the beginning of 1990s when P. Halloran studied the humoral mechanisms of rejection in greater depth. Feutch described the importance of C4d deposits as a marker that shows a humoral mechanism of allograft rejection in 1993. As a result of many studies carried out, the Banff consensus group established some diagnostic histopathological criteria of acute (ABMR) in 2003. These have been modified slightly in later meetings of the group. Furthermore, in 2005 this same working group looked at the physiopathological mechanisms causing chronic allograft failure in more detail and established the criteria defining chronic humoral rejection. In this review, we are trying to update any useful histopathological criteria for diagnosing acute and chronic ABMR.

  18. Protein disulfide isomerases are antibody targets during immune-mediated tumor destruction

    PubMed Central

    Fonseca, Catia; Soiffer, Robert; Ho, Vincent; Vanneman, Matthew; Jinushi, Masahisa; Ritz, Jerome; Neuberg, Donna; Stone, Richard; DeAngelo, Dan

    2009-01-01

    The identification of cancer antigens that contribute to transformation and are linked with immune-mediated tumor destruction is an important goal for immunotherapy. Toward this end, we screened a murine renal cell carcinoma cDNA expression library with sera from mice vaccinated with irradiated tumor cells engineered to secrete granulocyte macrophage colony-stimulating factor (GM-CSF). Multiple nonmutated, overexpressed proteins that function in tumor cell migration, protein/nucleic acid homeostasis, metabolism, and stress responses were detected. Among these, the most frequently recognized clone was protein disulfide isomerase (PDI). High titer antibodies to human PDI were similarly induced in an acute myeloid leukemia patient who achieved a complete response after vac-cination with irradiated, autologous GM-CSF–secreting tumor cells in the setting of nonmyeloablative allogeneic bone marrow transplantation. Moreover, ERp5, a closely related disulfide isomerase involved in major histocompatibility complex (MHC) class I chain-related protein A (MICA) shedding, also evoked potent humoral reactions in diverse solid and hematologic malignancy patients who responded to GM-CSF–secreting tumor cell vaccines or antibody blockade of cytotoxic T lymphocyte–associated antigen 4 (CTLA-4). Together, these findings reveal the unexpected immunogenicity of PDIs and raise the possibility that these gene products might serve as targets for therapeutic monoclonal antibodies. PMID:19008459

  19. Design considerations for liposomal vaccines: influence of formulation parameters on antibody and cell-mediated immune responses to liposome associated antigens.

    PubMed

    Watson, Douglas S; Endsley, Aaron N; Huang, Leaf

    2012-03-16

    Liposomes (phospholipid bilayer vesicles) are versatile and robust delivery systems for induction of antibody and T lymphocyte responses to associated subunit antigens. In the last 15 years, liposome vaccine technology has matured and now several vaccines containing liposome-based adjuvants have been approved for human use or have reached late stages of clinical evaluation. Given the intensifying interest in liposome-based vaccines, it is important to understand precisely how liposomes interact with the immune system and stimulate immunity. It has become clear that the physicochemical properties of liposomal vaccines - method of antigen attachment, lipid composition, bilayer fluidity, particle charge, and other properties - exert dramatic effects on the resulting immune response. Here, we present a comprehensive review of the physicochemical properties of liposomal vaccines and how they influence immune responses. A discussion of novel and emerging immunomodulators that are suitable for inclusion in liposomal vaccines is also presented. Through a comprehensive analysis of the body of liposomal vaccine literature, we enumerate a series of principles that can guide the rational design of liposomal vaccines to elicit immune responses of a desired magnitude and quality. We also identify major unanswered questions in the field, pointing the direction for future study.

  20. Design considerations for liposomal vaccines: Influence of formulation parameters on antibody and cell-mediated immune responses to liposome associated antigens

    PubMed Central

    Watson, Douglas S.; Endsley, Aaron N.; Huang, Leaf

    2012-01-01

    Liposomes (phospholipid bilayer vesicles) are versatile and robust delivery systems for induction of antibody and T lymphocyte responses to associated subunit antigens. In the last 15 years, liposome vaccine technology has matured and now several vaccines containing liposome-based adjuvants have been approved for human use or have reached late stages of clinical evaluation. Given the intensifying interest in liposome-based vaccines, it is important to understand precisely how liposomes interact with the immune system and stimulate immunity. It has become clear that the physicochemical properties of liposomal vaccines – method of antigen attachment, lipid composition, bilayer fluidity, particle charge, and other properties – exert dramatic effects on the resulting immune response. Here, we present a comprehensive review of the physicochemical properties of liposomal vaccines and how they influence immune responses. A discussion of novel and emerging immunomodulators that are suitable for inclusion in liposomal vaccines is also presented. Through a comprehensive analysis of the body of liposomal vaccine literature, we enumerate a series of principles that can guide the rational design of liposomal vaccines to elicit immune responses of a desired magnitude and quality. We also identify major unanswered questions in the field, pointing the direction for future study. PMID:22306376

  1. Isolation and characterization of cytotoxic effector cells and antibody producing cells from human intestine.

    PubMed

    MacDermott, R P

    1985-01-01

    We have examined the ability of intestinal and peripheral blood mononuclear cells isolated from patients with inflammatory bowel disease to mediate killing against cell line targets in spontaneous, antibody-dependent, lectin-induced, and interferon-induced cell-mediated cytotoxicity assays, as well as responsiveness in the allogeneic mixed leukocyte reaction, and effector capabilities in cell-mediated lympholysis. IMC were poor mediators of spontaneous or antibody-dependent cellular cytotoxicity with cell line cells as targets (in comparison to normal PBMC, but were capable of killing antibody coated chicken red blood cells. Although IMC were capable of responding to allogeneic cell surface antigens in the mixed leukocyte reaction, they did not exhibit effector function in cell-mediated lympholysis. Mitogenic lectins induced cell-mediated cytotoxicity by isolated intestinal mononuclear cells from controls and patients. HFIF induces cytotoxicity by control but not inflammatory bowel disease intestinal cells. Pokeweed mitogen was the lectin which induced the greatest amount of killing against human cell line targets. We therefore speculate that exogenous agents, or endogenous factors released during viral infection, could play a role in inducing cell mediated cytotoxic damage to the intestine in inflammatory bowel disease patients. In addition, the functional differences between IMC and PBMC indicate that intestinal MNC may have unique cell capabilities which must be better understood prior to the delineation of immunopathologic events in solid organ tissues. We have also examined the secretion of IgA, IgM, and IgG by isolated human IMC, human bone marrow MNC from rib specimens, and PBMC from patients with CD, UC, SLE, or Henoch-Schoenlein purpura (HSP). Control IMC exhibited high spontaneous secretion of IgA, while intestinal MNC from UC and CD patients exhibited only modest increases in IgA secretion. PBMC from patients with CD, UC, SLE, or HSP exhibited markedly

  2. Access of protective antiviral antibody to neuronal tissues requires CD4 T-cell help.

    PubMed

    Iijima, Norifumi; Iwasaki, Akiko

    2016-05-26

    Circulating antibodies can access most tissues to mediate surveillance and elimination of invading pathogens. Immunoprivileged tissues such as the brain and the peripheral nervous system are shielded from plasma proteins by the blood-brain barrier and blood-nerve barrier, respectively. Yet, circulating antibodies must somehow gain access to these tissues to mediate their antimicrobial functions. Here we examine the mechanism by which antibodies gain access to neuronal tissues to control infection. Using a mouse model of genital herpes infection, we demonstrate that both antibodies and CD4 T cells are required to protect the host after immunization at a distal site. We show that memory CD4 T cells migrate to the dorsal root ganglia and spinal cord in response to infection with herpes simplex virus type 2. Once inside these neuronal tissues, CD4 T cells secrete interferon-γ and mediate local increase in vascular permeability, enabling antibody access for viral control. A similar requirement for CD4 T cells for antibody access to the brain is observed after intranasal challenge with vesicular stomatitis virus. Our results reveal a previously unappreciated role of CD4 T cells in mobilizing antibodies to the peripheral sites of infection where they help to limit viral spread. PMID:27225131

  3. Access of protective antiviral antibody to neuronal tissues requires CD4 T-cell help.

    PubMed

    Iijima, Norifumi; Iwasaki, Akiko

    2016-05-26

    Circulating antibodies can access most tissues to mediate surveillance and elimination of invading pathogens. Immunoprivileged tissues such as the brain and the peripheral nervous system are shielded from plasma proteins by the blood-brain barrier and blood-nerve barrier, respectively. Yet, circulating antibodies must somehow gain access to these tissues to mediate their antimicrobial functions. Here we examine the mechanism by which antibodies gain access to neuronal tissues to control infection. Using a mouse model of genital herpes infection, we demonstrate that both antibodies and CD4 T cells are required to protect the host after immunization at a distal site. We show that memory CD4 T cells migrate to the dorsal root ganglia and spinal cord in response to infection with herpes simplex virus type 2. Once inside these neuronal tissues, CD4 T cells secrete interferon-γ and mediate local increase in vascular permeability, enabling antibody access for viral control. A similar requirement for CD4 T cells for antibody access to the brain is observed after intranasal challenge with vesicular stomatitis virus. Our results reveal a previously unappreciated role of CD4 T cells in mobilizing antibodies to the peripheral sites of infection where they help to limit viral spread.

  4. Establishment of Stable, Cell-Mediated Immunity that Makes "Susceptible" Mice Resistant to Leishmania major

    NASA Astrophysics Data System (ADS)

    Bretscher, Peter A.; Wei, Guojian; Menon, Juthika N.; Bielefeldt-Ohmann, Helle

    1992-07-01

    Cell-mediated, but not antibody-mediated, immune responses protect humans against certain pathogens that produce chronic diseases such as leishmaniasis. Effective vaccination against such pathogens must therefore produce an immunological "imprint" so that stable, cell-mediated immunity is induced in all individuals after natural infection. BALB/c mice "innately susceptible" to Leishmania major produce antibodies after substantial infection. In the present study, "susceptible" mice injected with a small number of parasites mounted a cell-mediated response and acquired resistance to a larger, normally pathogenic, challenge. This vaccination strategy may be applicable in diseases in which protection is dependent on cell-mediated immunity.

  5. Understanding the causes of kidney transplant failure: the dominant role of antibody-mediated rejection and nonadherence.

    PubMed

    Sellarés, J; de Freitas, D G; Mengel, M; Reeve, J; Einecke, G; Sis, B; Hidalgo, L G; Famulski, K; Matas, A; Halloran, P F

    2012-02-01

    We prospectively studied kidney transplants that progressed to failure after a biopsy for clinical indications, aiming to assign a cause to every failure. We followed 315 allograft recipients who underwent indication biopsies at 6 days to 32 years posttransplant. Sixty kidneys progressed to failure in the follow-up period (median 31.4 months). Failure was rare after T-cell-mediated rejection and acute kidney injury and common after antibody-mediated rejection or glomerulonephritis. We developed rules for using biopsy diagnoses, HLA antibody and clinical data to explain each failure. Excluding four with missing information, 56 failures were attributed to four causes: rejection 36 (64%), glomerulonephritis 10 (18%), polyoma virus nephropathy 4 (7%) and intercurrent events 6 (11%). Every rejection loss had evidence of antibody-mediated rejection by the time of failure. Among rejection losses, 17 of 36 (47%) had been independently identified as nonadherent by attending clinicians. Nonadherence was more frequent in patients who progressed to failure (32%) versus those who survived (3%). Pure T-cell-mediated rejection, acute kidney injury, drug toxicity and unexplained progressive fibrosis were not causes of loss. This prospective cohort indicates that many actual failures after indication biopsies manifest phenotypic features of antibody-mediated or mixed rejection and also underscores the major role of nonadherence.

  6. Social Support Mediates Loneliness and Human Herpesvirus Type 6 (HHV-6) Antibody Titers

    PubMed Central

    Dixon, Denise; Cruess, Stacy; Kilbourn, Kristin; Klimas, Nancy; Fletcher, Mary Ann; Ironson, Gail; Baum, Andrew; Schneiderman, Neil; Antoni, Michael H.

    2009-01-01

    The current study investigated the impact of a severe environmental stressor and the role that declining social integration played in mediating its effect on loneliness and immune status. Increased loneliness and decreased social support in the months following the stressor (storm) were significantly associated with increased HHV-6 antibody titers, reflecting poorer control over the virus. Poorer social integration mediated the relationship between loneliness and HHV-6, even after controlling for nonspecific polyclonal B-cell activation, disease status (CD3+CD4+ cell counts), living arrangements, acute social losses (bereavement), and potential disruptions in social-support resources. These findings suggest that specific elements of social support may explain the oft-noted negative effects of loneliness on the immune system, and generalized to a medically vulnerable population. PMID:20407593

  7. Oncolytic reovirus enhances rituximab-mediated antibody-dependent cellular cytotoxicity against chronic lymphocytic leukaemia.

    PubMed

    Parrish, C; Scott, G B; Migneco, G; Scott, K J; Steele, L P; Ilett, E; West, E J; Hall, K; Selby, P J; Buchanan, D; Varghese, A; Cragg, M S; Coffey, M; Hillmen, P; Melcher, A A; Errington-Mais, F

    2015-09-01

    The naturally occurring oncolytic virus (OV), reovirus, replicates in cancer cells causing direct cytotoxicity, and can activate innate and adaptive immune responses to facilitate tumour clearance. Reovirus is safe, well tolerated and currently in clinical testing for the treatment of multiple myeloma, in combination with dexamethasone/carfilzomib. Activation of natural killer (NK) cells has been observed after systemic delivery of reovirus to cancer patients; however, the ability of OV to potentiate NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) is unexplored. This study elucidates the potential of oncolytic reovirus for the treatment of chronic lymphocytic leukaemia (CLL), both as a direct cytotoxic agent and as an immunomodulator. We demonstrate that reovirus: (i) is directly cytotoxic against CLL, which requires replication-competent virus; (ii) phenotypically and functionally activates patient NK cells via a monocyte-derived interferon-α (IFNα)-dependent mechanism; and (iii) enhances ADCC-mediated killing of CLL in combination with anti-CD20 antibodies. Our data provide strong preclinical evidence to support the use of reovirus in combination with anti-CD20 immunotherapy for the treatment of CLL.

  8. Cell-mediated Protection in Influenza Infection

    PubMed Central

    Thomas, Paul G.; Keating, Rachael; Hulse-Post, Diane J.

    2006-01-01

    Current vaccine strategies against influenza focus on generating robust antibody responses. Because of the high degree of antigenic drift among circulating influenza strains over the course of a year, vaccine strains must be reformulated specifically for each influenza season. The time delay from isolating the pandemic strain to large-scale vaccine production would be detrimental in a pandemic situation. A vaccine approach based on cell-mediated immunity that avoids some of these drawbacks is discussed here. Specifically, cell-mediated responses typically focus on peptides from internal influenza proteins, which are far less susceptible to antigenic variation. We review the literature on the role of CD4+ and CD8+ T cell–mediated immunity in influenza infection and the available data on the role of these responses in protection from highly pathogenic influenza infection. We discuss the advantages of developing a vaccine based on cell-mediated immune responses toward highly pathogenic influenza virus and potential problems arising from immune pressure. PMID:16494717

  9. Effects of Bifidobacterium longum BB536 administration on influenza infection, influenza vaccine antibody titer, and cell-mediated immunity in the elderly.

    PubMed

    Namba, Kazuyoshi; Hatano, Michiko; Yaeshima, Tomoko; Takase, Mitsunori; Suzuki, Kunihiko

    2010-01-01

    Twenty-seven elderly subjects (mean age 86.7+/-6.6 years) were pre-administered a test food containing 1x10(11) cfu of BB536 daily for 5 weeks (P1), during which they also received influenza vaccination at week 3. The subjects were then randomized to a BB536 group and a placebo group for 14 weeks (P2). The proportion of subjects who contracted influenza was significantly lower in BB536 group than in the to placebo group. The proportion of subjects with fever was also significantly lower in the BB536 group than in the placebo group. In the P1 period, the NK cell activity and the bactericidal activity of the neutrophils were significantly higher at week 5 than to before BB536 administration. In the P2 period, although NK cell activity and neutrophilic activities declined at the end of the study in both the placebo and the BB536 group, neutrophil phagocytic activity and NK cell activity tended to maintain slightly higher levels in the BB536 group than in the placebo group. These results suggest that continuous ingestion of BB536 reduces the incidence of influenza and fever, probably by potentiating innate immunity.

  10. Persistent expression of biologically active anti-HER2 antibody by AAVrh.10-mediated gene transfer.

    PubMed

    Wang, G; Qiu, J; Wang, R; Krause, A; Boyer, J L; Hackett, N R; Crystal, R G

    2010-08-01

    Trastuzumab (Herceptin) is a recombinant humanized monoclonal antibody (mAb) directed against an extracellular region of the human epidermal growth-factor receptor type 2 (HER2) protein. We hypothesized that a single adeno-associated virus (AAV)-mediated genetic delivery of an anti-HER2 antibody should be effective in mediating long-term production of anti-HER2 and in suppressing the growth of human tumors in a xenograft model in nude mice. The adeno-associated virus gene transfer vector AAVrh.10alphaHER2 was constructed based on a non-human primate AAV serotype rh.10 to express the complementary DNAs for the heavy and light chains of mAb 4D5, the murine precursor to trastuzumab. The data show that genetically transferred anti-HER2 selectively bound human HER2 protein and suppressed the proliferation of HER2(+) tumor cell lines. A single administration of AAVrh.10alphaHER2 provided long-term therapeutic levels of anti-HER2 antibody expression without inducing an anti-idiotype response, suppressed the growth of HER2(+) tumors and increased the survival of tumor bearing mice. In the context that trastuzumab therapy requires frequent and repeated administration, this strategy might be developed as an alternate platform for delivery of anti-HER2 therapy.

  11. Additive cytotoxicity of different monoclonal antibody-cobra venom factor conjugates for human neuroblastoma cells.

    PubMed

    Juhl, H; Petrella, E C; Cheung, N K; Bredehorst, R; Vogel, C W

    1997-11-01

    Insufficient numbers of antigen molecules and heterogeneity of antigen expression on tumor cells are major factors limiting the immunotherapeutic potential of the few clinically useful monoclonal antibodies capable of mediating complement cytotoxicity and antibody-dependent cellular cytotoxicity. To overcome this limitation, we converted two non-cytotoxic monoclonal anti-neuroblastoma antibodies, designated 3E7 (IgG2b) and 8H9 (IgG1), and the non-cytotoxic F(ab')2 fragment of the cytotoxic monoclonal anti-GD2 antibody 3F8 (IgG3) into cytotoxic antibody conjugates by covalent attachment of cobra venom factor (CVF), a structural and functional homologue of the activated third component of complement. Competitive binding experiments confirmed the different specificities of the three antibodies. In the presence of human complement, all three antibody-CVF conjugates mediated selective complement-dependent lysis of human neuroblastoma cells. Consistent with the kinetics of the alternative pathway of complement, approximately seven hours incubation were required to reach maximum cytotoxicity of up to 25% for the 3E7-CVF conjugate, up to 60% for the 8H9-CVF conjugate, and up to 95% for the 3F8 F(ab')2-CVF conjugate. The different extent of maximal cytotoxic activity of the three conjugates was reflected by corresponding differences in the extent of binding of both unconjugated antibodies and the respective conjugates. Any combination of the three antibody-CVF conjugates caused an additive effect in complement-mediated lysis. Using a cocktail of all three conjugates, the extent of complement-mediated killing could be increased up to 100%. These data demonstrate that by coupling of CVF the relative large number of non-cytotoxic monoclonal anti-tumor antibodies of interesting specificity can be used to design cocktails of cytotoxic conjugates and, thereby, to overcome the problem of insufficient and heterogeneous antigen expression on tumor cells for immunotherapy.

  12. Current and future challenges in therapy for antibody-mediated rejection.

    PubMed

    Nair, Nandini; Ball, Timothy; Uber, Patricia A; Mehra, Mandeep R

    2011-06-01

    Antibody-mediated rejection (AMR) continues to present a challenge for the survival of the cardiac allograft. AMR appears to be on the rise, likely secondary to changing trends in clinical practice, including selection of patients for transplantation on mechanical circulatory support and development of more effective combinations of immunosuppressive drugs against acute cellular rejection. Most current strategies are aimed at treating acute AMR, but the treatment of chronic AMR is still not well defined. Clinically, AMR can often be more severe than cellular rejection and more difficult to treat, often not responding to typical protocols of increased immunosuppression. Complex steps involved in the antibody response allows for several potential targets for therapeutic intervention, including suppression of T and B cells, elimination of circulating antibodies, and inhibition of residual antibodies. Existing evidence suggests a multiregimen approach is the best option. Sustenance of accommodation and induction of tolerance could be viewed as viable options if adequate immune surveillance can be achieved in this setting. This review discusses the challenges in treating AMR and provides a critical analysis of current and possible future therapies.

  13. Current and future challenges in therapy for antibody-mediated rejection.

    PubMed

    Nair, Nandini; Ball, Timothy; Uber, Patricia A; Mehra, Mandeep R

    2011-06-01

    Antibody-mediated rejection (AMR) continues to present a challenge for the survival of the cardiac allograft. AMR appears to be on the rise, likely secondary to changing trends in clinical practice, including selection of patients for transplantation on mechanical circulatory support and development of more effective combinations of immunosuppressive drugs against acute cellular rejection. Most current strategies are aimed at treating acute AMR, but the treatment of chronic AMR is still not well defined. Clinically, AMR can often be more severe than cellular rejection and more difficult to treat, often not responding to typical protocols of increased immunosuppression. Complex steps involved in the antibody response allows for several potential targets for therapeutic intervention, including suppression of T and B cells, elimination of circulating antibodies, and inhibition of residual antibodies. Existing evidence suggests a multiregimen approach is the best option. Sustenance of accommodation and induction of tolerance could be viewed as viable options if adequate immune surveillance can be achieved in this setting. This review discusses the challenges in treating AMR and provides a critical analysis of current and possible future therapies. PMID:21474341

  14. Decitabine enhances anti-CD33 monoclonal antibody BI 836858–mediated natural killer ADCC against AML blasts

    PubMed Central

    Vasu, Sumithira; He, Shun; Cheney, Carolyn; Gopalakrishnan, Bhavani; Mani, Rajeswaran; Lozanski, Gerard; Mo, Xiaokui; Groh, Veronica; Whitman, Susan P.; Konopitzky, Renate; Kössl, Christian; Bucci, Donna; Lucas, David M.; Yu, Jianhua; Caligiuri, Michael A.; Blum, William; Adam, Paul J.; Borges, Eric; Rueter, Bjoern; Heider, Karl-Heinz; Marcucci, Guido

    2016-01-01

    Acute myeloid leukemia (AML) is the most common type of acute leukemia, affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence, novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (gemtuzumab ozogamicin) have been shown to improve overall survival, validating CD33 as a target for antibody-based therapy of AML. Here, we report the in vitro efficacy of BI 836858, a fully human, Fc-engineered, anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858–opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK-cell–mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine, 2 hypomethylating agents that show efficacy in older patients, did not compromise BI 836858–induced NK-cell–mediated ADCC. Evaluation of BI 836858–mediated ADCC in serial marrow AML aspirates in patients who received a 10-day course of DAC (pre-DAC, days 4, 11, and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared with pre-DAC treatment. Analysis of ligands to activating receptors (NKG2D) showed significantly increased NKG2D ligand [NKG2DL] expression in day 28 post-DAC samples compared with pre-DAC samples; when NKG2DL receptor was blocked using antibodies, BI 836858–mediated ADCC was significantly decreased, suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML. PMID:27013443

  15. CD69-mediated pathway of lymphocyte activation: anti-CD69 monoclonal antibodies trigger the cytolytic activity of different lymphoid effector cells with the exception of cytolytic T lymphocytes expressing T cell receptor alpha/beta

    PubMed Central

    1991-01-01

    The effect of anti-CD69 monoclonal antibodies (mAbs) on the induction of the cytolytic activity in different types of lymphoid effector cells has been investigated. Three anti-CD69 mAbs, including the reference mAb MLR3 and two new mAbs (c227 and 31C4), have been used. All cloned CD3-CD16+ natural killer (NK) cells belonging to different subsets (as defined by the surface expression of GL183 and/or EB6 antigens) were efficiently triggered by anti-CD69 mAbs and lysed P815 mastocytoma cells in a redirected killing assay. Triggering of the cytolytic activity could also be induced in CD3-CD16- NK clones, which fail to respond to other stimuli (including anti-CD16, anti-CD2 mAbs, or phytohemagglutinin). A similar triggering effect was detected in T cell receptor (TCR) gamma/delta+ clones belonging to different subsets. On the other hand, anti-CD69 mAbs could not induce triggering of the cytolytic activity in TCR alpha/beta+ cytolytic clones. Since all thymocytes are known to express CD69 antigen after cell activation, we analyzed a series of phenotypically different cytolytic thymocyte populations and clones for their responsiveness to anti-CD69 mAb in a redirected killing assay. Again, anti-CD69 mAb triggered TCR gamma/delta+ but not TCR alpha/beta+ thymocytes. Anti-CD69 mAb efficiently triggered the cytolytic activity of "early" thymocytes lines or clones (CD3-4-8-7+), which lack all other known pathways of cell activation. Thus, it appears that CD69 molecules may initiate a pathway of activation of cytolytic functions common to a number of activated effector lymphocytes with the remarkable exception of TCR alpha/beta+ cytolytic cells. PMID:1720808

  16. JC polyomavirus mutants escape antibody-mediated neutralization.

    PubMed

    Ray, Upasana; Cinque, Paola; Gerevini, Simonetta; Longo, Valeria; Lazzarin, Adriano; Schippling, Sven; Martin, Roland; Buck, Christopher B; Pastrana, Diana V

    2015-09-23

    JC polyomavirus (JCV) persistently infects the urinary tract of most adults. Under conditions of immune impairment, JCV causes an opportunistic brain disease, progressive multifocal leukoencephalopathy (PML). JCV strains found in the cerebrospinal fluid of PML patients contain distinctive mutations in surface loops of the major capsid protein, VP1. We hypothesized that VP1 mutations might allow the virus to evade antibody-mediated neutralization. Consistent with this hypothesis, neutralization serology revealed that plasma samples from PML patients neutralized wild-type JCV strains but failed to neutralize patient-cognate PML-mutant JCV strains. This contrasted with serological results for healthy individuals, most of whom robustly cross-neutralized all tested JCV variants. Mice administered a JCV virus-like particle (VLP) vaccine initially showed neutralizing "blind spots" (akin to those observed in PML patients) that closed after booster immunization. A PML patient administered an experimental JCV VLP vaccine likewise showed markedly increased neutralizing titer against her cognate PML-mutant JCV. The results indicate that deficient humoral immunity is a common aspect of PML pathogenesis and that vaccination may overcome this humoral deficiency. Thus, vaccination with JCV VLPs might prevent the development of PML.

  17. Notch-Mediated Cell Adhesion

    PubMed Central

    Murata, Akihiko; Hayashi, Shin-Ichi

    2016-01-01

    Notch family members are generally recognized as signaling molecules that control various cellular responses in metazoan organisms. Early fly studies and our mammalian studies demonstrated that Notch family members are also cell adhesion molecules; however, information on the physiological roles of this function and its origin is limited. In this review, we discuss the potential present and ancestral roles of Notch-mediated cell adhesion in order to explore its origin and the initial roles of Notch family members dating back to metazoan evolution. We hypothesize that Notch family members may have initially emerged as cell adhesion molecules in order to mediate multicellularity in the last common ancestor of metazoan organisms. PMID:26784245

  18. Natural Killer Cell Mediated Cytotoxic Responses in the Tasmanian Devil

    PubMed Central

    Brown, Gabriella K.; Kreiss, Alexandre; Lyons, A. Bruce; Woods, Gregory M.

    2011-01-01

    The Tasmanian devil (Sarcophilus harrisii), the world's largest marsupial carnivore, is under threat of extinction following the emergence of an infectious cancer. Devil facial tumour disease (DFTD) is spread between Tasmanian devils during biting. The disease is consistently fatal and devils succumb without developing a protective immune response. The aim of this study was to determine if Tasmanian devils were capable of forming cytotoxic antitumour responses and develop antibodies against DFTD cells and foreign tumour cells. The two Tasmanian devils immunised with irradiated DFTD cells did not form cytotoxic or humoral responses against DFTD cells, even after multiple immunisations. However, following immunisation with xenogenic K562 cells, devils did produce cytotoxic responses and antibodies against this foreign tumour cell line. The cytotoxicity appeared to occur through the activity of natural killer (NK) cells in an antibody dependent manner. Classical NK cell responses, such as innate killing of DFTD and foreign cancer cells, were not observed. Cells with an NK-like phenotype comprised approximately 4 percent of peripheral blood mononuclear cells. The results of this study suggest that Tasmanian devils have NK cells with functional cytotoxic pathways. Although devil NK cells do not directly recognise DFTD cancer cells, the development of antibody dependent cell-mediated cytotoxicity presents a potential pathway to induce cytotoxic responses against the disease. These findings have positive implications for future DFTD vaccine research. PMID:21957452

  19. Mechanism of human antibody-mediated neutralization of Marburg virus.

    PubMed

    Flyak, Andrew I; Ilinykh, Philipp A; Murin, Charles D; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L; Hashiguchi, Takao; Bornholdt, Zachary A; Slaughter, James C; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G; Ward, Andrew B; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E

    2015-02-26

    The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition. PMID:25723164

  20. Modeling single cell antibody excretion on a biosensor.

    PubMed

    Stojanović, Ivan; Baumgartner, Wolfgang; van der Velden, Thomas J G; Terstappen, Leon W M M; Schasfoort, Richard B M

    2016-07-01

    We simulated, using Comsol Multiphysics, the excretion of antibodies by single hybridoma cells and their subsequent binding on a surface plasmon resonance imaging (SPRi) sensor. The purpose was to confirm that SPRi is suitable to accurately quantify antibody (anti-EpCAM) excretion. The model showed that antibody loss by diffusion away from the sensor was less than 1%. Unexpectedly, more than 99% of the excreted antibodies were captured on the sensor. These data prove the remarkable phenomenon that the SPRi output of cellular antibody excretion and its subsequent binding, performed under the conditions described here, is directly usable for quantification of single cell antibody production rates. PMID:27040182

  1. Modeling single cell antibody excretion on a biosensor.

    PubMed

    Stojanović, Ivan; Baumgartner, Wolfgang; van der Velden, Thomas J G; Terstappen, Leon W M M; Schasfoort, Richard B M

    2016-07-01

    We simulated, using Comsol Multiphysics, the excretion of antibodies by single hybridoma cells and their subsequent binding on a surface plasmon resonance imaging (SPRi) sensor. The purpose was to confirm that SPRi is suitable to accurately quantify antibody (anti-EpCAM) excretion. The model showed that antibody loss by diffusion away from the sensor was less than 1%. Unexpectedly, more than 99% of the excreted antibodies were captured on the sensor. These data prove the remarkable phenomenon that the SPRi output of cellular antibody excretion and its subsequent binding, performed under the conditions described here, is directly usable for quantification of single cell antibody production rates.

  2. Tregalizumab – A Monoclonal Antibody to Target Regulatory T Cells

    PubMed Central

    König, Martin; Rharbaoui, Faiza; Aigner, Silke; Dälken, Benjamin; Schüttrumpf, Jörg

    2016-01-01

    Regulatory T cells (Tregs) represent a subpopulation of CD4+ T cells, which are essential for the maintenance of immunological tolerance. The absence or dysfunction of Tregs can lead to autoimmunity and allergies. The restoration of functional Tregs and/or Treg cell numbers represents a novel and attractive approach for the treatment of autoimmune diseases, e.g., rheumatoid arthritis (RA). The CD4 cell surface receptor is a target for modulation of T cell function. Monoclonal antibodies (mAbs) against CD4 have previously been tested for the treatment of autoimmune diseases, including RA. Furthermore, in model systems, anti-CD4 antibodies are able to induce tolerance and mediate immunomodulatory effects through a variety of mechanisms. Despite the availability of innovative and effective therapies for RA, many patients still have persistently active disease or experience adverse events that can limit use. A growing body of evidence suggests that Treg modulation could offer a new therapeutic strategy in RA and other autoimmune disorders. Here, we describe tregalizumab (BT-061), which is a novel, non-depleting IgG1 mAb that binds to a unique epitope of CD4. Tregalizumab represents the first humanized anti-CD4 mAb that selectively induces Treg activation. PMID:26834751

  3. Siglecs as targets for therapy in immune cell mediated disease

    PubMed Central

    O’Reilly, Mary K.; Paulson, James C.

    2010-01-01

    The sialic acid-binding immunoglobulin-like lectins (siglecs) comprise a family of receptors that are differentially expressed on leukocytes and other immune cells. The restricted expression of several siglecs to one or a few cell types makes them attractive targets for cell-directed therapies. The anti-CD33 (Siglec-3) antibody Gemtuzumab (Mylotarg™) is approved for treatment of acute myeloid leukemia (AML), and antibodies targeting CD22 (Siglec-2) are currently in clinical trials for treatment of B cell non-Hodgkins lymphomas and autoimmune diseases. Because siglecs are endocytic receptors, they are well suited for a ‘Trojan horse’ strategy, whereby therapeutic agents conjugated to an antibody, or multimeric glycan ligand, bind to the siglec and are efficiently carried into the cell. Although the rapid internalization of unmodified siglec antibodies reduces their utility for induction of antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated cytotoxicity (CDC), antibody binding of Siglec-8, Siglec-9, and CD22 have been demonstrated to induce apoptosis of eosinophils, neutrophils, and depletion of B cells, respectively. Here we review the properties of siglecs that make them attractive for cell-targeted therapies. PMID:19359050

  4. Cell adhesion molecules: detection with univalent second antibody

    PubMed Central

    1980-01-01

    Identification of cell surface molecules that play a role in cell-cell adhesion (here called cell adhesion molecules) has been achieved by demonstrating the inhibitory effect of univalent antibodies that bind these molecules in an in vitro assay of cell-cell adhesion. A more convenient reagent, intact (divalent) antibody, has been avoided because it might agglutinate the cells rather than blocking cell-cell adhesion. In this report, we show that intact rabbit immunoglobulin directed against certain cell surface molecules of Dictyostelium discoideum blocks cell-cell adhesion when the in vitro assay is performed in the presence of univalent goat anti-rabbit antibody. Under appropriate experimental conditions, the univalent second antibody blocks agglutination induced by the rabbit antibody without significantly interfering with its effect on cell-cell adhesion. This method promises to be useful for screening monoclonal antibodies raised against potential cell adhesion molecules because: (a) it allows for the screening of large numbers of antibody samples without preparation of univalent fragments; and (b) it requires much less antibody because of the greater affinity of divalent antibodies for antigens. PMID:6970200

  5. A Highly Conserved Residue of the HIV-1 gp120 Inner Domain Is Important for Antibody-Dependent Cellular Cytotoxicity Responses Mediated by Anti-cluster A Antibodies

    PubMed Central

    Ding, Shilei; Veillette, Maxime; Coutu, Mathieu; Prévost, Jérémie; Scharf, Louise; Bjorkman, Pamela J.; Ferrari, Guido; Robinson, James E.; Stürzel, Christina; Hahn, Beatrice H.; Sauter, Daniel; Kirchhoff, Frank; Lewis, George K.; Pazgier, Marzena

    2015-01-01

    Previous studies have shown that sera from HIV-1-infected individuals contain antibodies able to mediate antibody-dependent cellular cytotoxicity (ADCC). These antibodies preferentially recognize envelope glycoprotein (Env) epitopes induced upon CD4 binding. Here, we show that a highly conserved tryptophan at position 69 of the gp120 inner domain is important for ADCC mediated by anti-cluster A antibodies and sera from HIV-1-infected individuals. PMID:26637462

  6. Antibody-Mediated Targeting of Tau In Vivo Does Not Require Effector Function and Microglial Engagement.

    PubMed

    Lee, Seung-Hye; Le Pichon, Claire E; Adolfsson, Oskar; Gafner, Valérie; Pihlgren, Maria; Lin, Han; Solanoy, Hilda; Brendza, Robert; Ngu, Hai; Foreman, Oded; Chan, Ruby; Ernst, James A; DiCara, Danielle; Hotzel, Isidro; Srinivasan, Karpagam; Hansen, David V; Atwal, Jasvinder; Lu, Yanmei; Bumbaca, Daniela; Pfeifer, Andrea; Watts, Ryan J; Muhs, Andreas; Scearce-Levie, Kimberly; Ayalon, Gai

    2016-08-01

    The spread of tau pathology correlates with cognitive decline in Alzheimer's disease. In vitro, tau antibodies can block cell-to-cell tau spreading. Although mechanisms of anti-tau function in vivo are unknown, effector function might promote microglia-mediated clearance. In this study, we investigated whether antibody effector function is required for targeting tau. We compared efficacy in vivo and in vitro of two versions of the same tau antibody, with and without effector function, measuring tau pathology, neuron health, and microglial function. Both antibodies reduced accumulation of tau pathology in Tau-P301L transgenic mice and protected cultured neurons against extracellular tau-induced toxicity. Only the full-effector antibody enhanced tau uptake in cultured microglia, which promoted release of proinflammatory cytokines. In neuron-microglia co-cultures, only effectorless anti-tau protected neurons, suggesting full-effector tau antibodies can induce indirect toxicity via microglia. We conclude that effector function is not required for efficacy, and effectorless tau antibodies may represent a safer approach to targeting tau. PMID:27475227

  7. Nanoliposome-mediated targeting of antibodies to tumors: IVIG antibodies as a model.

    PubMed

    Nikpoor, Amin Reza; Tavakkol-Afshari, Jalil; Gholizadeh, Zahra; Sadri, Kayvan; Babaei, Mohammad Hossein; Chamani, Jamshidkhan; Badiee, Ali; Jalali, Seyed Amir; Jaafari, Mahmoud Reza

    2015-11-10

    Monoclonal antibodies are routinely used as tools in immunotherapies against solid tumors. However, administration of monoclonal antibodies may cause undesired side effects due to their accumulation in non-targeted organs. Nanoliposomes of less than 200 nm can target antibodies to tumors by enhanced permeation and retention (EPR) mechanisms. To direct monoclonal antibodies to tumors, nanoliposomes encapsulating intravenous immunoglobulin (IVIG) as a model antibody were prepared. The liposomes had average diameters of 100 nm and encapsulation efficiencies of 31 to 46%. They showed less than 10% release in plasma at 37°C up to seven days. The secondary and tertiary structures of liposome-encapsulated antibodies were analyzed by circular dichroism (CD) spectroscopy. The near and far-UV spectra analyses revealed no obvious conformational changes in the structures of the encapsulated antibodies. The biodistribution of free and liposome-encapsulated iodinated antibodies was investigated in mice bearing C-26 colon carcinoma tumors. The accumulation of liposome-encapsulated antibodies in tumors was significantly greater than that of free antibodies due to the EPR effect. The PEGylated liposomes were more efficient in the delivery of antibodies to the tumor site than non-PEGylated liposomes. We conclude that administration of monoclonal antibodies in PEGylated liposomes is more efficient than administration of non-encapsulated monoclonal antibodies for solid tumor immunotherapy. PMID:26302860

  8. Anti-Inositolglycan Antibodies Selectively Block Some of the Actions of Insulin in Intact BC_3H1 Cells

    NASA Astrophysics Data System (ADS)

    Romero, Guillermo; Gamez, Graciela; Huang, Laura C.; Lilley, Kevin; Luttrell, Louis

    1990-02-01

    We have studied the mechanism of generation of insulin mediators by using specific antibodies raised against the oligosaccharide anchor of membrane proteins. These antibodies (i) block the in vitro effects of purified insulin mediators and (ii) block the insulin-induced stimulation of pyruvate dehydrogenase in intact BC_3H1 myocytes but not insulin-stimulated glucose uptake, generation of diacylglycerol, or generation of insulin mediators. When added to intact cells in the presence of insulin, these antibodies induce the accumulation of insulin mediator activity in the extracellular medium. We therefore conclude that these anti-inositolglycan antibodies block some of the effects of insulin by inhibiting the uptake of specific insulin mediators generated outside the cell.

  9. Anti-inositolglycan antibodies selectively block some of the actions of insulin in intact BC3H1 cells.

    PubMed Central

    Romero, G; Gámez, G; Huang, L C; Lilley, K; Luttrell, L

    1990-01-01

    We have studied the mechanism of generation of insulin mediators by using specific antibodies raised against the oligosaccharide anchor of membrane proteins. These antibodies (i) block the in vitro effects of purified insulin mediators and (ii) block the insulin-induced stimulation of pyruvate dehydrogenase in intact BC3H1 myocytes but not insulin-stimulated glucose uptake, generation of diacylglycerol, or generation of insulin mediators. When added to intact cells in the presence of insulin, these antibodies induce the accumulation of insulin mediator activity in the extracellular medium. We therefore conclude that these anti-inositolglycan antibodies block some of the effects of insulin by inhibiting the uptake of specific insulin mediators generated outside the cell. PMID:2137614

  10. Adenovirus-mediated delivery of an anti-V antigen monoclonal antibody protects mice against a lethal Yersinia pestis challenge.

    PubMed

    Sofer-Podesta, Carolina; Ang, John; Hackett, Neil R; Senina, Svetlana; Perlin, David; Crystal, Ronald G; Boyer, Julie L

    2009-04-01

    Pneumonic plague, caused by inhalation of Yersinia pestis, represents a major bioterrorism threat for which no vaccine is available. Based on the knowledge that genetic delivery of monoclonal antibodies (MAbs) with adenovirus (Ad) gene transfer vectors results in rapid, high-level antibody expression, we evaluated the hypothesis that Ad-mediated delivery of a neutralizing antibody directed against the Y. pestis V antigen would protect mice against a Y. pestis challenge. MAbs specific for the Y. pestis V antigen were generated, and the most effective in protecting mice against a lethal intranasal Y. pestis challenge was chosen for further study. The coding sequences for the heavy and light chains were isolated from the corresponding hybridoma and inserted into a replication-defective serotype 5 human Ad gene transfer vector (AdalphaV). Western analysis of AdalphaV-infected cell supernatants demonstrated completely assembled antibodies reactive with V antigen. Following AdalphaV administration to mice, high levels of anti-V antigen antibody titers were detectable as early as 1 day postadministration, peaked by day 3, and remained detectable through a 12-week time course. When animals that received AdalphaV were challenged with Y. pestis at day 4 post-AdalphaV administration, 80% of the animals were protected, while 0% of control animals survived (P < 0.01). Ad-mediated delivery of a V antigen-neutralizing antibody is an effective therapy against plague in experimental animals and could be developed as a rapidly acting antiplague therapeutic.

  11. Cytolysis of oligodendrocytes is mediated by killer (K) cells but not by natural killer (NK) cells.

    PubMed

    Satoh, J; Kim, S U; Kastrukoff, L F

    1991-03-01

    The cytotoxic activity of killer (K) cells against enriched cultures of bovine oligodendrocytes (BOL) was investigated in multiple sclerosis (MS) and controls. Human K cells mediated cytotoxicity to primary cultures of BOL in the presence of anti-BOL antiserum in all study groups, while BOL were resistant to human natural killer (NK) cells. Cytotoxic activity was significantly reduced in MS when compared to age-matched normal controls but not when compared to other neurologic disease (OND) patients. K cell-mediated lysis of BOL could also be induced with anti-galactocerebroside antibody but not with other antibodies including those specific for OL antigens (myelin basic protein, proteolipid apoprotein, and 2',3'-cyclic nucleotide 3'-phosphodiesterase). Enrichment of the effector population indicated that antibody-dependent cell-mediated cytotoxicity (ADCC) to BOL was mediated by large granular lymphocytes, and the effector population was further characterized by flow cytometry. The effector cells mediating ADCC could be inhibited by protein A of Staphylococcus aureus, and by K562 cells in cold competition assay. These observations indicate that oligodendrocytes are resistant to NK cells but are susceptible to cytolysis mediated by K cells. This may represent a potentially important immune mechanism in the pathogenesis of MS.

