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Sample records for humanized anti-her2 monoclonal

  1. Novel anti-HER2 monoclonal antibodies: synergy and antagonism with tumor necrosis factor-α

    PubMed Central

    2012-01-01

    Background One-third of breast cancers display amplifications of the ERBB2 gene encoding the HER2 kinase receptor. Trastuzumab, a humanized antibody directed against an epitope on subdomain IV of the extracellular domain of HER2 is used for therapy of HER2-overexpressing mammary tumors. However, many tumors are either natively resistant or acquire resistance against Trastuzumab. Antibodies directed to different epitopes on the extracellular domain of HER2 are promising candidates for replacement or combinatorial therapy. For example, Pertuzumab that binds to subdomain II of HER2 extracellular domain and inhibits receptor dimerization is under clinical trial. Alternative antibodies directed to novel HER2 epitopes may serve as additional tools for breast cancer therapy. Our aim was to generate novel anti-HER2 monoclonal antibodies inhibiting the growth of breast cancer cells, either alone or in combination with tumor necrosis factor-α (TNF-α). Methods Mice were immunized against SK-BR-3 cells and recombinant HER2 extracellular domain protein to produce monoclonal antibodies. Anti-HER2 antibodies were characterized with breast cancer cell lines using immunofluorescence, flow cytometry, immunoprecipitation, western blot techniques. Antibody epitopes were localized using plasmids encoding recombinant HER2 protein variants. Antibodies, either alone or in combination with TNF-α, were tested for their effects on breast cancer cell proliferation. Results We produced five new anti-HER2 monoclonal antibodies, all directed against conformational epitope or epitopes restricted to the native form of the extracellular domain. When tested alone, some antibodies inhibited modestly but significantly the growth of SK-BR-3, BT-474 and MDA-MB-361 cells displaying ERBB2 amplification. They had no detectable effect on MCF-7 and T47D cells lacking ERBB2 amplification. When tested in combination with TNF-α, antibodies acted synergistically on SK-BR-3 cells, but antagonistically on BT

  2. Labeling anti-HER2/neu monoclonal antibodies with 111In and 90Y using a bifunctional DTPA chelating agent.

    PubMed

    Blend, Michael J; Stastny, Jerry J; Swanson, Steven M; Brechbiel, Martin W

    2003-06-01

    The goal of this investigation was to develop stable radioimmunoconjugates (RICs) of anti-HER2/neu monoclonal antibodies (MoAbs) for imaging and therapy in an animal model bearing human breast tumor xenografts that express normal (MCF-7 cells) and increased amounts of HER2/neu receptors (HCC-1954, BT-474, SKBR-3 cells) on their cell surface membranes. Pharmacy-grade Herceptin, a murine anti-HER2/neu MoAb, and nonspecific mouse IgG protein were conjugated with the recently developed DTPA linker known as CHX-A"-DTPA. These immunoconjugates were labeled with (111)InCl(3) and (90)YCl(3). Using a molar excess of 10:1 CHX-A"-DTPA to immunoglobulin, average specific activities of 1.87 microCi (111)In/microg RIC and 2.71 microCi (90)Y/microg RIC were obtained. The purity of RICs was 96%+ for (111)In and 99%+ for (90)Y. Stability in human plasma at 37 degrees C for both RICs ranged from 98% at 24 h to 85% at 96 h. Binding capacity of the RICs was tested with human cancer cell lines MCF-7, HCC-1954, BT-474, and SKBR-3. Using (111)In-labeled nonspecific IgG protein as a control, (111)In-Herceptin RIC was found to bind to MCF-7 cells with a ratio of 2.5:1 and to SKBR-3 cells with a ratio of 85:1 after 3 h of incubation. (111)In anti-HER2/neu RIC bound to MCF-7 cells with a ratio of 6:1 and to SKBR-3 cells with a ratio of 115:1 after 3 h of incubation. (90)Y-anti-HER2/neu RIC bound 10-times greater to BT-474 cells than to MCF-7 cells. Thus, these MoAbs can be labeled with (111)In and (90)Y using the CHX-A"-DTPA linker. The resulting RICs ((111)In- and (90)Y-anti HER2/neu antibodies) are stable and bind significantly to HER2 overexpressing tumor cell lines.