  12. Anti-CD2 antibodies induce T cell unresponsiveness in vivo

    PubMed Central

    1991-01-01

    The CD2 receptor functions as an adhesion and signal molecule in T cell recognition. Multimeric binding of CD2 on T cells to its physiologic ligand LFA-3 on cognate partner cells in vitro efficiently augments the antigen-specific T cell signal delivered by the T cell receptor/CD3 complex. The precise contribution of the antigen-nonspecific CD2-LFA-3 interactions to T cell immune responses in vivo, however, has been difficult to assess. Here we analyzed the role of CD2 in the murine immune response using a nondepleting anti-CD2 monoclonal antibody that induces a marked, reversible modulation of CD2 expression on murine T and B cells in situ. This modulation is dose and time dependent, specific for CD2, and does not require the Fc portion of the antibody. Anti-CD2 antibodies [rat IgG1 or F(ab')2] significantly inhibit the CD4+ T cell-mediated response to hen egg lysozyme and the cytotoxic CD8+ T cell response to a syngeneic tumor cell line. In both cases, anti-CD2 antibodies are only effective when administered before or within 24 h after antigen priming. The suppression of the antitumor response corresponds to a sixfold reduction of specific cytotoxic T lymphocyte precursor cells and results in the abrogation of protective antitumor immunity. Anti-CD2 antibodies also affect the humoral immune response to oxazolone: the isotype switch from specific IgM to IgG1 antibodies is delayed, whereas the IgM response is unaltered. In addition, a single antibody injection results in sustained polyclonal unresponsiveness of T cells irrespective of antigen priming and CD2 modulation. These results document that CD2-mediated signals induce a state of T cell unresponsiveness in vivo. PMID:1682413

  13. Antibody secreting cell assay for influenza A virus in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An ELISPOT assay to enumerate B-cells producing antibodies specific to a given antigen, also known as an antibody secreting cell (ASC) assay, was adapted to detect B-cells specific for influenza A virus (IAV). The assay is performed ex vivo and enumerates ASC at a single cell level. A simple ASC det...

  14. Detection of anti-liver cell membrane antibody using a human hepatocellular carcinoma cell line

    SciTech Connect

    Lobo-Yeo, A.; McSorley, C.; McFarlane, B.M.; Mieli-Vergani, G.; Mowat, A.P.; Vergani, D.

    1989-02-01

    A radioimmunometric technique for the detection of autoantibodies to liver membrane antigens has been developed using Alexander cells, a human hepatocellular carcinoma cell line. After incubation of Alexander cells with serum, antimembrane antibodies were detected by addition of /sup 125/I-labeled Protein A. Binding ratios in 15 children with uncontrolled autoimmune chronic active hepatitis and in seven children with primary sclerosing cholangitis were significantly higher than in 18 age-matched normal controls. Nine patients with inactive autoimmune chronic active hepatitis, 13 with alpha 1-antitrypsin deficiency and five with fulminant hepatic failure had ratios similar to controls. In nine patients with Wilson's disease, there was a modest but significant increase in binding ratio. In four children with autoimmune chronic active hepatitis, binding ratios fell during effective immunosuppressive therapy. Sera from patients with systemic lupus erythematosus or rheumatoid arthritis gave normal results, excluding that binding derives from Fc-mediated immune complex capture. A positive correlation was found between Alexander cell binding values and anti-liver-specific protein antibody titers, suggesting that the two assays detect antibodies against shared antigenic determinants. The Alexander cell assay is a simple, rapid and sensitive technique to detect antibody to liver cell membrane antigens.

  15. A monoclonal antibody to human immunodeficiency virus type 1 which mediates cellular cytotoxicity and neutralization.

    PubMed Central

    Broliden, P A; Ljunggren, K; Hinkula, J; Norrby, E; Akerblom, L; Wahren, B

    1990-01-01

    Monoclonal antibodies (MAbs) were raised against human immunodeficiency virus type 1 gp120. One MAb, P4/D10, was found to mediate highly efficient antibody-dependent cellular cytotoxicity and virus neutralization. The reactivity was located to a major neutralizing region (amino acids 304 to 323) on gp120. Five other MAbs with a similar epitopic reactivity did not show any antibody-dependent cellulan cytotoxicity activity but had a virus-neutralizing capacity. PMID:2296090

  16. Naturally occurring anti-band-3 antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes

    SciTech Connect

    Lutz, H.U.; Bussolino, F.; Flepp, R.; Fasler, S.; Stammler, P.; Kazatchkine, M.D.; Arese, P.

    1987-11-01

    Treatment of erythrocytes with the thiol-specific oxidant azodicarboxylic acid bis(dimethylamide) (diamide) enhances their phagocytosis by adherent monocytes. Phagocytosis of diamide-treated erythrocytes required that the cells were opsonized with whole serum, since complement inactivation abolished phagocytosis. Opsonization with whole serum containing 20-100 times the physiological concentration of naturally occurring anti-band-3- antibodies enhanced phagocytosis of diamide-treated erythrocytes. High inputs of anti-band-3 also restored phagocytosis of erythrocytes that had been incubated with complement-inactivated serum. Elevated concentrations of anti-spectrin antibodies were ineffective in whole and complement-inactivated serum. Specific recognition of diamide-treated erythrocytes by anti-band-3 antibodies may be due to generation of anti-band-3 reactive protein oligomers on intact diamide-treated erythrocytes. Generation of such oligomers was dose-dependent with respect to diamide. Bound anti-band-3 alone was not sufficient to mediate phagocytosis. It resulted in deposition of complement component C3b on the cells through activation of the alternative complement pathway in amounts exceeding that of bound antibodies by two orders of magnitude. Thus, anti-band-3 and complement together mediate phagocytosis of oxidatively stressed erythrocytes, which simulate senescent erythrocytes with respect to bound antibody and complement.

  17. The Biological Effects of IL-21 Signaling on B-Cell-Mediated Responses in Organ Transplantation

    PubMed Central

    Wu, Yongkang; van Besouw, Nicole M.; Shi, Yunying; Hoogduijn, Martin J.; Wang, Lanlan; Baan, Carla C.

    2016-01-01

    Antibody-mediated rejection has emerged as one of the major issues limiting the success of organ transplantation. It exerts a highly negative impact on graft function and outcome, and effective treatment is lacking. The triggers for antibody development, and the mechanisms leading to graft dysfunction and failure, are incompletely understood. The production of antibodies is dependent on instructions from various immunocytes including CD4 T-helper cells that secrete interleukin (IL)-21 and interact with antigen-specific B-cells via costimulatory molecules. In this article, we discuss the role of IL-21 in the activation and differentiation of B-cells and consider the mechanisms of IL-21 and B-cell interaction. An improved understanding of the biological mechanisms involved in antibody-mediated complications after organ transplantation could lead to the development of novel therapeutic strategies, which control humoral alloreactivity, potentially preventing and treating graft-threatening antibody-mediated rejection.

  18. The Biological Effects of IL-21 Signaling on B-Cell-Mediated Responses in Organ Transplantation

    PubMed Central

    Wu, Yongkang; van Besouw, Nicole M.; Shi, Yunying; Hoogduijn, Martin J.; Wang, Lanlan; Baan, Carla C.

    2016-01-01

    Antibody-mediated rejection has emerged as one of the major issues limiting the success of organ transplantation. It exerts a highly negative impact on graft function and outcome, and effective treatment is lacking. The triggers for antibody development, and the mechanisms leading to graft dysfunction and failure, are incompletely understood. The production of antibodies is dependent on instructions from various immunocytes including CD4 T-helper cells that secrete interleukin (IL)-21 and interact with antigen-specific B-cells via costimulatory molecules. In this article, we discuss the role of IL-21 in the activation and differentiation of B-cells and consider the mechanisms of IL-21 and B-cell interaction. An improved understanding of the biological mechanisms involved in antibody-mediated complications after organ transplantation could lead to the development of novel therapeutic strategies, which control humoral alloreactivity, potentially preventing and treating graft-threatening antibody-mediated rejection. PMID:27602031

  19. The Biological Effects of IL-21 Signaling on B-Cell-Mediated Responses in Organ Transplantation.

    PubMed

    Wu, Yongkang; van Besouw, Nicole M; Shi, Yunying; Hoogduijn, Martin J; Wang, Lanlan; Baan, Carla C

    2016-01-01

    Antibody-mediated rejection has emerged as one of the major issues limiting the success of organ transplantation. It exerts a highly negative impact on graft function and outcome, and effective treatment is lacking. The triggers for antibody development, and the mechanisms leading to graft dysfunction and failure, are incompletely understood. The production of antibodies is dependent on instructions from various immunocytes including CD4 T-helper cells that secrete interleukin (IL)-21 and interact with antigen-specific B-cells via costimulatory molecules. In this article, we discuss the role of IL-21 in the activation and differentiation of B-cells and consider the mechanisms of IL-21 and B-cell interaction. An improved understanding of the biological mechanisms involved in antibody-mediated complications after organ transplantation could lead to the development of novel therapeutic strategies, which control humoral alloreactivity, potentially preventing and treating graft-threatening antibody-mediated rejection. PMID:27602031

  20. Identification of Aleutian Mink Disease Parvovirus Capsid Sequences Mediating Antibody-Dependent Enhancement of Infection, Virus Neutralization, and Immune Complex Formation

    PubMed Central

    Bloom, Marshall E.; Best, Sonja M.; Hayes, Stanley F.; Wells, Richard D.; Wolfinbarger, James B.; McKenna, Robert; Agbandje-McKenna, Mavis

    2001-01-01

    Aleutian mink disease parvovirus (ADV) causes a persistent infection associated with circulating immune complexes, immune complex disease, hypergammaglobulinemia, and high levels of antiviral antibody. Although antibody can neutralize ADV infectivity in Crandell feline kidney cells in vitro, virus is not cleared in vivo, and capsid-based vaccines have proven uniformly ineffective. Antiviral antibody also enables ADV to infect macrophages, the target cells for persistent infection, by Fc-receptor-mediated antibody-dependent enhancement (ADE). The antibodies involved in these unique aspects of ADV pathogenesis may have specific targets on the ADV capsid. Prominent differences exist between the structure of ADV and other, more-typical parvoviruses, which can be accounted for by short peptide sequences in the flexible loop regions of the capsid proteins. In order to determine whether these short sequences are targets for antibodies involved in ADV pathogenesis, we studied heterologous antibodies against several peptides present in the major capsid protein, VP2. Of these antibodies, a polyclonal rabbit antibody to peptide VP2:428-446 was the most interesting. The anti-VP2:428-446 antibody aggregated virus particles into immune complexes, mediated ADE, and neutralized virus infectivity in vitro. Thus, antibody against this short peptide can be implicated in key facets of ADV pathogenesis. Structural modeling suggested that surface-exposed residues of VP2:428-446 are readily accessible for antibody binding. The observation that antibodies against a single target peptide in the ADV capsid can mediate both neutralization and ADE may explain the failure of capsid-based vaccines. PMID:11602751

  1. Recombinant-antibody-mediated resistance against Tomato yellow leaf curl virus in Nicotiana benthamiana.

    PubMed

    Safarnejad, Mohammad Reza; Fischer, Rainer; Commandeur, Ulrich

    2009-01-01

    Tomato yellow leaf curl virus (TYLCV) is a geminivirus species whose members cause severe crop losses in the tropics and subtropics. We report the expression of a single-chain variable fragment (scFv) antibody that protected Nicotiana benthamiana plants from a prevalent Iranian isolate of the virus (TYLCV-Ir). Two recombinant antibodies (scFv-ScRep1 and scFv-ScRep2) interacting with the multifunctional replication initiator protein (Rep) were obtained from phage display libraries and expressed in plants, both as stand-alone proteins and as N-terminal GFP fusions. Initial results indicated that both scFvs and both fusions accumulated to a detectable level in the cytosol and nucleus of plant cells. Transgenic plants challenged with TYLCV-Ir showed that the scFv-ScRep1, but more so the fusion proteins, were able to suppress TYLCV-Ir replication. These results show that expression of a scFv-ScRep1-GFP fusion protein can attenuate viral DNA replication and prevent the development of disease symptoms. The present article describes the first successful application of a recombinant antibody-mediated resistance approach against a plant DNA virus. PMID:19234665

  2. The specialized proresolving mediator 17-HDHA enhances the antibody-mediated immune response against influenza virus: Anew class of adjuvant?a

    PubMed Central

    Ramon, Sesquile; Baker, Steven F.; Sahler, Julie M.; Kim, Nina; Feldsott, Eric A.; Serhan, Charles N.; Martínez-Sobrido, Luis; Topham, David J.; Phipps, Richard P.

    2014-01-01

    Influenza viruses remain a critical global health concern. More efficacious vaccines are needed to protect against influenza virus, yet few adjuvants are approved for routine use. Specialized proresolving mediators (SPMs) are powerful endogenous bioactive regulators of inflammation, with great clinical translational properties. Here, we investigated the ability of the SPM 17-HDHA to enhance the adaptive immune response using an OVA immunization model and a pre-clinical influenza vaccination mouse model. Our findings revealed that mice immunized with OVA plus 17-HDHA or with H1N1-derived HA protein plus 17-HDHA increased antigen-specific antibody titers. 17-HDHA increased the number of antibody-secreting cells in vitro as well as the number of HA-specific antibody secreting cells present in the bone marrow. Importantly, the 17-HDHA-mediated increased antibody production was more protective against live pH1N1 influenza infection in mice. This is the first report on the biological effects of omega-3-derived SPMs on the humoral immune response. These findings illustrate a previously unknown biological link between proresolution signals and the adaptive immune system. Furthermore, this work has important implications for the understanding of B cell biology, as well as the development of new potential vaccine adjuvants. PMID:25392529

  3. Antibody-mediated neutralization of myelin-associated EphrinB3 accelerates CNS remyelination.

    PubMed

    Syed, Yasir A; Zhao, Chao; Mahad, Don; Möbius, Wiebke; Altmann, Friedrich; Foss, Franziska; Sentürk, Aycan; Acker-Palmer, Amparo; Lubec, Gert; Lilley, Kathryn; Franklin, Robin J M; Nave, Klaus-A; Kotter, Mark R N

    2016-02-01

    Remyelination in multiple sclerosis (MS) lesions often remains incomplete despite the presence of oligodendrocyte progenitor cells (OPCs). Amongst other factors, successful remyelination depends on the phagocytic clearance of myelin debris. However, the proteins in myelin debris that act as potent and selective inhibitors on OPC differentiation and inhibit CNS remyelination remain unknown. Here, we identify the transmembrane signalling protein EphrinB3 as important mediator of this inhibition, using a protein analytical approach in combination with a primary rodent OPC assay. In the presence of EphrinB3, OPCs fail to differentiate. In a rat model of remyelination, infusion of EphrinB3 inhibits remyelination. In contrast, masking EphrinB3 epitopes using antibodies promotes remyelination. Finally, we identify EphrinB3 in MS lesions and demonstrate that MS lesion extracts inhibit OPC differentiation while antibody-mediated masking of EphrinB3 epitopes promotes it. Our findings suggest that EphrinB3 could be a target for therapies aiming at promoting remyelination in demyelinating disease.

  4. Antibody-mediated neutralization of myelin-associated EphrinB3 accelerates CNS remyelination.

    PubMed

    Syed, Yasir A; Zhao, Chao; Mahad, Don; Möbius, Wiebke; Altmann, Friedrich; Foss, Franziska; Sentürk, Aycan; Acker-Palmer, Amparo; Lubec, Gert; Lilley, Kathryn; Franklin, Robin J M; Nave, Klaus-A; Kotter, Mark R N

    2016-02-01

    Remyelination in multiple sclerosis (MS) lesions often remains incomplete despite the presence of oligodendrocyte progenitor cells (OPCs). Amongst other factors, successful remyelination depends on the phagocytic clearance of myelin debris. However, the proteins in myelin debris that act as potent and selective inhibitors on OPC differentiation and inhibit CNS remyelination remain unknown. Here, we identify the transmembrane signalling protein EphrinB3 as important mediator of this inhibition, using a protein analytical approach in combination with a primary rodent OPC assay. In the presence of EphrinB3, OPCs fail to differentiate. In a rat model of remyelination, infusion of EphrinB3 inhibits remyelination. In contrast, masking EphrinB3 epitopes using antibodies promotes remyelination. Finally, we identify EphrinB3 in MS lesions and demonstrate that MS lesion extracts inhibit OPC differentiation while antibody-mediated masking of EphrinB3 epitopes promotes it. Our findings suggest that EphrinB3 could be a target for therapies aiming at promoting remyelination in demyelinating disease. PMID:26687980

  5. A game of numbers: the stoichiometry of antibody-mediated neutralization of flavivirus infection

    PubMed Central

    Pierson, Theodore C.; Diamond, Michael S.

    2016-01-01

    The humoral response contributes to the protection against viral pathogens. Although antibodies have the potential to inhibit viral infections via several mechanisms, an ability to neutralize viruses directly may be particularly important. Neutralizing antibody titers are commonly used as predictors of protection from infection, especially in the context of vaccine responses and immunity. Despite the simplicity of the concept, how antibody binding results in virus inactivation is incompletely understood despite decades of research. Flaviviruses have been an attractive system in which to seek a structural and quantitative understanding of how antibody interactions with virions modulate infection because of the contribution of antibodies to both protection and pathogenesis. This review will present a stoichiometric model of antibody-mediated neutralization of flaviviruses and discuss how these concepts can inform the development of vaccines and antibody-based therapeutics. PMID:25595803

  6. Anti-DNA antibody mediated catalysis is isotype dependent.

    PubMed

    Xia, Yumin; Eryilmaz, Ertan; Zhang, Qiuting; Cowburn, David; Putterman, Chaim

    2016-01-01

    Anti-DNA antibodies are the serological hallmark of systemic lupus erythematosus, and participate in the pathogenesis of lupus nephritis by cross-reacting with multiple renal antigens. Previously, using a panel of murine anti-DNA IgGs that share identical variable regions but that differ in the constant regions, we demonstrated that the cross-reaction and renal pathogenicity of anti-DNA antibodies are isotype dependent. In this study, we investigated the catalytic potential of this anti-DNA antibody panel, and determined its isotype dependency. The three isotype switch variants (IgG1, IgG2a, IgG2b) and the parent IgG3 PL9-11 anti-DNA antibodies were compared in their catalysis of 500 base pair linear double stranded DNA and a 12-mer peptide (ALWPPNLHAWVP), by gel analysis, MALDI-TOF mass spectrometry, and nuclear magnetic resonance spectroscopy. The binding affinity of anti-DNA antibodies to double stranded DNA and peptide antigens were assessed by ELISA and surface plasmon resonance. We found that the PL9-11 antibody isotypes vary significantly in their potential to catalyze the cleavage of both linear and double stranded DNA and the proteolysis of peptides. The degree of the cleavage and proteolysis increases with the incubation temperature and time. While different PL9-11 isotypes have the same initial attack sites within the ALWPPNLHAWVP peptide, there was no correlation between binding affinity to the peptide and proteolysis rates. In conclusion, the catalytic properties of anti-DNA antibodies are isotype dependent. This finding provides further evidence that antibodies that share the same variable region, but which have different constant regions, are functionally distinct. The catalytic effects modulated by antibody constant regions need to be considered in the design of therapeutic antibodies (abzymes) and peptides designed to block pathogenic autoantibodies. PMID:26655427

  7. Anti-DNA antibody mediated catalysis is isotype dependent.

    PubMed

    Xia, Yumin; Eryilmaz, Ertan; Zhang, Qiuting; Cowburn, David; Putterman, Chaim

    2016-01-01

    Anti-DNA antibodies are the serological hallmark of systemic lupus erythematosus, and participate in the pathogenesis of lupus nephritis by cross-reacting with multiple renal antigens. Previously, using a panel of murine anti-DNA IgGs that share identical variable regions but that differ in the constant regions, we demonstrated that the cross-reaction and renal pathogenicity of anti-DNA antibodies are isotype dependent. In this study, we investigated the catalytic potential of this anti-DNA antibody panel, and determined its isotype dependency. The three isotype switch variants (IgG1, IgG2a, IgG2b) and the parent IgG3 PL9-11 anti-DNA antibodies were compared in their catalysis of 500 base pair linear double stranded DNA and a 12-mer peptide (ALWPPNLHAWVP), by gel analysis, MALDI-TOF mass spectrometry, and nuclear magnetic resonance spectroscopy. The binding affinity of anti-DNA antibodies to double stranded DNA and peptide antigens were assessed by ELISA and surface plasmon resonance. We found that the PL9-11 antibody isotypes vary significantly in their potential to catalyze the cleavage of both linear and double stranded DNA and the proteolysis of peptides. The degree of the cleavage and proteolysis increases with the incubation temperature and time. While different PL9-11 isotypes have the same initial attack sites within the ALWPPNLHAWVP peptide, there was no correlation between binding affinity to the peptide and proteolysis rates. In conclusion, the catalytic properties of anti-DNA antibodies are isotype dependent. This finding provides further evidence that antibodies that share the same variable region, but which have different constant regions, are functionally distinct. The catalytic effects modulated by antibody constant regions need to be considered in the design of therapeutic antibodies (abzymes) and peptides designed to block pathogenic autoantibodies.

  8. T lymphocytes expressing a CD16 signaling receptor exert antibody-dependent cancer cell killing.

    PubMed

    Kudo, Ko; Imai, Chihaya; Lorenzini, Paolo; Kamiya, Takahiro; Kono, Koji; Davidoff, Andrew M; Chng, Wee Joo; Campana, Dario

    2014-01-01

    To expand applications for T-cell-based immunotherapy in cancer, we designed a receptor that binds the Fc portion of human immunoglobulins and delivers activation signals. The construct included the high-affinity CD16 (FCGR3A) V158 variant, CD8α hinge, and transmembrane domains, along with signaling domains from CD3ζ and 4-1BB (TNFRSF9), forming a chimeric receptor termed CD16V-BB-ζ. After retrovirus-mediated expression in human T cells, CD16V-BB-ζ bound humanized antibodies with higher affinity than a control receptor containing the more common F158 variant. Engagement of CD16V-BB-ζ provoked T-cell activation, exocytosis of lytic granules, and sustained proliferation, with a mean cell recovery after 4-week coculture with Daudi lymphoma cells and rituximab of nearly 70-fold relative to input cells. In contrast, unbound antibody alone produced no effect. CD16V-BB-ζ T cells specifically killed lymphoma cells and primary chronic lymphocytic leukemia cells in combination with rituximab at a low effector:target ratio, even when assayed on mesenchymal cells. Trastuzumab triggered CD16V-BB-ζ-mediated killing of HER2 (ERBB2)(+) breast and gastric cancer cells; similar results were obtained with an anti-GD2 antibody in neuroblastoma and osteosarcoma cells. Furthermore, coadministration of CD16V-BB-ζ T cells with immunotherapeutic antibodies exerted considerable antitumor activity in vivo. Signaling mediated by 4-1BB-CD3ζ induced higher T-cell activation, proliferation, and cytotoxicity than CD3ζ or FcεRIγ, and the receptor was expressed effectively after mRNA electroporation without viral vectors, facilitating clinical translation. Our results offer preclinical proof of concept for CD16V-BB-ζ as a universal, next-generation chimeric receptor with the potential to augment the efficacy of antibody therapies for cancer.

  9. What Lies Beneath: Antibody Dependent Natural Killer Cell Activation by Antibodies to Internal Influenza Virus Proteins.

    PubMed

    Vanderven, Hillary A; Ana-Sosa-Batiz, Fernanda; Jegaskanda, Sinthujan; Rockman, Steven; Laurie, Karen; Barr, Ian; Chen, Weisan; Wines, Bruce; Hogarth, P Mark; Lambe, Teresa; Gilbert, Sarah C; Parsons, Matthew S; Kent, Stephen J

    2016-06-01

    The conserved internal influenza proteins nucleoprotein (NP) and matrix 1 (M1) are well characterised for T cell immunity, but whether they also elicit functional antibodies capable of activating natural killer (NK) cells has not been explored. We studied NP and M1-specific ADCC activity using biochemical, NK cell activation and killing assays with plasma from healthy and influenza-infected subjects. Healthy adults had antibodies to M1 and NP capable of binding dimeric FcγRIIIa and activating NK cells. Natural symptomatic and experimental influenza infections resulted in a rise in antibody dependent NK cell activation post-infection to the hemagglutinin of the infecting strain, but changes in NK cell activation to M1 and NP were variable. Although antibody dependent killing of target cells infected with vaccinia viruses expressing internal influenza proteins was not detected, opsonising antibodies to NP and M1 likely contribute to an antiviral microenvironment by stimulating innate immune cells to secrete cytokines early in infection. We conclude that effector cell activating antibodies to conserved internal influenza proteins are common in healthy and influenza-infected adults. Given the significance of such antibodies in animal models of heterologous influenza infection, the definition of their importance and mechanism of action in human immunity to influenza is essential. PMID:27428437

  10. CD4+ T Cells Promote Antibody Production but Not Sustained Affinity Maturation during Borrelia burgdorferi Infection

    PubMed Central

    Elsner, Rebecca A.; Hastey, Christine J.

    2014-01-01

    CD4 T cells are crucial for enhancing B cell-mediated immunity, supporting the induction of high-affinity, class-switched antibody responses, long-lived plasma cells, and memory B cells. Previous studies showed that the immune response to Borrelia burgdorferi appears to lack robust T-dependent B cell responses, as neither long-lived plasma cells nor memory B cells form for months after infection, and nonswitched IgM antibodies are produced continuously during this chronic disease. These data prompted us to evaluate the induction and functionality of B. burgdorferi infection-induced CD4 TFH cells. We report that CD4 T cells were effectively primed and TFH cells induced after B. burgdorferi infection. These CD4 T cells contributed to the control of B. burgdorferi burden and supported the induction of B. burgdorferi-specific IgG responses. However, while affinity maturation of antibodies against a prototypic T-dependent B. burgdorferi protein, Arthritis-related protein (Arp), were initiated, these increases were reversed later, coinciding with the previously observed involution of germinal centers. The cessation of affinity maturation was not due to the appearance of inhibitory or exhausted CD4 T cells or a strong induction of regulatory T cells. In vitro T-B cocultures demonstrated that T cells isolated from B. burgdorferi-infected but not B. burgdorferi-immunized mice supported the rapid differentiation of B cells into antibody-secreting plasma cells rather than continued proliferation, mirroring the induction of rapid short-lived instead of long-lived T-dependent antibody responses in vivo. The data further suggest that B. burgdorferi infection drives the humoral response away from protective, high-affinity, and long-lived antibody responses and toward the rapid induction of strongly induced, short-lived antibodies of limited efficacy. PMID:25312948

  11. Neuropeptide-mediated regulation of hapten-specific IgE responses in mice. I. Substance P-mediated isotype-specific suppression of BPO-specific IgE antibody-forming cell responses induced in vivo and in vitro.

    PubMed

    Carucci, J A; Auci, D L; Herrick, C A; Durkin, H G

    1995-01-01

    The ability of substance P (SP) to regulate peak benzyl-penicilloyl (BPO)-specific IgE antibody-forming cell (AFC) responses in vivo and the ability of SP and other neuropeptides to regulate BPO-specific memory IgE AFC responses induced in vitro was determined. SP injected subcutaneously into BPO-keyhole limpet hemocyanin (BPO-KLH)-sensitized mice at the time of peak IgE responses suppressed these responses within 48 h (> 90%). The suppression obtained was IgE isotype-specific, dose-dependent, and transient. When spleen cells from immunized mice were cultured for 5 days with BPO-KLH, peak memory IgE AFC responses were induced in vitro. Inclusion of either SP or vasoactive intestinal peptide (VIP), but not neurotensin, serotonin, somatostatin, or gastrin, in cultures suppressed these responses in isotype-specific, dose-dependent fashion (approximately 70%). SP-, but not VIP-mediated suppression of IgE responses was abrogated by inclusion of anti-IFN gamma culture.

  12. Monoclonal antibodies against plant cell wall polysaccharides

    SciTech Connect

    Hahn, M.G.; Bucheli, E.; Darvill, A.; Albersheim, P. )

    1989-04-01

    Monoclonal antibodies (McAbs) are useful tools to probe the structure of plant cell wall polysaccharides and to localize these polysaccharides in plant cells and tissues. Murine McAbs were generated against the pectic polysaccharide, rhamnogalacturonan I (RG-I), isolated from suspension-cultured sycamore cells. The McAbs that were obtained were grouped into three classes based upon their reactivities with a variety of plant polysaccharides and membrane glycoproteins. Eleven McAbs (Class I) recognize epitope(s) that appear to be immunodominant and are found in RG-I from sycamore and maize, citrus pectin, polygalacturonic acid, and membrane glycoproteins from suspension-cultured cells of sycamore, maize, tobacco, parsley, and soybean. A second group of five McAbs (Class II) recognize epitope(s) present in sycamore RG-I, but do not bind to any of the other polysaccharides or glycoproteins recognized by Class I. Lastly, one McAb (Class III) reacts with sycamore RG-I, sycamore and tamarind xyloglucan, and sycamore and rice glucuronoarabinoxylan, but does not bind to maize RG-I, polygalacturonic acid or the plant membrane glycoproteins recognized by Class I. McAbs in Classes II and III are likely to be useful in studies of the structure, biosynthesis and localization of plant cell wall polysaccharides.

  13. Antibody-mediated reduction of {alpha}-ketoamides

    DOEpatents

    Schultz, P.G.; Gallop, M.A.

    1998-06-09

    Monoclonal antibodies raised against a 4-nitrophenyl phosphonate hapten catalyze the stereospecific reduction of an {alpha}-ketoamide to the corresponding {alpha}-hydroxyamide in the presence of an appropriate reducing agent.

  14. Antibody-mediated reduction of .alpha.-ketoamides

    DOEpatents

    Schultz, Peter G.; Gallop, Mark A.

    1998-01-01

    Monoclonal antibodies raised against a 4-nitrophenyl phosphonate hapten catalyze the stereospecific reduction of an .alpha.-ketoamide to the corresponding .alpha.-hydroxyamide in the presence of an appropriate reducing agent.

  15. Expression of conformationally constrained adhesion peptide in an antibody CDR loop and inhibition of natural killer cell cytotoxic activity by an antibody antigenized with the RGD motif.

    PubMed Central

    Zanetti, M; Filaci, G; Lee, R H; del Guercio, P; Rossi, F; Bacchetta, R; Stevenson, F; Barnaba, V; Billetta, R

    1993-01-01

    We report that an antibody engineered to express three Arg-Gly-Asp (RGD) repeats in the third complementarity-determining region of the heavy chain (antigenized antibody) efficiently inhibits the lysis of human erythroleukemia K-562 cells by natural killer (NK) cells. Synthetic peptides containing RGD did not inhibit. Inhibition was specific for the (RGD)3-containing loop and required simultaneous occupancy of the Fc receptor (CD16) on effector cells. The antigenized antibody inhibited other forms of cytotoxicity mediated by NK cells but not cytotoxicity mediated by major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL). A three-dimensional model of the engineered antibody loop shows the structure and physicochemical characteristics probably required for the ligand activity. The results indicate that an RGD motif is involved in the productive interaction between NK and target cells. Moreover, they show that peptide expression in the hypervariable loops of an antibody molecule is an efficient procedure for stabilizing oligopeptides within a limited spectrum of tertiary structures. This is a new approach towards imparting ligand properties to antibody molecules and can be used to study the biological function and specificity of short peptide motifs, including those involved in cell adhesion. Images PMID:8223447

  16. Mucosal priming of newborn mice with S. Typhi Ty21a expressing anthrax protective antigen (PA) followed by parenteral PA-boost induces B and T cell-mediated immunity that protects against infection bypassing maternal antibodies

    PubMed Central

    Ramirez, Karina; Ditamo, Yanina; Galen, James E.; Baillie, Les W. J.; Pasetti, Marcela F.

    2010-01-01

    The currently licensed anthrax vaccine has several limitations and its efficacy has been proven only in adults. Effective immunization of newborns and infants requires adequate stimulation of their immune system, which is competent but not fully activated. We explored the use of the licensed live attenuated S. Typhi vaccine strain Ty21a expressing Bacillus anthracis protective antigen [Ty21a(PA)] followed PA-alum as a strategy for immunizing the pediatric population. Newborn mice primed with a single dose of Ty21a(PA) exhibited high frequencies of mucosal IgA-secreting B cells and IFN-γ-secreting T cells during the neonatal period, none of which was detected in newborns immunized with a single dose of PA-alum. Priming with Ty21a(PA) followed by PA-boost resulted in high levels of PA-specific IgG, toxin-neutralizing and opsonophagocytic antibodies and increased frequency of bone marrow IgG plasma cells and memory B cells compared with repeated immunization with PA-alum alone. Robust B and T cell responses developed even in the presence of maternal antibodies. The prime-boost protected against systemic and respiratory infection. Mucosal priming with a safe and effective S. Typhi-based anthrax vaccine followed by PA-boost could serve as a practical and effective prophylactic approach to prevent anthrax early in life. PMID:20619377

  17. Influence of IgG Subclass on Human Antimannan Antibody-Mediated Resistance to Hematogenously Disseminated Candidiasis in Mice.

    PubMed

    Nishiya, Casey T; Boxx, Gayle M; Robison, Kerry; Itatani, Carol; Kozel, Thomas R; Zhang, Mason X

    2015-11-16

    Candida albicans is a yeast-like pathogen and can cause life-threatening systemic candidiasis. Its cell surface is enriched with mannan that is resistant to complement activation. Previously, we developed the recombinant human IgG1 antimannan antibody M1g1. M1g1 was found to promote complement activation and phagocytosis and protect mice from systemic candidiasis. Here, we evaluate the influence of IgG subclass on antimannan antibody-mediated protection. Three IgG subclass variants of M1g1 were constructed: M1g2, M1g3, and M1g4. The IgG subclass identity for each variant was confirmed with DNA sequence and subclass-specific antibodies. These variants contain identical M1 Fabs and exhibited similar binding affinities for C. albicans yeast and purified mannan. Yeast cells and hyphae recovered from the kidney of antibody-treated mice with systemic candidiasis showed uniform binding of each variant, indicating constitutive expression of the M1 epitope and antibody opsonization in the kidney. All variants promoted deposition of both murine and human C3 onto the yeast cell surface, with M1g4 showing delayed activation, as determined by flow cytometry and immunofluorescence microscopy. M1g4-mediated complement activation was found to be associated with its M1 Fab that activates the alternative pathway in an Fc-independent manner. Treatment with each subclass variant extended the survival of mice with systemic candidiasis (P < 0.001). However, treatment with M1g1, M1g3, or M1g4, but not with M1g2, also reduced the kidney fungal burden (P < 0.001). Thus, the role of human antimannan antibody in host resistance to systemic candidiasis is influenced by its IgG subclass.

  18. A role for plasma cell targeting agents in immune tolerance induction in autoimmune disease and antibody responses to therapeutic proteins.

    PubMed

    Rosenberg, A S; Pariser, A R; Diamond, B; Yao, L; Turka, L A; Lacana, E; Kishnani, P S

    2016-04-01

    Antibody responses to life saving therapeutic protein products, such as enzyme replacement therapies (ERT) in the setting of lysosomal storage diseases, have nullified product efficacy and caused clinical deterioration and death despite treatment with immune-suppressive therapies. Moreover, in some autoimmune diseases, pathology is mediated by a robust antibody response to endogenous proteins such as is the case in pulmonary alveolar proteinosis, mediated by antibodies to Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF). In this work, we make the case that in such settings, when the antibody response is high titered, sustained, and refractory to immune suppressive treatments, the antibody response is mediated by long-lived plasma cells which are relatively unperturbed by immune suppressants including rituximab. However, long-lived plasma cells can be targeted by proteasome inhibitors such as bortezomib. Recent reports of successful reversal of antibody responses with bortezomib in the settings of ERT and Thrombotic Thrombocytopenic Purpura (TTP) argue that the safety and efficacy of such plasma cell targeting agents should be evaluated in larger scale clinical trials to delineate the risks and benefits of such therapies in the settings of antibody-mediated adverse effects to therapeutic proteins and autoantibody mediated pathology. PMID:26928739

  19. T cell tolerance induced by therapeutic antibodies.

    PubMed

    Cobbold, Stephen P

    2005-09-29

    Ever since the discovery of Medawar, over 50 years ago, that immunological tolerance was an acquired phenomenon that could be manipulated in neonatal mice, the ability to induce therapeutic tolerance against autoantigens, allergens and organ grafts has been a major driving force in immunology. Within the last 20 years we have found that a brief treatment with monoclonal antibodies that block certain functional molecules on the surface of the T cell is able to reprogramme the established immune repertoire of the adult mouse, allowing indefinite acceptance of allografts or effective curing of autoimmune diseases. We are only now just beginning to define many of the regulatory mechanisms that induce and maintain the tolerant state with the aim of being able to safely and reliably apply these technologies to human clinical situations. PMID:16147534

  20. Human T-cell subset requirements for the production of specific anti-influenza virus antibody in vitro

    SciTech Connect

    Yarchoan, R.; Biddison, W.E.; Schneider, H.S.; Nelson, D.L.

    1982-04-01

    Studies were undertaken to define the helper T-cell requirements for in vitro specific antibody production to influenza virus. Subpopulations of human T cells were separated on the basis of their reactivity with the monoclonal antibody OKT4. B cells cultured with OKT4+ T cells produced specific antibody to influenza virus, while B cells cultured with OKT4- T cells did not. Irradiation (1200 rads) of the OKT4- subset to potentially eliminate suppressor-cell activity did not augment the helper-cell function of that subset. Thus, unlike the cytotoxic T-cell response to influenza, help for an in vitro antibody response is mediated only by OKT4+ T cells.

  1. Anti-HIV-1 antibody-dependent cellular cytotoxicity mediated by hyperimmune bovine colostrum IgG.

    PubMed

    Kramski, Marit; Lichtfuss, Gregor F; Navis, Marjon; Isitman, Gamze; Wren, Leia; Rawlin, Grant; Center, Rob J; Jaworowski, Anthony; Kent, Stephen J; Purcell, Damian F J

    2012-10-01

    Antibodies with antibody-dependent cellular cytotoxicity (ADCC) activity play an important role in protection against HIV-1 infection, but generating sufficient amounts of antibodies to study their protective efficacy is difficult. HIV-specific IgG can be easily and inexpensively produced in large quantities using bovine colostrum. We previously vaccinated cows with HIV-1 envelope gp140 and elicited high titers of anti-gp140-binding IgG in colostrum. In the present study, we determined whether bovine antibodies would also demonstrate specific cytotoxic activity. We found that bovine IgG bind to Fcγ-receptors (FcγRs) on human neutrophils, monocytes, and NK cells in a dose-dependent manner. Antibody-dependent killing was observed in the presence of anti-HIV-1 colostrum IgG but not nonimmune colostrum IgG. Killing was dependent on Fc and FcγR interaction since ADDC activity was not seen with F(ab')(2) fragments. ADCC activity was primarily mediated by CD14(+) monocytes with FcγRIIa (CD32a) as the major receptor responsible for monocyte-mediated ADCC in response to bovine IgG. In conclusion, we demonstrate that bovine anti-HIV colostrum IgG have robust HIV-1-specific ADCC activity and therefore offer a useful source of antibodies able to provide a rapid and potent response against HIV-1 infection. This could assist the development of novel Ab-mediated approaches for prevention of HIV-1 transmission. PMID:22730083

  2. Cell-Mediated Drugs Delivery

    PubMed Central

    Batrakova, Elena V.; Gendelman, Howard E.; Kabanov, Alexander V.

    2011-01-01

    INTRODUCTION Drug targeting to sites of tissue injury, tumor or infection with limited toxicity is the goal for successful pharmaceutics. Immunocytes (including mononuclear phagocytes (dendritic cells, monocytes and macrophages), neutrophils, and lymphocytes) are highly mobile; they can migrate across impermeable barriers and release their drug cargo at sites of infection or tissue injury. Thus immune cells can be exploited as trojan horses for drug delivery. AREAS COVERED IN THIS REVIEW This paper reviews how immunocytes laden with drugs can cross the blood brain or blood tumor barriers, to facilitate treatments for infectious diseases, injury, cancer, or inflammatory diseases. The promises and perils of cell-mediated drug delivery are reviewed, with examples of how immunocytes can be harnessed to improve therapeutic end points. EXPERT OPINION Using cells as delivery vehicles enables targeted drug transport, and prolonged circulation times, along with reductions in cell and tissue toxicities. Such systems for drug carriage and targeted release represent a novel disease combating strategy being applied to a spectrum of human disorders. The design of nanocarriers for cell-mediated drug delivery may differ from those used for conventional drug delivery systems; nevertheless, engaging different defense mechanisms into drug delivery may open new perspectives for the active delivery of drugs. PMID:21348773

  3. TIM-1 signaling in B cells regulates antibody production

    SciTech Connect

    Ma, Juan; Usui, Yoshihiko; Takeda, Kazuyoshi; Harada, Norihiro; Yagita, Hideo; Okumura, Ko; Akiba, Hisaya

    2011-03-11

    Highlights: {yields} TIM-1 is highly expressed on anti-IgM + anti-CD40-stimulated B cells. {yields} Anti-TIM-1 mAb enhanced proliferation and Ig production on activated B cell in vitro. {yields} TIM-1 signaling regulates Ab production by response to TI-2 and TD antigens in vivo. -- Abstract: Members of the T cell Ig and mucin (TIM) family have recently been implicated in the control of T cell-mediated immune responses. In this study, we found TIM-1 expression on anti-IgM- or anti-CD40-stimulated splenic B cells, which was further up-regulated by the combination of anti-IgM and anti-CD40 Abs. On the other hand, TIM-1 ligand was constitutively expressed on B cells and inducible on anti-CD3{sup +} anti-CD28-stimulated CD4{sup +} T cells. In vitro stimulation of activated B cells by anti-TIM-1 mAb enhanced proliferation and expression of a plasma cell marker syndecan-1 (CD138). We further examined the effect of TIM-1 signaling on antibody production in vitro and in vivo. Higher levels of IgG2b and IgG3 secretion were detected in the culture supernatants of the anti-TIM-1-stimulated B cells as compared with the control IgG-stimulated B cells. When immunized with T-independent antigen TNP-Ficoll, TNP-specific IgG1, IgG2b, and IgG3 Abs were slightly increased in the anti-TIM-1-treated mice. When immunized with T-dependent antigen OVA, serum levels of OVA-specific IgG2b, IgG3, and IgE Abs were significantly increased in the anti-TIM-1-treated mice as compared with the control IgG-treated mice. These results suggest that TIM-1 signaling in B cells augments antibody production by enhancing B cell proliferation and differentiation.

  4. Idiotype and antigen-specific T cell responses in mice on immunization with antigen, antibody, and anti-idiotypic antibody.

    PubMed

    Mitra-Kaushik, S; Shaila, M S; Karande, A K; Nayak, R

    2001-05-01

    Idiotypic determinants of immunoglobulin molecules can evoke both CD4(+) and CD8(+) T responses and exist not only as the integral components of a bona fide antigen binding receptor but also as distinct molecular entities in the processed forms on the cell surface of B lymphocytes. The present work provides experimental evidence for the concept that regulation of memory B cell populations can be achieved through the presentation of idiotypic and anti-idiotypic determinants to helper and cytotoxic cell. The potential of B cells to present antigens to helper and cytotoxic T cells through class II and class I MHC suggests a mechanism by which both B and T cell homeostasis can be maintained. We provide evidence for the generation of idiotype- and antigen-specific Th and Tc cells upon immunization of syngenic mice with antigen or idiotypic antibody (Ab1) or anti-idiotypic antibody (Ab2). The selective activation and proliferation of the antigen-specific Th and Tc cells mediated by idiotypic stimulation observed in these experiments suggests a B-cell-driven mechanism for the maintenance of antigen-specific T cell memory in the absence of antigenic stimulation, under certain conditions.

  5. Monoclonal Antibody and an Antibody-Toxin Conjugate to a Cell Surface Proteoglycan of Melanoma Cells Suppress in vivo Tumor Growth

    NASA Astrophysics Data System (ADS)

    Bumol, T. F.; Wang, Q. C.; Reisfeld, R. A.; Kaplan, N. O.

    1983-01-01

    A monoclonal antibody directed against a cell surface chondroitin sulfate proteoglycan of human melanoma cells, 9.2.27, and its diphtheria toxin A chain (DTA) conjugate were investigated for their effects on in vitro protein synthesis and in vivo tumor growth of human melanoma cells. The 9.2.27 IgG and its DTA conjugate display similar serological activities against melanoma target cells but only the conjugate can induce consistent in vitro inhibition of protein synthesis and toxicity in M21 melanoma cells. However, both 9.2.27 IgG and its DTA conjugate effect significant suppression of M21 tumor growth in vivo in an immunotherapy model of a rapidly growing tumor in athymic nu/nu mice, suggesting that other host mechanisms may mediate monoclonal antibody-induced tumor suppression.

  6. Single-domain antibody-based and linker-free bispecific antibodies targeting FcγRIII induce potent antitumor activity without recruiting regulatory T cells.

    PubMed

    Rozan, Caroline; Cornillon, Amélie; Pétiard, Corinne; Chartier, Martine; Behar, Ghislaine; Boix, Charlotte; Kerfelec, Brigitte; Robert, Bruno; Pèlegrin, André; Chames, Patrick; Teillaud, Jean-Luc; Baty, Daniel

    2013-08-01

    Antibody-dependent cell-mediated cytotoxicity, one of the most prominent modes of action of antitumor antibodies, suffers from important limitations due to the need for optimal interactions with Fcγ receptors. In this work, we report the design of a new bispecific antibody format, compact and linker-free, based on the use of llama single-domain antibodies that are capable of circumventing most of these limitations. This bispecific antibody format was created by fusing single-domain antibodies directed against the carcinoembryonic antigen and the activating FcγRIIIa receptor to human Cκ and CH1 immunoglobulin G1 domains, acting as a natural dimerization motif. In vitro and in vivo characterization of these Fab-like bispecific molecules revealed favorable features for further development as a therapeutic molecule. They are easy to produce in Escherichia coli, very stable, and elicit potent lysis of tumor cells by human natural killer cells at picomolar concentrations. Unlike conventional antibodies, they do not engage inhibitory FcγRIIb receptor, do not compete with serum immunoglobulins G for receptor binding, and their cytotoxic activity is independent of Fc glycosylation and FcγRIIIa polymorphism. As opposed to anti-CD3 bispecific antitumor antibodies, they do not engage regulatory T cells as these latter cells do not express FcγRIII. Studies in nonobese diabetic/severe combined immunodeficient gamma mice xenografted with carcinoembryonic antigen-positive tumor cells showed that Fab-like bispecific molecules in the presence of human peripheral blood mononuclear cells significantly slow down tumor growth. This new compact, linker-free bispecific antibody format offers a promising approach for optimizing antibody-based therapies.

  7. CEA TCB: A novel head-to-tail 2:1 T cell bispecific antibody for treatment of CEA-positive solid tumors.

    PubMed

    Bacac, Marina; Klein, Christian; Umana, Pablo

    2016-08-01

    Carcinoembryonic antigen T cell bispecific antibody (CEA TCB) is a bispecific antibody used to recognize CEA and CD3e via a novel molecular format (2:1) that induces T cell-mediated killing of CEA over-expressing tumors while sparing primary cells with low CEA expression. CEA TCB treatment inhibits tumor growth and generates a highly inflamed tumor microenvironment. PMID:27622073

  8. Chimeric Antibody c.8B6 to O-Acetyl-GD2 Mediates the Same Efficient Anti-Neuroblastoma Effects as Therapeutic ch14.18 Antibody to GD2 without Antibody Induced Allodynia

    PubMed Central

    Cochonneau, Denis; Chaumette, Tanguy; Xiao, Wenhua; Diccianni, Mitchell B.; Barbet, Jacques; Yu, Alice L.; Paris, François; Sorkin, Linda S.; Birklé, Stéphane

    2014-01-01

    Background Anti-GD2 antibody is a proven therapy for GD2-postive neuroblastoma. Monoclonal antibodies against GD2, such as chimeric mAb ch14.18, have become benchmarks for neuroblastoma therapies. Pain, however, can limit immunotherapy with anti-GD2 therapeutic antibodies like ch14.18. This adverse effect is attributed to acute inflammation via complement activation on GD2-expressing nerves. Thus, new strategies are needed for the development of treatment intensification strategies to improve the outcome of these patients. Methodology/Principal Findings We established the mouse-human chimeric antibody c.8B6 specific to OAcGD2 in order to reduce potential immunogenicity in patients and to fill the need for a selective agent that can kill neuroblastoma cells without inducing adverse neurological side effects caused by anti-GD2 antibody immunotherapy. We further analyzed some of its functional properties compared with anti-GD2 ch14.18 therapeutic antibody. With the exception of allodynic activity, we found that antibody c.8B6 shares the same anti-neuroblastoma attributes as therapeutic ch14.18 anti-GD2 mAb when tested in cell-based assay and in vivo in an animal model. Conclusion/Significance The absence of OAcGD2 expression on nerve fibers and the lack of allodynic properties of c.8B6–which are believed to play a major role in mediating anti-GD2 mAb dose-limiting side effects–provide an important rationale for the clinical application of c.8B6 in patients with high-risk neuroblastoma. PMID:24520328

  9. CD13 mediates phagocytosis in human monocytic cells.

    PubMed

    Licona-Limón, Ileana; Garay-Canales, Claudia A; Muñoz-Paleta, Ofelia; Ortega, Enrique

    2015-07-01

    CD13 is a membrane-bound ectopeptidase, highly expressed on monocytes, macrophages, and dendritic cells. CD13 is involved in diverse functions, including degradation of peptide mediators, cellular adhesion, migration, viral endocytosis, signaling, and positive modulation of phagocytosis mediated by FcγRs and other phagocytic receptors. In this work, we explored whether besides acting as an accessory receptor, CD13 by itself is a primary phagocytic receptor. We found that hCD13 mediates efficient phagocytosis of large particles (erythrocytes) modified so as to interact with the cell only through CD13 in human macrophages and THP-1 monocytic cells. The extent of this phagocytosis is comparable with the phagocytosis mediated through the canonical phagocytic receptor FcγRI. Furthermore, we demonstrated that hCD13 expression in the nonphagocytic cell line HEK293 is sufficient to enable these cells to internalize particles bound through hCD13. CD13-mediated phagocytosis is independent of other phagocytic receptors, as it occurs in the absence of FcγRs, CR3, and most phagocytic receptors. Phagocytosis through CD13 is independent of its enzymatic activity but is dependent on actin rearrangement and activation of PI3K and is partially dependent on Syk activation. Moreover, the cross-linking of CD13 with antibodies rapidly induced pSyk in human macrophages. Finally, we observed that antibody-mediated cross-linking of hCD13, expressed in the murine macrophage-like J774 cell line, induces production of ROS. These results demonstrate that CD13 is a fully competent phagocytic receptor capable of mediating internalization of large particles.

  10. Late acute antibody mediated rejection after nine years of renal transplantation.

    PubMed

    Halim, Medhat Abdel; Al-Otaibi, Torki; Al-Waheeb, Salah; Tawab, Khaled Abdel; El Kholy, Osama; Nair, Prasad; Said, Tarek; Narayanan Nampoory, M R

    2010-11-01

    Acute antibody mediated rejection (AMR) is rarely reported as a long-term com-plication of renal transplantation, and it can present on top of another chronic pathology affecting the graft. A 45-year-old gentleman with chronic kidney disease due to unknown etiology received renal transplantation from his sister with 4 HLA mismatches. He received antithymocte globulin induction therapy and was maintained on steroids, azathioprine (AZA) and cyclosporine A (CsA). Up to eight years post-transplantation he was clinically and biochemically stable. He lost follow-up for about one year, and then presented with nephritic nephrotic syndrome and rise of serum creatinine (SCr.) to 210 μmol/L. Graft biopsy revealed picture suggestive of acute AMR on top of de novo membranoprolipherative glomerulonephritis (MPGN) with focal crescent formation, diffuse immune complex deposition and peritubular capillaries C4d positivity. Anti-HLA donor specific antibodies were highly positive for B and T cells class I and class II. The patient was treated with intravenous immunoglobulin, plasma exchange and anti-CD20 (rituximab). AZA was changed to mycophenolate mofetil and CsA to tacrolimus. He had partial response, but SCr. continued at 220 μmol/L.

  11. Univalent antibodies kill tumour cells in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Glennie, M. J.; Stevenson, G. T.

    1982-02-01

    Antibody molecules are bivalent, or less often multivalent, with each antibody site within a single molecule having the same specificity. Bivalency must enhance the tenacity of antibody attachment to cell surfaces, as dissociation will require simultaneous release at both sites. However, the bivalency of the antibody sometimes induces a target cell to undergo antigenic modulation1-3, thereby offering the cell a means of evading complement and the various effector cells recruited by the antibody. We have investigated the attack by univalent antibodies, which, despite removal of one antibody site, retain their Fc zones and hence their ability to recruit the killing agents, on neoplastic B lymphocytes of the guinea pig L2C line. Rabbit antibodies raised against surface immunoglobulin of these cells were partially digested with papain to yield the univalent Fab/c derivatives4,5. We report here that these derivatives showed enhanced cell killing both in vitro and in vivo, and that this enhancement appeared to derive from avoiding antigenic modulation.

  12. Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells.

    PubMed

    Meng, Weixu; Li, Leike; Xiong, Wei; Fan, Xuejun; Deng, Hui; Bett, Andrew J; Chen, Zhifeng; Tang, Aimin; Cox, Kara S; Joyce, Joseph G; Freed, Daniel C; Thoryk, Elizabeth; Fu, Tong-Ming; Casimiro, Danilo R; Zhang, Ningyan; A Vora, Kalpit; An, Zhiqiang

    2015-01-01

    Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.

  13. Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells.

    PubMed

    Meng, Weixu; Li, Leike; Xiong, Wei; Fan, Xuejun; Deng, Hui; Bett, Andrew J; Chen, Zhifeng; Tang, Aimin; Cox, Kara S; Joyce, Joseph G; Freed, Daniel C; Thoryk, Elizabeth; Fu, Tong-Ming; Casimiro, Danilo R; Zhang, Ningyan; A Vora, Kalpit; An, Zhiqiang

    2015-01-01

    Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs. PMID:25996084

  14. Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells

    PubMed Central

    Meng, Weixu; Li, Leike; Xiong, Wei; Fan, Xuejun; Deng, Hui; Bett, Andrew J; Chen, Zhifeng; Tang, Aimin; Cox, Kara S; Joyce, Joseph G; Freed, Daniel C; Thoryk, Elizabeth; Fu, Tong-Ming; Casimiro, Danilo R; Zhang, Ningyan; A Vora, Kalpit; An, Zhiqiang

    2015-01-01

    Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs. PMID:25996084

  15. B-cell memory and the persistence of antibody responses.

    PubMed Central

    MacLennan, I C; García de Vinuesa, C; Casamayor-Palleja, M

    2000-01-01

    Antigens such as viral envelope proteins and bacterial exotoxins induce responses which result in the production of neutralizing antibody. These responses persist for years and provide highly efficient defence against reinfection. During these antibody responses a proportion of participating B cells mutate the genes that encode their immunoglobulin variable regions. This can increase the affinity of the antibody, but can also induce autoreactive B cells. Selection mechanisms operate which allow the cells with high affinity for the provoking antigen to persist, while other B cells recruited into the response die. PMID:10794052

  16. Engineered Protease-resistant Antibodies with Selectable Cell-killing Functions*

    PubMed Central

    Kinder, Michelle; Greenplate, Allison R.; Grugan, Katharine D.; Soring, Keri L.; Heeringa, Katharine A.; McCarthy, Stephen G.; Bannish, Gregory; Perpetua, Meredith; Lynch, Frank; Jordan, Robert E.; Strohl, William R.; Brezski, Randall J.

    2013-01-01

    Molecularly engineered antibodies with fit-for-purpose properties will differentiate next generation antibody therapeutics from traditional IgG1 scaffolds. One requirement for engineering the most appropriate properties for a particular therapeutic area is an understanding of the intricacies of the target microenvironment in which the antibody is expected to function. Our group and others have demonstrated that proteases secreted by invasive tumors and pathological microorganisms are capable of cleaving human IgG1, the most commonly adopted isotype among monoclonal antibody therapeutics. Specific cleavage in the lower hinge of IgG1 results in a loss of Fc-mediated cell-killing functions without a concomitant loss of antigen binding capability or circulating antibody half-life. Proteolytic cleavage in the hinge region by tumor-associated or microbial proteases is postulated as a means of evading host immune responses, and antibodies engineered with potent cell-killing functions that are also resistant to hinge proteolysis are of interest. Mutation of the lower hinge region of an IgG1 resulted in protease resistance but also resulted in a profound loss of Fc-mediated cell-killing functions. In the present study, we demonstrate that specific mutations of the CH2 domain in conjunction with lower hinge mutations can restore and sometimes enhance cell-killing functions while still retaining protease resistance. By identifying mutations that can restore either complement- or Fcγ receptor-mediated functions on a protease-resistant scaffold, we were able to generate a novel protease-resistant platform with selective cell-killing functionality. PMID:23986451

  17. Antibody-dependent cellular cytotoxicity-mediated serotherapy against murine neuroblastoma. I. In vitro and in vivo treatment using normal, gamma-irradiated and immune-stimulated rat effector cells.

    PubMed

    Byfield, J E; Zerubavel, R; Fonkalsrud, E W

    1982-01-01

    The in vitro and in vivo activity has been investigated of antisera prepared against a murine (C-1300) neuroblastoma line (MNB) capable of differentiation. An antibody-dependent cellular cytotoxicity (ADCC) reaction was employed using rat spleen cells (RSC). ADCC activity in vitro (using 51Cr-release) was shown, but a maximum of only 50% of the immunologically releasable 51Cr was achieved. Nevertheless, in vivo (syngeneic mouse-tumor flank assay) significant delays were obtained in tumor onset and lethality. Under ideal circumstances, i.e., coating of tumor cells prior to inoculation and high RSC effector cell ratios, a significant number of animals could be cured of substantial tumor burdens (10(6) cells). While close proximity of the site of injection of effector cells was required (ectopic injections of RSC were ineffective), the anti-MNB ADCC was shown to be quite active in vivo without external precoating of the cells with antisera. RCS obtained from BCG-treated rats were more numerous and slightly more effective. RSC obtained from gamma-radiated animals retained normal activity. With appropriate antisera this approach could be useful under selected clinical circumstances.

  18. Iron as the Key Modulator of Hepcidin Expression in Erythroid Antibody-Mediated Hypoplasia

    PubMed Central

    Fernandes, J. C.; Garrido, P.; Ribeiro, S.; Rocha-Pereira, P.; Bronze-da-Rocha, E.; Belo, L.; Costa, E.; Reis, F.; Santos-Silva, A.

    2014-01-01

    Erythroid hypoplasia (EH) is a rare complication associated with recombinant human erythropoietin (rHuEPO) therapies, due to development of anti-rHuEPO antibodies; however, the underlying mechanisms remain poorly clarified. Our aim was to manage a rat model of antibody-mediated EH induced by rHuEPO and study the impact on iron metabolism and erythropoiesis. Wistar rats treated during 9 weeks with a high rHuEPO dose (200 IU) developed EH, as shown by anemia, reduced erythroblasts, reticulocytopenia, and plasmatic anti-rHuEPO antibodies. Serum iron was increased and associated with mRNA overexpression of hepatic hepcidin and other iron regulatory mediators and downregulation of matriptase-2; overexpression of divalent metal transporter 1 and ferroportin was observed in duodenum and liver. Decreased EPO expression was observed in kidney and liver, while EPO receptor was overexpressed in liver. Endogenous EPO levels were normal, suggesting that anti-rHuEPO antibodies blunted EPO function. Our results suggest that anti-rHuEPO antibodies inhibit erythropoiesis causing anemia. This leads to a serum iron increase, which seems to stimulate hepcidin expression despite no evidence of inflammation, thus suggesting iron as the key modulator of hepcidin synthesis. These findings might contribute to improving new therapeutic strategies against rHuEPO resistance and/or development of antibody-mediated EH in patients under rHuEPO therapy. PMID:25580431

  19. Enhanced antigen-antibody binding affinity mediated by an anti-idiotypic antibody

    SciTech Connect

    Sawutz, D.G.; Koury, R.; Homcy, C.J.

    1987-08-25

    The authors previously described the production of four monoclonal antibodies to the ..beta..-adrenergic receptor antagonist alprenolol. One of these antibodies, 5B7 (IgG/sub 2a/, kappa), was used to raise anti-idiotypic antisera in rabbits. In contrast to the expected results, one of the anti-idiotypic antisera (R9) promotes (/sup 125/I)iodocyanopinodolol (ICYP) binding to antibody 5B7. In the presence of R9, the dissociation constant decreases 100-fold from 20 to 0.3 nM. This increase in binding affinity of antibody 5B7 for ICYP is not observed in the presence of preimmune, rabbit anti-mouse or anti-idiotypic antisera generated to a monoclonal antibody of a different specificity. Furthermore, R9 in the absence of 5B7 does not bind ICYP. The F(ab) fragments of 5B7 and T9 behaved in a similar manner, and the soluble complex responsible for the high-affinity interaction with ICYP can be identified by gel filtration chromatography. The elution position of the complex is consistent with a 5B7 F(ab)-R9 F(ab) dimer, indicating that polyvalency is not responsible for the enhanced ligand binding. Kinetic analysis of ICYP-5B7 binding revealed that the rate of ICYP dissociation from 5B7 in the presence of R9 is approximately 100 times slower than in the absence of R9, consistent with the 100-fold change in binding affinity of 5B7 for ICYP. The available data best fit a model in which an anti-idiotypic antibody binds at or near the binding site of the idiotype participating in the formation of a hybrid ligand binding site. This would allow increased contact of the ligand with the idiotype-anti-idiotype complex and result in an enhanced affinity of the ligand interaction.

  20. Antibody-Mediated Oligodendrocyte Remyelination Promotes Axon Health in Progressive Demyelinating Disease.

    PubMed

    Wootla, Bharath; Denic, Aleksandar; Watzlawik, Jens O; Warrington, Arthur E; Rodriguez, Moses

    2016-10-01

    Demyelination underlies early neurological symptoms in multiple sclerosis (MS); however, axonal damage is considered critical for permanent chronic deficits. The precise mechanisms by which axonal injury occurs in MS are unclear; one hypothesis is the absence or failure of remyelination, suggesting that promoting remyelination may protect axons from death. This report provides direct evidence that promoting oligodendrocyte remyelination protects axons and maintains transport function. Persistent Theiler's virus infection of Swiss Jim Lambert (SJL)/J mice was used as a model of MS to assess the effects of remyelination on axonal injury following demyelination in the spinal cord. Remyelination was induced using an oligodendrocyte/myelin-specific recombinant human monoclonal IgM, rHIgM22. The antibody is endowed with strong anti-apoptotic and pro-proliferative effects on oligodendrocyte progenitor cells. We used (1)H-magnetic resonance spectroscopy (MRS) at the brainstem to measure N-acetyl-aspartate (NAA) as a surrogate of neuronal health and spinal cord integrity. We found increased brainstem NAA concentrations at 5 weeks post-treatment with rHIgM22, which remained stable out to 10 weeks. Detailed spinal cord morphology studies revealed enhanced remyelination in the rHIgM22-treated group but not in the isotype control antibody- or saline-treated groups. Importantly, we found rHIgM22-mediated remyelination protected small- and medium-caliber mid-thoracic spinal cord axons from damage despite similar demyelination and inflammation across all experimental groups. The most direct confirmation of remyelination-mediated protection of descending neurons was an improvement in retrograde transport. Treatment with rHIgM22 significantly increased the number of retrograde-labeled neurons in the brainstem, indicating that preserved axons are functionally competent. This is direct validation that remyelination preserves spinal cord axons and protects functional axon integrity

  1. Molecular mechanisms of antibody-mediated neutralisation of flavivirus infection.

    PubMed

    Pierson, Theodore C; Diamond, Michael S

    2008-01-01

    Flaviviruses are a group of positive-stranded RNA viruses that cause a spectrum of severe illnesses globally in more than 50 million individuals each year. While effective vaccines exist for three members of this group (yellow fever, Japanese encephalitis, and tick-borne encephalitis viruses), safe and effective vaccines for several other flaviviruses of clinical importance, including West Nile and dengue viruses, remain in development. An effective humoral immune response is critical for protection against flaviviruses and an essential goal of vaccine development. The effectiveness of virus-specific antibodies in vivo reflects their capacity to inhibit virus entry and spread through several mechanisms, including the direct neutralisation of virus infection. Recent advances in our understanding of the structural biology of flaviviruses, coupled with the use of small-animal models of flavivirus infection, have promoted significant advances in our appreciation of the factors that govern antibody recognition and inhibition of flaviviruses in vitro and in vivo. In this review, we discuss the properties that define the potency of neutralising antibodies and the molecular mechanisms by which they inhibit virus infection. How recent advances in this area have the potential to improve the development of safe and effective vaccines and immunotherapeutics is also addressed. PMID:18471342

  2. Molecular mechanisms of antibody-mediated neutralisation of flavivirus infection.

    PubMed

    Pierson, Theodore C; Diamond, Michael S

    2008-01-01

    Flaviviruses are a group of positive-stranded RNA viruses that cause a spectrum of severe illnesses globally in more than 50 million individuals each year. While effective vaccines exist for three members of this group (yellow fever, Japanese encephalitis, and tick-borne encephalitis viruses), safe and effective vaccines for several other flaviviruses of clinical importance, including West Nile and dengue viruses, remain in development. An effective humoral immune response is critical for protection against flaviviruses and an essential goal of vaccine development. The effectiveness of virus-specific antibodies in vivo reflects their capacity to inhibit virus entry and spread through several mechanisms, including the direct neutralisation of virus infection. Recent advances in our understanding of the structural biology of flaviviruses, coupled with the use of small-animal models of flavivirus infection, have promoted significant advances in our appreciation of the factors that govern antibody recognition and inhibition of flaviviruses in vitro and in vivo. In this review, we discuss the properties that define the potency of neutralising antibodies and the molecular mechanisms by which they inhibit virus infection. How recent advances in this area have the potential to improve the development of safe and effective vaccines and immunotherapeutics is also addressed.

  3. Monoclonal antibodies against the human leukemia cell line K 562.

    PubMed

    Böttger, V; Hering, S; Jantscheff, P; Micheel, B

    1985-01-01

    Three monoclonal antibodies raised against K 562, a cell line originally established from a patient with chronic myeloid leukemia (CML) in terminal blast crisis, were selected according to their distinct reaction pattern. Whereas two antibodies (ZIK-C1-A/C5 and ZIK-C1-A/H5 also designated C and H) recognized antigens, present on K 562 cells and other immature and mature hematopoietic cells (cell lines and normal blood and bone marrow cells), antibody ZIK-C1-A/D9 also designated Y showed an exclusive binding to K 562 cells. The results obtained (here and in the following paper) indicate, that antibody ZIK-C1-A/D9 defines an early differentiation antigen of hematopoiesis or a leukemia-associated antigen.

  4. Affinity enhancement of antibodies: how low-affinity antibodies produced early in immune responses are followed by high-affinity antibodies later and in memory B-cell responses.

    PubMed

    Eisen, Herman N

    2014-05-01

    The antibodies produced initially in response to most antigens are high molecular weight (MW) immunoglobulins (IgM) with low affinity for the antigen, while the antibodies produced later are lower MW classes (e.g., IgG and IgA) with, on average, orders of magnitude higher affinity for that antigen. These changes, often termed affinity maturation, take place largely in small B-cell clusters (germinal center; GC) in lymphoid tissues in which proliferating antigen-stimulated B cells express the highly mutagenic cytidine deaminase that mediates immunoglobulin class-switching and sequence diversification of the immunoglobulin variable domains of antigen-binding receptors on B cells (BCR). Of the large library of BCR-mutated B cells thus rapidly generated, a small minority with affinity-enhancing mutations are selected to survive and differentiate into long-lived antibody-secreting plasma cells and memory B cells. BCRs are also endocytic receptors; they internalize and cleave BCR-bound antigen, yielding peptide-MHC complexes that are recognized by follicular helper T cells. Imperfect correlation between BCR affinity for antigen and cognate T-cell engagement may account for the increasing affinity heterogeneity that accompanies the increasing average affinity of antibodies. Conservation of mechanisms underlying mutation and selection of high-affinity antibodies over the ≈200 million years of evolution separating bird and mammal lineages points to the crucial role of antibody affinity enhancement in adaptive immunity.

  5. Recombinant antibody mediated delivery of organelle-specific DNA pH sensors along endocytic pathways

    NASA Astrophysics Data System (ADS)

    Modi, Souvik; Halder, Saheli; Nizak, Clément; Krishnan, Yamuna

    2013-12-01

    DNA has been used to build nanomachines with potential in cellulo and in vivo applications. However their different in cellulo applications are limited by the lack of generalizable strategies to deliver them to precise intracellular locations. Here we describe a new molecular design of DNA pH sensors with response times that are nearly 20 fold faster. Further, by changing the sequence of the pH sensitive domain of the DNA sensor, we have been able to tune their pH sensitive regimes and create a family of DNA sensors spanning ranges from pH 4 to 7.6. To enable a generalizable targeting methodology, this new sensor design also incorporates a `handle' domain. We have identified, using a phage display screen, a set of three recombinant antibodies (scFv) that bind sequence specifically to the handle domain. Sequence analysis of these antibodies revealed several conserved residues that mediate specific interactions with the cognate DNA duplex. We also found that all three scFvs clustered into different branches indicating that their specificity arises from mutations in key residues. When one of these scFvs is fused to a membrane protein (furin) that traffics via the cell surface, the scFv-furin chimera binds the `handle' and ferries a family of DNA pH sensors along the furin endocytic pathway. Post endocytosis, all DNA nanodevices retain their functionality in cellulo and provide spatiotemporal pH maps of retrogradely trafficking furin inside living cells. This new molecular technology of DNA-scFv-protein chimeras can be used to site-specifically complex DNA nanostructures for bioanalytical applications.DNA has been used to build nanomachines with potential in cellulo and in vivo applications. However their different in cellulo applications are limited by the lack of generalizable strategies to deliver them to precise intracellular locations. Here we describe a new molecular design of DNA pH sensors with response times that are nearly 20 fold faster. Further, by changing

  6. Enhancement of antibody-dependent mechanisms of tumor cell lysis by a targeted activator of complement.

    PubMed

    Imai, Masaki; Ohta, Rieko; Varela, Juan C; Song, Hongbin; Tomlinson, Stephen

    2007-10-01

    Complement inhibitors expressed on tumor cells provide a hindrance to the therapeutic efficacy of some monoclonal antibodies (mAb). We investigated a novel strategy to overwhelm complement inhibitor activity and amplify complement activation on tumor cells. The C3-binding domain of human complement receptor 2 (CR2; CD21) was linked to the complement-activating Fc region of human IgG1 (CR2-Fc), and the ability of the construct to target and amplify complement deposition on tumor cells was investigated. CR2 binds C3 activation fragments, and CR2-Fc targeted tumor cells by binding to C3 initially deposited by a tumor-specific antibody. Complement deposition on Du145 cells (human prostate cancer cell line) and anti-MUC1 mAb-mediated complement-dependent lysis of Du145 cells were significantly enhanced by CR2-Fc. Anti-MUC1 antibody-dependent cell-mediated cytotoxicity of Du145 by human peripheral blood mononuclear cells was also significantly enhanced by CR2-Fc in both the presence and the absence of complement. Radiolabeled CR2-Fc targeted to s.c. Du145 tumors in nude mice treated with anti-MUC1 mAb, validating the targeting strategy in vivo. A metastatic model was used to investigate the effect of CR2-Fc in a therapeutic paradigm. Administration of CR2-Fc together with mAb therapy significantly improved long-term survival of nude mice challenged with an i.v. injection of EL4 cells. The data show that CR2-Fc enhances the therapeutic efficacy of antibody therapy, and the construct may provide particular benefits under conditions of limiting antibody concentration or low tumor antigen density.

  7. Enhancement of antibody-dependent mechanisms of tumor cell lysis by a targeted activator of complement.

    PubMed

    Imai, Masaki; Ohta, Rieko; Varela, Juan C; Song, Hongbin; Tomlinson, Stephen

    2007-10-01

    Complement inhibitors expressed on tumor cells provide a hindrance to the therapeutic efficacy of some monoclonal antibodies (mAb). We investigated a novel strategy to overwhelm complement inhibitor activity and amplify complement activation on tumor cells. The C3-binding domain of human complement receptor 2 (CR2; CD21) was linked to the complement-activating Fc region of human IgG1 (CR2-Fc), and the ability of the construct to target and amplify complement deposition on tumor cells was investigated. CR2 binds C3 activation fragments, and CR2-Fc targeted tumor cells by binding to C3 initially deposited by a tumor-specific antibody. Complement deposition on Du145 cells (human prostate cancer cell line) and anti-MUC1 mAb-mediated complement-dependent lysis of Du145 cells were significantly enhanced by CR2-Fc. Anti-MUC1 antibody-dependent cell-mediated cytotoxicity of Du145 by human peripheral blood mononuclear cells was also significantly enhanced by CR2-Fc in both the presence and the absence of complement. Radiolabeled CR2-Fc targeted to s.c. Du145 tumors in nude mice treated with anti-MUC1 mAb, validating the targeting strategy in vivo. A metastatic model was used to investigate the effect of CR2-Fc in a therapeutic paradigm. Administration of CR2-Fc together with mAb therapy significantly improved long-term survival of nude mice challenged with an i.v. injection of EL4 cells. The data show that CR2-Fc enhances the therapeutic efficacy of antibody therapy, and the construct may provide particular benefits under conditions of limiting antibody concentration or low tumor antigen density. PMID:17909064

  8. The induction of cytotoxicity by a bispecific antibody against CEA positive cell line, in vitro.

    PubMed

    Hideshima, T; Iwasaki, A; Baba, M; Yamashita, Y; Shirakusa, T; Okada, H

    1996-01-01

    A mouse anti-human carcinoembryonic antigen (CEA) x anti-human CD3 bispecific antibody, AB5C10*UCHT1, was developed. This antibody-heteroconjugate was chemically prepared by cross-linking the AB5C10 monoclonal antibody reactive with human CEA with the monoclonal antibody, UCHT1, which binds to CD3 on human T-lymphocytes. The AB5C10*UCHT1 recognized both CEA expressed on the KATOIII cell line and CD3 expressed on T-lymphocytes, as determined using flowcytometry. Next, AB5C10*UCHT1-mediated cytolysis was analyzed by 51Cr-release assay. When 51Cr-labeled target KATOIII cells were incubated for 6 h with effector cells that had been pretreated with AB5C10*UCHT1 for 60 min at 4 degrees C, the percentage specific lysis was significantly increased compared to that of untreated effector cells. Using peripheral blood mononuclear cells (PBMC) and lymphokine-activated killer (LAK) cells pretreated with AB5C10*UCHT1 for effector cells, the percentage specific lysis was determined to be 16.3% and 57.4% at effector: target (E:T) ratios of 100:1 and 12.5:1, respectively. On the other hand, the percentage specific lysis of untreated PBMC and LAK cells determined to be 3.0% and 35.8% at E:T ratios of 100:1 and 12.5:1, respectively. The minimum effective dose of AB5C10*UCHT1 required for antibody-mediated cytotoxicity was 0.1 mu g/ml. The results of this study suggest that AB5C10*UCHT1 could be useful for augmenting the cytotoxicity of CD3-positive T-cells against CEA-positive target cells in vitro. PMID:8919276

  9. The use of antibody to complement protein C5 for salvage treatment of severe antibody-mediated rejection.

    PubMed

    Locke, J E; Magro, C M; Singer, A L; Segev, D L; Haas, M; Hillel, A T; King, K E; Kraus, E; Lees, L M; Melancon, J K; Stewart, Z A; Warren, D S; Zachary, A A; Montgomery, R A

    2009-01-01

    Desensitized patients are at high risk of developing acute antibody-mediated rejection (AMR). In most cases, the rejection episodes are mild and respond to a short course of plasmapheresis (PP) / low-dose IVIg treatment. However, a subset of patients experience severe AMR associated with sudden onset oliguria. We previously described the utility of emergent splenectomy in rescuing allografts in patients with this type of severe AMR. However, not all patients are good candidates for splenectomy. Here we present a single case in which eculizumab, a complement protein C5 antibody that inhibits the formation of the membrane attack complex (MAC), was used combined with PP/IVIg to salvage a kidney undergoing severe AMR. We show a marked decrease in C5b-C9 (MAC) complex deposition in the kidney after the administration of eculizumab.

  10. Monitoring Protein Kinase Expression and Phosphorylation in Cell Lysates with Antibody Microarrays.

    PubMed

    Zhang, Hong; Shi, Xiaoqing; Pelech, Steven

    2016-01-01

    Fuelled by advances in our understanding of the human kinome and phosphoproteome and the increasing availability of pan- and phosphosite-specific antibodies, antibody microarrays have emerged as powerful tools for interrogating protein phosphorylation-mediated signaling systems in ex vivo studies. This economical platform permits ultra-sensitive, semiquantitative measurements of the levels of hundreds of protein kinases and their substrates along with their phosphorylation status simultaneously with minute amounts of specimens. Recent technological innovations in the design and fabrication of antibody microarrays and sample preparation have permitted further refinements of the technology to yield improvements in data quality. In this chapter, we describe a detailed protocol that we have developed for tracking the expression and phosphorylation of protein kinases and their substrates in crude cell lysate samples using a high-content antibody microarray.

  11. HA Antibody-Mediated FcγRIIIa Activity Is Both Dependent on FcR Engagement and Interactions between HA and Sialic Acids

    PubMed Central

    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. PMID:27746785

  12. Profiling human antibody responses by integrated single-cell analysis.

    PubMed

    Ogunniyi, Adebola O; Thomas, Brittany A; Politano, Timothy J; Varadarajan, Navin; Landais, Elise; Poignard, Pascal; Walker, Bruce D; Kwon, Douglas S; Love, J Christopher

    2014-05-19

    Comprehensive characterization of the antigen-specific B cells induced during infections or following vaccination would facilitate the discovery of novel antibodies and inform how interventions shape protective humoral responses. The analysis of human B cells and their antibodies has been performed using flow cytometry to evaluate memory B cells and expanded plasmablasts, while microtechnologies have also provided a useful tool to examine plasmablasts/plasma cells after vaccination. Here we present an integrated analytical platform, using arrays of subnanoliter wells (nanowells), for constructing detailed profiles for human B cells comprising the immunophenotypes of these cells, the distribution of isotypes of the secreted antibodies, the specificity and relative affinity for defined antigens, and for a subset of cells, the genes encoding the heavy and light chains. The approach combines on-chip image cytometry, microengraving, and single-cell RT-PCR. Using clinical samples from HIV-infected subjects, we demonstrate that the method can identify antigen-specific neutralizing antibodies, is compatible with both plasmablasts/plasma cells and activated memory B cells, and is well-suited for characterizing the limited numbers of B cells isolated from tissue biopsies (e.g., colon biopsies). The technology should facilitate detailed analyses of human humoral responses for evaluating vaccines and their ability to raise protective antibody responses across multiple anatomical compartments. PMID:24602776

  13. Complement-mediated, antibody-dependent enhancement of HIV-1 infection in vitro is characterized by increased protein and RNA syntheses and infectious virus release.

    PubMed

    Robinson, W E; Montefiori, D C; Gillespie, D H; Mitchell, W M

    1989-01-01

    Antibody-dependent enhancement (ADE) of human immunodeficiency virus type 1 (HIV-1) infection in vitro has been described recently and was shown to occur by two mechanisms: either participation of the alternative pathway of complement or to involve an Fc receptor-mediated, complement-independent mechanism. Complement-mediated ADE results in an accelerated cytopathic effect in target cells that can abrogate the protective properties of neutralizing antibodies. This study characterizes the surface antigens of MT-2 cells using flow cytometric analysis and shows that these cells express high levels of both CD4 and complement receptor type 2 (CR2) while several CD4+ cell lines that do not demonstrate complement-mediated ADE lack high levels of complement receptors. Further, utilizing MT-2 cell cultures, it is demonstrated that complement-mediated ADE of HIV-1 infection is conferred by the sera from more than 80% of HIV-1 antibody-positive individuals (N = 85). Complement-mediated ADE of HIV-1 infection causes an acceleration of several parameters indicative of HIV-1 infection in vitro including increased HIV-1 antigen synthesis as detected by indirect immunofluorescence, RNA accumulation as measured by a solution hybridization protocol, reverse transcriptase release, and progeny virus production. PMID:2465404

  14. Markers of Endothelial-to-Mesenchymal Transition: Evidence for Antibody-Endothelium Interaction during Antibody-Mediated Rejection in Kidney Recipients.

    PubMed

    Xu-Dubois, Yi-Chun; Peltier, Julie; Brocheriou, Isabelle; Suberbielle-Boissel, Caroline; Djamali, Arjang; Reese, Shannon; Mooney, Nuala; Keuylian, Zela; Lion, Julien; Ouali, Nacéra; Levy, Pierre P; Jouanneau, Chantal; Rondeau, Eric; Hertig, Alexandre

    2016-01-01

    Antibody-mediated rejection (ABMR) is a leading cause of allograft loss. Treatment efficacy depends on accurate diagnosis at an early stage. However, sensitive and reliable markers of antibody-endothelium interaction during ABMR are not available for routine use. Using immunohistochemistry, we retrospectively studied the diagnostic value of three markers of endothelial-to-mesenchymal transition (EndMT), fascin1, vimentin, and heat shock protein 47, for ABMR in 53 renal transplant biopsy specimens, including 20 ABMR specimens, 24 cell-mediated rejection specimens, and nine normal grafts. We validated our results in an independent set of 74 unselected biopsy specimens. Endothelial cells of the peritubular capillaries in grafts with ABMR expressed fascin1, vimentin, and heat shock protein 47 strongly, whereas those from normal renal grafts did not. The level of EndMT marker expression was significantly associated with current ABMR criteria, including capillaritis, glomerulitis, peritubular capillary C4d deposition, and donor-specific antibodies. These markers allowed us to identify C4d-negative ABMR and to predict late occurrence of disease. EndMT markers were more specific than capillaritis for the diagnosis and prognosis of ABMR and predicted late (up to 4 years after biopsy) renal graft dysfunction and proteinuria. In the independent set of 74 renal graft biopsy specimens, the EndMT markers for the diagnosis of ABMR had a sensitivity of 100% and a specificity of 85%. Fascin1 expression in peritubular capillaries was also induced in a rat model of ABMR. In conclusion, EndMT markers are a sensitive and reliable diagnostic tool for detecting endothelial activation during ABMR and predicting late loss of allograft function.

  15. Markers of Endothelial-to-Mesenchymal Transition: Evidence for Antibody-Endothelium Interaction during Antibody-Mediated Rejection in Kidney Recipients.

    PubMed

    Xu-Dubois, Yi-Chun; Peltier, Julie; Brocheriou, Isabelle; Suberbielle-Boissel, Caroline; Djamali, Arjang; Reese, Shannon; Mooney, Nuala; Keuylian, Zela; Lion, Julien; Ouali, Nacéra; Levy, Pierre P; Jouanneau, Chantal; Rondeau, Eric; Hertig, Alexandre

    2016-01-01

    Antibody-mediated rejection (ABMR) is a leading cause of allograft loss. Treatment efficacy depends on accurate diagnosis at an early stage. However, sensitive and reliable markers of antibody-endothelium interaction during ABMR are not available for routine use. Using immunohistochemistry, we retrospectively studied the diagnostic value of three markers of endothelial-to-mesenchymal transition (EndMT), fascin1, vimentin, and heat shock protein 47, for ABMR in 53 renal transplant biopsy specimens, including 20 ABMR specimens, 24 cell-mediated rejection specimens, and nine normal grafts. We validated our results in an independent set of 74 unselected biopsy specimens. Endothelial cells of the peritubular capillaries in grafts with ABMR expressed fascin1, vimentin, and heat shock protein 47 strongly, whereas those from normal renal grafts did not. The level of EndMT marker expression was significantly associated with current ABMR criteria, including capillaritis, glomerulitis, peritubular capillary C4d deposition, and donor-specific antibodies. These markers allowed us to identify C4d-negative ABMR and to predict late occurrence of disease. EndMT markers were more specific than capillaritis for the diagnosis and prognosis of ABMR and predicted late (up to 4 years after biopsy) renal graft dysfunction and proteinuria. In the independent set of 74 renal graft biopsy specimens, the EndMT markers for the diagnosis of ABMR had a sensitivity of 100% and a specificity of 85%. Fascin1 expression in peritubular capillaries was also induced in a rat model of ABMR. In conclusion, EndMT markers are a sensitive and reliable diagnostic tool for detecting endothelial activation during ABMR and predicting late loss of allograft function. PMID:25995444

  16. Antibody h-R3-dendrimer mediated siRNA has excellent endosomal escape and tumor targeted delivery ability, and represents efficient siPLK1 silencing and inhibition of cell proliferation, migration and invasion

    PubMed Central

    Li, Jun; Liu, Jing; Li, Shengnan; Hao, Yanli; Chen, Lei; Zhang, Xiaoning

    2016-01-01

    The major obstacle to developing siRNA delivery is their extracellular and intracellular barriers. Herein, a humanized anti-EGFR monoclonal antibody h-R3 was developed to modify the self-assembled binary complexes (dendriplexes) of PAMAM and siRNA via electrostatic interactions, and two common ligands HSA and EGF were used as a control. Compared to dendriplexes, h-R3/EGF/HSA-dendriplexes showed increased particle size, decreased zeta potentials and lower cytotoxicity. Moreover, h-R3-dendriplexes presented greater cellular uptake and excellent endosomal escape ability in HepG2 cells. Ex vivo fluorescence imaging revealed that h-R3-dendriplexes showed higher targeted delivery and gene expression in the tumors than dendriplexes, HSA-dendriplexes and EGF-dendriplexes, which was in agreement with confocal results of cryosections. Furthermore, h-R3-dendriplexes for siPLK1 delivery indicated efficient gene silencing, potentiated cell growth inhibition and cell apoptosis, and suppressed cellular migration/invasion. These results indicate that h-R3-dendriplexes represent a great potential to be used as efficient targeted siRNA delivery carriers. PMID:26883109

  17. Modalities for treatment of antisperm antibody mediated infertility: novel perspectives.

    PubMed

    Naz, Rajesh K

    2004-05-01

    Immunoinfertility because of antisperm antibodies (ASA) is an important cause of infertility in humans. The incidence of ASA in infertile couples is 9-36% depending on the reporting center. Early claims regarding the incidence and involvement of ASA in involuntary infertility were probably overemphasized, which has resulted in subsequent confusion, doubt, and underestimation of their clinical significance. No immunoglobulin that binds to sperm should be called an antisperm antibody in a strict sense unless it is directed against a sperm antigen that plays a role in fertilization and fertility. ASA directed against the fertilization-related antigens are more relevant to infertility than the immunoglobulins that bind to sperm associated antigens. Several methods have been reported for treatment of immunoinfertility. These include: immunosuppressive therapies using corticosteroids or cyclosporine; assisted reproductive technologies such as intrauterine insemination, gamete intrafallopian transfer, in vitro fertilization, and intracytoplasmic sperm injection; laboratory techniques such as sperm washing, immunomagnetic sperm separation, proteolytic enzyme treatment, and use of immunobeads. Most of the available techniques have side effects, are invasive and expensive, have low efficacy, or provide conflicting results. Recent findings using defined sperm antigens that have a role in fertilization/fertility have provided animal models and innovative novel perspectives for studying the mechanism of immunoinfertility and possible modalities for treatment. The better understanding of local immunity and latest advances in hybridoma and recombinant technologies, proteomics and genomics leading to characterization of sperm antigens relevant to fertility will help to clarify the controversy and to establish the significance of ASA in infertility. PMID:15212677

  18. Establishment of a cell model for screening antibody drugs against rheumatoid arthritis with ADCC and CDC.

    PubMed

    Yan, Li; Hu, Rui; Tu, Song; Cheng, Wen-Jun; Zheng, Qiong; Wang, Jun-Wen; Kan, Wu-Sheng; Ren, Yi-Jun

    2015-01-01

    TNFα played a dominant role in the development and progression of rheumatoid arthritis (RA). Clinical trials proved the efficacies of anti-TNFα agents for curing RA. However, most researchers were concentrating on their abilities of neutralizing TNFα, the potencies of different anti-TNFα agents varied a lot due to the antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). For better understanding and differentiating the potentiality of various candidate anti-TNF reagents at the stage of new drug research and development, present study established a cell model expressing the transmembrane TNFα for usage in in vitro ADCC or CDC assay, meanwhile, the assay protocol described here could provide guidelines for screening macromolecular antibody drugs. A stable cell subline bearing transmembrane TNFα was first established by conventional transfection method, the expression of transmembrane TNFα was approved by flow cytometer, and the performance of the stable subline in ADCC and CDC assay was evaluated, using human peripheral blood mononuclear cells as effector cells, and Adalimumab as the anti-TNFα reagent. The stable cell subline demonstrated high level of surface expression of transmembrane TNFα, and Adalimumab exerted both ADCC and CDC effects on this cell model. In conclusion, the stable cell line we established in present research could be used in ADCC or CDC assay for screening antibody drugs, which would provide in-depth understanding of the potencies of candidate antibody drugs in addition to the traditional TNFα neutralizing assay.