  3. H2Mab-77 is a Sensitive and Specific Anti-HER2 Monoclonal Antibody Against Breast Cancer.

    PubMed

    Itai, Shunsuke; Fujii, Yuki; Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Chang, Yao-Wen; Handa, Saori; Takahashi, Maki; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

    2017-08-01

    Human epidermal growth factor receptor 2 (HER2) plays a critical role in the progression of breast cancers, and HER2 overexpression is associated with poor clinical outcomes. Trastuzumab is an anti-HER2 humanized antibody that leads to significant survival benefits in patients with HER2-positive metastatic breast cancers. In this study, we developed novel anti-HER2 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. Initially, we expressed the full length or ectodomain of HER2 in LN229 glioblastoma cells and then immunized mice with ectodomain of HER2 or LN229/HER2, and performed the first screening by enzyme-linked immunosorbent assays using ectodomain of HER2. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). Among 100 mAb clones, only three mAbs reacted with HER2 in Western blot, and clone H2Mab-77 (IgG1, kappa) was selected. Finally, immunohistochemical analyses with H2Mab-77 showed sensitive and specific reactions against breast cancer cells, warranting the use of H2Mab-77 to detect HER2 in pathological analyses of breast cancers.

  4. Development and Characterization of a Humanized Anti-HER2 Antibody HuA21 with Potent Anti-Tumor Properties in Breast Cancer Cells.

    PubMed

    Li, Ruilin; Hu, Siyi; Chang, Yan; Zhang, Zhihui; Zha, Zhao; Huang, Hui; Shen, Guodong; Liu, Jing; Song, Lihua; Wei, Wei

    2016-04-15

    Human epidermal growth factor receptor 2 (HER2) is one of the most studied tumor-associated antigens for cancer immunotherapy. An engineered anti-HER-2 chimeric A21 antibody (chA21) is a chimeric antibody targeted to subdomain I of the HER2 extracellular domain. Here, we report the anti-tumor activity of the novel engineered monoclonal antibody humanized chA21 (HuA21) that targets HER2 on the basis of chA21, and we describe the underlying mechanisms. Our results reveal that HuA21 markedly inhibits the proliferation and migration of HER2-overexpressing breast cancer cells and causes enhanced antibody-dependent cell-mediated cytotoxicity potency against HER2-overexpressing tumor cells. In particular, HuA21, but not trastuzumab (Tra), markedly suppresses growth and enhances the internalization of the antibody in Tra-resistant BT-474 breast cancer cells. These characteristics are highly associated with the intrinsic ability of HuA21 to down-regulate HER2 activation and inhibit the extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (Akt) signaling pathways. Furthermore, the combination of HuA21 with Tra synergistically enhances the anti-tumor effects in vitro and in vivo and inhibits HER2 activation and the ERK1/2 and Akt signaling pathways. Altogether, our results suggest that HuA21 may represent a unique anti-HER2 antibody with potential as a therapeutic candidate alone or in combination with other anti-HER2 reagents in cancer therapy.

  5. Development and Characterization of a Humanized Anti-HER2 Antibody HuA21 with Potent Anti-Tumor Properties in Breast Cancer Cells

    PubMed Central

    Li, Ruilin; Hu, Siyi; Chang, Yan; Zhang, Zhihui; Zha, Zhao; Huang, Hui; Shen, Guodong; Liu, Jing; Song, Lihua; Wei, Wei