  19. Establishment of a cell model for screening antibody drugs against rheumatoid arthritis with ADCC and CDC.

    PubMed

    Yan, Li; Hu, Rui; Tu, Song; Cheng, Wen-Jun; Zheng, Qiong; Wang, Jun-Wen; Kan, Wu-Sheng; Ren, Yi-Jun

    2015-01-01

    TNFα played a dominant role in the development and progression of rheumatoid arthritis (RA). Clinical trials proved the efficacies of anti-TNFα agents for curing RA. However, most researchers were concentrating on their abilities of neutralizing TNFα, the potencies of different anti-TNFα agents varied a lot due to the antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). For better understanding and differentiating the potentiality of various candidate anti-TNF reagents at the stage of new drug research and development, present study established a cell model expressing the transmembrane TNFα for usage in in vitro ADCC or CDC assay, meanwhile, the assay protocol described here could provide guidelines for screening macromolecular antibody drugs. A stable cell subline bearing transmembrane TNFα was first established by conventional transfection method, the expression of transmembrane TNFα was approved by flow cytometer, and the performance of the stable subline in ADCC and CDC assay was evaluated, using human peripheral blood mononuclear cells as effector cells, and Adalimumab as the anti-TNFα reagent. The stable cell subline demonstrated high level of surface expression of transmembrane TNFα, and Adalimumab exerted both ADCC and CDC effects on this cell model. In conclusion, the stable cell line we established in present research could be used in ADCC or CDC assay for screening antibody drugs, which would provide in-depth understanding of the potencies of candidate antibody drugs in addition to the traditional TNFα neutralizing assay. PMID:26884918

  20. Adhesion between peptides/antibodies and breast cancer cells

    NASA Astrophysics Data System (ADS)

    Meng, J.; Paetzell, E.; Bogorad, A.; Soboyejo, W. O.

    2010-06-01

    Atomic force microscopy (AFM) techniques were used to measure the adhesion forces between the receptors on breast cancer cells specific to human luteinizing hormone-releasing hormone (LHRH) peptides and antibodies specific to the EphA2 receptor. The adhesion forces between LHRH-coated AFM tips and human MDA-MB-231 cells (breast cancer cells) were shown to be about five times greater than those between LHRH-coated AFM tips and normal Hs578Bst breast cells. Similarly, those between EphA2 antibody-coated AFM tips and breast cancer cells were over five times greater than those between EphA2 antibody-coated AFM tips and normal breast cells. The results suggest that AFM can be used for the detection of breast cancer cells in biopsies. The implications of the results are also discussed for the early detection and localized treatment of cancer.

  1. 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

  2. Antibody-mediated disruption of the mechanics of CS20 fimbriae of enterotoxigenic Escherichia coli

    PubMed Central

    Singh, Bhupender; Mortezaei, Narges; Uhlin, Bernt Eric; Savarino, Stephen J.; Bullitt, Esther; Andersson, Magnus

    2015-01-01

    Preventive vaccines against enterotoxigenic Escherichia coli (ETEC) are being developed, many of which target common fimbrial colonization factors as the major constituent, based on empirical evidence that these function as protective antigens. Particularly, passive oral administration of ETEC anti-fimbrial antibodies prevent ETEC diarrhea. Little is, however, known regarding the specific mechanisms by which intestinal antibodies against ETEC fimbriae function to prevent disease. Using coli surface antigen 20 (CS20) fimbriae as a model ETEC colonization factor, we show using force spectroscopy that anti-fimbrial antibodies diminish fimbrial elasticity by inhibiting their natural capacity to unwind and rewind. In the presence of anti-CS20 antibodies the force required to unwind a single fimbria was increased several-fold and the extension length was shortened several-fold. Similar measurements in the presence of anti-CS20 Fab fragments did not show any effect, indicating that bivalent antibody binding is required to reduce fimbrial elasticity. Based on these findings, we propose a model for an in-vivo mechanism whereby antibody-mediated disruption of the biomechanical properties of CS20 fimbriae impedes sustained adhesion of ETEC to the intestinal mucosal surface. Further elucidation of the role played by intestinal antibodies in mechanical disruption of fimbrial function may provide insights relevant to ETEC vaccine development. PMID:26411657

  3. Antibody-mediated disruption of the mechanics of CS20 fimbriae of enterotoxigenic Escherichia coli.

    PubMed

    Singh, Bhupender; Mortezaei, Narges; Uhlin, Bernt Eric; Savarino, Stephen J; Bullitt, Esther; Andersson, Magnus

    2015-01-01

    Preventive vaccines against enterotoxigenic Escherichia coli (ETEC) are being developed, many of which target common fimbrial colonization factors as the major constituent, based on empirical evidence that these function as protective antigens. Particularly, passive oral administration of ETEC anti-fimbrial antibodies prevent ETEC diarrhea. Little is, however, known regarding the specific mechanisms by which intestinal antibodies against ETEC fimbriae function to prevent disease. Using coli surface antigen 20 (CS20) fimbriae as a model ETEC colonization factor, we show using force spectroscopy that anti-fimbrial antibodies diminish fimbrial elasticity by inhibiting their natural capacity to unwind and rewind. In the presence of anti-CS20 antibodies the force required to unwind a single fimbria was increased several-fold and the extension length was shortened several-fold. Similar measurements in the presence of anti-CS20 Fab fragments did not show any effect, indicating that bivalent antibody binding is required to reduce fimbrial elasticity. Based on these findings, we propose a model for an in-vivo mechanism whereby antibody-mediated disruption of the biomechanical properties of CS20 fimbriae impedes sustained adhesion of ETEC to the intestinal mucosal surface. Further elucidation of the role played by intestinal antibodies in mechanical disruption of fimbrial function may provide insights relevant to ETEC vaccine development. PMID:26411657

  4. Nephritogenic Lupus Antibodies Recognize Glomerular Basement Membrane-Associated Chromatin Fragments Released from Apoptotic Intraglomerular Cells

    PubMed Central

    Kalaaji, Manar; Mortensen, Elin; Jørgensen, Leif; Olsen, Randi; Rekvig, Ole Petter

    2006-01-01

    Antibodies to dsDNA represent a classification criterion for systemic lupus erythematosus. Subpopulations of these antibodies are involved in lupus nephritis. No known marker separates nephritogenic from non-nephritogenic anti-dsDNA antibodies. It is not clear whether specificity for glomerular target antigens or intrinsic antibody-affinity for dsDNA or nucleosomes is a critical parameter. Furthermore, it is still controversial whether glomerular target antigen(s) is constituted by nucleosomes or by non-nucleosomal glomerular structures. Previously, we have demonstrated that antibodies eluted from murine nephritic kidneys recognize nucleosomes, but not other glomerular antigens. In this study, we determined the structures that bind nephritogenic autoantibodies in vivo by transmission electron microscopy, immune electron microscopy, and colocalization immune electron microscopy using experimental antibodies to dsDNA, to histones and transcription factors, or to laminin. The data obtained are consistent and point at glomerular basement membrane-associated nucleosomes as target structures for the nephritogenic autoantibodies. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling or caspase-3 assays demonstrate that lupus nephritis is linked to intraglomerular cell apoptosis. The data suggest that nucleosomes are released by apoptosis and associate with glomerulus basement membranes, which may then be targeted by pathogenic anti-nucleosome antibodies. Thus, apoptotic nucleosomes may represent both inducer and target structures for nephritogenic autoantibodies in systemic lupus erythematosus. PMID:16723695

  5. Nephritogenic lupus antibodies recognize glomerular basement membrane-associated chromatin fragments released from apoptotic intraglomerular cells.

    PubMed

    Kalaaji, Manar; Mortensen, Elin; Jørgensen, Leif; Olsen, Randi; Rekvig, Ole Petter

    2006-06-01

    Antibodies to dsDNA represent a classification criterion for systemic lupus erythematosus. Subpopulations of these antibodies are involved in lupus nephritis. No known marker separates nephritogenic from non-nephritogenic anti-dsDNA antibodies. It is not clear whether specificity for glomerular target antigens or intrinsic antibody-affinity for dsDNA or nucleosomes is a critical parameter. Furthermore, it is still controversial whether glomerular target antigen(s) is constituted by nucleosomes or by non-nucleosomal glomerular structures. Previously, we have demonstrated that antibodies eluted from murine nephritic kidneys recognize nucleosomes, but not other glomerular antigens. In this study, we determined the structures that bind nephritogenic autoantibodies in vivo by transmission electron microscopy, immune electron microscopy, and colocalization immune electron microscopy using experimental antibodies to dsDNA, to histones and transcription factors, or to laminin. The data obtained are consistent and point at glomerular basement membrane-associated nucleosomes as target structures for the nephritogenic autoantibodies. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling or caspase-3 assays demonstrate that lupus nephritis is linked to intraglomerular cell apoptosis. The data suggest that nucleosomes are released by apoptosis and associate with glomerulus basement membranes, which may then be targeted by pathogenic anti-nucleosome antibodies. Thus, apoptotic nucleosomes may represent both inducer and target structures for nephritogenic autoantibodies in systemic lupus erythematosus.

  6. Antibody binding to Streptococcus mitis and Streptococcus oralis cell fractions

    PubMed Central

    Wirth, Katherine A.; Bowden, George H.; Richmond, Dorothy A.; Sheridan, Michael J.; Cole, Michael F.

    2008-01-01

    Summary Objective To determine which cell fraction(s) of Streptococcus mitis biovar 1 serve as the best source of antigens recognized by salivary SIgA antibodies in infants. Design Whole cells of 38 reference and wild-type isolates of Streptococcus mitis, S. oralis, S. gordonii, Enterococcus casseliflavus, and E. faecalis were fractionated into cell walls CW), protease-treated cell walls (PTCW), cell membranes (CM) and cell protein (CP). Whole cells and these fractions were tested for binding by rabbit anti-S. mitis SK145 and anti-S. oralis SK100 sera, and also by salivary SIgA antibodies from infants and adults. Results Anti-SK145 and anti-SK100 sera bound whole cells and fractions of all strains of S. mitis and S. oralis variably. Cluster analysis of antibody binding data placed the strains into S. mitis, S. oralis and ‘Non-S. mitis/non-S. oralis’ clusters. Antigens from CW and CM best discriminated S. mitis from S. oralis. CM bound the most infant salivary SIgA antibody and PTCW bound the least. In contrast, adult salivary SIgA antibody bound all of the cell fractions and at higher levels. Conclusions Presumably the relatively short period of immune stimulation and immunological immaturity in infants, in contrast to adults, result in low levels of salivary SIgA antibody that preferentially bind CM of S. mitis but not PTCW. By utilizing isolated cell walls and membranes as sources of antigens for proteomics it may be possible to identify antigens common to oral streptococci and dissect the fine specificity of salivary SIgA antibodies induced by oral colonization by S. mitis. PMID:17904095

  7. Detection of opsonic antibodies against Enterococcus faecalis cell wall carbohydrates in immune globulin preparations.

    PubMed

    Hufnagel, M; Sixel, K; Hammer, F; Kropec, A; Sava, I G; Theilacker, C; Berner, R; Huebner, J

    2014-08-01

    Three different commercially available polyvalent immune globulins (IG) were investigated for the existence of antibodies against cell wall carbohydrates of four different E. faecalis serotypes (using a cell wall carbohydrate-enzyme-linked immunosorbent assay), and whether these antibodies mediated opsonic killing (using an opsonic-killing assay). All three IG preparations contained antibodies against all four serotypes (CPS-A to CPS-D). However, only one of the three IG preparations showed opsonic killing against all four serotypes. Average killing was higher against serotypes A and B (72 and 79 %, respectively) than against serotypes C and D (30 and 37 %, respectively). Such IG preparations could play a role as an adjuvant therapeutic option in life-threatening infections with E. faecalis, particularly when resistant strains are involved.

  8. Antidrug Antibodies: B Cell Immunity Against Therapy.

    PubMed

    Fogdell-Hahn, A

    2015-09-01

    Chronic inflammatory diseases are now treated with a range of different biopharmaceuticals, often requiring lifelong parenteral administrations. This exposure to drugs is unnatural and can trigger the immune system and result in the formation of antidrug antibodies. Drug-specific antibodies will, if of sufficiently high titre and affinity, block the intended effect of the drug, increase its clearance and make continued treatment worthless. We expect the immune system to react towards therapies against which tolerance has never been established, which is the case for factor VIII treatment in patients with haemophilia A. However, even biopharmaceutical molecules that we should be tolerant against can elicit antidrug antibodies, for instance in treatment of multiple sclerosis patients with recombinant human interferon-beta. Possible immunological mechanisms behind the breaking of tolerance against drugs, the impact this has on continuous treatment success, clinical practice and drug development, will be discussed in this review.

  9. Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions.

    PubMed

    Purushothaman, Anurag; Bandari, Shyam Kumar; Liu, Jian; Mobley, James A; Brown, Elizabeth E; Sanderson, Ralph D

    2016-01-22

    Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression. PMID:26601950

  10. Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions.

    PubMed

    Purushothaman, Anurag; Bandari, Shyam Kumar; Liu, Jian; Mobley, James A; Brown, Elizabeth E; Sanderson, Ralph D

    2016-01-22

    Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression.

  11. The Complement System and Antibody-Mediated Transplant Rejection.

    PubMed

    Stites, Erik; Le Quintrec, Moglie; Thurman, Joshua M

    2015-12-15

    Complement activation is an important cause of tissue injury in patients with Ab-mediated rejection (AMR) of transplanted organs. Complement activation triggers a strong inflammatory response, and it also generates tissue-bound and soluble fragments that are clinically useful markers of inflammation. The detection of complement proteins deposited within transplanted tissues has become an indispensible biomarker of AMR, and several assays have recently been developed to measure complement activation by Abs reactive to specific donor HLA expressed within the transplant. Complement inhibitors have entered clinical use and have shown efficacy for the treatment of AMR. New methods of detecting complement activation within transplanted organs will improve our ability to diagnose and monitor AMR, and they will also help guide the use of complement inhibitory drugs.

  12. The Complement System and Antibody-Mediated Transplant Rejection.

    PubMed

    Stites, Erik; Le Quintrec, Moglie; Thurman, Joshua M

    2015-12-15

    Complement activation is an important cause of tissue injury in patients with Ab-mediated rejection (AMR) of transplanted organs. Complement activation triggers a strong inflammatory response, and it also generates tissue-bound and soluble fragments that are clinically useful markers of inflammation. The detection of complement proteins deposited within transplanted tissues has become an indispensible biomarker of AMR, and several assays have recently been developed to measure complement activation by Abs reactive to specific donor HLA expressed within the transplant. Complement inhibitors have entered clinical use and have shown efficacy for the treatment of AMR. New methods of detecting complement activation within transplanted organs will improve our ability to diagnose and monitor AMR, and they will also help guide the use of complement inhibitory drugs. PMID:26637661

  13. Mitochondrial Pyruvate Import Promotes Long-Term Survival of Antibody-Secreting Plasma Cells.

    PubMed

    Lam, Wing Y; Becker, Amy M; Kennerly, Krista M; Wong, Rachel; Curtis, Jonathan D; Llufrio, Elizabeth M; McCommis, Kyle S; Fahrmann, Johannes; Pizzato, Hannah A; Nunley, Ryan M; Lee, Jieun; Wolfgang, Michael J; Patti, Gary J; Finck, Brian N; Pearce, Erika L; Bhattacharya, Deepta

    2016-07-19

    Durable antibody production after vaccination or infection is mediated by long-lived plasma cells (LLPCs). Pathways that specifically allow LLPCs to persist remain unknown. Through bioenergetic profiling, we found that human and mouse LLPCs could robustly engage pyruvate-dependent respiration, whereas their short-lived counterparts could not. LLPCs took up more glucose than did short-lived plasma cells (SLPCs) in vivo, and this glucose was essential for the generation of pyruvate. Glucose was primarily used to glycosylate antibodies, but glycolysis could be promoted by stimuli such as low ATP levels and the resultant pyruvate used for respiration by LLPCs. Deletion of Mpc2, which encodes an essential component of the mitochondrial pyruvate carrier, led to a progressive loss of LLPCs and of vaccine-specific antibodies in vivo. Thus, glucose uptake and mitochondrial pyruvate import prevent bioenergetic crises and allow LLPCs to persist. Immunizations that maximize these plasma cell metabolic properties might thus provide enduring antibody-mediated immunity. PMID:27396958

  14. Rebmab200, a humanized monoclonal antibody targeting the sodium phosphate transporter NaPi2b displays strong immune mediated cytotoxicity against cancer: a novel reagent for targeted antibody therapy of cancer.

    PubMed

    Lopes dos Santos, Mariana; Yeda, Fernanda Perez; Tsuruta, Lilian Rumi; Horta, Bruno Brasil; Pimenta, Alécio A; Degaki, Theri Leica; Soares, Ibere C; Tuma, Maria Carolina; Okamoto, Oswaldo Keith; Alves, Venancio A F; Old, Lloyd J; Ritter, Gerd; Moro, Ana Maria

    2013-01-01

    NaPi2b, a sodium-dependent phosphate transporter, is highly expressed in ovarian carcinomas and is recognized by the murine monoclonal antibody MX35. The antibody had shown excellent targeting to ovarian cancer in several early phase clinical trials but being murine the antibody's full therapeutic potential could not be explored. To overcome this impediment we developed a humanized antibody version named Rebmab200, expressed in human PER.C6® cells and cloned by limiting dilution. In order to select a clone with high therapeutic potential clones were characterized using a series of physicochemical assays, flow cytometry, real-time surface plasmon resonance, glycosylation analyses, immunohistochemistry, antibody-dependent cell-mediated cytotoxicity, complement-dependent-cytotoxicity assays and quantitative PCR. Comparative analyses of Rebmab200 and MX35 monoclonal antibodies demonstrated that the two antibodies had similar specificity for NaPi2b by flow cytometry with a panel of 30 cell lines and maintained similar kinetic parameters. Robust and high producer cell clones potentially suitable for use in manufacturing were obtained. Rebmab200 antibodies were assessed by immunohistochemistry using a large panel of tissues including human carcinomas of ovarian, lung, kidney and breast origin. An assessment of its binding towards 33 normal human organs was performed as well. Rebmab200 showed selected strong reactivity with the tested tumor types but little or no reactivity with the normal tissues tested confirming its potential for targeted therapeutics strategies. The remarkable cytotoxicity shown by Rebmab200 in OVCAR-3 cells is a significant addition to the traits of stability and productivity displayed by the top clones of Rebmab200. Antibody-dependent cell-mediated toxicity functionality was confirmed in repeated assays using cancer cell lines derived from ovary, kidney and lung as targets. To explore use of this antibody in clinical trials, GMP production of Rebmab

  15. Bispecific-antibody-mediated targeting of radiolabeled bivalent haptens: theoretical, experimental and clinical results.

    PubMed

    Le Doussal, J M; Barbet, J; Delaage, M

    1992-01-01

    Chemically conjugated bispecific (anti-cell surface antigen, anti-hapten) Fab'-Fab antibodies (Bs-MAbs) have been used to target 125I-, 111In- and 99mTc-labeled haptens to cell sub-sets. In vitro, bivalent haptens were found to bind more strongly than their monovalent analogs to the Bs-MAbs bound to ("ordered" on) the cell surface, or than to free ("disordered") Bs-MAbs: they are selective for cell-bound Bs-MAbs. In tumor-grafted nude mice models, the sequential injections of microgram amounts of Bs-MAb, and 1 day later, of microC amounts of bivalent haptens permits to sharply delineate small tumors (using a gamma camera), hours after injection. Further, the isotope biodistribution was found to be at least 3 times more selective for the tumor than that obtained with directly labeled anti-CEA F(ab)'2 or with monovalent haptens. This better in vivo selectivity of the 2-step targeting of bivalent haptens was also demonstrated in a pharmacokinetic study using therapeutic amounts of reagents. In primary-colon-carcinoma patients, a similar comparative immunoscintigraphy study confirmed the better selectivity of bivalent hapten targeting over direct targeting, on the basis of image quality and ex vivo tissue counting. In patients with medullary carcinoma of the thyroid, bivalent hapten targeting allowed us to confirm tumor extension and to find occult lesions. Interestingly, radio-immunoguided surgery was necessary to resect these small lesions. These experimental results, together with technological and theoretical considerations, suggest that Bs-MAb-mediated targeting of isotopes (or other agents) is one of the major ways to increase the clinical performance of MAb-based targeting diagnostic and therapeutic tools.

  16. Sialylated intravenous immunoglobulin suppress anti-ganglioside antibody mediated nerve injury.

    PubMed

    Zhang, Gang; Massaad, Cynthia A; Gao, Tong; Pillai, Laila; Bogdanova, Nataliia; Ghauri, Sameera; Sheikh, Kazim A

    2016-08-01

    The precise mechanisms underlying the efficacy of intravenous immunoglobulin (IVIg) in autoimmune neurological disorders including Guillain-Barré syndrome (GBS) are not known. Anti-ganglioside antibodies have been reported to be pathogenic in some variants of GBS, and we have developed passive transfer animal models to study anti-ganglioside antibody mediated-endoneurial inflammation and associated neuropathological effects and to evaluate the efficacy of new therapeutic approaches. Some studies indicate that IVIg's anti-inflammatory activity resides in a minor sialylated IVIg (sIVIg) fractions and is dependent on an innate Th2 response via binding to a specific ICAM3-grabbing nonintegrin related 1 receptor (SIGN-R1). Therefore the efficacy of IVIg, IVIg fractions with various IgG Fc sialylation status, and the involvement of Th2 pathway were examined in one of our animal model of antibody-mediated inhibition of axonal regeneration. We demonstrate that both IVIg and sIVIg ameliorated anti-glycan antibody mediated-pathological effect, whereas, the unsialylated fractions of IVIg were not beneficial in our model. Tenfold lower doses of sIVIg compared to whole IVIg provided equivalent efficacy in our studies. Moreover, we found that whole IVIg and sIVIg significantly upregulates the gene expression of IL-33, which itself can provide protection from antibody-mediated nerve injury in our model. Our results support that the SIGN-R1-Th2 pathway is involved in the anti-inflammatory effects of IVIg on endoneurium in our model and elements of this pathway including IL-33 can provide novel therapeutics in inflammatory neuropathies. PMID:27208700

  17. Delivery of Antibody Mimics into Mammalian Cells via Anthrax Toxin Protective Antigen

    PubMed Central

    Liao, Xiaoli; Rabideau, Amy E; Pentelute, Bradley L

    2014-01-01

    Antibody mimics have significant scientific and therapeutic utility for the disruption of protein–protein interactions inside cells; however, their delivery to the cell cytosol remains a major challenge. Here we show that protective antigen (PA), a component of anthrax toxin, efficiently transports commonly used antibody mimics to the cytosol of mammalian cells when conjugated to the N-terminal domain of LF (LFN). In contrast, a cell-penetrating peptide (CPP) was not able to deliver any of these antibody mimics into the cell cytosol. The refolding and binding of a transported tandem monobody to Bcr-Abl (its protein target) in chronic myeloid leukemia cells were confirmed by co-immunoprecipitation. We also observed inhibition of Bcr-Abl kinase activity and induction of apoptosis caused by the monobody. In a separate case, we show disruption of key interactions in the MAPK signaling pathway after PA-mediated delivery of an affibody binder that targets hRaf-1. We show for the first time that PA can deliver bioactive antibody mimics to disrupt intracellular protein–protein interactions. This technology adds a useful tool to expand the applications of these modern agents to the intracellular milieu. PMID:25250705

  18. Retargeting cytokine-induced killer cell activity by CD16 engagement with clinical-grade antibodies

    PubMed Central

    Cappuzzello, Elisa; Tosi, Anna; Zanovello, Paola; Sommaggio, Roberta; Rosato, Antonio

    2016-01-01

    ABSTRACT Cytokine-induced Killer (CIK) cells are a heterogeneous population of ex vivo expanded T lymphocytes capable of MHC-unrestricted antitumor activity, which share phenotypic and functional features with both NK and T cells. Preclinical data and initial clinical studies demonstrated their high tolerability in vivo, supporting CIK cells as a promising cell population for adoptive cell immunotherapy. In this study, we report for the first time that CIK cells display a donor-dependent expression of CD16, which can be engaged by trastuzumab or cetuximab to exert a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against ovarian and breast cancer cell lines, leading to an increased lytic activity in vitro, and an enhanced therapeutic efficacy in vivo. Thus, an efficient tumor antigen-specific retargeting can be achieved by a combination therapy with clinical-grade monoclonal antibodies already widely used in cancer therapy, and CIK cell populations that are easily expandable in very large numbers, inexpensive, safe and do not require genetic manipulations. Overall, these data provide a new therapeutic strategy for the treatment of Her2 and EGFR expressing tumors by adoptive cell therapy, which could find wide implementation and application, and could also be expanded to the use of additional therapeutic antibodies. PMID:27622068

  19. Retargeting cytokine-induced killer cell activity by CD16 engagement with clinical-grade antibodies.

    PubMed

    Cappuzzello, Elisa; Tosi, Anna; Zanovello, Paola; Sommaggio, Roberta; Rosato, Antonio

    2016-08-01

    Cytokine-induced Killer (CIK) cells are a heterogeneous population of ex vivo expanded T lymphocytes capable of MHC-unrestricted antitumor activity, which share phenotypic and functional features with both NK and T cells. Preclinical data and initial clinical studies demonstrated their high tolerability in vivo, supporting CIK cells as a promising cell population for adoptive cell immunotherapy. In this study, we report for the first time that CIK cells display a donor-dependent expression of CD16, which can be engaged by trastuzumab or cetuximab to exert a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against ovarian and breast cancer cell lines, leading to an increased lytic activity in vitro, and an enhanced therapeutic efficacy in vivo. Thus, an efficient tumor antigen-specific retargeting can be achieved by a combination therapy with clinical-grade monoclonal antibodies already widely used in cancer therapy, and CIK cell populations that are easily expandable in very large numbers, inexpensive, safe and do not require genetic manipulations. Overall, these data provide a new therapeutic strategy for the treatment of Her2 and EGFR expressing tumors by adoptive cell therapy, which could find wide implementation and application, and could also be expanded to the use of additional therapeutic antibodies. PMID:27622068

  20. Antibody

    MedlinePlus

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  1. Salmonella Modulates B Cell Biology to Evade CD8+ T Cell-Mediated Immune Responses

    PubMed Central

    Lopez-Medina, Marcela; Perez-Lopez, Araceli; Alpuche-Aranda, Celia; Ortiz-Navarrete, Vianney

    2014-01-01

    Although B cells and antibodies are the central effectors of humoral immunity, B cells can also produce and secrete cytokines and present antigen to helper T cells. The uptake of antigen is mainly mediated by endocytosis; thus, antigens are often presented by MHC-II molecules. However, it is unclear if B cells can present these same antigens via MHC-I molecules. Recently, Salmonella bacteria were found to infect B cells, allowing possible antigen cross-processing that could generate bacterial peptides for antigen presentation via MHC-I molecules. Here, we will discuss available knowledge regarding Salmonella antigen presentation by infected B cell MHC-I molecules and subsequent inhibitory effects on CD8+ T cells for bacterial evasion of cell-mediated immunity. PMID:25484884

  2. Monoclonal Antibody-Directed Effector Cells Selectively Lyse Human Melanoma Cells in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Schulz, Gregor; Bumol, Thomas F.; Reisfeld, Ralph A.

    1983-09-01

    Monoclonal antibody 9.2.27 (mAb 9.2.27) directed to a chondroitin sulfate proteoglycan on human melanoma cells was able to suppress tumor growth in athymic (nu/nu) mice more effectively when bound with polyethylene glycol to murine effector cells than when injected alone. These ``armed'' effector cells also proved more effective than the monoclonal antibody in eliciting antibody-dependent cellular cytotoxicity against human melanoma target cells in vitro.

  3. Mechanistic insights into the impairment of memory B cells and antibody production in the elderly.

    PubMed

    Aberle, Judith H; Stiasny, Karin; Kundi, Michael; Heinz, Franz X

    2013-04-01

    It is well established that immunologic memory generated early in life can be maintained into old age and mediate robust anamnestic antibody responses. Little is known, however, about the initiation of memory B cells in the elderly. We have conducted a prospective analysis of the quantities and functionalities of antigen-specific B cell responses and its association with the functional helper CD4(+)T cell responses. The ability of naïve B cells from old (60-80 years) and young (20-31 years) humans to establish functional memory was examined following primary and booster vaccination with an inactivated-virus vaccine against tick-borne encephalitis. Our data show that the number of antigen-specific memory B cells generated during primary vaccination was ~3-fold lower in old than in young individuals. The maintenance and booster responsiveness of these memory B cells were not compromised, as evidenced by similar increases in specific memory B cell frequencies upon revaccination in old and young adults. In contrast, the Ab response mediated per memory B cell after revaccination was dramatically diminished in the elderly. Also, antigen-specific IL-2-positive CD4(+)T cell responses were strongly reduced in the elderly and displayed an excellent correlation with Ab titres. The data suggest that the dramatically lower antibody response in the elderly could only partially be accounted for by the reduced B cell numbers and was strongly correlated with profound functional defects in CD4 help.

  4. 131-iodine conjugated antibody cell kill enhanced by bromodeoxyuridine

    SciTech Connect

    Morstyn, G.; Miller, R.; Russo, A.; Mitchell, J.

    1984-08-01

    /sup 131/I-conjugated monoclonal and polyclonal antibodies are being administered in vivo to kill human tumor cells. Although these conjugates deliver relatively large doses of radiation to tumors (10 to 50 Gy), the rate of dose delivery is low (0.05 to 0.2 Gy/hour). To determine whether the cell kill produced by low dose rate radiation is enhanced by a radiation sensitizer (bromodeoxyuridine, BUdR) the authors studied the cell kill produced by an /sup 131/I-conjugated polyclonal antibody against Chinese hamster V79 cells. Cells not containing BUdR were also studied. The /sup 131/I-conjugated antibody produced up to 40% cell kill and BUdR increased the fraction of cells killed to 75%. These studies demonstrate that cells frozen at -196/sup 0/C are useful for the investigation of the cell kill produced by isotopes that deliver radiation at low dose rates and that the cell kill caused by /sup 131/I-conjugated antibodies can be enhanced by BUdR.

  5. Protein Delivery of Thymidylate Kinase Mediated by Tumor-Specific Antibody-Precoated Microvesicles.

    PubMed

    Mohammadzadeh-Vardin, Mohammad; Panahpour, Hamdollah; Golmohammadi, Mohammad Ghasem; Sagha, Mohsen

    2016-01-01

    Molecular targeted therapy is an important, novel approach in the treatment of cancer because it interferes with certain molecules involved in carcinogenesis and tumor growth. Examples include monoclonal antibodies, microvesicles, and suicide genes. Several studies have focused on targeted therapies in prostate cancer, which is a serious cause of cancer death in men. We hypothesize that antibody-coated microvesicles can deliver thymidylate kinase, a suicide protein, to prostate cancer cells, potentiating them to death following azidothymidine (AZT) treatment. PMID:27278881

  6. 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

  7. Engineering multivalent antibodies to target heregulin-induced HER3 signaling in breast cancer cells

    PubMed Central

    Kang, Jeffrey C; Poovassery, Jayakumar S; Bansal, Pankaj; You, Sungyong; Manjarres, Isabel M; Ober, Raimund J; Ward, E Sally

    2014-01-01

    The use of antibodies in therapy and diagnosis has undergone an unprecedented expansion during the past two decades. This is due in part to innovations in antibody engineering that now offer opportunities for the production of “second generation” antibodies with multiple specificities or altered valencies. The targeting of individual components of the human epidermal growth factor receptor (HER)3-PI3K signaling axis, including the preferred heterodimerization partner HER2, is known to have limited anti-tumor effects. The efficacy of antibodies or small molecule tyrosine kinase inhibitors (TKIs) in targeting this axis is further reduced by the presence of the HER3 ligand, heregulin. To address these shortcomings, we performed a comparative analysis of two distinct approaches toward reducing the proliferation and signaling in HER2 overexpressing tumor cells in the presence of heregulin. These strategies both involve the use of engineered antibodies in combination with the epidermal growth factor receptor (EGFR)/HER2 specific TKI, lapatinib. In the first approach, we generated a bispecific anti-HER2/HER3 antibody that, in the presence of lapatinib, is designed to sequester HER3 into inactive HER2-HER3 dimers that restrain HER3 interactions with other possible dimerization partners. The second approach involves the use of a tetravalent anti-HER3 antibody with the goal of inducing efficient HER3 internalization and degradation. In combination with lapatinib, we demonstrate that although the multivalent HER3 antibody is more effective than its bivalent counterpart in reducing heregulin-mediated signaling and growth, the bispecific HER2/HER3 antibody has increased inhibitory activity. Collectively, these observations provide support for the therapeutic use of bispecifics in combination with TKIs to recruit HER3 into complexes that are functionally inert. PMID:24492289

  8. A type I interferon signature characterizes chronic antibody-mediated rejection in kidney transplantation.

    PubMed

    Rascio, Federica; Pontrelli, Paola; Accetturo, Matteo; Oranger, Annarita; Gigante, Margherita; Castellano, Giuseppe; Gigante, Maddalena; Zito, Anna; Zaza, Gianluigi; Lupo, Antonio; Ranieri, Elena; Stallone, Giovanni; Gesualdo, Loreto; Grandaliano, Giuseppe

    2015-09-01

    Chronic antibody-mediated rejection (CAMR) represents the main cause of kidney graft loss. To uncover the molecular mechanisms underlying this condition, we characterized the molecular signature of peripheral blood mononuclear cells (PBMCs) and, separately, of CD4(+) T lymphocytes isolated from CAMR patients, compared to kidney transplant recipients with normal graft function and histology. We enrolled 29 patients with biopsy-proven CAMR, 29 stable transplant recipients (controls), and 8 transplant recipients with clinical and histological evidence of interstitial fibrosis/tubular atrophy. Messenger RNA and microRNA profiling of PBMCs and CD4(+) T lymphocytes was performed using Agilent microarrays in eight randomly selected patients per group from CAMR and control subjects. Results were evaluated statistically and by functional pathway analysis (Ingenuity Pathway Analysis) and validated in the remaining subjects. In PBMCs, 45 genes were differentially expressed between the two groups, most of which were up-regulated in CAMR and were involved in type I interferon signalling. In the same patients, 16 microRNAs were down-regulated in CAMR subjects compared to controls: four were predicted modulators of six mRNAs identified in the transcriptional analysis. In silico functional analysis supported the involvement of type I interferon signalling. To further confirm this result, we investigated the transcriptomic profiles of CD4(+) T lymphocytes in an independent group of patients, observing that the activation of type I interferon signalling was a specific hallmark of CAMR. In addition, in CAMR patients, we detected a reduction of circulating BDCA2(+) dendritic cells, the natural type I interferon-producing cells, and their recruitment into the graft along with increased expression of MXA, a type I interferon-induced protein, at the tubulointerstitial and vascular level. Finally, interferon alpha mRNA expression was significantly increased in CAMR compared to control

  9. Competency development in antibody production in cancer cell biology

    SciTech Connect

    Park, M.S.

    1998-12-01

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at Los Alamos National Laboratory (LANL). The main objective of this project was to develop a rapid recombinant antibody production technology. To achieve the objective, the authors employed (1) production of recombinant antigens that are important for cell cycle regulation and DNA repair, (2) immunization and specific selection of antibody-producing lymphocytes using the flow cytometry and magnetic bead capturing procedure, (3) construction of single chain antibody library, (4) development of recombinant vectors that target, express, and regulate the expression of intracellular antibodies, and (5) specific inhibition of tumor cell growth in tissue culture. The authors have accomplished (1) optimization of a selection procedure to isolate antigen-specific lymphocytes, (2) optimization of the construction of a single-chain antibody library, and (3) development of a new antibody expression vector for intracellular immunization. The future direction of this research is to continue to test the potential use of the intracellular immunization procedure as a tool to study functions of biological molecules and as an immuno-cancer therapy procedure to inhibit the growth of cancer cells.

  10. Neutralizing human monoclonal antibodies to conformational epitopes of human T-cell lymphotropic virus type 1 and 2 gp46.

    PubMed Central

    Hadlock, K G; Rowe, J; Perkins, S; Bradshaw, P; Song, G Y; Cheng, C; Yang, J; Gascon, R; Halmos, J; Rehman, S M; McGrath, M S; Foung, S K

    1997-01-01

    Ten human monoclonal antibodies derived from peripheral B cells of a patient with human T-cell lymphotropic virus (HTLV)-associated myelopathy are described. One monoclonal antibody recognized a linear epitope within the carboxy-terminal 43 amino acids of HTLV gp21, and two monoclonal antibodies recognized linear epitopes within HTLV type 1 (HTLV-1) gp46. The remaining seven monoclonal antibodies recognized denaturation-sensitive epitopes within HTLV-1 gp46 that were expressed on the surfaces of infected cells. Two of these antibodies also bound to viable HTLV-2 infected cells and immunoprecipitated HTLV-2 gp46. Virus neutralization was determined by syncytium inhibition assays. Eight monoclonal antibodies, including all seven that recognized denaturation-sensitive epitopes within HTLV-1 gp46, possessed significant virus neutralization activity. By competitive inhibition analysis it was determined that these antibodies recognized at least four distinct conformational epitopes within HTLV-1 gp46. These findings indicate the importance of conformational epitopes within HTLV-1 gp46 in mediating a neutralizing antibody response to HTLV infection. PMID:9223472

  11. Elucidation of the potential disease-promoting influence of IgM apoptotic cell-reactive antibodies in lupus.

    PubMed

    Malik, M; Arora, P; Sachdeva, R; Sharma, L; Ramachandran, V G; Pal, R

    2016-06-01

    The undigested remnants of apoptosis are believed to stimulate the generation of autoantibodies in lupus. The biological properties of initiator, disease-specific IgM antibodies that specifically recognize apoptotic cells, readily detected in the sera of lupus patients, remain unclear. Apoptotic cell-reactive IgM monoclonal antibodies (generated from lupus-prone mice), as opposed to control IgM, preferentially stimulated maturation of bone marrow-derived dendritic cells (BMDCs) derived from such mice, relative to BMDCs derived from healthy mice. An influence of both antibody specificity and cell genotype was also apparent in the secretion of signature inflammatory cytokines. Immunization of such antibodies in lupus-prone animals induced increases in total serum IgG levels, with the elicited antibodies also preferentially recognizing moieties on dying cells. An expanded specificity was apparent both upon Western blot on cellular lysate and from the enhanced recognition of dsDNA, Ro60, RNP68 and Sm; the antibody most efficient in mediating autoreactive diversity, while being germline encoded, also induced the highest degree of phenotypic changes on BMDCs. Apoptotic cell-reactive IgM antibodies may therefore be potentially capable of influencing the course of systemic autoimmune disease by affecting both innate and adaptive immunity.

  12. Nanomedicine-mediated cancer stem cell therapy.

    PubMed

    Shen, Song; Xia, Jin-Xing; Wang, Jun

    2016-01-01

    Circumstantial evidence suggests that most tumours are heterogeneous and contain a small population of cancer stem cells (CSCs) that exhibit distinctive self-renewal, proliferation and differentiation capabilities, which are believed to play a crucial role in tumour progression, drug resistance, recurrence and metastasis in multiple malignancies. Given that the existence of CSCs is a primary obstacle to cancer therapy, a tremendous amount of effort has been put into the development of anti-CSC strategies, and several potential approaches to kill therapeutically-resistant CSCs have been explored, including inhibiting ATP-binding cassette transporters, blocking essential signalling pathways involved in self-renewal and survival of CSCs, targeting CSCs surface markers and destroying the tumour microenvironment. Meanwhile, an increasing number of therapeutic agents (e.g. small molecule drugs, nucleic acids and antibodies) to selectively target CSCs have been screened or proposed in recent years. Drug delivery technology-based approaches hold great potential for tackling the limitations impeding clinical applications of CSC-specific agents, such as poor water solubility, short circulation time and inconsistent stability. Properly designed nanocarrier-based therapeutic agents (or nanomedicines) offer new possibilities of penetrating CSC niches and significantly increasing therapeutic drug accumulation in CSCs, which are difficult for free drug counterparts. In addition, intelligent nanomedicine holds great promise to overcome pump-mediated multidrug resistance which is driven by ATP and to decrease detrimental effects on normal somatic stem cells. In this review, we summarise the distinctive biological processes related to CSCs to highlight strategies against inherently drug-resistant CSCs. We then focus on some representative examples that give a glimpse into state-of-the-art nanomedicine approaches developed for CSCs elimination. A perspective on innovative therapeutic

  13. Inhibitory Effect of Individual or Combinations of Broadly Neutralizing Antibodies and Antiviral Reagents against Cell-Free and Cell-to-Cell HIV-1 Transmission

    PubMed Central

    Kolodkin-Gal, Dror; Eslamizar, Leila; Owuor, Joshua O.; Mazzola, Emanuele; Gonzalez, Ana M.; Korioth-Schmitz, Birgit; Gelman, Rebecca S.; Montefiori, David C.; Haynes, Barton F.; Schmitz, Joern E.

    2015-01-01

    ABSTRACT To date, most therapeutic and vaccine candidates for human immunodeficiency virus type 1 (HIV-1) are evaluated preclinically for efficacy against cell-free viral challenges. However, cell-associated HIV-1 is suggested to be a major contributor to sexual transmission by mucosal routes. To determine if neutralizing antibodies or inhibitors block cell-free and cell-associated virus transmission of diverse HIV-1 strains with different efficiencies, we tested 12 different antibodies and five inhibitors against four green fluorescent protein (GFP)-labeled HIV-1 envelope (Env) variants from transmitted/founder (T/F) or chronic infection isolates. We evaluated antibody/inhibitor-mediated virus neutralization using either TZM-bl target cells, in which infectivity was determined by virus-driven luciferase expression, or A3R5 lymphoblastoid target cells, in which infectivity was evaluated by GFP expression. In both the TZM-bl and A3R5 assays, cell-free virus or infected CD4+ lymphocytes were used as targets for neutralization. We further hypothesized that the combined use of specific neutralizing antibodies targeting HIV-1 Env would more effectively prevent cell-associated virus transmission than the use of individual antibodies. The tested antibody combinations included two gp120-directed antibodies, VRC01 and PG9, or VRC01 with the gp41-directed antibody 10E8. Our results demonstrated that cell-associated virus was less sensitive to neutralizing antibodies and inhibitors, particularly using the A3R5 neutralization assay, and the potencies of these neutralizing agents differed among Env variants. A combination of different neutralizing antibodies that target specific sites on gp120 led to a significant reduction in cell-associated virus transmission. These assays will help identify ideal combinations of broadly neutralizing antibodies to use for passive preventive antibody administration and further characterize targets for the most effective neutralizing antibodies

  14. Antibody-Mediated Neutralization of the Exotoxin Mycolactone, the Main Virulence Factor Produced by Mycobacterium ulcerans

    PubMed Central

    Gersbach, Philipp; Hug, Melanie N.; Bieri, Raphael; Bomio, Claudio; Li, Jun; Huber, Sylwia; Altmann, Karl-Heinz; Pluschke, Gerd

    2016-01-01

    Background Mycolactone, the macrolide exotoxin produced by Mycobacterium ulcerans, causes extensive tissue destruction by inducing apoptosis of host cells. In this study, we aimed at the production of antibodies that could neutralize the cytotoxic activities of mycolactone. Methodology/Principal Findings Using the B cell hybridoma technology, we generated a series of monoclonal antibodies with specificity for mycolactone from spleen cells of mice immunized with the protein conjugate of a truncated synthetic mycolactone derivative. L929 fibroblasts were used as a model system to investigate whether these antibodies can inhibit the biological effects of mycolactone. By measuring the metabolic activity of the fibroblasts, we found that anti-mycolactone mAbs can completely neutralize the cytotoxic activity of mycolactone. Conclusions/Significance The toxin neutralizing capacity of anti-mycolactone mAbs supports the concept of evaluating the macrolide toxin as vaccine target. PMID:27351976

  15. Ptch2 mediates the Shh response in Ptch1-/- cells.

    PubMed

    Alfaro, Astrid C; Roberts, Brock; Kwong, Lina; Bijlsma, Maarten F; Roelink, Henk

    2014-09-01

    The Hedgehog (Hh) signaling response is regulated by the interaction of three key components that include the sonic hedgehog (Shh) ligand, its receptor patched 1 (Ptch1) and the pathway activator smoothened (Smo). Under the prevailing model of Shh pathway activation, the binding of Shh to Ptch1 (the key Shh receptor) results in the release of Ptch1-mediated inhibition of Smo, leading to Smo activation and subsequent cell-autonomous activation of the Shh response. Consistent with this model, Ptch1(-/-) cells show a strong upregulation of the Shh response. Our finding that this response can be inhibited by the Shh-blocking antibody 5E1 indicates that the Shh response in Ptch1(-/-) cells remains ligand dependent. Furthermore, we find that Shh induces a strong response in Ptch1(-/-);Shh(-/-) cells, and that Ptch1(-/-) fibroblasts retain their ability to migrate towards Shh, demonstrating that Ptch1(-/-) cells remain sensitive to Shh. Expression of a dominant-negative Ptch1 mutant in the developing chick neural tube had no effect on Shh-mediated patterning, but expression of a dominant-negative form of patched 2 (Ptch2) caused an activation of the Shh response. This indicates that, at early developmental stages, Ptch2 functions to suppress Shh signaling. We found that Ptch1(-/-);Ptch2(-/-) cells cannot further activate the Shh response, demonstrating that Ptch2 mediates the response to Shh in the absence of Ptch1. PMID:25085974

  16. Immunoglobulins, antibody repertoire and B cell development.

    PubMed

    Butler, J E; Zhao, Y; Sinkora, M; Wertz, N; Kacskovics, I

    2009-03-01

    Swine share with most placental mammals the same five antibody isotypes and same two light chain types. Loci encoding lambda, kappa and Ig heavy chains appear to be organized as they are in other mammals. Swine differ from rodents and primates, but are similar to rabbits in using a single VH family (VH3) to encode their variable heavy chain domain, but not the family used by cattle, another artiodactyl. Distinct from other hoofed mammals and rodents, Ckappa:Clambda usage resembles the 1:1 ratio seen in primates. Since IgG subclasses diversified after speciation, same name subclass homologs do not exist among swine and other mammals unless very closely related. Swine possess six putative IgG subclasses that appear to have diversified by gene duplication and exon shuffle while retaining motifs that can bind to FcgammaRs, FcRn, C1q, protein A and protein G. The epithelial chorial placenta of swine and the precosial nature of their offspring have made piglets excellent models for studies on fetal antibody repertoire development and on the postnatal role of gut colonization, maternal colostrum and neonatal infection on the development of adaptive immunity during the "critical window" of immunological development. This chapter traces the study of the humoral immune system of this species through its various eras of discovery and compiles the results in tables and figures that should be a useful reference for educators and investigators.

  17. Polyanhydride Nanovaccines Induce Germinal Center B Cell Formation and Sustained Serum Antibody Responses.

    PubMed

    Vela Ramirez, Julia E; Tygrett, Lorraine T; Hao, Jihua; Habte, Habtom H; Cho, Michael W; Greenspan, Neil S; Waldschmidt, Thomas J; Narasimhan, Balaji

    2016-06-01

    Biodegradable polymeric nanoparticle-based subunit vaccines have shown promising characteristics by enhancing antigen presentation and inducing protective immune responses when compared with soluble protein. Specifically, polyanhydride nanoparticle-based vaccines (i.e., nanovaccines) have been shown to successfully encapsulate and release antigens, activate B and T cells, and induce both antibody- and cell-mediated immunity towards a variety of immunogens. One of the characteristics of strong thymus-dependent antibody responses is the formation of germinal centers (GC) and the generation of GC B cells, which is part of the T helper cell driven cellular response. In order to further understand the role of nanovaccines in the induction of antigen-specific immune responses, their ability to induce germinal center B cell formation and isotype switching and the effects thereof on serum antibody responses were investigated in these studies. Polyanhydride nanovaccines based on 1,6-bis(p-carboxyphenoxy)hexane and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane were used to subcutaneously administer a viral antigen. GC B cell formation and serum antibody responses induced by the nanovaccines were compared to that induced by alum-based vaccine formulations. It was demonstrated that a single dose of polyanhydride nanovaccines resulted in the formation of robust GCs and serum antibody in comparison to that induced by the alum-based formulation. This was attributed to the sustained release of antigen provided by the nanovaccines. When administered in a multiple dose regimen, the highest post-immunization titer and GC B cell number was enhanced, and the immune response induced by the nanovaccines was further sustained. These studies provide foundational information on the mechanism of action of polyanhydride nanovaccines. PMID:27319223

  18. Facilitation of syngeneic stem cell engraftment by anti-class I monoclonal antibody pretreatment of unirradiated recipients

    SciTech Connect

    Voralia, M.; Semeluk, A.; Wegmann, T.G.

    1987-10-01

    We have established a murine model of syngeneic bone marrow transplantation based on the use of monoclonal antibody as the sole conditioning regimen in unirradiated recipients. Administration of a single injection of monoclonal antibody directed against major histocompatibility complex-encoded class I determinants facilitated permanent hemopoietic stem cell engraftment without any apparent side-effects. Whereas untreated hosts exhibited a maximal chimerism of 15% at donor cell doses of up to 12 X 10(7) bone marrow cells, pretreatment by 2 mg of anti-class I antibody one week prior to transplantation of 3 X 10(7) syngeneic bone marrow cells resulted in a mean donor representation of about 80%. The antibody can be given up to four weeks prior to transplantation, and the degree of donor engraftment observed is a function of the dose of antibody administered. The fact that specific antibody enhanced engraftment in two strain combinations indicates that antibody is the active agent in facilitating engraftment and that facilitation is not strain-restricted. Anti-class I antibodies of the IgG2a, but not IgG1, isotype are effective in promoting engraftment. Although the isotype requirement suggests a role for antibody-mediated cytotoxicity in promoting stem cell engraftment, the extensive time-frame of facilitation suggests that other effects of the antibody may also be involved. The model of syngeneic bone marrow transplantation we describe here will be useful in studying the mechanisms regulating stem cell engraftment and may have potential clinical application as an approach to autologous marrow transplantation.

  19. Novel mutations in Marburg virus glycoprotein associated with viral evasion from antibody mediated immune pressure.

    PubMed

    Kajihara, Masahiro; Nakayama, Eri; Marzi, Andrea; Igarashi, Manabu; Feldmann, Heinz; Takada, Ayato

    2013-04-01

    Marburg virus (MARV) and Ebola virus, members of the family Filoviridae, cause lethal haemorrhagic fever in humans and non-human primates. Although the outbreaks are concentrated mainly in Central Africa, these viruses are potential agents of imported infectious diseases and bioterrorism in non-African countries. Recent studies demonstrated that non-human primates passively immunized with virus-specific antibodies were successfully protected against fatal filovirus infection, highlighting the important role of antibodies in protective immunity for this disease. However, the mechanisms underlying potential evasion from antibody mediated immune pressure are not well understood. To analyse possible mutations involved in immune evasion in the MARV envelope glycoprotein (GP) which is the major target of protective antibodies, we selected escape mutants of recombinant vesicular stomatitis virus (rVSV) expressing MARV GP (rVSVΔG/MARVGP) by using two GP-specific mAbs, AGP127-8 and MGP72-17, which have been previously shown to inhibit MARV budding. Interestingly, several rVSVΔG/MARVGP variants escaping from the mAb pressure-acquired amino acid substitutions in the furin-cleavage site rather than in the mAb-specific epitopes, suggesting that these epitopes are recessed, not exposed on the uncleaved GP molecule, and therefore inaccessible to the mAbs. More surprisingly, some variants escaping mAb MGP72-17 lacked a large proportion of the mucin-like region of GP, indicating that these mutants efficiently escaped the selective pressure by deleting the mucin-like region including the mAb-specific epitope. Our data demonstrate that MARV GP possesses the potential to evade antibody mediated immune pressure due to extraordinary structural flexibility and variability.

  20. Heparan sulfate mediates trastuzumab effect in breast cancer cells

    PubMed Central

    2013-01-01

    these resistant cells. Conclusion Trastuzumab action is dependent on the availability of heparan sulfate on the surface of breast cancer cells. Furthermore, our data suggest that high levels of heparan sulfate shed to the medium are able to capture trastuzumab, blocking the antibody action mediated by HER2. In addition to HER2 levels, heparan sulfate synthesis and shedding determine breast cancer cell susceptibility to trastuzumab. PMID:24083474

  1. Cell-Mediated Immune Responses in Paraneoplastic Neurological Syndromes

    PubMed Central

    Zaborowski, Mikolaj Piotr

    2013-01-01

    Paraneoplastic neurological syndromes (PNS) are disorders of the nervous system that are associated with remote effects of malignancy. PNS are considered to have an autoimmune pathology. It has been suggested that immune antitumor responses are the origin of improved outcome in PNS. We describe cell-mediated immune responses in PNS and their potential contributions to antitumor reactions. Experimental and neuropathological studies have revealed infiltrates in nervous tissue and disturbances in lymphocyte populations in both cerebrospinal fluid and peripheral blood. A predominance of cytotoxic T lymphocytes (CTLs) over T helper cells has been observed. CTLs can be specifically aggressive against antigens shared by tumors and nervous tissue. Based on genetic studies, a common clonal origin of lymphocytes from blood, tumor, and nervous tissue is suggested. Suppressive regulatory T (Treg) lymphocytes are dysfunctional. Simultaneously, in tumor tissue, more intense cell-mediated immune responses are observed, which often coincide with a less aggressive course of neoplastic disease. An increased titer of onconeural antibodies is also related to better prognoses in patients without PNS. The evaluation of onconeural and neuronal surface antibodies was recommended in current guidelines. The link between PNS emergence and antitumor responses may result from more active CTLs and less functional Treg lymphocytes. PMID:24575143

  2. Fas and its ligand in a general mechanism of T-cell-mediated cytotoxicity.

    PubMed Central

    Hanabuchi, S; Koyanagi, M; Kawasaki, A; Shinohara, N; Matsuzawa, A; Nishimura, Y; Kobayashi, Y; Yonehara, S; Yagita, H; Okumura, K

    1994-01-01

    To investigate the mechanisms of T-cell-mediated cytotoxicity, we estimated the involvement of apoptosis-inducing Fas molecule on the target cells and its ligand on the effector cells. When redirected by ConA or anti-CD3 monoclonal antibody, a CD4+ T-cell clone, BK1, could lyse the target cells expressing wild-type Fas molecule but not those expressing death signaling-deficient mutants. This indicates the involvement of Fas-mediated signal transduction in the target cell lysis by BK1. Anti-CD3-activated but not resting BK1 expressed Fas ligand as detected by binding of a soluble Fas-Ig fusion protein, and the BK1-mediated cytotoxicity was blocked by the addition of Fas-Ig, implicating the inducible Fas ligand in the BK1 cytotoxicity. Ability to exert the Fas-mediated cytotoxicity was not confined to BK1, but splenic CD4+ T cells and, to a lesser extent, CD8+ T cells could also exert the Fas-dependent target cell lysis. This indicates that the Fas-mediated target cell lytic pathway can be generally involved in the T-cell-mediated cytotoxicity. Interestingly, CD4+ T cells prepared from gld/gld mice did not mediate the Fas-mediated cytotoxicity, indicating defective expression of functional Fas ligand in gld mice. PMID:7515183

  3. Antibody mediated therapy targeting CD47 inhibits tumor progression of hepatocellular carcinoma

    PubMed Central

    Xiao, Zhenyu; Chung, Haniee; Banan, Babak; Manning, Pamela T.; Ott, Katherine C.; Lin, Shin; Capoccia, Benjamin J.; Subramanian, Vijay; Hiebsch, Ronald R.; Upadhya, Gundumi A.; Mohanakumar, Thalachallour; Frazier, William A.; Lin, Yiing; Chapman, William C.

    2016-01-01

    Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival times. The efficacy of current systemic therapies for HCC is limited. In this study, we used xenograft tumor models to investigate the use of antibodies that block CD47 and inhibit HCC tumor growth. Immunostaining of tumor tissue and HCC cell lines demonstrated CD47 over-expression in HCC as compared to normal hepatocytes. Macrophage phagocytosis of HCC cells was increased after treatment with CD47 antibodies (CD47mAbs) that block CD47 binding to SIRPα. Further, CD47 blockade inhibited tumor growth in both heterotopic and orthotopic models of HCC, and promoted the migration of macrophages into the tumor mass. Our results demonstrate that targeting CD47 by specific antibodies has potential immunotherapeutic efficacy in human HCC. PMID:25721088

  4. A-CAM: a 135-kD receptor of intercellular adherens junctions. II. Antibody-mediated modulation of junction formation

    PubMed Central

    1986-01-01

    Intercellular adherens junctions between cultured lens epithelial cells are highly Ca2+-dependent and are readily dissociated upon chelation of extracellular Ca2+ ions. Addition of Ca2+ to EGTA-treated cells results in the recovery of cell-cell junctions including the reorganization of adherens junction-specific cell adhesion molecule (A-CAM), vinculin, and actin (Volk, T., and B. Geiger, 1986, J. Cell Biol., 103:000-000). Incubation of cells during the recovery phase with Fab' fragments of anti-A-CAM specifically inhibited the re-formation of cell-cell adherens junctions. This inhibition was accompanied by remarkable changes in microfilament organization manifested by an apparent deterioration of stress fibers and the appearance of fragmented actin bundles throughout the cytoplasm. Incubation of EGTA-dissociated cells with intact divalent anti-A-CAM antibodies in normal medium had no apparent inhibitory effect on junction formation and did not affect the assembly of actin microfilament bundles. Moreover, adherens junctions formed in the presence of the divalent antibodies became essentially Ca2+-independent, suggesting that cell-cell adhesion between them was primarily mediated by the antibodies. These studies suggest that A-CAM participates in intercellular adhesion in adherens-type junctions and point to its involvement in microfilament bundle assembly. PMID:3095334

  5. M-cadherin-mediated intercellular interactions activate satellite cell division.

    PubMed

    Marti, Merce; Montserrat, Núria; Pardo, Cristina; Mulero, Lola; Miquel-Serra, Laia; Rodrigues, Alexandre Miguel Cavaco; Andrés Vaquero, José; Kuebler, Bernd; Morera, Cristina; Barrero, María José; Izpisua Belmonte, Juan Carlos

    2013-11-15

    Adult muscle stem cells and their committed myogenic precursors, commonly referred to as the satellite cell population, are involved in both muscle growth after birth and regeneration after damage. It has been previously proposed that, under these circumstances, satellite cells first become activated, divide and differentiate, and only later fuse to the existing myofiber through M-cadherin-mediated intercellular interactions. Our data show that satellite cells fuse with the myofiber concomitantly to cell division, and only when the nuclei of the daughter cells are inside the myofiber, do they complete the process of differentiation. Here we demonstrate that M-cadherin plays an important role in cell-to-cell recognition and fusion, and is crucial for cell division activation. Treatment of satellite cells with M-cadherin in vitro stimulates cell division, whereas addition of anti-M-cadherin antibodies reduces the cell division rate. Our results suggest an alternative model for the contribution of satellite cells to muscle development, which might be useful in understanding muscle regeneration, as well as muscle-related dystrophies.

  6. CEA TCB: A novel head-to-tail 2:1 T cell bispecific antibody for treatment of CEA-positive solid tumors

    PubMed Central

    Bacac, Marina; Klein, Christian; Umana, Pablo

    2016-01-01

    ABSTRACT Carcinoembryonic antigen T cell bispecific antibody (CEA TCB) is a bispecific antibody used to recognize CEA and CD3e via a novel molecular format (2:1) that induces T cell-mediated killing of CEA over-expressing tumors while sparing primary cells with low CEA expression. CEA TCB treatment inhibits tumor growth and generates a highly inflamed tumor microenvironment. PMID:27622073

  7. Intravenous immunoglobulins and rituximab therapy for severe transplant glomerulopathy in chronic antibody-mediated rejection: a pilot study.

    PubMed

    Bachelet, Thomas; Nodimar, Celine; Taupin, Jean-Luc; Lepreux, Sebastien; Moreau, Karine; Morel, Delphine; Guidicelli, Gwendaline; Couzi, Lionel; Merville, Pierre

    2015-05-01

    Outcome of patients with transplant glomerulopathy (TG) is poor. Using B-cell targeting molecules represent a rational strategy to treat TG during chronic antibody-mediated rejection. In this pilot study, 21 patients with this diagnosis received four doses of intravenous immunoglobulins and two doses of rituximab (IVIG/RTX group). They were retrospectively compared with a untreated control group of 10 patients. At 24 months post-biopsy, graft survival was similar and poor between the treated and the untreated group, 47% vs. 40%, respectively, p = 0.69. This absence of response of IVIG/RTX treatment was observed, regardless the phenotype of TG. Baseline estimated glomerular filtration rate (eGFR) and decline in eGFR during the first six months after the treatment were risk factors associated with 24-month graft survival. The IVIG/RTX therapy had a modest effect on the kinetics of donor-specific alloantibodies at M24, compared to the untreated group, not associated with an improvement in graft survival. The mean number of adverse events per patient was higher in the IVIG/RTX group than in the control group (p = 0.03). Taken together, IVIG/RTX treatment for severe TG during chronic antibody-mediated rejection does not seem to change the natural history of TG and is associated with a high incidence of adverse events.

  8. Design of a stable cell line producing a recombinant monoclonal anti-TNFα antibody based on a CHO cell line.

    PubMed

    Voronina, E V; Seregin, Y A; Litvinova, N A; Shvets, V I; Shukurov, R R

    2016-01-01

    Recombinant monoclonal antibodies (mAbs) against tumor necrosis factor alpha are widely used in the biopharmaceutical therapy of autoimmune diseases. Currently, a large number of drugs based on these antibodies are available. Accordingly, the development of these products for the Russian market is an important goal. The aim of the current study is to describe the development of one such technology. CHO-DG44-derived cell lines producing mAb were developed using two strategies, one based on individual clones and the other based on cell pools. To obtain recombinant cell lines with highly amplified genes of interest, the clones underwent dihydrofolate reductase-mediated gene amplification. Using the best strategy for the selection and amplification of mAb-producing clones, we achieved the production of more than 1 g/L in small scale, non-optimized conditions. PMID:27652157

  9. A complement-dependent model of thrombotic thrombocytopenic purpura induced by antibodies reactive with endothelial cells.

    PubMed

    Ren, Guohui; Hack, Bradley K; Minto, Andrew W; Cunningham, Patrick N; Alexander, Jessy J; Haas, Mark; Quigg, Richard J

    2002-04-01

    Thrombotic thrombocytopenic purpura (TTP) is an immunologically mediated disease characterized by thrombocytopenia, hemolytic anemia, and pathologic changes in various organs, including the kidney, which are secondary to widespread thromboses. Central to TTP is platelet activation, which may occur from a variety of mechanisms, including endothelial cell activation or injury. In this study, injection of K6/1, a monoclonal antibody with widespread reactivity toward endothelia, led to dose-dependent thrombocytopenia in rats. This was magnified if animals were preimmunized with mouse IgG, thereby resulting in an accelerated autologous phase of injury. In this setting, significant anemia also resulted. Rats injected with K6/1 developed renal injury, consisting of tubular damage and glomerular thrombi. Thrombocytopenia and renal morphological abnormalities were eliminated if animals were complement depleted with cobra venom factor prior to K6/1 injection and worsened when the activity of the ubiquitous complement regulator Crry was inhibited with function-neutralizing antibodies. Therefore, we have developed a complement-dependent model of TTP in rats by injecting monoclonal antibodies reactive with endothelial cells. Antibody-directed complement activation leads to stimulation of platelets, through direct interactions with complement fragments and/or indirectly through endothelial cell activation or injury, with the subsequent development of TTP.

  10. 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

  11. Elimination of human T cell leukemia virus type-1-infected cells by neutralizing and antibody-dependent cellular cytotoxicity-inducing antibodies against human t cell leukemia virus type-1 envelope gp46.

    PubMed

    Tanaka, Yuetsu; Takahashi, Yoshiaki; Tanaka, Reiko; Kodama, Akira; Fujii, Hideki; Hasegawa, Atsuhiko; Kannagi, Mari; Ansari, Aftab A; Saito, Mineki

    2014-06-01

    Human T cell leukemia virus type-1 (HTLV-1) is prevalent worldwide with foci of high prevalence. However, to date no effective vaccine or drug against HTLV-1 infection has been developed. In efforts to define the role of antibodies in the control of HTLV-1 infection, we capitalized on the use of our previously defined anti-gp46 neutralizing monoclonal antibody (mAb) (clone LAT-27) and high titers of human anti-HTLV-1 IgG purified from HAM/TSP patients (HAM-IgG). LAT-27 and HAM-IgG completely blocked syncytium formation and T cell immortalization mediated by HTLV-1 in vitro. The addition of these antibodies to cultures of CD8(+) T cell-depleted peripheral blood mononuclear cells (PBMCs) from HAM/TSP patients at the initiation of culture not only decreased the numbers of Tax-expressing cells and the production of HTLV-1 p24 but also inhibited the spontaneous immortalization of T cells. Coculture of in vitro-HTLV-1-immortalized T cell lines with autologous PBMCs in the presence of LAT-27 or HAM-IgG, but not an F(ab')2 fragment of LAT-27 or nonneutralizing anti-gp46 mAbs, resulted in depletion of HTLV-1-infected cells. A 24-h (51)Cr release assay showed the presence of significant antibody-dependent cellular cytotoxicity (ADCC) activity in LAT-27 and HAM-IgG, but not F(ab')2 of LAT-27, resulting in the depletion of HTLV-1-infected T cells by autologous PBMCs. The depletion of natural killer (NK) cells from the effector PBMCs reduced this ADCC activity. Altogether, the present data demonstrate that the neutralizing and ADCC-inducing activities of anti-HTLV-1 antibodies are capable of reducing infection and eliminating HTLV-1-infected cells in the presence of autologous PBMCs. PMID:24524420

  12. Elimination of HIV-1-infected cells by broadly neutralizing antibodies.

    PubMed

    Bruel, Timothée; Guivel-Benhassine, Florence; Amraoui, Sonia; Malbec, Marine; Richard, Léa; Bourdic, Katia; Donahue, Daniel Aaron; Lorin, Valérie; Casartelli, Nicoletta; Noël, Nicolas; Lambotte, Olivier; Mouquet, Hugo; Schwartz, Olivier

    2016-01-01

    The Fc region of HIV-1 Env-specific broadly neutralizing antibodies (bNAbs) is required for suppressing viraemia, through mechanisms which remain poorly understood. Here, we identify bNAbs that exert antibody-dependent cellular cytotoxicity (ADCC) in cell culture and kill HIV-1-infected lymphocytes through natural killer (NK) engagement. These antibodies target the CD4-binding site, the glycans/V3 and V1/V2 loops on gp120, or the gp41 moiety. The landscape of Env epitope exposure at the surface and the sensitivity of infected cells to ADCC vary considerably between viral strains. Efficient ADCC requires sustained cell surface binding of bNAbs to Env, and combining bNAbs allows a potent killing activity. Furthermore, reactivated infected cells from HIV-positive individuals expose heterogeneous Env epitope patterns, with levels that are often but not always sufficient to trigger killing by bNAbs. Our study delineates the parameters controlling ADCC activity of bNAbs, and supports the use of the most potent antibodies to clear the viral reservoir. PMID:26936020

  13. Elimination of HIV-1-infected cells by broadly neutralizing antibodies

    PubMed Central

    Bruel, Timothée; Guivel-Benhassine, Florence; Amraoui, Sonia; Malbec, Marine; Richard, Léa; Bourdic, Katia; Donahue, Daniel Aaron; Lorin, Valérie; Casartelli, Nicoletta; Noël, Nicolas; Lambotte, Olivier; Mouquet, Hugo; Schwartz, Olivier

    2016-01-01

    The Fc region of HIV-1 Env-specific broadly neutralizing antibodies (bNAbs) is required for suppressing viraemia, through mechanisms which remain poorly understood. Here, we identify bNAbs that exert antibody-dependent cellular cytotoxicity (ADCC) in cell culture and kill HIV-1-infected lymphocytes through natural killer (NK) engagement. These antibodies target the CD4-binding site, the glycans/V3 and V1/V2 loops on gp120, or the gp41 moiety. The landscape of Env epitope exposure at the surface and the sensitivity of infected cells to ADCC vary considerably between viral strains. Efficient ADCC requires sustained cell surface binding of bNAbs to Env, and combining bNAbs allows a potent killing activity. Furthermore, reactivated infected cells from HIV-positive individuals expose heterogeneous Env epitope patterns, with levels that are often but not always sufficient to trigger killing by bNAbs. Our study delineates the parameters controlling ADCC activity of bNAbs, and supports the use of the most potent antibodies to clear the viral reservoir. PMID:26936020

  14. Microtubule-dependent control of cell shape and pseudopodial activity is inhibited by the antibody to kinesin motor domain

    PubMed Central

    1993-01-01

    One of the major functions of cytoplasmic microtubules is their involvement in maintenance of asymmetric cell shape. Microtubules were considered to perform this function working as rigid structural elements. At the same time, microtubules play a critical role in intracellular organelle transport, and this fact raises the possibility that the involvement of microtubules in maintenance of cell shape may be mediated by directed transport of certain cellular components to a limited area of the cell surface (e.g., to the leading edge) rather than by their functioning as a mechanical support. To test this hypothesis we microinjected cultured human fibroblasts with the antibody (called HD antibody) raised against kinesin motor domain highly conserved among the different members of kinesin superfamily. As was shown before this antibody inhibits kinesin-dependent microtubule gliding in vitro and interferes with a number of microtubule-dependent transport processes in living cells. Preimmune IgG fraction was used for control experiments. Injections of fibroblasts with HD antibody but not with preimmune IgG significantly reduced their asymmetry, resulting in loss of long processes and elongated cell shape. In addition, antibody injection suppressed pseudopodial activity at the leading edge of fibroblasts moving into an experimentally made wound. Analysis of membrane organelle distribution showed that kinesin antibody induced clustering of mitochondria in perinuclear region and their withdrawal from peripheral parts of the cytoplasm. HD antibody does not affect either density or distribution of cytoplasmic microtubules. The results of our experiments show that many changes of phenotype induced in cells by microtubule-depolymerizing agents can be mimicked by the inhibition of motor proteins, and therefore microtubule functions in maintaining of the cell shape and polarity are mediated by motor proteins rather than by being provided by rigidity of tubulin polymer itself. PMID

  15. Novel anti–B-cell maturation antigen antibody-drug conjugate (GSK2857916) selectively induces killing of multiple myeloma

    PubMed Central

    Mayes, Patrick A.; Acharya, Chirag; Zhong, Mike Y.; Cea, Michele; Cagnetta, Antonia; Craigen, Jenny; Yates, John; Gliddon, Louise; Fieles, William; Hoang, Bao; Tunstead, James; Christie, Amanda L.; Kung, Andrew L.; Richardson, Paul; Munshi, Nikhil C.; Anderson, Kenneth C.

    2014-01-01

    B-cell maturation antigen (BCMA), highly expressed on malignant plasma cells in human multiple myeloma (MM), has not been effectively targeted with therapeutic monoclonal antibodies. We here show that BCMA is universally expressed on the MM cell surface and determine specific anti-MM activity of J6M0-mcMMAF (GSK2857916), a novel humanized and afucosylated antagonistic anti-BCMA antibody-drug conjugate via a noncleavable linker. J6M0-mcMMAF specifically blocks cell growth via G2/M arrest and induces caspase 3–dependent apoptosis in MM cells, alone and in coculture with bone marrow stromal cells or various effector cells. It strongly inhibits colony formation by MM cells while sparing surrounding BCMA-negative normal cells. J6M0-mcMMAF significantly induces effector cell-mediated lysis against allogeneic or autologous patient MM cells, with increased potency and efficacy compared with the wild-type J6M0 without Fc enhancement. The antibody-dependent cell-mediated cytotoxicity and apoptotic activity of J6M0-mcMMAF is further enhanced by lenalidomide. Importantly, J6M0-mcMMAF rapidly eliminates myeloma cells in subcutaneous and disseminated mouse models, and mice remain tumor-free up to 3.5 months. Furthermore, J6M0-mcMMAF recruits macrophages and mediates antibody-dependent cellular phagocytosis of MM cells. Together, these results demonstrate that GSK2857916 has potent and selective anti-MM activities via multiple cytotoxic mechanisms, providing a promising next-generation immunotherapeutic in this cancer. PMID:24569262

  16. Immunosuppressive human anti-CD83 monoclonal antibody depletion of activated dendritic cells in transplantation.

    PubMed

    Seldon, T A; Pryor, R; Palkova, A; Jones, M L; Verma, N D; Findova, M; Braet, K; Sheng, Y; Fan, Y; Zhou, E Y; Marks, J D; Munro, T; Mahler, S M; Barnard, R T; Fromm, P D; Silveira, P A; Elgundi, Z; Ju, X; Clark, G J; Bradstock, K F; Munster, D J; Hart, D N J

    2016-03-01

    Current immunosuppressive/anti-inflammatory agents target the responding effector arm of the immune response and their nonspecific action increases the risk of infection and malignancy. These effects impact on their use in allogeneic haematopoietic cell transplantation and other forms of transplantation. Interventions that target activated dendritic cells (DCs) have the potential to suppress the induction of undesired immune responses (for example, graft versus host disease (GVHD) or transplant rejection) and to leave protective T-cell immune responses intact (for example, cytomegalovirus (CMV) immunity). We developed a human IgG1 monoclonal antibody (mAb), 3C12, specific for CD83, which is expressed on activated but not resting DC. The 3C12 mAb and an affinity improved version, 3C12C, depleted CD83(+) cells by CD16(+) NK cell-mediated antibody-dependent cellular cytotoxicity, and inhibited allogeneic T-cell proliferation in vitro. A single dose of 3C12C prevented human peripheral blood mononuclear cell-induced acute GVHD in SCID mouse recipients. The mAb 3C12C depleted CMRF-44(+)CD83(bright) activated DC but spared CD83(dim/-) DC in vivo. It reduced human T-cell activation in vivo and maintained the proportion of CD4(+) FoxP3(+) CD25(+) Treg cells and also viral-specific CD8(+) T cells. The anti-CD83 mAb, 3C12C, merits further evaluation as a new immunosuppressive agent in transplantation.

  17. Functional advantage of educated KIR2DL1(+) natural killer cells for anti-HIV-1 antibody-dependent activation.

    PubMed

    Gooneratne, S L; Center, R J; Kent, S J; Parsons, M S

    2016-04-01

    Evidence from the RV144 HIV-1 vaccine trial implicates anti-HIV-1 antibody-dependent cellular cytotoxicity (ADCC) in vaccine-conferred protection from infection. Among effector cells that mediate ADCC are natural killer (NK) cells. The ability of NK cells to be activated in an antibody-dependent manner is reliant upon several factors. In general, NK cell-mediated antibody-dependent activation is most robust in terminally differentiated CD57(+) NK cells, as well as NK cells educated through ontological interactions between inhibitory killer immunoglobulin-like receptors (KIR) and their major histocompatibility complex class I [MHC-I or human leucocyte antigen (HLA-I)] ligands. With regard to anti-HIV-1 antibody-dependent NK cell activation, previous research has demonstrated that the epidemiologically relevant KIR3DL1/HLA-Bw4 receptor/ligand combination confers enhanced activation potential. In the present study we assessed the ability of the KIR2DL1/HLA-C2 receptor/ligand combination to confer enhanced activation upon direct stimulation with HLA-I-devoid target cells or antibody-dependent stimulation with HIV-1 gp140-pulsed CEM.NKr-CCR5 target cells in the presence of an anti-HIV-1 antibody source. Among donors carrying the HLA-C2 ligand for KIR2DL1, higher interferon (IFN)-γ production was observed within KIR2DL1(+) NK cells than in KIR2DL1(-) NK cells upon both direct and antibody-dependent stimulation. No differences in KIR2DL1(+) and KIR2DL1(-) NK cell activation were observed in HLA-C1 homozygous donors. Additionally, higher activation in KIR2DL1(+) than KIR2DL1(-) NK cells from HLA-C2 carrying donors was observed within less differentiated CD57(-) NK cells, demonstrating that the observed differences were due to education and not an overabundance of KIR2DL1(+) NK cells within differentiated CD57(+) NK cells. These observations are relevant for understanding the regulation of anti-HIV-1 antibody-dependent NK cell responses.

  18. Growth control of genetically modified cells using an antibody/c-Kit chimera.

    PubMed

    Kaneko, Etsuji; Kawahara, Masahiro; Ueda, Hiroshi; Nagamune, Teruyuki

    2012-05-01

    Gene therapy has been regarded as an innovative potential treatment against serious congenital diseases. However, applications of gene therapy remain limited, partly because its clinical success depends on therapeutic gene-transduced cells acquiring a proliferative advantage. To address this problem, we have developed the antigen-mediated genetically modified cell amplification (AMEGA) system, which uses chimeric receptors to enable the selective proliferation of gene-transduced cells. In this report, we describe mimicry of c-Kit signaling and its application to the AMEGA system. We created an antibody/c-Kit chimera in which the extracellular domain of c-Kit is replaced with an anti-fluorescein single-chain Fv antibody fragment and the extracellular D2 domain of the erythropoietin receptor. A genetically modified mouse pro-B cell line carrying this chimera showed selective expansion in the presence of fluorescein-conjugated BSA (BSA-FL) as a growth inducer. By further engineering the transmembrane domain of the chimera to reduce interchain interaction we attained stricter ligand-dependency. Since c-Kit is an important molecule in the expansion of hematopoietic stem cells (HSCs), this antibody/c-Kit chimera could be a promising tool for gene therapy targeting HSCs.

  19. 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

  20. Inhibition of B cell growth factor (BCGF) by monoclonal antibodies directed against the C3d receptor (CR2).

    PubMed

    Perri, R T; Wilson, B S; Kay, N E

    1986-04-01

    Normal human B cell proliferation is controlled by various immunoregulatory signals including the T cell-derived lymphokine B cell growth factor (BCGF). Human BCGF provides the final proliferative signal to normal, activated B cells. We herein show that anti-CR2 monoclonal antibodies inhibit human B cell responsiveness to purified BCGF. Addition of anti-CR2 antibody, AB5, was capable of completely inhibiting BCGF-mediated enhancement of either anti-mu or staphylococcal protein A-activated human B cells (191 +/- 21 cpm vs. 3942 +/- 622 cpm, mean +/- SEM). Inhibition of B cell response to BCGF by AB5 occurred in a dose-dependent manner. Monoclonal antibody anti-B2, which recognizes the same 140-kDa glycoprotein as AB5, in comparable concentrations also inhibited B cell responsiveness to BCGF. Monoclonal antibodies of the same subclass (IgG1) showed no inhibitory effect on BCGF enhancement of B cell proliferation. The F(ab')2 fragment of AB5 generated by pepsin digestion was similarly inhibitory as was the intact Ig. AB5-mediated inhibition was independent of the target B cell state of activation. Both resting and activated B cells (anti-mu or staphylococcal protein A activated) incubated with similar concentrations of AB5 were unresponsive to BCGF. The ability of anti-CR2 antibodies to block BCGF-dependent B cell proliferation suggests that occupancy of C3d membrane receptors may result in modulation of B cell proliferation in physiologic or clinical disease states. PMID:2938967

  1. A new class of bispecific antibodies to redirect T cells for cancer immunotherapy.

    PubMed

    Rossi, Diane L; Rossi, Edmund A; Cardillo, Thomas M; Goldenberg, David M; Chang, Chien-Hsing

    2014-01-01

    Various constructs of bispecific antibodies (bsAbs) to redirect effector T cells for the targeted killing of tumor cells have shown considerable promise in both preclinical and clinical studies. The single-chain variable fragment (scFv)-based formats, including bispecific T-cell engager (BiTE) and dual-affinity re-targeting (DART), which provide monovalent binding to both CD3 on T cells and to the target antigen on tumor cells, can exhibit rapid blood clearance and neurological toxicity due to their small size (~55 kDa). Herein, we describe the generation, by the modular DOCK-AND-LOCK™) (DNL™) method, of novel T-cell redirecting bispecific antibodies, each comprising a monovalent anti-CD3 scFv covalently conjugated to a stabilized dimer of different anti-tumor Fabs. The potential advantages of this design include bivalent binding to tumor cells, a larger size (~130 kDa) to preclude renal clearance and penetration of the blood-brain barrier, and potent T-cell mediated cytotoxicity. These prototypes were purified to near homogeneity, and representative constructs were shown to provoke the formation of immunological synapses between T cells and their target tumor cells in vitro, resulting in T-cell activation and proliferation, as well as potent T-cell mediated anti-tumor activity. In addition, in vivo studies in NOD/SCID mice bearing Raji Burkitt lymphoma or Capan-1 pancreatic carcinoma indicated statistically significant inhibition of tumor growth compared with untreated controls. PMID:24492297

  2. Maternal IgG and IgA Antibodies Dampen Mucosal T Helper Cell Responses in Early Life.

    PubMed

    Koch, Meghan A; Reiner, Gabrielle L; Lugo, Kyler A; Kreuk, Lieselotte S M; Stanbery, Alison G; Ansaldo, Eduard; Seher, Thaddeus D; Ludington, William B; Barton, Gregory M

    2016-05-01

    To maintain a symbiotic relationship between the host and its resident intestinal microbiota, appropriate mucosal T cell responses to commensal antigens must be established. Mice acquire both IgG and IgA maternally; the former has primarily been implicated in passive immunity to pathogens while the latter mediates host-commensal mutualism. Here, we report the surprising observation that mice generate T cell-independent and largely Toll-like receptor (TLR)-dependent IgG2b and IgG3 antibody responses against their gut microbiota. We demonstrate that maternal acquisition of these antibodies dampens mucosal T follicular helper responses and subsequent germinal center B cell responses following birth. This work reveals a feedback loop whereby T cell-independent, TLR-dependent antibodies limit mucosal adaptive immune responses to newly acquired commensal antigens and uncovers a broader function for maternal IgG. PMID:27153495

  3. Photosensitizers form in histidine buffer and mediate the photodegradation of a monoclonal antibody.

    PubMed

    Stroop, Steven D; Conca, David M; Lundgard, Robert P; Renz, Mark E; Peabody, Lynn M; Leigh, Scott D

    2011-12-01

    Fluorescent light (FL) photodegradation of a monoclonal antibody (mAb) formulated in histidine buffer is mediated by histidine-derived photosensitizers that accumulate and greatly increase with light exposure. Histidine-derived photosensitizers are the primary mediators of Trp photooxidation. FL-photodegradation requires light exposure and is pH dependent. It is significantly reduced or eliminated by buffer exchanges, by oxygen depletion, or at pH values greater than 7. Antibody-fragment MS ion counts reveal that oxidation of a single light chain Trp in CDR1 correlates with binding loss. Multiple heavy chain methionines oxidize, but poorly correlate with binding loss. Photosensitizers extracted from photo-aged histidine buffer are potent mediators of FL-photodegradation including oxidation and, to a lesser degree, fragmentation and aggregation of the mAb. These photosensitizers absorb visible light and have neutral mass of 187.1- 386.1 Da. They are also fluorescent with ex/em at 360/450 nm. When spiked into histidine or MES buffered mAb formulations they produce a concentration dependent and pronounced increase in FL-photodegradation; however, no oxidation or loss of antibody function occurs in the dark and hydrogen peroxide does not oxidize Trp. The major component is consistent with histidine oxidation to 6a-hydroxy-2-oxo-octahydro-pyrollo[2,3-d]imidazole-5-carboxylic acid. Photosensitizer levels measured in the formulation prior to light exposure, are linearly related to the FL-photodegradation observed and can predict degradation in photostability testing.

  4. Acute Antibody-Mediated Rejection in Renal Transplantation: Current Clinical Management

    PubMed Central

    Schinstock, Carrie; Stegall, Mark D.

    2014-01-01

    Acute antibody mediated rejection (AMR) is recognized as a major cause of graft loss in renal transplant recipients. Early acute AMR in the first few days after transplantation occurs primarily in sensitized renal transplant recipients with donor-specific alloantibody at the time of transplant and is a relatively “pure” form of acute AMR. Late acute AMR occurs months to years after transplantation and is commonly a mixed cellular and humoral rejection. While there is no consensus regarding optimum treatment, we contend that rational therapeutic approaches are emerging and the acute episode can be managed in most instances. However, new therapies are needed to prevent ongoing chronic injury in these patients.