    2016-01-01

    Human epidermal growth factor receptor 2 (HER2) is one of the most studied tumor-associated antigens for cancer immunotherapy. An engineered anti-HER-2 chimeric A21 antibody (chA21) is a chimeric antibody targeted to subdomain I of the HER2 extracellular domain. Here, we report the anti-tumor activity of the novel engineered monoclonal antibody humanized chA21 (HuA21) that targets HER2 on the basis of chA21, and we describe the underlying mechanisms. Our results reveal that HuA21 markedly inhibits the proliferation and migration of HER2-overexpressing breast cancer cells and causes enhanced antibody-dependent cell-mediated cytotoxicity potency against HER2-overexpressing tumor cells. In particular, HuA21, but not trastuzumab (Tra), markedly suppresses growth and enhances the internalization of the antibody in Tra-resistant BT-474 breast cancer cells. These characteristics are highly associated with the intrinsic ability of HuA21 to down-regulate HER2 activation and inhibit the extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (Akt) signaling pathways. Furthermore, the combination of HuA21 with Tra synergistically enhances the anti-tumor effects in vitro and in vivo and inhibits HER2 activation and the ERK1/2 and Akt signaling pathways. Altogether, our results suggest that HuA21 may represent a unique anti-HER2 antibody with potential as a therapeutic candidate alone or in combination with other anti-HER2 reagents in cancer therapy. PMID:27092488

  6. An anti-HER2 antibody conjugated with monomethyl auristatin E is highly effective in HER2-positive human gastric cancer

    PubMed Central

    Li, Hongwen; Yu, Chao; Jiang, Jing; Huang, Changjiang; Yao, Xuejing; Xu, Qiaoyu; Yu, Fang; Lou, Liguang; Fang, Jianmin

    2016-01-01

    ABSTRACT Antibody-drug conjugate (ADC) is a novel class of therapeutics for cancer target therapy. This study assessed antitumor activity of ADC with an antimitotic agent, monomethyl auristatin E (MMAE) and a humanized monoclonal anti-HER2 antibody, hertuzumab, in gastric cancer. The efficacy of hertuzumab-MC-Val-Cit-PAB-MMAE (hertuzumab-vcMMAE) on human epidermal growth factor receptor 2 (HER2) positive human gastric cancer cells, NCI-N87, was evaluated in vitro and in vivo. The cytotoxicity of hertuzumab was significantly enhanced after conjugation with MMAE. Compared to trastuzumab, hertuzumab had a higher affinity to HER2 and had more potent antibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitro. After conjugation with MMAE, the binding specificity for HER2 was not affected. Furthermore, the internalization of hertuzumab-vcMMAE in HER2 positive gastric cancer cells was verified. Although the conjugation of hertuzumab and MMAE decreased the ADCC effect, the overall cytotoxicity was dramatically increased in HER2 positive gastric cancer cells. In vitro data on this hertuzumab-vcMMAE has exerted much stronger antitumor activity compared to trastuzumab-DM1 in HER2 positive gastric cancer cells. A single administration of hertuzumab-vcMMAE at 5 or 10 mg/kg showed high potency and a sustained tumor inhibitory effect on NCI-N87 xenografts in mice. In conclusion, hertuzumab-vcMMAE conjugate is a highly effective anti-HER2 targeted therapy for HER2-positive gastric cancer. PMID:26853765

  7. 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.

  8. Tumor targeting using anti-her2 immunoliposomes.

    PubMed

    Park, J W; Kirpotin, D B; Hong, K; Shalaby, R; Shao, Y; Nielsen, U B; Marks, J D; Papahadjopoulos, D; Benz, C C

    2001-07-06

    We have generated anti-HER2 (ErbB2) immunoliposomes (ILs), consisting of long circulating liposomes linked to anti-HER2 monoclonal antibody (MAb) fragments, to provide targeted drug delivery to HER2-overexpressing cells. Immunoliposomes were constructed using a modular strategy in which components were optimized for internalization and intracellular drug delivery. Parameters included choice of antibody construct, antibody density, antibody conjugation procedure, and choice of liposome construct. Anti-HER2 immunoliposomes bound efficiently to and internalized in HER2-overexpressing cells in vitro as determined by fluorescence microscopy, electron microscopy, and quantitative analysis of fluorescent probe delivery. Delivery via ILs in HER2-overexpressing cells yielded drug uptake that was up to 700-fold greater than with non-targeted sterically stabilized liposomes. In vivo, anti-HER2 ILs showed extremely long circulation as stable constructs in normal adult rats after a single i.v. dose, with pharmacokinetics that were indistinguishable from sterically stabilized liposomes. Repeat administrations revealed no increase in clearance, further confirming that ILs retain the long circulation and non-immunogenicity of sterically stabilized liposomes. In five different HER2-overexpressing xenograft models, anti-HER2 ILs loaded with doxorubicin (dox) showed potent anticancer activity, including tumor inhibition, regressions, and cures (pathologic complete responses). ILs were significantly superior vs. all other treatment conditions tested: free dox, liposomal dox, free MAb (trastuzumab), and combinations of dox+MAb or liposomal dox+MAb. For example, ILs produced significantly superior antitumor effects vs. non-targeted liposomes (P values from <0.0001 to 0.04 in eight separate experiments). In a non-HER2-overexpressing xenograft model (MCF7), ILs and non-targeted liposomal dox produced equivalent antitumor effects. Detailed studies of tumor localization indicated a novel