  5. Maternal Antibody-Mediated Disease Enhancement in Type I Interferon-Deficient Mice Leads to Lethal Disease Associated with Liver Damage

    PubMed Central

    Lam, Jian Hang; Binte Aman, Siti Amanlina; Libau, Eshele Anak; Lee, Pei Xuan; St. John, Ashley L.; Alonso, Sylvie

    2016-01-01

    Epidemiological studies have reported that most of the severe dengue cases occur upon a secondary heterologous infection. Furthermore, babies born to dengue immune mothers are at greater risk of developing severe disease upon primary infection with a heterologous or homologous dengue virus (DENV) serotype when maternal antibodies reach sub-neutralizing concentrations. These observations have been explained by the antibody mediated disease enhancement (ADE) phenomenon whereby heterologous antibodies or sub-neutralizing homologous antibodies bind to but fail to neutralize DENV particles, allowing Fc-receptor mediated entry of the virus-antibody complexes into host cells. This eventually results in enhanced viral replication and heightened inflammatory responses. In an attempt to replicate this ADE phenomenon in a mouse model, we previously reported that upon DENV2 infection 5-week old type I and II interferon (IFN) receptors-deficient mice (AG129) born to DENV1-immune mothers displayed enhancement of disease severity characterized by increased virus titers and extensive vascular leakage which eventually led to the animals’ death. However, as dengue occurs in immune competent individuals, we sought to reproduce this mouse model in a less immunocompromised background. Here, we report an ADE model that is mediated by maternal antibodies in type I IFN receptor-deficient A129 mice. We show that 5-week old A129 mice born to DENV1-immune mothers succumbed to a DENV2 infection within 4 days that was sub-lethal in mice born to naïve mothers. Clinical manifestations included extensive hepatocyte vacuolation, moderate vascular leakage, lymphopenia, and thrombocytopenia. Anti-TNFα therapy totally protected the mice and correlated with healthy hepatocytes. In contrast, blocking IL-6 did not impact the virus titers or disease outcome. This A129 mouse model of ADE may help dissecting the mechanisms involved in dengue pathogenesis and evaluate the efficacy of vaccine and

  6. Maternal Antibody-Mediated Disease Enhancement in Type I Interferon-Deficient Mice Leads to Lethal Disease Associated with Liver Damage.

    PubMed

    Martínez Gómez, Julia María; Ong, Li Ching; Lam, Jian Hang; Binte Aman, Siti Amanlina; Libau, Eshele Anak; Lee, Pei Xuan; St John, Ashley L; Alonso, Sylvie

    2016-03-01

    Epidemiological studies have reported that most of the severe dengue cases occur upon a secondary heterologous infection. Furthermore, babies born to dengue immune mothers are at greater risk of developing severe disease upon primary infection with a heterologous or homologous dengue virus (DENV) serotype when maternal antibodies reach sub-neutralizing concentrations. These observations have been explained by the antibody mediated disease enhancement (ADE) phenomenon whereby heterologous antibodies or sub-neutralizing homologous antibodies bind to but fail to neutralize DENV particles, allowing Fc-receptor mediated entry of the virus-antibody complexes into host cells. This eventually results in enhanced viral replication and heightened inflammatory responses. In an attempt to replicate this ADE phenomenon in a mouse model, we previously reported that upon DENV2 infection 5-week old type I and II interferon (IFN) receptors-deficient mice (AG129) born to DENV1-immune mothers displayed enhancement of disease severity characterized by increased virus titers and extensive vascular leakage which eventually led to the animals' death. However, as dengue occurs in immune competent individuals, we sought to reproduce this mouse model in a less immunocompromised background. Here, we report an ADE model that is mediated by maternal antibodies in type I IFN receptor-deficient A129 mice. We show that 5-week old A129 mice born to DENV1-immune mothers succumbed to a DENV2 infection within 4 days that was sub-lethal in mice born to naïve mothers. Clinical manifestations included extensive hepatocyte vacuolation, moderate vascular leakage, lymphopenia, and thrombocytopenia. Anti-TNFα therapy totally protected the mice and correlated with healthy hepatocytes. In contrast, blocking IL-6 did not impact the virus titers or disease outcome. This A129 mouse model of ADE may help dissecting the mechanisms involved in dengue pathogenesis and evaluate the efficacy of vaccine and

  7. Nonthermal Plasma-Mediated Cancer Cell Death; Targeted Cancer Treatment

    NASA Astrophysics Data System (ADS)

    Choi, Byul-Bora; Choi, Yeon-Sik; Lee, Hae-Jun; Lee, Jae-Koo; Kim, Uk-Kyu; Kim, Gyoo-Cheon

    Non-thermal air plasma can kill cancer cells. However, there is no selectivity between normal and cancer cells. Therefore, cancer specific antibody conjugated gold nanoparticle (GNP) was pretreated before plasma irradiation. Stimulation of antibody conjugated GNP by plasma treatment resulted in a significant decrease in viability of cancer cells. This technology shows the feasibility of using plasma therapy for killing cancer cells selectively.

  8. Fibronectin mediates mesendodermal cell fate decisions

    PubMed Central

    Cheng, Paul; Andersen, Peter; Hassel, David; Kaynak, Bogac L.; Limphong, Pattraranee; Juergensen, Lonny; Kwon, Chulan; Srivastava, Deepak

    2013-01-01

    Non-cell-autonomous signals often play crucial roles in cell fate decisions during animal development. Reciprocal signaling between endoderm and mesoderm is vital for embryonic development, yet the key signals and mechanisms remain unclear. Here, we show that endodermal cells efficiently promote the emergence of mesodermal cells in the neighboring population through signals containing an essential short-range component. The endoderm-mesoderm interaction promoted precardiac mesoderm formation in mouse embryonic stem cells and involved endodermal production of fibronectin. In vivo, fibronectin deficiency resulted in a dramatic reduction of mesoderm accompanied by endodermal expansion in zebrafish embryos. This event was mediated by regulation of Wnt signaling in mesodermal cells through activation of integrin-β1. Our findings highlight the importance of the extracellular matrix in mediating short-range signals and reveal a novel function of endoderm, involving fibronectin and its downstream signaling cascades, in promoting the emergence of mesoderm. PMID:23715551

  9. Neutrophil-mediated killing of Dipetalonema viteae microfilariae: simultaneous presence of IgE, IgG antibodies and complement is required.

    PubMed Central

    Aime, N; Haque, A; Bonnel, B; Torpier, G; Capron, A

    1984-01-01

    Neutrophils from the peripheral washings of normal rats in the presence of sera obtained from rats immune to circulating microfilariae adhered to and killed the microfilariae of Dipetalonema viteae in vitro within 16-24 hr. No significant adherence or cytotoxicity was mediated by sera collected from animals with a high microfilaraemia or from normal rats. Ultrastructural studies show that neutrophils, which are bigger than microfilariae, can easily internalize the small larvae resulting in the disintegration of the parasite. Immunoadsorption and inhibition experiments showed that the adherence-promoting activity resides both in IgG and IgE classes of antibody. However, the mere participation of these two antibodies is not sufficient to effect neutrophil adherence towards microfilariae, the presence of complement is also required. Samples of fresh immune rat serum (fIRS) depleted in alternative pathway components of complement by treatment with zymosan A failed to mediate cell adherence to the parasite. fIRS inactivated for the classical pathway of complement by the chelating agent EGTA partially retains its activity in mediating cytotoxicity to microfilariae. The striking antigenic specificity of D. viteae antibodies was shown by their ability to mediate cytotoxicity only to D. viteae but not towards Brugia malayi microfilariae. Images Figure 2 PMID:6538183

  10. Expansion of natural killer cells in mice transgenic for IgM antibody to ganglioside GD2: demonstration of prolonged survival after challenge with syngeneic tumor cells.

    PubMed

    Kawashima, Ikuo; Yoshida, Yukiko; Taya, Chouji; Shitara, Hiroshi; Yonekawa, Hiromichi; Karasuyama, Hajime; Tada, Nobuhiko; Furukawa, Koichi; Tai, Tadashi

    2003-08-01

    IgM antibodies to gangliosides, sialic acid-containing glycosphingolipids, have been shown to mediate anti-tumor effects in cancer patients with melanoma and neuroblastoma and to correlate with survival. Mechanisms by which the antibodies induce tumor suppression, however, have not been systematically studied. To investigate this point, we produced and characterized C57BL/6 mice transgenic for IgM antibody to ganglioside GD2. The transgenic (TG) mice showed high IgM, but not IgG antibody titers against GD2 in their sera. No significant clinical symptoms were observed. When EL4 cells, syngeneic T lymphoma that express ganglioside GD2, were injected into TG mice, prolonged survival was observed. Complement-dependent cytotoxicity (CDC) of EL4 cells was mediated with TG mice sera. Neither antibody-dependent cellular cytotoxicity with their sera nor cytotoxic T lymphocyte activity to EL4 cells was shown in TG mice. Spleen lymphocytes from TG mice had increased numbers of natural killer (NK) cells, but not T cells, B cells, or macrophages compared with wild-type mice. Depletion of NK cells with anti-asialo GM1 rabbit serum reduced or abrogated the observed anti-tumor effects, suggesting that NK cells play a major role in tumor eradication or suppression. NK cell activity in TG mice was much higher than wild-type mice. Moreover, TG mice showed prolonged survival after injection with syngeneic B16 melanoma cells, which express GM3, but not GD2 or GD3. Taking these results together, our studies demonstrate that the TG mice have significant anti-tumor characteristics, probably due to CDC and NK cell expansion and activation with anti-ganglioside GD2 antibody.

  11. Efficient Methods To Isolate Human Monoclonal Antibodies from Memory B Cells and Plasma Cells.

    PubMed

    Corti, Davide; Lanzavecchia, Antonio

    2014-10-01

    In this article, we highlight the advantages of isolating human monoclonal antibodies from the human memory B cells and plasma cell repertoires by using high-throughput cellular screens. Memory B cells are immortalized with high efficiency using Epstein-Barr virus (EBV) in the presence of a toll-like receptor (TLR) agonist, while plasma cells are maintained in single-cell cultures by using interleukin 6 (IL-6) or stromal cells. In both cases, multiple parallel assays, including functional assays, can be used to identify rare cells that produce antibodies with unique properties. Using these methods, we have isolated potent and broadly neutralizing antibodies against a variety of viruses, in particular, a pan-influenza-A-neutralizing antibody and an antibody that neutralizes four different paramyxoviruses. Given the high throughput and the possibility of directly screening for function (rather than just binding), these methods are instrumental to implement a target-agnostic approach to identify the most effective antibodies and, consequently, the most promising targets for vaccine design. This approach is exemplified by the identification of unusually potent cytomegalovirus-neutralizing antibodies that led to the identification of the target, a pentameric complex that we are developing as a candidate vaccine. PMID:26104354

  12. Anti-4-1BB monoclonal antibodies attenuate concanavalin A-induced immune-mediated liver injury in mice

    PubMed Central

    Xia, Guangtao; Wu, Sensen; Zhang, Yuanchao

    2016-01-01

    Effective therapies for the treatment of immune-mediated liver disease are currently lacking. As a member of the tumor necrosis factor receptor superfamily, 4-1BB has a key role in T-cell activation and has been implicated in the development of autoimmune disorders. The purpose of the present study was to evaluate the potential therapeutic or preventive function of an anti-4-1BB monoclonal antibody (mAb) in a mouse model of concanavalin (Con) A-induced immune-mediated liver injury. A mouse model of immune-mediated liver injury was established by tail vein injection of Con A (20 mg/kg). 4-1BB mAb (100 µg), with or without methylprednisolone (MEP; 3 mg/kg), was intraperitoneally injected into the tail vein 2 h prior to or 2 h following Con A injection. Con A induced marked hepatocyte necrosis, significantly reduced CD 4+/CD25+ T-cell levels, and increased the serum levels of aspartate transaminase (AST) and alanine transaminase (ALT), in addition to the percentage of 4-1BB+ T-cells, compared with the control (all P<0.05). The administration of 4-1BB mAb prior to or following Con A injection was able to attenuate Con A-induced liver tissue damage and significantly reduce serum AST and ALT levels (P<0.05). A combination of MEP and 4-1BB mAb further reduced serum AST and ALT levels, compared with either treatment alone. In addition, administration of 4-1BB mAb and MEP alone or in combination significantly increased CD4+/CD25+ T-cell levels, compared with the control (P<0.05). These results suggested that 4-1BB mAb was able to attenuate liver injury and preserve liver function in a mouse model of Con A-induced immune-mediated liver injury by promoting the expansion of CD4+/CD25+ T-cells. Furthermore, a combination of 4-1BB mAb with MEP was associated with greater beneficial effects than either treatment alone. The clinical significance of 4-1BB mAb in immune-mediated liver disease remains to be elucidated in future studies. PMID:27588047

  13. FLIP switches Fas-mediated glucose signaling in human pancreatic cells from apoptosis to cell replication

    NASA Astrophysics Data System (ADS)

    Maedler, Kathrin; Fontana, Adriano; Ris, Frédéric; Sergeev, Pavel; Toso, Christian; Oberholzer, José; Lehmann, Roger; Bachmann, Felix; Tasinato, Andrea; Spinas, Giatgen A.; Halban, Philippe A.; Donath, Marc Y.

    2002-06-01

    Type 2 diabetes mellitus results from an inadequate adaptation of the functional pancreatic cell mass in the face of insulin resistance. Changes in the concentration of glucose play an essential role in the regulation of cell turnover. In human islets, elevated glucose concentrations impair cell proliferation and induce cell apoptosis via up-regulation of the Fas receptor. Recently, it has been shown that the caspase-8 inhibitor FLIP may divert Fas-mediated death signals into those for cell proliferation in lymphatic cells. We observed expression of FLIP in human pancreatic cells of nondiabetic individuals, which was decreased in tissue sections of type 2 diabetic patients. In vitro exposure of islets from nondiabetic organ donors to high glucose levels decreased FLIP expression and increased the percentage of apoptotic terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL)-positive cells; FLIP was no longer detectable in such TUNEL-positive cells. Up-regulation of FLIP, by incubation with transforming growth factor or by transfection with an expression vector coding for FLIP, protected cells from glucose-induced apoptosis, restored cell proliferation, and improved cell function. The beneficial effects of FLIP overexpression were blocked by an antagonistic anti-Fas antibody, indicating their dependence on Fas receptor activation. The present data provide evidence for expression of FLIP in the human cell and suggest a novel approach to prevent and treat diabetes by switching Fas signaling from apoptosis to proliferation.

  14. Adhesion of Fusobacterium necrophorum to bovine endothelial cells is mediated by outer membrane proteins.

    PubMed

    Kumar, Amit; Gart, Elena; Nagaraja, T G; Narayanan, Sanjeev

    2013-03-23

    Fusobacterium necrophorum, a Gram-negative anaerobe, is frequently associated with suppurative and necrotic infections of animals and humans. The organism is a major bovine pathogen, and in cattle, the common fusobacterial infections are hepatic abscesses, foot rot, and necrotic laryngitis. The species comprises two subspecies: F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme. Bacterial adhesion to the host cell surface is a critical initial step in the pathogenesis, and outer membrane proteins (OMP) play an important role in adhesion and establishment of certain Gram-negative bacterial infections. The means by which F. necrophorum attaches to epithelial or endothelial cells has not been determined. We evaluated whether OMP of F. necrophorum, isolated from a liver abscess, mediated adhesion to bovine endothelial cells (adrenal gland capillary endothelial cell line). The extent of binding of subsp. necrophorum to the endothelial cells was higher than that of F. necrophorum subsp. funduliforme. Trypsin treatment of bacterial cells decreased their binding to endothelial cells indicating the protein nature of adhesins. Preincubation of endothelial cells with OMP extracted from F. necrophorum decreased the binding of bacterial cells. In addition, binding of each subspecies to endothelial cells was inhibited by polyclonal antibodies raised against respective OMP and the antibody-mediated inhibition was subspecies specific. The western blot analysis of OMP bound to endothelial cells with anti-OMP antibodies showed four OMP of 17, 24, 40 and 74 kDa. We conclude that OMP of F. necrophorum play a role in adhesion of bacterial cells to the endothelial cells.

  15. Monoclonal antibody to human endothelial cell surface internalization and liposome delivery in cell culture.

    PubMed

    Trubetskaya, O V; Trubetskoy, V S; Domogatsky, S P; Rudin, A V; Popov, N V; Danilov, S M; Nikolayeva, M N; Klibanov, A L; Torchilin, V P

    1988-02-01

    A monoclonal antibody (mAb), E25, is described that binds to the surface of cultured human endothelial cells. Upon binding E25 is rapidly internalized and digested intracellularly. Selective liposome targeting to the surface of the cells is performed using a biotinylated E25 antibody and an avidin-biotin system. Up to 30% of the cell-adherent liposomal lipid is internalized.

  16. Target-mediated drug disposition model and its approximations for antibody-drug conjugates.

    PubMed

    Gibiansky, Leonid; Gibiansky, Ekaterina

    2014-02-01

    Antibody-drug conjugate (ADC) is a complex structure composed of an antibody linked to several molecules of a biologically active cytotoxic drug. The number of ADC compounds in clinical development now exceeds 30, with two of them already on the market. However, there is no rigorous mechanistic model that describes pharmacokinetic (PK) properties of these compounds. PK modeling of ADCs is even more complicated than that of other biologics as the model should describe distribution, binding, and elimination of antibodies with different toxin load, and also the deconjugation process and PK of the released toxin. This work extends the target-mediated drug disposition (TMDD) model to describe ADCs, derives the rapid binding (quasi-equilibrium), quasi-steady-state, and Michaelis-Menten approximations of the TMDD model as applied to ADCs, derives the TMDD model and its approximations for ADCs with load-independent properties, and discusses further simplifications of the system under various assumptions. The developed models are shown to describe data simulated from the available clinical population PK models of trastuzumab emtansine (T-DM1), one of the two currently approved ADCs. Identifiability of model parameters is also discussed and illustrated on the simulated T-DM1 examples.

  17. Antibody-mediated complement C3b/iC3b binding to group B Streptococcus in paired mother and baby serum samples in a refugee population on the Thailand-Myanmar border.

    PubMed

    Herbert, Jenny; Thomas, Stephen; Brookes, Charlotte; Turner, Claudia; Turner, Paul; Nosten, Francois; Le Doare, Kirsty; Hudson, Michael; Heath, Paul T; Gorringe, Andrew; Taylor, Stephen

    2015-03-01

    Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal sepsis and meningitis. In this study, we determined antibody-mediated deposition of complement C3b/iC3b onto the bacterial cell surface of GBS serotypes Ia, Ib, II, III, and V. This was determined for 520 mother and umbilical cord serum sample pairs obtained at the time of birth from a population on the Thailand-Myanmar border. Antibody-mediated deposition of complement C3b/iC3b was detected to at least one serotype in 91% of mothers, despite a known carriage rate in this population of only 12%. Antibody-mediated C3b/iC3b deposition corresponded to known carriage rates, with the highest levels of complement deposition observed onto the most prevalent serotype (serotype II) followed by serotypes Ia, III, V, and Ib. Finally, neonates born to mothers carrying serotype II GBS at the time of birth showed higher antibody-mediated C3b/iC3b deposition against serotype II GBS than neonates born to mothers with no serotype II carriage. Assessment of antibody-mediated C3b/iC3b deposition against GBS may provide insights into the seroepidemiology of anti-GBS antibodies in mothers and infants in different populations. PMID:25589553

  18. Antibody-Mediated Complement C3b/iC3b Binding to Group B Streptococcus in Paired Mother and Baby Serum Samples in a Refugee Population on the Thailand-Myanmar Border

    PubMed Central

    Herbert, Jenny; Thomas, Stephen; Brookes, Charlotte; Turner, Claudia; Turner, Paul; Nosten, Francois; Le Doare, Kirsty; Hudson, Michael; Heath, Paul T.; Gorringe, Andrew

    2015-01-01

    Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal sepsis and meningitis. In this study, we determined antibody-mediated deposition of complement C3b/iC3b onto the bacterial cell surface of GBS serotypes Ia, Ib, II, III, and V. This was determined for 520 mother and umbilical cord serum sample pairs obtained at the time of birth from a population on the Thailand-Myanmar border. Antibody-mediated deposition of complement C3b/iC3b was detected to at least one serotype in 91% of mothers, despite a known carriage rate in this population of only 12%. Antibody-mediated C3b/iC3b deposition corresponded to known carriage rates, with the highest levels of complement deposition observed onto the most prevalent serotype (serotype II) followed by serotypes Ia, III, V, and Ib. Finally, neonates born to mothers carrying serotype II GBS at the time of birth showed higher antibody-mediated C3b/iC3b deposition against serotype II GBS than neonates born to mothers with no serotype II carriage. Assessment of antibody-mediated C3b/iC3b deposition against GBS may provide insights into the seroepidemiology of anti-GBS antibodies in mothers and infants in different populations. PMID:25589553

  19. Enzyme-mediated site-specific bioconjugation of metal complexes to proteins: sortase-mediated coupling of copper-64 to a single-chain antibody.

    PubMed

    Paterson, Brett M; Alt, Karen; Jeffery, Charmaine M; Price, Roger I; Jagdale, Shweta; Rigby, Sheena; Williams, Charlotte C; Peter, Karlheinz; Hagemeyer, Christoph E; Donnelly, Paul S

    2014-06-10

    The enzyme-mediated site-specific bioconjugation of a radioactive metal complex to a single-chain antibody using the transpeptidase sortase A is reported. Cage amine sarcophagine ligands that were designed to function as substrates for the sortase A mediated bioconjugation to antibodies were synthesized and enzymatically conjugated to a single-chain variable fragment. The antibody fragment scFv(anti-LIBS) targets ligand-induced binding sites (LIBS) on the glycoprotein receptor GPIIb/IIIa, which is present on activated platelets. The immunoconjugates were radiolabeled with the positron-emitting isotope (64)Cu. The new radiolabeled conjugates were shown to bind selectively to activated platelets. The diagnostic potential of the most promising conjugate was demonstrated in an in vivo model of carotid artery thrombosis using positron emission tomography. This approach gives homogeneous products through site-specific enzyme-mediated conjugation and should be broadly applicable to other metal complexes and proteins. PMID:24777818

  20. Migration of antibody secreting cells towards CXCL12 depends on the isotype that forms the BCR

    PubMed Central

    Achatz-Straussberger, Gertrude; Zaborsky, Nadja; Königsberger, Sebastian; Luger, Elke O.; Lamers, Marinus; Crameri, Reto; Achatz, Gernot

    2010-01-01

    Truncation of the cytoplasmic tail of membrane-bound IgE in vivo results in lower serum IgE levels, decreased numbers of IgE-secreting plasma cells and the abrogation of specific secondary immune responses. Here we present mouse strain KN1 that expresses a chimeric ε-γ1 BCR, consisting of the extracellular domains of the ε gene and the trans-membrane and cytoplasmic domains of the γ1 gene. Thus, differences in the IgE immune response of KN1 mice reflect the influence of the “γ1-mediated signalling” of mIgE bearing B cells. KN1 mice show an increased serum IgE level, resulting from an elevated number of IgE-secreting cells. Although the primary IgE immune response in KN1 mice is inconspicuous, the secondary response is far more robust. Most strikingly, IgE-antibody secreting cells with “γ1-signalling history” migrate more efficiently towards the chemokine CXCL12, which guides plasmablasts to plasma cell niches, than IgE-antibody secreting cells with WT “ε-signalling history”. We conclude that IgE plasmablasts have an intrinsic, lower chance to contribute to the long-lived plasma cell pool than IgG1 plasmablasts. PMID:18925577

  1. Anti-Glycoprotein G Antibodies of Herpes Simplex Virus 2 Contribute to Complete Protection after Vaccination in Mice and Induce Antibody-Dependent Cellular Cytotoxicity and Complement-Mediated Cytolysis

    PubMed Central

    Görander, Staffan; Ekblad, Maria; Bergström, Tomas; Liljeqvist, Jan-Åke

    2014-01-01

    We investigated the role of antibodies against the mature portion of glycoprotein G (mgG-2) of herpes simplex virus 2 (HSV-2) in protective immunity after vaccination. Mice were immunized intramuscularly with mgG-2 and oligodeoxynucleotides containing two CpG motifs plus alum as adjuvant. All C57BL/6 mice survived and presented no genital or systemic disease. High levels of immunoglobulin G subclass 1 (IgG1) and IgG2 antibodies were detected and re-stimulated splenic CD4+ T cells proliferated and produced IFN-γ. None of the sera from immunized mice exhibited neutralization, while all sera exerted antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis (ACMC) activity. Passive transfer of anti-mgG-2 monoclonal antibodies, or immune serum, to naive C57BL/6 mice did not limit disease progression. Immunized B‑cell KO mice presented lower survival rate and higher vaginal viral titers, as compared with vaccinated B-cell KO mice after passive transfer of immune serum and vaccinated C57BL/6 mice. Sera from mice that were vaccinated subcutaneously and intranasally with mgG-2 presented significantly lower titers of IgG antibodies and lower ADCC and ACMC activity. We conclude that anti-mgG-2 antibodies were of importance to limit genital HSV‑2 infection. ADCC and ACMC activity are potentially important mechanisms in protective immunity, and could tentatively be evaluated in future animal vaccine studies and in clinical trials. PMID:25398047

  2. Chip-based platform for dynamic analysis of NK cell cytolysis mediated by a triplebody.

    PubMed

    Chatzopoulou, Elisavet I; Roskopf, Claudia C; Sekhavati, Farzad; Braciak, Todd A; Fenn, Nadja C; Hopfner, Karl-Peter; Oduncu, Fuat S; Fey, Georg H; Rädler, Joachim O

    2016-04-01

    Cancer therapy via redirected lysis mediated by antibodies and antibody-derived agents relies on the availability of substantial numbers of sufficiently active immune effector cells. To monitor antitumor responses before and during therapy, sensitive methods are needed, capable of quantitating specific lysis of target cells. Here we present a chip-based single-cell cytometric assay, which uses adherent human target cells arrayed in structured micro-fields. Using a fluorescent indicator of cell death and time-lapse microscopy in an automated high-throughput mode, we measured specific target cell lysis by activated human NK cells, mediated by the therapeutic single chain triplebody SPM-2 (33-16-123). This antibody-derived tri-specific fusion protein carries binding sites for the myeloid antigens CD33 and CD123 and recruits NK cells via a binding site for the Fc-receptor CD16. Specific lysis increased with increasing triplebody concentration, and the single-cell assay was validated by direct comparison with a standard calcein-release assay. The chip-based approach allowed measurement of lysis events over 16 hours (compared to 4 hours for the calcein assay) and required far smaller numbers of primary cells. In addition, dynamic properties inaccessible to conventional methods provide new details about the activation of cytolytic effector cells by antibody-derived agents. Thus, the killing rate exhibited a dose-dependent maximum during the reaction interval. In clinical applications ex vivo monitoring of NK activity of patient's endogenous cells will likely help to choose appropriate therapy, to detect impaired or recovered NK function, and possibly to identify rare subsets of cancer cells with particular sensitivity to effector-cell mediated lysis. PMID:26958659

  3. Anti-huCD20 Antibody Therapy for Antibody-Mediated Rejection of Renal Allografts in a Mouse Model

    PubMed Central

    Abe, Toyofumi; Ishii, Daisuke; Gorbacheva, Victoria; Kohei, Naoki; Tsuda, Hidetoshi; Tanaka, Toshiaki; Dvorina, Nina; Nonomura, Norio; Takahara, Shiro; Valujskikh, Anna; Baldwin, William M.; Fairchild, Robert L.

    2016-01-01

    We have reported that B6.CCR5−/− mice reject renal allografts with high serum donor-specific antibody (DSA) titers and marked C4d deposition in grafts, features consistent with AMR. B6.huCD20/CCR5−/− mice, where human CD20 expression is restricted to B cells, rejected A/J renal allografts by day 26 post-transplant with DSA first detected in serum on day 5 post-transplant and increased thereafter. Recipient treatment with anti-huCD20 mAb prior to the transplant and weekly up to 7 weeks post-transplant promoted long-term allograft survival (> 100 days) with low DSA titers. To investigate the effect of B cell depletion at the time serum DSA was first detected, recipients were treated with anti-huCD20 mAb on days 5, 8 and 12 post-transplant. This regimen significantly reduced DSA titers and graft inflammation on day 15 post-transplant and prolonged allograft survival > 60 days. However, DSA returned to the titers observed in control treated recipients by day 30 post-transplant and histological analyses on day 60 post-transplant indicated severe interstitial fibrosis. These results indicate that anti-huCD20 mAb had the greatest effect as a prophylactic treatment and that the distinct kinetics of DSA responses accounts for acute renal allograft failure versus the development of fibrosis. PMID:25731734

  4. Outcome of subclinical antibody-mediated rejection in kidney transplant recipients with preformed donor-specific antibodies.

    PubMed

    Loupy, A; Suberbielle-Boissel, C; Hill, G S; Lefaucheur, C; Anglicheau, D; Zuber, J; Martinez, F; Thervet, E; Méjean, A; Charron, D; Duong van Huyen, J P; Bruneval, P; Legendre, C; Nochy, D

    2009-11-01

    This study describes clinical relevance of subclinical antibody-mediated rejection (SAMR) in a cohort of 54 DSA-positive kidney transplant recipients receiving a deceased donor. In 3 months screening biopsies, 31.1% of patients met the criteria of SAMR. A total of 48.9% had an incomplete form of SAMR (g+/ptc+/C4d-negative) whereas 20% had no humoral lesions. Patients with SAMR at 3 months had at 1 year: a higher C4d score, ptc score, and arteriosclerosis score, higher rate of IFTA (100% vs. 33.3%, p < 0.01) and a higher rate of transplant glomerulopathy (43% vs. 0%, p = 0.02) compared to patients without 3-month SAMR. Patients with SAMR at 3 months exhibited at 1 year a higher class II MFImax-DSA and a lower mGFR compared to patients without SAMR (39.2 +/- 13.9 vs. 61.9 +/- 19.2 mL/min/1.73 m(2) respectively, p < 0.01). The group of patients with C4d-negative SAMR at 3 months developed more ptc and IFTA lesions, and lower GFR at 1 year in comparison to biopsies without humoral lesions. SAMR is a frequent entity in KTR with preexisting DSAs and promotes subsequent GFR impairment and development of chronic AMR. C4d-negative SAMR patients displayed an intermediate course between the no-SAMR group and the C4d+ SAMR group. Screening biopsies may be useful to recognize patients more likely to develop SAMR. PMID:19775320

  5. Antibody-Mediated and Cellular Immune Responses Induced in Naive Volunteers by Vaccination with Long Synthetic Peptides Derived from the Plasmodium vivax Circumsporozoite Protein

    PubMed Central

    Arévalo-Herrera, Myriam; Soto, Liliana; Perlaza, Blanca Liliana; Céspedes, Nora; Vera, Omaira; Lenis, Ana Milena; Bonelo, Anilza; Corradin, Giampietro; Herrera, Sócrates

    2011-01-01

    Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate. We describe the characterization of specific immune responses induced in 21 malaria-naive volunteers vaccinated with long synthetic peptides derived from the CS protein formulated in Montanide ISA 720. Both antibody- and cell-mediated immune responses were analyzed. Antibodies were predominantly of IgG1 and IgG3 isotypes, recognized parasite proteins on the immunofluorescent antibody test, and partially blocked sporozoite invasion of hepatoma cell lines in vitro. Peripheral blood mononuclear cells from most volunteers (94%) showed IFN-γ production in vitro upon stimulation with both long signal peptide and short peptides containing CD8+ T-cell epitopes. The relatively limited sample size did not allow conclusions about HLA associations with the immune responses observed. In summary, the inherent safety and tolerability together with strong antibody responses, invasion blocking activity, and the IFN-γ production induced by these vaccine candidates warrants further testing in a phase II clinical trial. PMID:21292876

  6. A novel antibody discovery platform identifies anti-influenza A broadly neutralizing antibodies from human memory B cells.

    PubMed

    Xiao, Xiaodong; Chen, Yan; Varkey, Reena; Kallewaard, Nicole; Koksal, Adem C; Zhu, Qing; Wu, Herren; Chowdhury, Partha S; Dall'Acqua, William F

    2016-07-01

    Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here, memory B cells are activated and amplified using Epstein-Barr virus infection, co-cultured with CHO-muCD40L cells, and then assessed by functional screenings. An in vitro transcription and translation (IVTT) approach was used to analyze variable (V) genes recovered from each B cell sample and identify the relevant heavy/light chain pair(s). We achieved efficient amplification and activation of memory B cells, and eliminated the need to: 1) seed B cells at clonal level (≤1 cell/well) or perform limited dilution cloning; 2) immortalize B cells; or 3) assemble V genes into an IgG expression vector to confirm the relevant heavy/light chain pairing. Cross-reactive antibodies targeting a conserved epitope on influenza A hemagglutinin were successfully isolated from a healthy donor. In-depth analysis of the isolated antibodies suggested their potential uses as anti-influenza A antibody therapeutics and uncovered a distinct affinity maturation pathway. Importantly, our results showed that cognate heavy/light chain pairings contributed to both the expression level and binding abilities of our newly isolated VH1-69 family, influenza A neutralizing antibodies, contrasting with previous observations that light chains do not significantly contribute to the function of this group of antibodies. Our results further suggest the potential use of the IVTT as a powerful antibody developability assessment tool. PMID:27049174

  7. Target cell specific antibody-based photosensitizers for photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Rosenblum, Lauren T.; Mitsunaga, Makoto; Kakareka, John W.; Morgan, Nicole Y.; Pohida, Thomas J.; Choyke, Peter L.; Kobayashi, Hisataka

    2011-03-01

    In photodynamic therapy (PDT), localized monochromatic light is used to activate targeted photosensitizers (PS) to induce cellular damage through the generation of cytotoxic species such as singlet oxygen. While first-generation PS passively targeted malignancies, a variety of targeting mechanisms have since been studied, including specifically activatable agents. Antibody internalization has previously been employed as a fluorescence activation system and could potentially enable similar activation of PS. TAMRA, Rhodamine-B and Rhodamine-6G were conjugated to trastuzumab (brand name Herceptin), a humanized monoclonal antibody with specificity for the human epidermal growth factor receptor 2 (HER2), to create quenched PS (Tra-TAM, Tra-RhoB, and Tra-Rho6G). Specific PDT with Tra-TAM and Tra-Rho6G, which formed covalently bound H-dimers, was demonstrated in HER2+ cells: Minimal cell death (<6%) was observed in all treatments of the HER2- cell line (BALB/3T3) and in treatments the HER2+ cell line (3T3/HER2) with light or trastuzumab only. There was significant light-induced cell death in HER2 expressing cells using Tra-TAM (3% dead without light, 20% at 50 J/cm2, 46% at 100 J/cm2) and Tra-Rho6G (5% dead without light, 22% at 50 J/cm2, 46% at 100 J/cm2). No efficacy was observed in treatment with Tra-RhoB, which was also non-specifically taken up by BALB/3T3 cells and which had weaker PS-antibody interactions (as demonstrated by visualization of protein and fluorescence on SDS-PAGE).

  8. Enzymatic removal of alpha-galactosyl epitopes from porcine endothelial cells diminishes the cytotoxic effect of natural antibodies.

    PubMed

    LaVecchio, J A; Dunne, A D; Edge, A S

    1995-10-27

    Xenotransplantation of tissues between discordant species such as pig into human is not yet feasible due to the problem of hyperacute rejection. This rapid response to xenogeneic tissue is mediated by natural antibodies that react with antigens on the xenograft. A number of xenoantigens consist of carbohydrate residues, and a terminal galactose in alpha linkage has been shown to be involved in hyperacute rejection of pig-to-human xenografts. We show that alpha-linked galactose on porcine endothelial cells is a major epitope recognized by IgG and IgM antibodies present in monkey and human sera. Endothelial cells that had been treated with alpha-galactosidase did not react with fluorescein-labeled Griffonia simplicifolia I B4 (GS-IB4), a lectin that detects the alpha-galactosyl epitope on intact cells. The reactivity of both human and cynomolgus monkey serum with endothelial cells was decreased by 59% to 90% after treatment with coffee bean alpha-galactosidase. Using a colorimetric assay for cell viability, we show that natural antibodies present in the sera of cynomolgus monkey and humans are cytotoxic to porcine endothelial cells in the presence of exogenously added complement. When the terminal alpha-galactosy residues were removed by enzymatic digestion, the cytotoxic effect of natural antibodies on porcine endothelial cells was diminished by > 80%. Evaluation of the time course of reappearance of the alpha-galactosyl epitope at the cell surface revealed that 48 hr after alpha-galactosidase treatment, binding of GS-IB4 was diminished by 60%. These results suggest that glycosidase treatment of cells to be transplanted could prevent hyperacute rejection mediated by natural antibodies.

  9. Proteomic differences in recombinant CHO cells producing two similar antibody fragments

    PubMed Central

    Sommeregger, Wolfgang; Mayrhofer, Patrick; Steinfellner, Willibald; Reinhart, David; Henry, Michael; Clynes, Martin

    2016-01-01

    ABSTRACT Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. “Omics” studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label‐free LC‐MS proteomic analyses to investigate product‐specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single‐chain Fv‐Fc homodimeric antibody fragments (scFv‐Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase‐mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label‐free proteomic analysis. LC‐MS‐MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902–1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26913574

  10. Peroxisome proliferator-activated receptor gamma B cell-specific deficient mice have an impaired antibody response1

    PubMed Central

    Ramon, Sesquile; Bancos, Simona; Thatcher, Thomas H.; Murant, Thomas I.; Moshkani, Safiehkhatoon; Sahler, Julie M.; Bottaro, Andrea; Sime, Patricia J.; Phipps, Richard P.

    2012-01-01

    Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. PPARγ, a ligand activated transcription factor, has important anti-inflammatory and anti-proliferative functions and it has been associated with diseases including diabetes, scarring and atherosclerosis among others. PPARγ is expressed in most bone marrow derived cells and influences their function. PPARγ ligands can stimulate human B cell differentiation and promote antibody production. A knowledge gap is that the role of PPARγ in B cells under physiological conditions is not known. We developed a new B cell-specific PPARγ (B-PPARγ) knockout mouse and explored the role of PPARγ during both the primary and secondary immune response. Here, we show that PPARγ deficiency in B cells decreases germinal center B cells and plasma cell development as well as the levels of circulating antigen-specific antibodies during a primary challenge. Inability to generate germinal center B cells and plasma cells is correlated to decreased MHC class II expression and decreased Bcl-6 and Blimp-1 levels. Furthermore, B-PPARγ-deficient mice have an impaired memory response, characterized by low titers of antigen-specific antibodies and low numbers of antigen-experienced antibody-secreting cells. However, B-PPARγ-deficient mice have no differences in B cell population distribution within neither primary nor secondary lymphoid organs during development. This is the first report to show under physiological conditions that PPARγ expression in B cells is required for an efficient B cell-mediated immune response as it regulates B cell differentiation and antibody production. PMID:23041568

  11. Carboxypeptidase D is the only enzyme responsible for antibody C-terminal lysine cleavage in Chinese hamster ovary (CHO) cells.

    PubMed

    Hu, Zhilan; Zhang, Henry; Haley, Benjamin; Macchi, Frank; Yang, Feng; Misaghi, Shahram; Elich, Joseph; Yang, Renee; Tang, Yun; Joly, John C; Snedecor, Bradley R; Shen, Amy

    2016-10-01

    Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a different carboxypeptidase mRNA level was knocked down suggesting that carboxypeptidase D is the main contributor for C-terminal lysine processing. Most importantly, when CpD expression was knocked out by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, C-terminal lysine cleavage was completely abolished in CpD knockout cells based on mass spectrometry analysis, demonstrating that CpD is the only endogenous carboxypeptidase that cleaves antibody heavy chain C-terminal lysine in CHO cells. Hence, our work showed for the first time that the cleavage of antibody heavy chain C-terminal lysine is solely mediated by the carboxypeptidase D in CHO cells and our finding provides one solution to eliminating C-terminal lysine heterogeneity for therapeutic antibody production by knocking out CpD gene expression. Biotechnol. Bioeng. 2016;113: 2100-2106. © 2016 Wiley Periodicals, Inc.

  12. Yeast-Derived β-Glucan Augments the Therapeutic Efficacy Mediated by Anti–Vascular Endothelial Growth Factor Monoclonal Antibody in Human Carcinoma Xenograft Models

    PubMed Central

    Salvador, Carolina; Li, Bing; Hansen, Richard; Cramer, Daniel E.; Kong, Maiying; Jun, Yan

    2008-01-01

    Purpose Bevacizumab is a recombinant IgG1humanized monoclonal antibody against vascular endothelial growth factor (VEGF). Its proposed mechanism of action is independent of immune effector functions. Many human carcinomas not only secrete VEGF but also express membrane-bound VEGF. In addition, VEGF receptors are expressed on tumor cells. It is hypothesized that bevacizumab could bind membrane-bound VEGF or VEGF-VEGF receptor complexes on tumors, thereby initiating potential immunologic consequences. We previously showed that yeast-derived β-glucan functions with antitumor antibodies that activate complement to recruit complement receptor 3– expressing leukocytes capable of mediating complement receptor 3– dependent cellular cytotoxicity of tumors opsonized with iC3b. In the current study, the therapeutic efficacy mediated by combining bevacizumab with yeast-derived β-glucan was studied in human carcinoma xenograft models. Experimental Design Human tumor cell lines were screened for membrane-bound VEGF expression both in vitro and in vivo. Complement activation mediated by bevacizumab was examined. Tumor cell lines positive or negative for membrane-bound VEGF expression were implanted in severe combined immunodeficient mice to establish xenograft models. Tumor-bearing mice were treated with different regimens. Tumor regression and long-term survival were recorded. Results Human ovarian carcinoma SKOV-3 cells expressed membrane-bound VEGF both in vitro and in vivo. Bevacizumab was bound to membrane-bound VEGF, activated complement, and synergized with β-glucan to elicit cellular cytotoxicity in vitro. In vivo study showed that β-glucan could significantly augment the therapeutic efficacy mediated by bevacizumab. Conclusions Yeast-derived β-glucan can synergize with anti-VEGF monoclonal antibody bevacizumab for the treatment of cancer with membrane-bound VEGF expression. PMID:18281559

  13. Antibody-dependent antitumor cytotoxicity by human monocytes cultured with recombinant macrophage colony-stimulating factor. Induction of efficient antibody-mediated antitumor cytotoxicity not detected by isotope release assays.

    PubMed

    Munn, D H; Cheung, N K

    1989-08-01

    Macrophage colony-stimulating factor (M-CSF) is known to stimulate proliferation of monocyte/macrophage progenitors and enhance in vitro antitumor cytotoxicity by murine macrophages. In this paper we have shown that recombinant human M-CSF causes human peripheral blood monocytes to differentiate in culture into metabolically active macrophage-like cells. These cells mediate very efficient antibody-dependent cellular cytotoxicity (ADCC) against human melanoma and neuroblastoma cell lines in the presence of two murine IgG3 mAbs (3F8 and R24). They also mediate antibody-independent cytotoxicity (or cytostasis) to a lesser extent. Human serum had an inconsistent effect on ADCC, but often induced similar high levels of ADCC. Cytotoxicity was measured using a novel ELISA to detect surviving tumor cells after ADCC. Two conventional isotope-release assays (51Cr and [3H]TdR) underestimated or entirely failed to detect ADCC by M-CSF-activated monocytes. Optimal activation occurred with 100-300 U/ml of M-CSF, and required 9-11 d for completion. Most of the M-CSF cultured monocytes expressed the low-affinity Fc receptor (CD16). ADCC by cells of the monocyte/macrophage lineage using murine IgG3 mAbs may have significance for the immunotherapy of human malignancies.

  14. Immune-Mediated Necrotizing Myopathy, Associated With Antibodies to Signal Recognition Particle, Together With Lupus Nephritis: Case Presentation and Management

    PubMed Central

    O’Grady, John; Harty, Len; Mayer, Nick; Critcher, Val; Ryan, John

    2015-01-01

    A male patient with limb weakness, myalgia and edema was subsequently found to have an immune-mediated necrotizing myopathy (IMNM) on biopsy. Targeted myopathic antibody analysis revealed antibodies to signal recognition particle (SRP). Anti-SRP-associated necrotizing myopathy was diagnosed. This case was complicated by the concurrent development of class III lupus nephritis. We discuss an interesting case progression and development as well as the management of these difficult to treat conditions. PMID:25883715

  15. Eculizumab for the Treatment of Severe Antibody-Mediated Rejection: A Case Report and Review of the Literature

    PubMed Central

    Boucher, Anne; Collette, Suzon; Senécal, Lynne

    2016-01-01

    In renal transplantation, treatment options for antibody-mediated rejection are limited. Here, we report a case of severe AMR treated with eculizumab. A 50-year-old woman known for end stage kidney disease secondary to IgA nephropathy received a kidney transplant from a 50-year-old deceased donor. At 5 months after transplantation, she presented with acute graft dysfunction and biopsy showed a severe antibody-mediated rejection associated with thrombotic microangiopathy. Despite an aggressive conventional immunosuppressive regimen, signs of rejection persisted and the patient was treated with 3 doses of eculizumab. Following the therapy, markers of TMA improved and graft function stabilized. However, ongoing signs of rejection remained in the repeated biopsy. In kidney transplantation, eculizumab is an expensive treatment and its role in the treatment of antibody-mediated rejection remains to be determined. PMID:27478676

  16. Neutralization capacity of measles virus H protein specific IgG determines the balance between antibody-enhanced infectivity and protection in microglial cells

    PubMed Central

    Iankov, Ianko D.; Penheiter, Alan R.; Griesmann, Guy E.; Carlson, Stephanie K.; Federspiel, Mark J.; Galanis, Evanthia

    2013-01-01

    Neutralizing antibodies directed against measles virus (MV) surface glycoproteins prevent viral attachment and entry through the natural receptors. H protein specific IgG can enhance MV infectivity in macrophages via Fcγ receptor (FcγR)-dependent mechanism. H-specific IgM, anti-F antibodies and complement cascade activation are protective against antibody-mediated enhancement of MV infection. However, protective role of anti-H IgG against antibody-enhanced infection is not well understood. Here we designed a set of experiments to test the protective effect of H-specific IgG against FcγR-mediated infection in microglial cells. Microglial cells are also potential target of the antibody-mediated enhancement and spread of MV infection in the central nervous system. A partially neutralizing IgG monoclonal antibody (MAb) CL55, specific for MV H protein, at 10 μg/ml enhanced MV infection in mouse microglial cells by 13-14-fold. Infection-enhancing antibody concentrations induced large multinucleated syncytia formation 48-72 h post inoculation. We generated anti-H IgG MAb 20H6 with a strong neutralization capacity >1:80,000 at 1 mg/ml concentration in MV plaque-reduction neutralization assay. In contrast to the partially protective MAb CL55, enhancement of MV infectivity by MAb 20H6 required dilutions below the 1:120 serum titer considered protective against measles infection in humans. At a concentration of 10 μg/ml MAb 20H6 exhibited a dominant protective effect and prevented MAb CL55-mediated enhancement of MV infection and virus-mediated fusion. These results indicate that neutralization capacity of the H-specific IgG determines the balance between antibody enhancement and protection against MV infection in microglial cells. PMID:23266401

  17. Ah receptor mediated suppression of the antibody response in mice is primarily dependent on the Ah phenotype of lymphoid tissue.

    PubMed

    Silkworth, J B; Antrim, L A; Sack, G

    1986-12-01

    Halogenated aromatic hydrocarbons act through the aromatic hydrocarbon (Ah) receptor in mice to produce a series of toxic effects of the immune system. The receptor protein is a product of the Ah gene locus. Ah responsive (Ahb/Ahb) mice express a high affinity receptor in both lymphoid and nonlymphoid tissues whereas nonresponsive Ahd/Ahd mice express a poor affinity receptor. To determine the role of the Ah receptor of lymphoid tissue relative to that of nonlymphoid tissue in the induction of immune impairment, bone marrow was used to reconstitute lethally irradiated mice of the same or opposite Ah phenotype. All mice were given 3,3',4,4'-tetrachlorobiphenyl (35 and 350 mumol/kg) ip 2 days before immunization with sheep erythrocytes (SRBC). The immune response to this T dependent antigen and organ weights were determined 5 or 7 days later in normal or chimeric mice, respectively. Monoclonal Lyt 1.1 and Lyt 1.2 antibodies were used to establish the origin of the cells which repopulated the chimeric thymuses. The immune responses of both BALB/cBy (Ahb/Ahb) and the BALB/cBy X DBA/2 hybrid, CByD2F1 (Ahb/Ahd), were significantly suppressed but DBA/2 mice were unaffected. The immune responses of chimeric BALB/cBy----BALB/cBy and BALB/cBy----DBA/2 (donor----recipient) mice were also significantly suppressed and thymic atrophy was observed in both cases. The serum anti-SRBC antibody titers of DBA/2----BALB/cBy chimeras were also significantly decreased although not to the same extent as in BALB/cBy----DBA/2 mice. Chimeric DBA/2----DBA/2 mice were not affected. These results indicate that the sensitivity to Ah receptor mediated suppression of the antibody response is primarily determined by the Ah phenotype of the lymphoid tissue.

  18. PEROXIDASE-MEDIATED MAMMALIAN CELL CYTOTOXICITY

    PubMed Central

    Edelson, Paul J.; Cohn, Zanvil A.

    1973-01-01

    Lactoperoxidase, in the presence of hydrogen peroxide and iodide is cytotoxic for human and mouse lymphoid cells, and human erythrocytes. Myeloperoxidase, in amounts equivalent to 1.5 x 106 neutrophils, readily replaces lactoperoxidase, and allows the substitution of the iodide ion by chloride. The myeloperoxidase-mediated reaction is rapid, and highly efficient, leading to 85–90% cell death in 90 min, as measured by 51chromium release and dye exclusion. The mixture of granulocytes. monocytes, and lymphocytes present in an inflammatory exudate, and the intimate cell-to-cell association characteristic of cytotoxic phenomena may provide the in vivo requirements for such a system. PMID:4717124

  19. Natural antiband 3 antibodies in patients with sickle cell disease.

    PubMed

    Villaescusa, Rinaldo; Arce, Ada Amalia; Lalanne-Mistrih, Marie-Laure; Lamarre, Yann; Hierso, Régine; Hernández, Carlos; Hardy-Dessources, Marie-Dominique

    2013-03-01

    Band 3 oligomers, precociously formed in the membrane of sickle red blood cells (SS RBC) as a result of oxidative damage, induce two significant changes: (1) contribution to the adhesive nature of these cells to endothelial cells; (2) production of recognition sites for natural antiband 3 antibodies (antiband 3 Nabs). The inhibition of the adhesion of SS RBC to endothelial cells by band 3 peptides suggests a participation of antiband 3 Nabs in the etiology and prevention of vaso-occlusive crises (VOC). To address this question, we measured the levels of antiband 3 Nabs in sickle cell anaemia (SCA) patients (45 in steady state, 35 in VOC) and in controls (27 sickle trait, 30 normal AA subjects). A significant decreased of antiband 3 Nabs in the VOC group was demonstrated as compared with the steady state group, the sickle trait and healthy controls. This study provides data suggesting that Antiband 3 Nabs are likely to play a role in the SCA VOC.

  20. Fluorescent-Antibody Measurement Of Cancer-Cell Urokinase

    NASA Technical Reports Server (NTRS)

    Morrison, Dennis R.

    1993-01-01

    Combination of laboratory techniques provides measurements of amounts of urokinase in and between normal and cancer cells. Includes use of fluorescent antibodies specific against different forms of urokinase-type plasminogen activator, (uPA), fluorescence microscopy, quantitative analysis of images of sections of tumor tissue, and flow cytometry of different uPA's and deoxyribonucleic acid (DNA) found in suspended-tumor-cell preparations. Measurements provide statistical method for indicating or predicting metastatic potentials of some invasive tumors. Assessments of metastatic potentials based on such measurements used in determining appropriate follow-up procedures after surgical removal of tumors.

  1. A Quantitative Method for Comparing the Brightness of Antibody-dye Reagents and Estimating Antibodies Bound per Cell.

    PubMed

    Kantor, Aaron B; Moore, Wayne A; Meehan, Stephen; Parks, David R

    2016-01-01

    We present a quantitative method for comparing the brightness of antibody-dye reagents and estimating antibodies bound per cell. The method is based on complementary binding of test and fill reagents to antibody capture microspheres. Several aliquots of antibody capture beads are stained with varying amounts of the test conjugate. The remaining binding sites on the beads are then filled with a second conjugate containing a different fluorophore. Finally, the fluorescence of the test conjugate compared to the fill conjugate is used to measure the relative brightness of the test conjugate. The fundamental assumption of the test-fill method is that if it takes X molecules of one test antibody to lower the fill signal by Y units, it will take the same X molecules of any other test antibody to give the same effect. We apply a quadratic fit to evaluate the test-fill signal relationship across different amounts of test reagent. If the fit is close to linear, we consider the test reagent to be suitable for quantitative evaluation of antibody binding. To calibrate the antibodies bound per bead, a PE conjugate with 1 PE molecule per antibody is used as a test reagent and the fluorescence scale is calibrated with Quantibrite PE beads. When the fluorescence per antibody molecule has been determined for a particular conjugate, that conjugate can be used for measurement of antibodies bound per cell. This provides comparisons of the brightness of different conjugates when conducted on an instrument whose statistical photoelectron (Spe) scales are known. © 2016 by John Wiley & Sons, Inc.

  2. A systematic review of the role of C4d in the diagnosis of acute antibody-mediated rejection.

    PubMed

    Sapir-Pichhadze, Ruth; Curran, Simon P; John, Rohan; Tricco, Andrea C; Uleryk, Elizabeth; Laupacis, Andreas; Tinckam, Kathryn; Sis, Banu; Beyene, Joseph; Logan, Alexander G; Kim, S Joseph

    2015-01-01

    In this study, we conducted a systematic review of the literature to re-evaluate the role of C4d in the diagnosis of acute antibody-mediated rejection of kidney allografts. Electronic databases were searched until September 2013. Eligible studies allowed derivation of diagnostic tables for the performance of C4d by immunofluorescence or immunohistochemistry with comparison to histopathological features of acute antibody-mediated rejection and/or donor-specific antibody (DSA) assays. Of 3492 unique abstracts, 29 studies encompassing 3485 indication and 868 surveillance biopsies were identified. Assessment of C4d by immunofluorescence and immunohistochemistry exhibited slight to moderate agreement with glomerulitis, peritubular capillaritis, solid-phase DSA assays, DSA with glomerulitis, and DSA with peritubular capillaritis. The sensitivity and specificity of C4d varied as a function of C4d and comparator test thresholds. Prognostically, the presence of C4d was associated with inferior allograft survival compared with DSA or histopathology alone. Thus, our findings support the presence of complement-dependent and -independent phenotypes of acute antibody-mediated rejection. Whether the presence of C4d in combination with histopathology or DSA should be considered for the diagnosis of acute antibody-mediated rejection warrants further study. PMID:24827778

  3. Anti-CD25 monoclonal antibody Fc variants differentially impact regulatory T cells and immune homeostasis.

    PubMed

    Huss, David J; Pellerin, Alex F; Collette, Brian P; Kannan, Arun K; Peng, Liaomin; Datta, Abhishek; Wipke, Brian T; Fontenot, Jason D

    2016-07-01

    Interleukin-2 (IL-2) is a critical regulator of immune homeostasis through its non-redundant role in regulatory T (Treg) cell biology. There is major interest in therapeutic modulation of the IL-2 pathway to promote immune activation in the context of tumour immunotherapy or to enhance immune suppression in the context of transplantation, autoimmunity and inflammatory diseases. Antibody-mediated targeting of the high-affinity IL-2 receptor α chain (IL-2Rα or CD25) offers a direct mechanism to target IL-2 biology and is being actively explored in the clinic. In mouse models, the rat anti-mouse CD25 clone PC61 has been used extensively to investigate the biology of IL-2 and Treg cells; however, there has been controversy and conflicting data on the exact in vivo mechanistic function of PC61. Engineering antibodies to alter Fc/Fc receptor interactions can significantly alter their in vivo function. In this study, we re-engineered the heavy chain constant region of an anti-CD25 monoclonal antibody to generate variants with highly divergent Fc effector function. Using these anti-CD25 Fc variants in multiple mouse models, we investigated the in vivo impact of CD25 blockade versus depletion of CD25(+) Treg cells on immune homeostasis. We report that immune homeostasis can be maintained during CD25 blockade but aberrant T-cell activation prevails when CD25(+) Treg cells are actively depleted. These results clarify the impact of PC61 on Treg cell biology and reveal an important distinction between CD25 blockade and depletion of CD25(+) Treg cells. These findings should inform therapeutic manipulation of the IL-2 pathway by targeting the high-affinity IL-2R.

  4. Secretome identification of immune cell factors mediating metastatic cell homing

    PubMed Central

    Aguado, Brian A.; Wu, Jia J.; Azarin, Samira M.; Nanavati, Dhaval; Rao, Shreyas S.; Bushnell, Grace G.; Medicherla, Chaitanya B.; Shea, Lonnie D.

    2015-01-01

    Metastatic cell homing is a complex process mediated in part by diffusible factors secreted from immune cells found at a pre-metastatic niche. We report on connecting secretomics and TRanscriptional Activity CEll aRray (TRACER) data to identify functional paracrine interactions between immune cells and metastatic cells as novel mediators of homing. Metastatic breast cancer mouse models were used to generate a diseased splenocyte conditioned media (D-SCM) containing immune cell secreted factors. MDA-MB-231 metastatic cell activity including cell invasion, migration, transendothelial migration, and proliferation were increased in D-SCM relative to control media. Our D-SCM secretome analysis yielded 144 secreted factor candidates that contribute to increased metastatic cell activity. The functional mediators of homing were identified using MetaCore software to determine interactions between the immune cell secretome and the TRACER-identified active transcription factors within metastatic cells. Among the 5 candidate homing factors identified, haptoglobin was selected and validated in vitro and in vivo as a key mediator of homing. Our studies demonstrate a novel systems biology approach to identify functional signaling factors associated with a cellular phenotype, which provides an enabling tool that complements large-scale protein identification provided by proteomics. PMID:26634905

  5. Immunologic injury of cultured cells infected with measles virus. I. role of IfG antibody and the alternative complement pathway

    PubMed Central

    1975-01-01

    In these studies, a number of human cell lines including epithelial, neural, glial, and lymphoid cells infected with several strains of measles virus were found to be lysed upon incubation with fresh sera from humans containing antibody measles virus. In all instances, the cytolytic event was mediated by alternative complement (C) pathway without a significant contribution from classical pathway. In contrast, isolated measles virus in conjunction with antibody was found to selectively activate the classical C pathway. Measles antibodies of the IgG class, but not the IgA class, possessed cytolytic potential against cells infected with measles virus. Human IgG antibodies could directly activate the alternative C pathway. No defect was found in cytolytic measles antibody in sera or cerebrospinal fluid from patients with subacute sclerosing panencephalitis, nor was the alternative C pathway impaired in sera from these patients. Sera from newborn humans exhibited a functional alternative C pathway. PMID:1092789

  6. The application of anti-Toso antibody enhances CD8(+) T cell responses in experimental malaria vaccination and disease.

    PubMed

    Lapke, Nina; Tartz, Susanne; Lee, Kyeong-Hee; Jacobs, Thomas

    2015-11-27

    Toso is a molecule highly expressed on B cells. It influences their survival and was identified as an IgM binding molecule. B cells and natural antibodies play a role in vaccination-induced CD8(+) T cell responses. We investigated the impact of an anti-Toso antibody on vaccination efficiency in a malaria vaccination model. In this model, CD8(+) T cells exert antiparasitic functions on infected hepatocytes in the liver stage of the disease. In vaccinated anti-Toso treated mice, more antigen-specific CD8(+) T cells were induced than in control mice and after infection with Plasmodium berghei ANKA (PbA) sporozoites, the liver parasite burden was lower. In B cell deficient mice, the anti-Toso antibody did not stimulate the CD8(+) T cell response, indicating that B cells were mediating this effect. Furthermore, we analyzed the influence of anti-Toso treatment on non-vaccinated mice in the PbA infection model, in which CD8(+) T cells cause brain pathology. Anti-Toso treatment increased cerebral pathology and the accumulation of CD8(+) T cells in the brain. Thus, anti-Toso treatment enhanced the CD8(+) T cell response against PbA in a vaccination and in an infection model. Our findings indicate that Toso may be a novel target to boost vaccine-induced CD8(+) T cell responses.

  7. Chemokine-mediated B cell trafficking during early rabbit GALT development

    PubMed Central

    Zhai, Shi-Kang; Volgina, Veronica V.; Sethupathi, Periannan; Knight, Katherine L.; Lanning, Dennis K.

    2014-01-01

    Microbial and host cell interactions stimulate rabbit B cells to diversify the primary antibody repertoire in gut-associated lymphoid tissues (GALT). B cells at the base of appendix follicles begin proliferating and diversifying their V-(D)-J genes around 1 week of age, ∼5 days after B cells first begin entering appendix follicles, To gain insight into the microbial and host cell interactions that stimulate B cells to diversify the primary antibody repertoire, we analyzed B cell trafficking within follicles during the first week of life. We visualized B cells, as well as chemokines that mediate B cell homing in lymphoid tissues, by in situ hybridization, and examined B cell chemokine receptor expression by flow cytometry. We found that B cells were activated, and began downregulating their BCRs, well before a detectable B cell proliferative region appeared at the follicle base. The proliferative region was similar to germinal center dark zones, in that it exhibited elevated CXCL12 mRNA expression, and B cells that upregulated CXCR4 mRNA in response to signals acquired from select intestinal commensals localized in this region. Our results suggest that, after entering appendix follicles, B cells home sequentially to the FAE, the FDC network, the B cell:T cell boundary and, ultimately, the base of the follicle, where they enter a proliferative program and diversify the primary antibody repertoire. PMID:25385821

  8. Defective TFH Cell Function and Increased TFR Cells Contribute to Defective Antibody Production in Aging.

    PubMed

    Sage, Peter T; Tan, Catherine L; Freeman, Gordon J; Haigis, Marcia; Sharpe, Arlene H

    2015-07-14

    Defective antibody production in aging is broadly attributed to immunosenescence. However, the precise immunological mechanisms remain unclear. Here, we demonstrate an increase in the ratio of inhibitory T follicular regulatory (TFR) cells to stimulatory T follicular helper (TFH) cells in aged mice. Aged TFH and TFR cells are phenotypically distinct from those in young mice, exhibiting increased programmed cell death protein-1 expression but decreased ICOS expression. Aged TFH cells exhibit defective antigen-specific responses, and programmed cell death protein-ligand 1 blockade can partially rescue TFH cell function. In contrast, young and aged TFR cells have similar suppressive capacity on a per-cell basis in vitro and in vivo. Together, these studies reveal mechanisms contributing to defective humoral immunity in aging: an increase in suppressive TFR cells combined with impaired function of aged TFH cells results in reduced T-cell-dependent antibody responses in aged mice.

  9. Lepidopteran cells, an alternative for the production of recombinant antibodies?

    PubMed Central

    Cérutti, Martine; Golay, Josée

    2012-01-01

    Monoclonal antibodies are used with great success in many different therapeutic domains. In order to satisfy the growing demand and to lower the production cost of these molecules, many alternative systems have been explored. Among them, the baculovirus/insect cells system is a good candidate. This system is very safe, given that the baculoviruses have a highly restricted host range and they are not pathogenic to vertebrates or plants. But the major asset is the speed with which it is possible to obtain very stable recombinant viruses capable of producing fully active proteins whose glycosylation pattern can be modulated to make it similar to the human one. These features could ultimately make the difference by enabling the production of antibodies with very low costs. However, efforts are still needed, in particular to increase production rates and thus make this system commercially viable for the production of these therapeutic agents. PMID:22531440

  10. The Ch14.18-GM-CSF fusion protein is effective at mediating antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in vitro.

    PubMed

    Batova, A; Kamps, A; Gillies, S D; Reisfeld, R A; Yu, A L

    1999-12-01

    Granulocyte/macrophage-colony stimulating factor (GM-CSF) is very effective at enhancing antibody-dependent cellular cytotoxicity (ADCC) mediated by granulocytes and monocytes. Recently, a fusion protein consisting of GM-CSF and chimeric human/mouse anti-ganglioside G(D2) antibody Ch14.18 (Ch14.18-GM-CSF) has been generated to improve the effectiveness of immunotherapy by directing GM-CSF to the tumor microenvironment and prolonging its relatively short half-life. In this study, we examined the ability of this fusion protein to enhance the in vitro killing of G(D2)-expressing human neuroblastoma cells by granulocytes and mononuclear cells, as well as by complement. The Ch14.18-GM-CSF fusion protein was equally effective as the combination of equivalent amounts of free Ch14.18 and GM-CSF in mediating the killing of NMB7 neuroblastoma cells by granulocytes from seven of eight neuroblastoma patients. The fusion protein was also equally effective as the combination of Ch14.18 and GM-CSF in mediating ADCC by neuroblastoma patients' mononuclear cells. In addition, the fusion protein was as effective as Ch14.18 alone in directing complement-dependent cytotoxicity against NMB7 cells. Our results demonstrate that the biological activities expressed by ADCC and complement-dependent cytotoxicity of both monoclonal antibody Ch14.18 and GM-CSF are retained by the Ch14.18-GM-CSF fusion protein and lend further support for future clinical trials of this fusion protein in patients with neuroblastoma.

  11. Affinity maturation of T-cell receptor-like antibodies for Wilms tumor 1 peptide greatly enhances therapeutic potential

    PubMed Central

    Zhao, Qi; Ahmed, Mahiuddin; Tassev, Dimiter V.; Hasan, Aisha; Kuo, Tzu-Yun; Guo, Hong-fen; O’Reilly, Richard J.; Cheung, Nai-Kong V.

    2016-01-01

    WT1126 (RMFPNAPYL) is a human leukocyte antigen-A2 (HLA-A2) restricted peptide derived from Wilms tumor protein (WT1), which is widely expressed in a broad spectrum of leukemias, lymphomas and solid tumors. A novel T-cell-receptor (TCR)-like single chain variable fragment (scFv) antibody specific for the T cell epitope consisting of the WT1/HLA-A2 complex was isolated from a human scFv phage library. This scFv was affinity-matured by mutagenesis combined with yeast display, and structurally analyzed using a homology model. This monovalent scFv showed a 100-fold affinity improvement (dissociation constant [KD]= 3nM) and exquisite specificity towards its targeted epitope or HLA-A2+/WT1+ tumor cells. Bivalent scFv-huIgG1-Fc fusion protein demonstrated an even higher avidity (KD = 2pM) binding to the T cell epitope and to tumor targets, and was capable of mediating antibody-dependent cell-mediated cytotoxicity or tumor lysis by chimeric antigen receptor (CAR)-expressing human T or NK-92-MI transfected cells. This antibody demonstrated specific and potent cytotoxicity in vivo towards WT1-positive leukemia xenograft that was HLA-A2 restricted. In summary, T cell epitopes can provide novel targets for antibody-based therapeutics. By combining phage and yeast displays and scFv-Fc fusion platforms, a strategy for developing high affinity TCR-like antibodies could be rapidly explored for potential clinical development. PMID:25987253

  12. Selecting antagonistic antibodies that control differentiation through inducible expression in embryonic stem cells

    PubMed Central

    Melidoni, Anna N.; Dyson, Michael R.; Wormald, Sam; McCafferty, John

    2013-01-01

    Antibodies that modulate receptor function have great untapped potential in the control of stem cell differentiation. In contrast to many natural ligands, antibodies are stable, exquisitely specific, and are unaffected by the regulatory mechanisms that act on natural ligands. Here we describe an innovative system for identifying such antibodies by introducing and expressing antibody gene populations in ES cells. Following induced antibody expression and secretion, changes in differentiation outcomes of individual antibody-expressing ES clones are monitored using lineage-specific gene expression to identify clones that encode and express signal-modifying antibodies. This in-cell expression and reporting system was exemplified by generating blocking antibodies to FGF4 and its receptor FGFR1β, identified through delayed onset of ES cell differentiation. Functionality of the selected antibodies was confirmed by addition of exogenous antibodies to three different ES reporter cell lines, where retained expression of pluripotency markers Oct4, Nanog, and Rex1 was observed. This work demonstrates the potential for discovery and utility of functional antibodies in stem cell differentiation. This work is also unique in constituting an example of ES cells carrying an inducible antibody that causes a functional protein “knock-down” and allows temporal control of stable signaling components at the protein level. PMID:24082130

  13. Platelet antibodies.

    PubMed

    Pulkrabek, S M

    1996-12-01

    The proper diagnosis of patients with immune-mediated thrombocytopenias can be accomplished by using the advances made in the field of platelet serology. These techniques range from solid phase red cell adherence to sequencing platelet antigen amino acids by polymerase chain reaction. This article describes platelet antigens, the clinical tests available to detect platelet antigens and antibodies, and the value of these tests in supporting clinical diagnoses.

  14. The role of antigen specificity in the binding of murine monoclonal anti-DNA antibodies to microparticles from apoptotic cells.

    PubMed

    Ullal, Anirudh J; Marion, Tony N; Pisetsky, David S

    2014-10-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and markers of underlying immune system disturbances. These antibodies bind to both single-stranded and double-stranded DNA, mediating pathogenesis by forming immune complexes. As shown recently, DNA in blood exists in both free and particulate forms, with DNA representing an important component of microparticles. Microparticles are membrane-bound vesicles containing nuclear molecules, released by membrane blebbing during cell death and activation. A panel of monoclonal NZB/NZW F1 anti-DNA antibodies was tested for binding to microparticles generated from apoptotic THP-1 and Jurkat cells. These studies showed that only certain anti-DNA antibodies in the panel, specific for double-stranded DNA, bound to microparticles. Binding to particles was reduced by soluble DNA or DNase treatment. Together, these results indicate that particle binding is a feature of only certain anti-DNA antibodies, reflecting immunochemical properties of the antibodies and the nature of the exposed DNA antigens.

  15. B-Cell Deficiency Predisposes Mice to Disseminating Anaerobic Infections: Protection by Passive Antibody Transfer

    PubMed Central

    Hou, Linda; Sasakj, Hajime; Stashenko, Philip

    2000-01-01

    We have previously demonstrated that a high proportion of RAG-2 SCID knockout mice, which lack T and B cells, develop orofacial abscesses and disseminated infections following pulpal infection, whereas immunocompetent control mice do not. In the present study, we sought to identify the components of the adaptive immune response which contribute to protection against disseminating anaerobic infections and sepsis. For this purpose, various genetically engineered immunodeficient mice were employed, including RAG-2 SCID, Igh-6 (B-cell deficient), Tcrb Tcrd (T-cell deficient) and Hc0 (C5 deficient). For abscess induction, the mandibular first molars were subjected to pulp exposure on day 0. Teeth were infected with a mixture of four anaerobic pathogens, including Prevotella intermedia, Streptococcus intermedius, Fusobacterium nucleatum, and Peptostreptococcus micros, and teeth were sealed to prevent communication with the oral cavity. The findings demonstrate that both RAG-2 SCID and B-cell-deficient mice, but not T-cell- or C5-deficient mice, have increased susceptibility to the development of disseminating anaerobic infections. Abscess-susceptible RAG-2 SCID and B-cell-deficient mice also showed a significant loss of body weight, splenomegaly, and absent antibacterial antibody production. Furthermore, dissemination was significantly reduced, from 74 to 25%, in susceptible RAG-2 mice by passively transferred antibody, predominantly immunoglobulin G2b (IgG2b) and IgM, against the infecting bacterial innoculum. Fractionated IgG-enriched preparations were more efficient in transferring protection than IgM preparations. We conclude that an antibody-mediated mechanism(s), most likely bacterial opsonization, is of importance in localizing anaerobic root canal infections and in preventing their systemic spread. PMID:10992465

  16. A strong antibody reacting with enzyme modified E positive red blood cells.

    PubMed

    Heistø, H; Fagerhol, M K

    1979-01-01

    A high titer antibody was discovered in a healthy young man of blood group A1 R2r. The antibody strongly agglutinated all E positive red blood cells including his own, which had been modified by papain, ficin and bromelin, but only very weakly when modified by trypsin. The antibody was shown to be an IgM antibody. It did not react with unmodified red blood cells.

  17. A strong antibody reacting with enzyme modified E positive red blood cells.

    PubMed

    Heistø, H; Fagerhol, M K

    1979-01-01

    A high titer antibody was discovered in a healthy young man of blood group A1 R2r. The antibody strongly agglutinated all E positive red blood cells including his own, which had been modified by papain, ficin and bromelin, but only very weakly when modified by trypsin. The antibody was shown to be an IgM antibody. It did not react with unmodified red blood cells. PMID:116398

  18. Inhibition of Pneumococcal Adherence to Human Nasopharyngeal Epithelial Cells by Anti-PsaA Antibodies

    PubMed Central

    Romero-Steiner, Sandra; Pilishvili, Tamar; Sampson, Jacquelyn S.; Johnson, Scott E.; Stinson, Annie; Carlone, George M.; Ades, Edwin W.

    2003-01-01

    The role of pneumococcal (Pnc) surface adhesin A (PsaA) in the adherence of Streptococcus pneumoniae (pneumococcus) to host cells is not well defined. We examined the effect of anti-PsaA antibodies in an inhibition of adherence assay using Detroit 562 nasopharyngeal human epithelial cells. Rabbit polyclonal (Pab) anti-recombinant PsaA (rPsaA) sera, a purified mouse monoclonal antibody (MAb) (MAb 6F62G8E12), and 22 healthy adult sera with known anti-PsaA IgG levels (obtained by enzyme-linked immunosorbent assay) were evaluated for their abilities to inhibit Pnc adherence to confluent monolayers (measured as percent reduction in CFU counts compared to those of uninhibited controls). Pnc adherence was dependent on capsular phenotype (no or low adherence for opaque strains). With an inoculum of 104 to 105 bacteria/well, the mean ± standard deviation count in controls was 163 ± 32 CFU/well for transparent strains. Low adherence was observed for a PsaA-minus mutant even at higher inoculum doses. Mean percent inhibitions of adherence with Pab and MAb were 54 and 50%, respectively. Adult sera showed inhibition in a dose-response fashion with a range of 98 to 8%, depending on the serum anti-PsaA antibody concentration. Absorption of Pab with rPsaA restored Pnc adherence to control levels. Absorption of sera with a PsaA-minus mutant did not result in a significant decrease (P >0.05) of inhibition of adherence activity. Additionally, nearly 100% of Pnc adherence was inhibited by lipidated rPsaA at 2.5 μg/ml. Our data support the argument that PsaA is an adhesin that mediates Pnc adherence to human nasopharyngeal cells. This functional assay may be useful in evaluating antibodies elicited in response to PsaA vaccination. PMID:12626450

  19. Membrane-bound hemagglutinin mediates antibody and complement-dependent lysis of influenza virus-treated human platelets in autologous serum.

    PubMed Central

    Kazatchkine, M D; Lambré, C R; Kieffer, N; Maillet, F; Nurden, A T

    1984-01-01

    Influenza A virus-treated human platelets were lyzed in autologous serum. Lysis required the presence of antibody and occurred predominantly through activation of the classical complement pathway. Binding of the virus followed by its elution at 37 degrees C resulted in a dose-dependent desialation of the cells with a maximal release of 45% of total platelet sialic acid. In contrast, platelets that had been treated with Vibrio cholerae neuraminidase and from which 55% of total sialic acid had been removed were not lyzed in autologous serum and did not bind C3 as shown in binding assays using radiolabeled monoclonal anti-C3 antibody. Thus, the immune-mediated lysis of virus-treated platelets in autologous serum did not involve neoantigens expressed by desialated cells. To assess the effect of viruses on the platelet surface, treated platelets were incubated with galactose oxidase and sodium [3H]borohydride prior to separation and analysis of the labeled glycoproteins by SDS-PAGE. Viral treatment resulted in a desialation of each of the surface glycoproteins. At the same time, a labeled component of Mr 72,000 (nonreduced) and Mr 55,000 (reduced) was observed that was not present when V. cholerae-desialated platelets were examined in the same way. Immunoblotting experiments performed using antiwhole virus and anti-hemagglutinin antibodies demonstrated this component to be viral hemagglutinin. Involvement of membrane-bound hemagglutinin in antibody and in complement-mediated lysis of virus-treated platelets in autologous serum was supported by the increased lytic activity of a postvaccinal serum containing an elevated titer of complement fixing anti-hemagglutinin antibodies. Binding of a viral protein to the platelet surface provides a model for immune thrombocytopenias occurring during acute viral infections at the time of the specific immune response. Images PMID:6470149

  20. Activation of NLRC4 downregulates TLR5-mediated antibody immune responses against flagellin

    PubMed Central

    Li, Wei; Yang, Jingyi; Zhang, Ejuan; Zhong, Maohua; Xiao, Yang; Yu, Jie; Zhou, Dihan; Cao, Yuan; Yang, Yi; Li, Yaoming; Yan, Huimin

    2016-01-01

    Bacterial flagellin is a unique pathogen-associated molecular pattern (PAMP), which can be recognized by surface localized Toll-like receptor 5 (TLR5) and the cytosolic NOD-like receptor (NLR) protein 4 (NLRC4) receptors. Activation of the TLR5 and/or NLRC4 signaling pathways by flagellin and the resulting immune responses play important roles in anti-bacterial immunity. However, it remains unclear how the dual activities of flagellin that activate the TLR5 and/or NLRC4 signaling pathways orchestrate the immune responses. In this study, we assessed the effects of flagellin and its mutants lacking the ability to activate TLR5 and NLRC4 alone or in combination on the adaptive immune responses against flagellin. Flagellin that was unable to activate NLRC4 induced a significantly higher antibody response than did wild-type flagellin. The increased antibody response could be eliminated when macrophages were depleted in vivo. The activation of NLRC4 by flagellin downregulated the flagellin-induced and TLR5-mediated immune responses against flagellin. PMID:25914934

  1. Cell-mediated destruction of cells grown on artificial capillaries.

    PubMed

    Zwilling, B S; Clayman, D A

    1978-11-01

    This investigation was designed to determine the conditions required to assess cell-mediated destruction of target L-cells grown on artificial capillaries. In control cultures that contained L-cells alone, solid nodules with a diameter of 1 mm as well as dense cellular growth could be visually observed by the 12th day of culture. Alloimmune spleen cells from both immunized and normal C57BL/10 mice were shown to be capable of destroying tritiated thymidine-labeled L-cells growing on artificial capillaries. The destruction of target cells grown as monolayers in capillary culture correlated well with monolayer cultures incubated in 16-mm plastic tissue culture wells. When target cells were grown in capillary culture for 5 days before the addition of effector cells, significant destruction by normal effector cells was not observed until the 15th day of culture whereas that mediated by immune cells was observed by the 7th day. The possible effects of cell-culturing conditions on the kinetics of cell-mediated destruction in capillary chambers are discussed.

  2. Antibody-Dependent NK Cell Activation Is Associated with Late Kidney Allograft Dysfunction and the Complement-Independent Alloreactive Potential of Donor-Specific Antibodies.

    PubMed

    Legris, Tristan; Picard, Christophe; Todorova, Dilyana; Lyonnet, Luc; Laporte, Cathy; Dumoulin, Chloé; Nicolino-Brunet, Corinne; Daniel, Laurent; Loundou, Anderson; Morange, Sophie; Bataille, Stanislas; Vacher-Coponat, Henri; Moal, Valérie; Berland, Yvon; Dignat-George, Francoise; Burtey, Stéphane; Paul, Pascale

    2016-01-01

    Although kidney transplantation remains the best treatment for end-stage renal failure, it is limited by chronic humoral aggression of the graft vasculature by donor-specific antibodies (DSAs). The complement-independent mechanisms that lead to the antibody-mediated rejection (ABMR) of kidney allografts remain poorly understood. Increasing lines of evidence have revealed the relevance of natural killer (NK) cells as innate immune effectors of antibody-dependent cellular cytotoxicity (ADCC), but few studies have investigated their alloreactive potential in the context of solid organ transplantation. Our study aimed to investigate the potential contribution of the antibody-dependent alloreactive function of NK cells to kidney graft dysfunction. We first conducted an observational study to investigate whether the cytotoxic function of NK cells is associated with chronic allograft dysfunction. The NK-Cellular Humoral Activation Test (NK-CHAT) was designed to evaluate the recipient and antibody-dependent reactivity of NK cells against allogeneic target cells. The release of CD107a/Lamp1(+) cytotoxic granules, resulting from the recognition of rituximab-coated B cells by NK cells, was analyzed in 148 kidney transplant recipients (KTRs, mean graft duration: 6.2 years). Enhanced ADCC responsiveness was associated with reduced graft function and identified as an independent risk factor predicting a decline in the estimated glomerular filtration rate over a 1-year period (hazard ratio: 2.83). In a second approach, we used the NK-CHAT to reveal the cytotoxic potential of circulating alloantibodies in vitro. The level of CD16 engagement resulting from the in vitro recognition of serum-coated allogeneic B cells or splenic cells was further identified as a specific marker of DSA-induced ADCC. The NK-CHAT scoring of sera obtained from 40 patients at the time of transplant biopsy was associated with ABMR diagnosis. Our findings indicate that despite the administration of

  3. Antibody-Dependent NK Cell Activation Is Associated with Late Kidney Allograft Dysfunction and the Complement-Independent Alloreactive Potential of Donor-Specific Antibodies

    PubMed Central

    Legris, Tristan; Picard, Christophe; Todorova, Dilyana; Lyonnet, Luc; Laporte, Cathy; Dumoulin, Chloé; Nicolino-Brunet, Corinne; Daniel, Laurent; Loundou, Anderson; Morange, Sophie; Bataille, Stanislas; Vacher-Coponat, Henri; Moal, Valérie; Berland, Yvon; Dignat-George, Francoise; Burtey, Stéphane; Paul, Pascale

    2016-01-01

    Although kidney transplantation remains the best treatment for end-stage renal failure, it is limited by chronic humoral aggression of the graft vasculature by donor-specific antibodies (DSAs). The complement-independent mechanisms that lead to the antibody-mediated rejection (ABMR) of kidney allografts remain poorly understood. Increasing lines of evidence have revealed the relevance of natural killer (NK) cells as innate immune effectors of antibody-dependent cellular cytotoxicity (ADCC), but few studies have investigated their alloreactive potential in the context of solid organ transplantation. Our study aimed to investigate the potential contribution of the antibody-dependent alloreactive function of NK cells to kidney graft dysfunction. We first conducted an observational study to investigate whether the cytotoxic function of NK cells is associated with chronic allograft dysfunction. The NK-Cellular Humoral Activation Test (NK-CHAT) was designed to evaluate the recipient and antibody-dependent reactivity of NK cells against allogeneic target cells. The release of CD107a/Lamp1+ cytotoxic granules, resulting from the recognition of rituximab-coated B cells by NK cells, was analyzed in 148 kidney transplant recipients (KTRs, mean graft duration: 6.2 years). Enhanced ADCC responsiveness was associated with reduced graft function and identified as an independent risk factor predicting a decline in the estimated glomerular filtration rate over a 1-year period (hazard ratio: 2.83). In a second approach, we used the NK-CHAT to reveal the cytotoxic potential of circulating alloantibodies in vitro. The level of CD16 engagement resulting from the in vitro recognition of serum-coated allogeneic B cells or splenic cells was further identified as a specific marker of DSA-induced ADCC. The NK-CHAT scoring of sera obtained from 40 patients at the time of transplant biopsy was associated with ABMR diagnosis. Our findings indicate that despite the administration of

  4. Rituximab in Combination With Bortezomib, Plasmapheresis, and High-Dose IVIG to Treat Antibody-Mediated Renal Allograft Rejection

    PubMed Central

    Waiser, Johannes; Duerr, Michael; Schönemann, Constanze; Rudolph, Birgit; Wu, Kaiyin; Halleck, Fabian; Budde, Klemens; Lachmann, Nils

    2016-01-01

    Background Current treatment strategies for antibody-mediated renal allograft rejection (AMR) are not sufficiently effective. In most centers, “standard of care” treatment includes plasmapheresis (PPH) and IVIG preparations. Since several years, modern therapeutics targeting B cells and plasma cells have become available. We investigated, whether combined administration of rituximab and bortezomib in addition to PPH and high-dose IVIG is useful. Methods Between November 2011 and January 2013, we treated 10 consecutive patients with biopsy-proven AMR with rituximab (500 mg), bortezomib (4× 1.3 mg/m2), PPH (6×), and high-dose IVIG (1.5 g/kg) (group A). This group was compared with a group of 11 consecutive patients treated with an identical regimen without rituximab between July 2010 and November 2011 (group B). Results Median follow-up was 41(33-46) months in group A and 55(47-63) months in group B. At 40 months after treatment, graft survival was 60% in group A and 64% in group B, respectively (P = 0.87). Before and after treatment, serum creatinine, estimated glomerular filtration rate, and proteinuria were not different between groups. A significant reduction in donor-specific HLA antibody mean fluorescence intensity was observed in group A (25.2%, P = 0.046) and B (38.3%, P = 0.01) at 3 months posttreatment. In group A, more patients suffered from side effects compared with group B (infections: 70% vs 18%, P = 0.02). Conclusions The addition of rituximab to bortezomib, PPH, and high-dose IVIG did not further improve graft survival. Instead, we observed an increase of side effects. Therefore, combined administration of bortezomib and rituximab in addition to PPH and IVIG should be regarded with caution.

  5. P-selectin mediates adhesion of platelets to neuroblastoma and small cell lung cancer.

    PubMed Central

    Stone, J P; Wagner, D D

    1993-01-01

    Activated platelets and stimulated endothelial cells express P-selectin, an integral membrane protein receptor that binds monocytes and neutrophils. P-selectin mediates adhesion to glycoproteins with carbohydrate structures containing sialyl-Lewis X. Since many carcinoma cells also express these carbohydrate structures and are known to interact with platelets, we asked whether P-selectin may mediate this interaction. Both small cell lung cancer and neuroblastoma cell lines bound to activated platelets, and this interaction was blocked with inhibitory anti-P-selectin antibodies and by pretreatment of these cancer cells with neuraminidase or trypsin. Platelet binding to the small cell lung cancer cells was not inhibited with anti-GP IIb-IIIa antibody or Arg-Gly-Asp-Ser peptide. Pretreatment of the neuroblastoma cells with inhibitors of N-linked carbohydrate biosynthesis had little effect on binding to P-selectin, indicating that relevant carbohydrate ligand(s) may be O-linked. In addition, lipospheres containing P-selectin specifically bound to cryostat sections derived from a small cell lung tumor and two neuroblastoma tumors, but not to sections of normal lung. These observations demonstrate that P-selectin mediates binding of platelets to small cell lung cancer and to neuroblastoma and suggest a possible role for this lectin in metastasis. Images PMID:7688763

  6. [Antibody adsorption to tetrodotoxin-sensitive cytoplasmic proteins by murine neuroblastoma cells].

    PubMed

    Pinchuk, G V; Pinchuk, L N; Gerasimenko, O V

    1988-01-01

    The study was aimed to examine properties of polyclonal antibodies elicited after immunization by cytoplasmic nerve cell glycoproteins forming sodium channels in liposomes. It was shown that intact murine neuroblastoma cells can absorb these antibodies. Absorbing cell dose-effect curves were found to have a characteristic form able to shift when the cell culture time or serum concentration in the growth medium varied.

  7. Inhibition of Hepatitis C Virus-Like Particle Binding to Target Cells by Antiviral Antibodies in Acute and Chronic Hepatitis C

    PubMed Central

    Steinmann, Daniel; Barth, Heidi; Gissler, Bettina; Schürmann, Peter; Adah, Mohammed I.; Gerlach, J. Tilman; Pape, Gerd R.; Depla, Erik; Jacobs, Dirk; Maertens, Geert; Patel, Arvind H.; Inchauspé, Geneviève; Liang, T. Jake; Blum, Hubert E.; Baumert, Thomas F.