  9. A Conjugate Based on Anti-HER2 Diaffibody and Auristatin E Targets HER2-Positive Cancer Cells

    PubMed Central

    Serwotka-Suszczak, Anna M.; Sochaj-Gregorczyk, Alicja M.; Pieczykolan, Jerzy; Krowarsch, Daniel; Jelen, Filip; Otlewski, Jacek

    2017-01-01

    Antibody-drug conjugates (ADCs) have recently emerged as efficient and selective cancer treatment therapeutics. Currently, alternative forms of drug carriers that can replace monoclonal antibodies are under intensive investigation. Here, a cytotoxic conjugate of an anti-HER2 (Human Epidermal Growth Factor Receptor 2) diaffibody with monomethyl-auristatin E (MMAE) is proposed as a potential anticancer therapeutic. The anti-HER2 diaffibody was based on the ZHER2:4 affibody amino acid sequence. The anti-HER2 diaffibody has been expressed as a His-tagged protein in E. coli and purified by Ni-nitrilotriacetyl (Ni-NTA) agarose chromatography. The molecule was properly folded, and the high affinity and specificity of its interaction with HER2 was confirmed by surface plasmon resonance (SPR) and flow cytometry, respectively. The (ZHER2:4)2DCS-MMAE conjugate was obtained by coupling the maleimide group linked with MMAE to cysteines, which were introduced in a drug conjugation sequence (DCS). Cytotoxicity of the conjugate was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide MTT assay and the xCELLigence Real-Time Cell Analyzer. Our experiments demonstrated that the conjugate delivered auristatin E specifically to HER2-positive tumor cells, which finally led to their death. These results indicate that the cytotoxic diaffibody conjugate is a highly potent molecule for the treatment of various types of cancer overexpressing HER2 receptors. PMID:28216573

  10. Anti-HER2 cancer therapy and cardiotoxicity.

    PubMed

    Babar, Tania; Blomberg, Christopher; Hoffner, Eileen; Yan, Xinhua

    2014-01-01

    A significant milestone in the treatment of breast cancer is the identification of the HER2 receptor as a drug target for cancer therapies. Trastuzumab (Herceptin), a monoclonal antibody that blocks the HER2 receptor, is among the first of such drugs approved by the US Food and Drug Administration for targeted cancer therapy. Clinical studies have shown that Trastuzumab significantly improves the overall survival of breast cancer patients. However, an unforeseen significant side-effect of cardiotoxicity manifested as left ventricular dysfunction and heart failure. Concurrent studies have demonstrated the essential role of the HER2 receptor in cardiac development and maintaining the physiological function of an adult heart. The HER2 receptor, therefore, has become a critical link between the oncology and cardiology fields. In addition to Trastuzumab, new drugs targeting the HER2 receptor, such as Lapatinib, Pertuzumab and Afatinib, are either approved or being evaluated in clinical trials for cancer therapy. With the concern of cardiotoxicity caused by HER2 inhibition, it becomes clear that new therapeutic strategies for preventing such cardiac side effects need to be developed. It is the intent of this paper to review the potential cardiac impact of anti-HER2 cancer therapy.