    2004-01-01

    Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis worldwide. The study of antibody-mediated virus neutralization has been hampered by the lack of an efficient and high-throughput cell culture system for the study of virus neutralization. The HCV structural proteins have been shown to assemble into noninfectious HCV-like particles (HCV-LPs). Similar to serum-derived virions, HCV-LPs bind and enter human hepatocytes and hepatoma cell lines. In this study, we developed an HCV-LP-based model system for a systematic functional analysis of antiviral antibodies from patients with acute or chronic hepatitis C. We demonstrate that cellular HCV-LP binding was specifically inhibited by antiviral antibodies from patients with acute or chronic hepatitis C in a dose-dependent manner. Using a library of homologous overlapping envelope peptides covering the entire HCV envelope, we identified an epitope in the N-terminal E2 region (SQKIQLVNTNGSWHI; amino acid positions 408 to 422) as one target of human antiviral antibodies inhibiting cellular particle binding. Using a large panel of serum samples from patients with acute and chronic hepatitis C, we demonstrated that the presence of antibodies with inhibition of binding activity was not associated with viral clearance. In conclusion, antibody-mediated inhibition of cellular HCV-LP binding represents a convenient system for the functional characterization of human anti-HCV antibodies, allowing the mapping of envelope neutralization epitopes targeted by naturally occurring antiviral antibodies. PMID:15308699

  8. Antibody-dependent complement-mediated cytotoxicity in sera from patients with HIV-1 infection is controlled by CD55 and CD59.

    PubMed Central

    Schmitz, J; Zimmer, J P; Kluxen, B; Aries, S; Bögel, M; Gigli, I; Schmitz, H

    1995-01-01

    Various immune mechanisms have been reported to contribute to the progressive destruction of Th cells in HIV-1-infected patients. Among these, complement mediated lysis of infected cells has been suggested. An increased sensitivity of lymphocytes from HIV-1-infected patients to lysis by monoclonal antibodies directed to MHC class I antigen and complement has been directly correlated with a decreased expression of the decay accelerating factor (CD55). It also has been reported that the expression of the membrane inhibitor of reactive lysis (CD59) is decreased during HIV-1 infection. We examined the effect of antibodies in the serum of HIV-1-positive individuals and normal human serum (NHS) as source of complement on several HIV-1-infected cell lines differing in their expression of CD55 and CD59. When HIV-1-infected target cells without membrane expression of CD55 and CD59 were used, a highly significant cytotoxic effect was observed in the presence of heat inactivated anti-HIV-1-positive sera and NHS, while heat-inactivated anti-HIV-1-negative sera and NHS were unable to induce cytolysis. Similar results were obtained using purified IgG isolated from HIV-1-positive sera and either NHS or guinea pig serum as source of complement. Lysis of HIV-1-infected cells correlated with expression of viral antigens on the cell surface. HIV-1-infected CD55 and CD59 positive target cells showed specific lysis, when the function of these molecules was abrogated by blocking antibodies to CD55 and CD59. The finding of anti-HIV-1-specific cytotoxic antibodies in sera from HIV-1-infected patients should be considered in the pathogenesis of the HIV-1-infection. PMID:7544808

  9. Curcumin Attenuates Staurosporine-Mediated Death of Retinal Ganglion Cells

    PubMed Central

    Burugula, Balabharathi; Ganesh, Bhagyalaxmi S.

    2011-01-01

    Purpose. Staurosporine (SS) causes retinal ganglion cell (RGC) death in vivo, but the underlying mechanisms have been unclear. Since previous studies on RGC-5 cells indicated that SS induces cell death by elevating proteases, this study was undertaken to investigate whether SS induces RGC loss by elevating proteases in the retina, and curcumin prevents SS-mediated death of RGCs. Methods. Transformed mouse retinal ganglion-like cells (RGC-5) were treated with 2.0 μM SS and various doses of curcumin. Two optimal doses of SS (12.5 and 100 nM) and curcumin (2.5 and 10 μM) were injected into the vitreous of C57BL/6 mice. Matrix metalloproteinase (MMP)-9, tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA) activities were assessed by zymography assays. Viability of RGC-5 cells was assessed by MTT assays. RGC and amacrine cell loss in vivo was assessed by immunostaining with Brn3a and ChAT antibodies, respectively. Frozen retinal cross sections were immunostained for nuclear factor-κB (NF-κB). Results. Staurosporine induced uPA and tPA levels in RGC-5 cells, and MMP-9, uPA, and tPA levels in the retinas and promoted the death of RGC-5 cells in vitro and RGCs and amacrine cells in vivo. In contrast, curcumin attenuated RGC and amacrine cell loss, despite elevated levels of proteases. An NF-κB inhibitory peptide reversed curcumin-mediated protective effect on RGC-5 cells, but did not inhibit protease levels. Curcumin did not inhibit protease levels in vivo, but attenuated RGC and amacrine cell loss by restoring NF-κB expression. Conclusions. The results show that curcumin attenuates RGC and amacrine cell death despite elevated levels of proteases and raises the possibility that it may be used as a plausible adjuvant therapeutic agent to prevent the loss of these cells in retinal degenerative conditions. PMID:21498608

  10. Mast Cell-Mediated Mechanisms of Nociception

    PubMed Central

    Aich, Anupam; Afrin, Lawrence B.; Gupta, Kalpna

    2015-01-01

    Mast cells are tissue-resident immune cells that release immuno-modulators, chemo-attractants, vasoactive compounds, neuropeptides and growth factors in response to allergens and pathogens constituting a first line of host defense. The neuroimmune interface of immune cells modulating synaptic responses has been of increasing interest, and mast cells have been proposed as key players in orchestrating inflammation-associated pain pathobiology due to their proximity to both vasculature and nerve fibers. Molecular underpinnings of mast cell-mediated pain can be disease-specific. Understanding such mechanisms is critical for developing disease-specific targeted therapeutics to improve analgesic outcomes. We review molecular mechanisms that may contribute to nociception in a disease-specific manner. PMID:26690128

  11. R-phycoerythrin-conjugated antibodies are inappropriate for intracellular staining of murine plasma cells.

    PubMed

    Kim, Myun Soo; Kim, Tae Sung

    2013-05-01

    Phycoerythrin (PE) is a type of phycobiliproteins found in cyanobacteria and red algae. PE-conjugated antibodies are broadly used for flow cytometry and immunofluorescence microscopy. Because nonspecific binding of antibodies results in decreased analytic accuracy, numerous efforts have been made to unveil cases and mechanisms of nonspecific bindings. However, nonspecific binding of specific cell types by a fluorescent dye-conjugated form of antibody has been rarely reported. In the present study, we discovered that PE-conjugated antibodies, but not FITC- or APC-antibodies, selectively stained lamina propria plasma cells (LP-PCs) from the murine small intestine after membrane permeabilization. We demonstrated that LP-PC-selective staining with PE-antibodies was not due to interactions of antibody-epitope or antibody-Fc receptor. This unexpected staining by PE-antibody was not dependent on the mouse strain of LP-PCs, experimental methods, or origin species of the antibody, but dependent on PE itself. This phenomenon was also observed in plasma cells isolated from bone marrow, spleen, and mesenteric lymph nodes. Furthermore, in vitro activated B cells and in vivo generated LP-PCs were also selectively stained by PE-conjugated antibodies. Taken together, these results show that PE-conjugated antibodies are inappropriate for intracellular staining of murine plasma cells.

  12. Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule

    PubMed Central

    1984-01-01

    By means of a multistage quantitative assay, we have identified a new kind of cell adhesion molecule (CAM) on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which we name neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAMs separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously found to be inhibited by Fab' fragments prepared from antisera against neuronal membranes but not by Fab' fragments against N-CAM, the neural cell adhesion molecule. This suggested that neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAMs . To verify that this was the case, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and gamma spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells were also found to inhibit neuronal vesicle binding to glial cells. The inhibitory activity of the Fab' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab' fragment inhibition; after chromatography, neutralizing activity was enriched 50- fold. This fraction was injected into mice to produce monoclonal

  13. Target deletion of complement component 9 attenuates antibody-mediated hemolysis and lipopolysaccharide (LPS)-induced acute shock in mice

    PubMed Central

    Fu, Xiaoyan; Ju, Jiyu; Lin, Zhijuan; Xiao, Weiling; Li, Xiaofang; Zhuang, Baoxiang; Zhang, Tingting; Ma, Xiaojun; Li, Xiangyu; Ma, Chao; Su, Weiliang; Wang, Yuqi; Qin, Xuebin; Liang, Shujuan

    2016-01-01

    Terminal complement membrane attack complex (MAC) formation is induced initially by C5b, followed by the sequential condensation of the C6, C7, C8. Polymerization of C9 to the C5b-8 complex forms the C5b-9 (or MAC). The C5b-9 forms lytic or non lytic pores in the cell membrane destroys membrane integrity. The biological functionalities of MAC has been previously investigated by using either the mice deficient in C5 and C6, or MAC’s regulator CD59. However, there is no available C9 deficient mice (mC9−/−) for directly dissecting the role of C5b-9 in the pathogenesis of human diseases. Further, since C5b-7 and C5b-8 complexes form non lytic pore, it may also plays biological functionality. To better understand the role of terminal complement cascades, here we report a successful generation of mC9−/−. We demonstrated that lack of C9 attenuates anti-erythrocyte antibody-mediated hemolysis or LPS-induced acute shock. Further, the rescuing effect on the acute shock correlates with the less release of IL-1β in mC9−/−, which is associated with suppression of MAC-mediated inflammasome activation in mC9−/−. Taken together, these results not only confirm the critical role of C5b-9 in complement-mediated hemolysis and but also highlight the critical role of C5b-9 in inflammasome activation. PMID:27444648

  14. Cooperativity between CD8+ T cells, non-neutralizing antibodies, and alveolar macrophages is important for heterosubtypic influenza virus immunity.

    PubMed

    Laidlaw, Brian J; Decman, Vilma; Ali, Mohammed-Alkhatim A; Abt, Michael C; Wolf, Amaya I; Monticelli, Laurel A; Mozdzanowska, Krystyna; Angelosanto, Jill M; Artis, David; Erikson, Jan; Wherry, E John

    2013-03-01

    Seasonal epidemics of influenza virus result in ∼36,000 deaths annually in the United States. Current vaccines against influenza virus elicit an antibody response specific for the envelope glycoproteins. However, high mutation rates result in the emergence of new viral serotypes, which elude neutralization by preexisting antibodies. T lymphocytes have been reported to be capable of mediating heterosubtypic protection through recognition of internal, more conserved, influenza virus proteins. Here, we demonstrate using a recombinant influenza virus expressing the LCMV GP33-41 epitope that influenza virus-specific CD8+ T cells and virus-specific non-neutralizing antibodies each are relatively ineffective at conferring heterosubtypic protective immunity alone. However, when combined virus-specific CD8 T cells and non-neutralizing antibodies cooperatively elicit robust protective immunity. This synergistic improvement in protective immunity is dependent, at least in part, on alveolar macrophages and/or other lung phagocytes. Overall, our studies suggest that an influenza vaccine capable of eliciting both CD8+ T cells and antibodies specific for highly conserved influenza proteins may be able to provide heterosubtypic protection in humans, and act as the basis for a potential "universal" vaccine. PMID:23516357

  15. Redox polymer mediation for enzymatic biofuel cells

    NASA Astrophysics Data System (ADS)

    Gallaway, Joshua

    Mediated biocatalytic cathodes prepared from the oxygen-reducing enzyme laccase and redox-conducting osmium hydrogels were characterized for use as cathodes in enzymatic biofuel cells. A series of osmium-based redox polymers was synthesized with redox potentials spanning the range from 0.11 V to 0.85 V (SHE), and the resulting biocatalytic electrodes were modeled to determine reaction kinetic constants using the current response, measured osmium concentration, and measured apparent electron diffusion. As in solution-phase systems, the bimolecular rate constant for mediation was found to vary greatly with mediator potential---from 250 s-1M-1 when mediator and enzyme were close in potential to 9.4 x 10 4 s-1M-1 when this overpotential was large. Optimum mediator potential for a cell operating with a non-limiting platinum anode and having no mass transport limitation from bulk solution was found to be 0.66 V (SHE). Redox polymers were synthesized under different concentrations, producing osmium variation. An increase from 6.6% to 7.2% osmium increased current response from 1.2 to 2.1 mA/cm2 for a planar film in 40°C oxygen-saturated pH 4 buffer, rotating at 900 rpm. These results translated to high surface area electrodes, nearly doubling current density to 13 mA/cm2, the highest to date for such an electrode. The typical fungal laccase from Trametes versicolor was replaced by a bacterially-expressed small laccase from Streptomyces coelicolor, resulting in biocatalytic films that reduced oxygen at increased pH, with full functionality at pH 7, producing 1.5 mA/cm 2 in planar configuration. Current response was biphasic with pH, matching the activity profile of the free enzyme in solution. The mediated enzyme electrode system was modeled with respect to apparent electron diffusion, mediator concentration, and transport of oxygen from bulk solution, all of which are to some extent controlled by design. Each factor was found to limit performance in certain circumstances

  16. Use of erythrocytes sensitized with purified enterotoxin from Vibrio cholerae for the assay of antibody and antibody-forming cells.

    PubMed

    Kateley, J R; Friedman, H

    1975-02-01

    A method was developed for the sensitization of ovine erythrocytes with a purified enterotoxin from Vibrio cholerae. Sensitized cells were used for the titration of serum antibody by passive hemagglutination and in a hemolytic plaque assay for both IgM and IgG antibody-secreting cells. Inhibition experiments with various antigens of V. cholerae indicated that the toxin, whether unheated or heat-inactivated, significantly reduced the expected antitoxic plaque-forming cell response, whereas a lipopolysaccharide-rich extract from homologous vibiros was not inhibitory.

  17. Mediation of immunity to intracellular infection (Toxoplasma and Besnoitia) within somatic cells.

    PubMed Central

    Chinchilla, M; Frenkel, J K

    1978-01-01

    Antigen-treated lymphocytes from immune hamsters specifically protected not only macrophages, but also cultured fibroblasts and kidney cells infected with Toxoplasma gondii or Besnoitia jellisoni. Macrophages were not necessary for the protection of fibroblasts and kidney cells. A mediator that inhibited the intracellular proliferation of these microbes was obtained from immune lymphocytes in contact with specific antigen. Again, macrophages were not necessary for the elaboration of this mediator or its activity in kidney cells or fibroblasts. The mediator was microbe and host specific, had a molecular weight between 4,000 and 5,000, was resistant to heating at 56 degrees C for 30 min, and was sensitive to chymotrypsin, but resistant to ribonuclease and deoxyribonuclease. A single injection of Besnoitia mediator afforded better protection to hamsters infected with Besnoitia than did antibody. Whereas antibody lysed extracellular organisms, the microbe-specific mediators conferred immunity not only on macrophages, but also on other cells of the body, apparently the first such demonstration. Images PMID:640741

  18. Mouse Invariant Monoclonal Antibody NKT14: A Novel Tool to Manipulate iNKT Cell Function In Vivo

    PubMed Central

    Scheuplein, Felix; Lamont, Deanna J.; Poynter, Matthew E.; Boyson, Jonathan E.; Serreze, David; Lundblad, Lennart K. A.; Mashal, Robert; Schaub, Robert

    2015-01-01

    Invariant Natural Killer T (iNKT) cells are a T cell subset expressing an invariant T Cell Receptor (TCR) that recognizes glycolipid antigens rather than peptides. The cells have both innate-like rapid cytokine release, and adaptive-like thymic positive selection. iNKT cell activation has been implicated in the pathogenesis of allergic asthma and inflammatory diseases, while reduced iNKT cell activation promotes infectious disease, cancer and certain autoimmune diseases such as Type 1 diabetes (T1D). Therapeutic means to reduce or deplete iNKT cells could treat inflammatory diseases, while approaches to promote their activation may have potential in certain infectious diseases, cancer or autoimmunity. Thus, we developed invariant TCR-specific monoclonal antibodies to better understand the role of iNKT cells in disease. We report here the first monoclonal antibodies specific for the mouse invariant TCR that by modifying the Fc construct can specifically deplete or activate iNKT cells in vivo in otherwise fully immuno-competent animals. We have used both the depleting and activating version of the antibody in the NOD model of T1D. As demonstrated previously using genetically iNKT cell deficient NOD mice, and in studies of glycolipid antigen activated iNKT cells in standard NOD mice, we found that antibody mediated depletion or activation of iNKT cells respectively accelerated and retarded T1D onset. In BALB/c mice, ovalbumin (OVA) mediated airway hyper-reactivity (AHR) was abrogated with iNKT cell depletion prior to OVA sensitization, confirming studies in knockout mice. Depletion of iNKT cells after sensitization had no effect on AHR in the conducting airways but did reduce AHR in the lung periphery. This result raises caution in the interpretation of studies that use animals that are genetically iNKT cell deficient from birth. These activating and depleting antibodies provide a novel tool to assess the therapeutic potential of iNKT cell manipulation. PMID:26474487

  19. ApoE Receptor 2 mediates trophoblast dysfunction and pregnancy complications induced by antiphospholipid antibodies in mice

    PubMed Central

    Ulrich, Victoria; Gelber, Shari E.; Vukelic, Milena; Sacharidou, Anastasia; Herz, Joachim; Urbanus, Rolf T.; de Groot, Philip G.; Natale, David R.; Harihara, Anirudha; Redecha, Patricia; Abrahams, Vikki M.; Shaul, Philip W.

    2015-01-01

    Objective Pregnancies in women with the antiphospholipid syndrome (APS) are frequently complicated by fetal loss and intrauterine growth restriction (IUGR). How circulating antiphospholipid antibodies (aPL) cause pregnancy complications in APS is poorly understood. We sought to determine if the LDL receptor family member apoE receptor 2 (apoER2) mediates trophoblast dysfunction and pregnancy complications induced by aPL. Methods Placental and trophoblast apoER2 expression was evaluated by immunohistochemistry and immunoblotting. Normal human IgG (NHIgG) and aPL were purified from healthy individuals and APS patients, respectively. The role of apoER2 in aPL-induced changes in trophoblast proliferation, migration and kinase activation was assessed using RNA interference in HTR-8/SVneo cells. The participation of apoER2 in aPL-induced pregnancy loss and IUGR was evaluated in pregnant apoER2+/+ and apoER2−/− mice injected with aPL or NHIgG. Results We found that apoER2 is abundant in human and mouse placental trophoblasts, and in multiple trophoblast-derived cell lines including HTR-8/SVneo cells. ApoER2 and its interaction with the cell surface protein β2-glycoprotein I were required for aPL-induced inhibition of cultured trophoblast proliferation and migration. In parallel, aPL antagonism of Akt kinase activation by EGF in trophoblasts was mediated by apoER2. Furthermore, in a murine passive transfer model of pregnancy complications of APS, apoER2−/− mice were protected from both aPL-induced fetal loss and aPL-induced IUGR. Conclusion ApoER2 plays a major role in the attenuation of trophoblast function by aPL, and the receptor mediates aPL-induced pregnancy complications in vivo in mice. ApoER2-directed interventions can now potentially be developed to combat the pregnancy complications associated with APS. PMID:26474194

  20. Nature of "memory" in T-cell-mediated antibacterial immunity: anamnestic production of mediator T cells.

    PubMed Central

    North, R J

    1975-01-01

    Mice that survived an immunizing infection with Listeria monocytogenes remained specifically resistant to lethal secondary infection for several months. This acquired, long-lived state of resistance was not dependent on activated macrophages that remained after the primary response. It depended, instead, on an acquired long-lived capacity on the part of immunized mice for generating mediator T cells faster and in larger numbers than normal mice. The number of mediator T cells generated in response to secondary infection was proportional to the level of infection. The results suggest that the accelerated production of mediator T cells that occurs in response to secondary infection represents the expression of a state of immunological T-cell memory. PMID:811558

  1. NK Cells and γδ T Cells Mediate Resistance to Polyomavirus–Induced Tumors

    PubMed Central

    Mishra, Rabinarayan; Chen, Alex T.; Welsh, Raymond M.; Szomolanyi-Tsuda, Eva

    2010-01-01

    NK and γδ T cells can eliminate tumor cells in many experimental models, but their effect on the development of tumors caused by virus infections in vivo is not known. Polyomavirus (PyV) induces tumors in neonatally infected mice of susceptible strains and in adult mice with certain immune deficiencies, and CD8+ αβ T cells are regarded as the main effectors in anti-tumor immunity. Here we report that adult TCRβ knockout (KO) mice that lack αβ but have γδ T cells remain tumor-free after PyV infection, whereas TCRβ×δ KO mice that lack all T cells develop tumors. In addition, E26 mice, which lack NK and T cells, develop the tumors earlier than TCRβ×δ KO mice. These observations implicate γδ T and NK cells in the resistance to PyV-induced tumors. Cell lines established from PyV-induced tumors activate NK and γδ T cells both in culture and in vivo and express Rae-1, an NKG2D ligand. Moreover, these PyV tumor cells are killed by NK cells in vitro, and this cytotoxicity is prevented by treatment with NKG2D-blocking antibodies. Our findings demonstrate a protective role for NK and γδ T cells against naturally occurring virus-induced tumors and suggest the involvement of NKG2D-mediated mechanisms. PMID:20523894

  2. Anti-Her-2/neu antibody induces apoptosis in Her-2/neu overexpressing breast cancer cells independently from p53 status

    PubMed Central

    Brodowicz, T; Kandioler, D; Tomek, S; Ludwig, C; Rudas, M; Kunstfeld, R; Koestler, W; Hejna, M; Budinsky, A; Wiltschke, C; Zielinski, C C

    2001-01-01

    Anti-Her-2/neu antibody is known to induce apoptosis in HER-2/neu overexpressing breast cancer cells. However, exact regulatory mechanisms mediating and controlling this phenomenon are still unknown. In the present study, we have investigated the effect of anti-Her-2/neu antibody on apoptosis of HER-2/neu overexpressing human breast cancer cell lines SK-BR-3, HTB-24, HTB-25, HTB-27, HTB-128, HTB-130 and HTB-131 in relation to p53 genotype and bcl-2 status. SK-BR-3, HTB-24, HTB-128 and HTB-130 cells exhibited mutant p53, whereas wild type p53 was found in HTB-25, HTB-27 and HTB-131 cells. All seven cell lines weakly expressed bcl-2 protein (10–20%). Anti-Her-2/neu antibody, irrespective of p53 and bcl-2 status, induced apoptosis in all 7 cell lines dose- and time-dependently and correlated with Her-2/neu overexpression. In addition, incubation of cell lines with anti-Her-2/neu antibody did not alter p53 or bcl-2 expression. Anti-HER-2/neu antibody did not induce apoptosis in HER-2/neu negative HBL-100 and HTB-132 cell lines. Our results indicate that within the panel of tested breast cancer cell lines, anti-Her-2/neu antibody-induced apoptosis was independent from the presence of intact p53. © 2001 Cancer Research Compaign http://www.bjcancer.com PMID:11742500

  3. MicroRNA-mediated somatic cell reprogramming.

    PubMed

    Kuo, Chih-Hao; Ying, Shao-Yao

    2013-02-01

    Since the first report of induced pluripotent stem cells (iPSCs) using somatic cell nuclear transfer (SCNT), much focus has been placed on iPSCs due to their great therapeutic potential for diseases such as abnormal development, degenerative disorders, and even cancers. Subsequently, Takahashi and Yamanaka took a novel approach by using four defined transcription factors to generate iPSCs in mice and human fibroblast cells. Scientists have since been trying to refine or develop better approaches to reprogramming, either by using different combinations of transcription factors or delivery methods. However, recent reports showed that the microRNA expression pattern plays a crucial role in somatic cell reprogramming and ectopic introduction of embryonic stem cell-specific microRNAs revert cells back to an ESC-like state, although, the exact mechanism underlying this effect remains unclear. This review describes recent work that has focused on microRNA-mediated approaches to somatic cell reprogramming as well as some of the pros and cons to these approaches and a possible mechanism of action. Based on the pivotal role of microRNAs in embryogenesis and somatic cell reprogramming, studies in this area must continue in order to gain a better understanding of the role of microRNAs in stem cells regulation and activity. PMID:22961769

  4. Gene therapy for colorectal cancer using adenovirus-mediated full-length antibody, cetuximab

    PubMed Central

    Xing, Man; Wang, Xiang; Chi, Yudan; Zhou, Dongming

    2016-01-01

    Cetuximab is a chimeric monoclonal antibody, approved to treat patients with metastatic colorectal cancer (mCRC), head and neck squamous cell carcinoma (HNSCC), non-small-cell lung cancer (NSCLC) for years. It functions by blocking the epidermal growth factor receptor (EGFR) from receiving signals or interacting with other proteins. Although the demand for cetuximab for the treatment of cancer patients in clinics is increasing, the complicated techniques involved and its high cost limit its wide applications. Here, a new, cheaper form of cetuximab was generated for cancer gene therapy. This was achieved by cloning the full-length cetuximab antibody into two serotypes of adenoviral vectors, termed as AdC68-CTB and Hu5-CTB. In vivo studies showed that a single dose of AdC68-CTB or Hu5-CTB induced sustained cetuximab expression and dramatically suppressed tumor growth in NCI-H508– or DiFi-inoculated nude mice. In conclusion, gene therapy using adenovirus expressing full-length cetuximab could be a novel alternative method for the effective treatment of colorectal cancer. PMID:27058423

  5. A new cell line for high throughput HIV-specific antibody-dependent cellular cytotoxicity (ADCC) and cell-to-cell virus transmission studies

    PubMed Central

    Orlandi, Chiara; Flinko, Robin; Lewis, George K.

    2016-01-01

    Several lines of evidence indicate that antibody-dependent cellular cytotoxicity (Wren et al., 2013) is important in the pathogenesis of HIV-1 infection. Namely, ADCC is induced during natural HIV-1 infection or in HIV-1 vaccine studies, the latter demonstrated by the RV144 vaccine trial. To expedite the assessment of ADCC in studies of HIV, we have developed a high throughput assay. We have optimized the rapid fluorometric antibody-mediated cytotoxicity assay (RFADCC) by transfecting the EGFP-CEM-NKr cell line to constitutively express SNAP-tagged CCR5. This cell line can then serve as a source of HIV-specific targets when coated with monomeric gp120, spinoculated with inactivated intact virions, infected by cell-free viral diffusion or infected by cell-to-cell transmission of virus. The optimized strategy has two significant advantages over the original RFADCC method: First, the preparation of detectable target cells is less labor intensive and faster as it does not rely on multiple staining and washing steps for target cells. Second, because the target cell markers GFP and SNAP are constitutively expressed, the assay provides highly reproducible data. These strengths make the optimized RFADCC assay suitable not only for studies of HIV-1 specific cytotoxicity but also for studies of cell–cell transmission of virus. In conclusion, this assay provides a new generation T cell line that can expedite large clinical studies as well as research studies in humans or non-human primates. PMID:26969387

  6. Encephalitis and antibodies to synaptic and neuronal cell surface proteins

    PubMed Central

    Lancaster, Eric; Martinez-Hernandez, Eugenia

    2011-01-01

    The identification of encephalitis associated with antibodies against cell surface and synaptic proteins, although recent, has already had a substantial impact in clinical neurology and neuroscience. The target antigens are receptors and proteins that have critical roles in synaptic transmission and plasticity, including the NMDA receptor, the AMPA receptor, the GABAB receptor, and the glycine receptor. Other autoantigens, such as leucine-rich glioma-inactivated 1 and contactin-associated protein-like 2, form part of trans-synaptic complexes and neuronal cell adhesion molecules involved in fine-tuning synaptic transmission and nerve excitability. Syndromes resulting from these immune responses resemble those of pharmacologic or genetic models in which the antigens are disrupted. For some immune responses, there is evidence that the antibodies alter the structure and function of the antigen, suggesting a direct pathogenic effect. These disorders are important because they can affect children and young adults, are severe and protracted, occur with or without tumor association, and respond to treatment but may relapse. This review provides an update on these syndromes and autoantigens with special emphasis on clinical diagnosis and treatment. PMID:21747075

  7. Heme Oxygenase-1 Inhibits HLA Class I Antibody-Dependent Endothelial Cell Activation

    PubMed Central

    Vijayan, Vijith; Hiller, Oliver; Figueiredo, Constanca; Aljabri, Abid; Blasczyk, Rainer; Theilmeier, Gregor; Becker, Jan Ulrich; Larmann, Jan; Immenschuh, Stephan

    2015-01-01

    Antibody-mediated rejection (AMR) is a key limiting factor for long-term graft survival in solid organ transplantation. Human leukocyte antigen (HLA) class I (HLA I) antibodies (Abs) play a major role in the pathogenesis of AMR via their interactions with HLA molecules on vascular endothelial cells (ECs). The antioxidant enzyme heme oxygenase (HO)-1 has anti-inflammatory functions in the endothelium. As complement-independent effects of HLA I Abs can activate ECs, it was the goal of the current study to investigate the role of HO-1 on activation of human ECs by HLA I Abs. In cell cultures of various primary human macro- and microvascular ECs treatment with monoclonal pan- and allele-specific HLA I Abs up-regulated the expression of inducible proinflammatory adhesion molecules and chemokines (vascular cell adhesion molecule-1 [VCAM-1], intercellular cell adhesion molecule-1 [ICAM-1], interleukin-8 [IL-8] and monocyte chemotactic protein 1 [MCP-1]). Pharmacological induction of HO-1 with cobalt-protoporphyrin IX reduced, whereas inhibition of HO-1 with either zinc-protoporphyrin IX or siRNA-mediated knockdown increased HLA I Ab-dependent up-regulation of VCAM-1. Treatment with two carbon monoxide (CO)-releasing molecules, which liberate the gaseous HO product CO, blocked HLA I Ab-dependent EC activation. Finally, in an in vitro adhesion assay exposure of ECs to HLA I Abs led to increased monocyte binding, which was counteracted by up-regulation of HO-1. In conclusion, HLA I Ab-dependent EC activation is modulated by endothelial HO-1 and targeted induction of this enzyme may be a novel therapeutic approach for the treatment of AMR in solid organ transplantation. PMID:26690352

  8. Clearance of Human IgG1-Sensitised Red Blood Cells In Vivo in Humans Relates to the In Vitro Properties of Antibodies from Alternative Cell Lines

    PubMed Central

    Armour, Kathryn L.; Smith, Cheryl S.; Ip, Natasha C. Y.; Ellison, Cara J.; Kirton, Christopher M.; Wilkes, Anthony M.; Williamson, Lorna M.; Clark, Michael R.

    2014-01-01

    We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance. PMID:25302805

  9. Automation of cross-matching and red cell antibody screening.

    PubMed

    Wattar, B; Lambermont, M; Govaerts, A

    1982-01-01

    This automatic system combines the major cross-match with screening for allo- and autoantibodies. Moreover, the detected antibodies can be identified on a panel of frozen and thawed red blood cells (RBC). The system is made up of two connected samplers, three channels working, respectively, with bromelin PVP, LISP and saline PVP at 4 degrees C, three colorimeters or three red cell autocounters and their recorders. The optimal speed is 50 samples/h and one whole test requires 19 min. Our experience indicates that this automatic system is appreciably more sensitive and much more rapid and efficient than manual techniques. In spite of increased sensitivity, the ratio of rejected bags does not exceed 2.7%. PMID:7090333

  10. Lysis of human tumor cell lines by canine complement plus monoclonal antiganglioside antibodies or natural canine xenoantibodies.

    PubMed

    Helfand, S C; Hank, J A; Gan, J; Sondel, P M

    1996-01-10

    Because certain antiganglioside monoclonal antibodies can facilitate antibody-dependent cellular cytotoxicity against GD2+ ganglioside-bearing human and canine tumor cells, we wished to determine if clinically relevant antiganglioside monoclonal antibodies (Mabs) could also fix canine complement to lyse tumor cells in vitro. Using flow cytometry, human tumor cell lines (M21 melanoma and OHS osteosarcoma) were shown to highly express ganglioside GD2 and, to a lesser degree, GD3. In 51Cr release assays, M21 cells were lysed with canine serum, as a source of complement, plus either Mab 14.G2a or its mouse-human chimera, ch 14.18, specific for GD2. Heating canine serum abrogated its lytic activity and addition of rabbit complement reconstituted M21 lysis. Similar results were obtained with M21 cells when Mab R24 (against GD3) and canine serum were used. OHS cells were also lysed with canine serum plus Mab 14.G2a and lytic activity was abolished by heating canine serum but reconstituted with rabbit complement. Alone, canine serum or Mabs were not lytic to M21 or OHS cells. Conversely, human neuroblastoma (LAN-5) and K562 erythroleukemia cells were lysed by canine serum alone which was shown by flow cytometry to contain naturally occurring canine IgM antibodies that bound LAN-5 and K562 cells. The lytic activity of canine serum for LAN-5 or K562 cells was abolished by heating and restored by addition of either human or rabbit complement. Thus, human tumor cell lines can be lysed with antiganglioside Mabs through fixation and activation of canine complement-dependent lytic pathways. Canine xenoantibodies also mediate complement-dependent cytotoxicity of some human tumor cell lines. Together, these results are significant because they demonstrate an antitumor effect of the canine immune system which is of potential importance for cancer immunotherapy in a promising animal model.

  11. Antibody-dependent enhancement of dengue virus infection inhibits RLR-mediated Type-I IFN-independent signalling through upregulation of cellular autophagy.

    PubMed

    Huang, Xinwei; Yue, Yaofei; Li, Duo; Zhao, Yujiao; Qiu, Lijuan; Chen, Junying; Pan, Yue; Xi, Juemin; Wang, Xiaodan; Sun, Qiangming; Li, Qihan

    2016-01-01

    Antibody dependent enhancement (ADE) of dengue virus (DENV) infection is identified as the main risk factor of severe Dengue diseases. Through opsonization by subneutralizing or non-neutralizing antibodies, DENV infection suppresses innate cell immunity to facilitate viral replication. However, it is largely unknown whether suppression of type-I IFN is necessary for a successful ADE infection. Here, we report that both DENV and DENV-ADE infection induce an early ISG (NOS2) expression through RLR-MAVS signalling axis independent of the IFNs signaling. Besides, DENV-ADE suppress this early antiviral response through increased autophagy formation rather than induction of IL-10 secretion. The early induced autophagic proteins ATG5-ATG12 participate in suppression of MAVS mediated ISGs induction. Our findings suggest a mechanism for DENV to evade the early antiviral response before IFN signalling activation. Altogether, these results add knowledge about the complexity of ADE infection and contribute further to research on therapeutic strategies. PMID:26923481

  12. Response to abdominal hysterectomy with bilateral salpingo-oophorectomy in postmenopausal woman with anti-yo antibody mediated paraneoplastic cerebellar degeneration.

    PubMed

    Bhargava, Amita; Bhushan, Bharat; Kasundra, Gaurav M; Shubhakaran, Khichar; Pujar, Guruprasad S; Banakar, Basavaraj

    2014-07-01

    Paraneoplastic cerebellar degeneration (PCD) is a rare neurological disorder characterized by a widespread loss of Purkinje cells associated with a progressive pancerebellar dysfunction. PCD often precedes the cancer diagnosis by months to years. Here, we report a case of 44-year old postmenopausal woman who presented with PCD symptoms and high levels of anti-Yo antibodies titer since 8 months. We failed to conclude any neoplastic focus after thorough laboratory and imaging study. She minimally responded to methylprednisolone and immunoglobulin therapies. Despite therapy she was severely disabled. Planned abdominal hysterectomy with bilateral salpingo-oophorectomy (AHBSO) was done, histology revealed grade IIA borderline serous papillary carcinoma of ovary. Her neurological deficit responded dramatically to AHBSO. It is first case report who emphasize the response of AHBSO with presentation of anti-Yo antibody-mediated PCD and hidden nidus in post menopausal women.

  13. Antibody-dependent enhancement of dengue virus infection inhibits RLR-mediated Type-I IFN-independent signalling through upregulation of cellular autophagy

    PubMed Central

    Huang, Xinwei; Yue, Yaofei; Li, Duo; Zhao, Yujiao; Qiu, Lijuan; Chen, Junying; Pan, Yue; Xi, Juemin; Wang, Xiaodan; Sun, Qiangming; Li, Qihan

    2016-01-01

    Antibody dependent enhancement (ADE) of dengue virus (DENV) infection is identified as the main risk factor of severe Dengue diseases. Through opsonization by subneutralizing or non-neutralizing antibodies, DENV infection suppresses innate cell immunity to facilitate viral replication. However, it is largely unknown whether suppression of type-I IFN is necessary for a successful ADE infection. Here, we report that both DENV and DENV-ADE infection induce an early ISG (NOS2) expression through RLR-MAVS signalling axis independent of the IFNs signaling. Besides, DENV-ADE suppress this early antiviral response through increased autophagy formation rather than induction of IL-10 secretion. The early induced autophagic proteins ATG5-ATG12 participate in suppression of MAVS mediated ISGs induction. Our findings suggest a mechanism for DENV to evade the early antiviral response before IFN signalling activation. Altogether, these results add knowledge about the complexity of ADE infection and contribute further to research on therapeutic strategies. PMID:26923481

  14. T cell-mediated paraneoplastic ganglionitis--an autopsy case.

    PubMed

    Drlicek, M; Bodenteich, A; Setinek, U; Tucek, G; Urbanits, S; Grisold, W

    2000-05-01

    A 57-year-old woman presented with subacute sensory, ataxic neuronopathy. Clinical investigation revealed a right-sided non-small-cell lung cancer. Serum investigation for specific antineuronal antibodies was negative. Histology showed T lymphocytic infiltrates in dorsal root ganglia. The observed histological pattern is similar to that described in antibody-positive cases. Thus, these findings suggest similar pathways in specific antineuronal antibody-negative and -positive cases of paraneoplastic subacute sensory neuronopathy.

  15. CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer.

    PubMed

    Weiskopf, Kipp; Jahchan, Nadine S; Schnorr, Peter J; Cristea, Sandra; Ring, Aaron M; Maute, Roy L; Volkmer, Anne K; Volkmer, Jens-Peter; Liu, Jie; Lim, Jing Shan; Yang, Dian; Seitz, Garrett; Nguyen, Thuyen; Wu, Di; Jude, Kevin; Guerston, Heather; Barkal, Amira; Trapani, Francesca; George, Julie; Poirier, John T; Gardner, Eric E; Miles, Linde A; de Stanchina, Elisa; Lofgren, Shane M; Vogel, Hannes; Winslow, Monte M; Dive, Caroline; Thomas, Roman K; Rudin, Charles M; van de Rijn, Matt; Majeti, Ravindra; Garcia, K Christopher; Weissman, Irving L; Sage, Julien

    2016-07-01

    Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers.

  16. CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer

    PubMed Central

    Weiskopf, Kipp; Jahchan, Nadine S.; Schnorr, Peter J.; Ring, Aaron M.; Maute, Roy L.; Volkmer, Anne K.; Volkmer, Jens-Peter; Liu, Jie; Lim, Jing Shan; Yang, Dian; Seitz, Garrett; Nguyen, Thuyen; Wu, Di; Guerston, Heather; Trapani, Francesca; George, Julie; Poirier, John T.; Gardner, Eric E.; Miles, Linde A.; de Stanchina, Elisa; Lofgren, Shane M.; Vogel, Hannes; Winslow, Monte M.; Dive, Caroline; Thomas, Roman K.; Rudin, Charles M.; van de Rijn, Matt; Majeti, Ravindra; Garcia, K. Christopher; Weissman, Irving L.

    2016-01-01

    Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers. PMID:27294525

  17. Conserved natural IgM antibodies mediate innate and adaptive immunity against the opportunistic fungus Pneumocystis murina.

    PubMed

    Rapaka, Rekha R; Ricks, David M; Alcorn, John F; Chen, Kong; Khader, Shabaana A; Zheng, Mingquan; Plevy, Scott; Bengtén, Eva; Kolls, Jay K

    2010-12-20

    Host defense against opportunistic fungi requires coordination between innate and adaptive immunity for resolution of infection. Antibodies generated in mice vaccinated with the fungus Pneumocystis prevent growth of Pneumocystis organisms within the lungs, but the mechanisms whereby antibodies enhance antifungal host defense are poorly defined. Nearly all species of fungi contain the conserved carbohydrates β-glucan and chitin within their cell walls, which may be targets of innate and adaptive immunity. In this study, we show that natural IgM antibodies targeting these fungal cell wall carbohydrates are conserved across many species, including fish and mammals. Natural antibodies bind fungal organisms and enhance host defense against Pneumocystis in early stages of infection. IgM antibodies influence recognition of fungal antigen by dendritic cells, increasing their migration to draining pulmonary lymph nodes. IgM antibodies are required for adaptive T helper type 2 (Th2) and Th17 cell differentiation and guide B cell isotype class-switch recombination during host defense against Pneumocystis. These experiments suggest a novel role for the IgM isotype in shaping the earliest steps in recognition and clearance of this fungus. We outline a mechanism whereby serum IgM, containing ancient specificities against conserved fungal antigens, bridges innate and adaptive immunity against fungal organisms.

  18. Dengue vascular leakage is augmented by mast cell degranulation mediated by immunoglobulin Fcγ receptors

    PubMed Central

    Syenina, Ayesa; Jagaraj, Cyril J; Aman, Siti AB; Sridharan, Aishwarya; St John, Ashley L

    2015-01-01

    Dengue virus (DENV) is the most significant human arboviral pathogen and causes ∼400 million infections in humans each year. In previous work, we observed that mast cells (MC) mediate vascular leakage during DENV infection in mice and that levels of MC activation are correlated with disease severity in human DENV patients (St John et al., 2013b). A major risk factor for developing severe dengue is secondary infection with a heterologous serotype. The dominant theory explaining increased severity during secondary DENV infection is that cross-reactive but non-neutralizing antibodies promote uptake of virus and allow enhanced replication. Here, we define another mechanism, dependent on FcγR-mediated enhanced degranulation responses by MCs. Antibody-dependent mast cell activation constitutes a novel mechanism to explain enhanced vascular leakage during secondary DENV infection. DOI: http://dx.doi.org/10.7554/eLife.05291.001 PMID:25783751

  19. Dengue vascular leakage is augmented by mast cell degranulation mediated by immunoglobulin Fcγ receptors.

    PubMed

    Syenina, Ayesa; Jagaraj, Cyril J; Aman, Siti A B; Sridharan, Aishwarya; St John, Ashley L

    2015-01-01

    Dengue virus (DENV) is the most significant human arboviral pathogen and causes ∼400 million infections in humans each year. In previous work, we observed that mast cells (MC) mediate vascular leakage during DENV infection in mice and that levels of MC activation are correlated with disease severity in human DENV patients (St John et al., 2013b). A major risk factor for developing severe dengue is secondary infection with a heterologous serotype. The dominant theory explaining increased severity during secondary DENV infection is that cross-reactive but non-neutralizing antibodies promote uptake of virus and allow enhanced replication. Here, we define another mechanism, dependent on FcγR-mediated enhanced degranulation responses by MCs. Antibody-dependent mast cell activation constitutes a novel mechanism to explain enhanced vascular leakage during secondary DENV infection.

  20. Inhibitory effects of mast cell-mediated allergic reactions by cell cultured Siberian Ginseng.

    PubMed

    Jeong, H J; Koo, H N; Myung, N I; Shin, M K; Kim, J W; Kim, D K; Kim, K S; Kim, H M; Lee, Y M

    2001-02-01

    The crude drug "Siberian Ginseng (SG)" has long been used in empirical Oriental medicine for the nonspecific enhancement of resistance in humans and animals. In this study, we investigated the effect of cell cultured SG by oral administration in