  11. Current therapeutic strategies of anti-HER2 treatment in advanced breast cancer patients

    PubMed Central

    Nowara, Elżbieta

    2016-01-01

    The HER2/neu (ERBB2) oncogene is amplified and/or overexpressed in approximately 20% of breast cancers, and is a strong prognostic factor for relapse and poor overall survival, particularly in node-positive patients. It is also an important predictor for response to trastuzumab, which has established efficacy against breast cancer with overexpression or amplification of the HER2 oncogene. Treatment with the anti-HER2 humanized monoclonal antibody – trastuzumab significantly improves progression-free and overall survival among patients with HER2-positive breast cancer. However, in most patients with HER2-positive metastatic breast cancer, the disease progresses occurred, what cause the need for new targeted therapies for advanced disease. In clinical trials, there are tested new drugs to improve the results of treatment for this group of patients. This paper presents new drugs introduced into clinical practice for treatment of advanced breast cancer, whose molecular target are receptors of the HER2 family. In addition, new therapeutic strategies and drugs that are currently in clinical researches are discussed. PMID:27095932

  12. [Targeted detecting HER2 expression with recombinant anti HER2 ScFv-GFP fusion antibody].

    PubMed

    Gao, Guohui; Chen, Chong; Yang, Yanmei; Yang, Han; Wang, Jindan; Zheng, Yi; Huang, Qidi; Hu, Xiaoqu

    2012-08-01

    To verify the reliability of targeted detecting HER2 positive cancer cells and clinical pathological tissue specimens with a recombinant anti HER2 single chain antibody in single chain Fv fragment (scFv) format, we have constructed the fusion variable regions of the ScFv specific for HER2/neu. labeled a green-fluorescent protein(GFP). The humanized recombinant Anti HER2 ScFv-GFP gene was inserted into pFast Bac HT A, and expressed in insect cells sf9. Then the recombinant fusion protein Anti HER2 ScFv-GFP was properly purified with Ni2+-NTA affinity chromatography from the infected sf9 cells used to test the specificity of the fusion antibody for HER2 positive cancer cells. Firstly, the purified antibody incubated with HER2 positive breast cancer cells SKBR3, BT474 and HER2 negative breast cancer cells MCF7 for 12 h/24 h/48 h at 37 degrees C, in order to confirm targeted detecting HER2 positive breast cancer cells by Laser Confocal Microscopy. Furthermore, the same clinical pathological tissue samples were assessed by immunohistochemistry (IHC) and the fusion antibody Anti HER2 ScFv-GFP in the meanwhile. The data obtained indicated that the recombinant eukaryotic expression plasmid pFast Bac HT A/Anti HER2 ScFv-GFP was constructed successfully In addition, obvious green fluorescent was observed in insect cells sf9. When the purified fusion antibody was incubated with different cancer cells, much more green fluorescent was observed on the surface of the HER2 positive cancer cells SKBR3 and BT474. In contrast, no green fluorescent on the surface of the HER2 negative cancer cells MCF7 was detected. The concentration of the purified fusion antibody was 115.5 microg/mL, of which protein relative molecular weight was 60 kDa. The analysis showed the purity was about 97% and the titer was about 1:64. The detection results of IHC and fusion antibody testing indicated the conformity. In summary, the study showed that the new fusion antibody Anti HER2 ScFv-GFP can test HER2

  13. [Anti-HER2 vaccines: The HER2 immunotargeting future?].

    PubMed

    Ladjemi, M Z; Jacot, W; Pèlegrin, A; Navarro-Teulon, I

    2011-06-01

    Breast cancer is a widely spread women's disease. In spite of progress in the field of surgery and adjuvant therapies, the risk of breast cancer metastatic relapses remains high especially in those overexpressing HER2. Different studies have shown cellular and/or humoral immune responses against HER2 in patients with HER2-overexpressing tumors. This immune response is associated with a lower tumor development at early stages of the disease. These observations, associated with the efficiency today demonstrated by a trastuzumab-based anti-HER2 immunotherapy, allowed to envisage various vaccinal strategies against HER2. These findings have so led to the hypothesis that the generation of an anti-HER2 immune response should protect patients from HER2-overexpressing tumor growth, and induction of a stable and strong immunity by cancer vaccines is expected to lead to establishment of immune memory, thereby preventing tumor recurrence. However, an immunological tolerance against HER2 antigen exists representing a barrier to effective vaccination against this oncoprotein. As a consequence, the current challenge for vaccines is to find the best conditions to break this immunological tolerance. In this review, we will discuss the different anti-HER2 vaccine strategies currently developed; considering the strategies having reached the clinical phases as well as those still in preclinical development. The used antigen can be composed of tumoral allogenic cells or autologous cells or be specific of HER2. It can be delivered by denditric cells or in a DNA, peptidic or proteic form. Another area of the research concerns the use of anti-idiotypic antibodies mimicking HER2.

  14. Effective treatment of established human breast tumor xenografts in immunodeficient mice with a single dose of the alpha-emitting radioisotope astatine-211 conjugated to anti-HER2/neu diabodies.

    PubMed

    Robinson, Matthew K; Shaller, Calvin; Garmestani, Kayhan; Plascjak, Paul S; Hodge, Kathryn M; Yuan, Qing-An; Marks, James D; Waldmann, Thomas A; Brechbiel, Martin W; Adams, Gregory P

    2008-02-01

    Successful radioimmunotherapy strategies depend on selecting radioisotopes with physical properties complementary to the biological properties of the targeting vehicle. Small, engineered antitumor antibody fragments are capable of rapid, highly specific tumor targeting in immunodeficient mouse models. We hypothesized that the C6.5 diabody, a noncovalent anti-HER2 single-chain Fv dimer, would be an ideal radioisotope carrier for the radioimmunotherapy of established tumors using the short-lived alpha-emitting radioisotope (211)At. Immunodeficient nude mice bearing established HER2/neu-positive MDA-MB-361/DYT2 tumors treated with N-succinimidyl N-(4-[(211)At]astatophenethyl)succinamate ((211)At-SAPS)-C6.5 diabody. Additional cohorts of mice were treated with (211)At-SAPS T84.66 diabody targeting the carcinoembryonic antigen or (211)At-SAPS on a diabody specific for the Müllerian inhibiting substance type II receptor, which is minimally expressed on this tumor cell line. A single i.v. injection of (211)At-SAPS C6.5 diabody led to a 30-day delay in tumor growth when a 20 muCi dose was administered and a 57-day delay in tumor growth (60% tumor-free after 1 year) when a 45 muCi dose was used. Treatment of mice bearing the same tumors with (211)At-SAPS T84.66 diabody at the same doses led to a delay in tumor growth, but no complete responses, likely due to substantially lower expression of this antigen on the MDA-MB-361/DYT2 tumors. In contrast, a dose of 20 muCi of (211)At-SAPS on the anti-Müllerian-inhibiting substance type II receptor diabody did not affect tumor growth rate, demonstrating specificity of the therapeutic effect. These findings indicate that diabody molecules can be effective agents for targeted radioimmunotherapy of solid tumors using powerful, short-lived alpha-emitting radioisotopes.

  15. Effective Treatment of Established Human Breast Tumor Xenografts in Immunodeficient Mice with a Single Dose of the α-Emitting Radioisotope Astatine-211 Conjugated to Anti-HER2/neu Diabodies

    PubMed Central

    Robinson, Matthew K.; Shaller, Calvin; Garmestani, Kayhan; Plascjak, Paul S.; Hodge, Kathryn M.; Yuan, Qing-An; Marks, James D.; Waldmann, Thomas A.; Brechbiel, Martin W.; Adams, Gregory P.

    2008-01-01

    Successful RAIT strategies depend upon selecting radioisotopes with physical properties complementary to the biological properties of the targeting vehicle. Small, engineered anti-tumor antibody fragments are capable of rapid, highly specific tumor targeting in immunodeficient mouse models. We hypothesized that their rapid systemic elimination would make them ideal radioisotope carriers for the radioimmunotherapy (RAIT) of established tumors. The C6.5 diabody, a non-covalent anti-HER2 single-chain Fv dimer, has a T1/2 α (equilibration phase) of 0.7 hrs, a T1/2 β (elimination phase) of 6 hrs, and a T1/2 in tumor of approximately 30 hrs that favors the use of short-lived radioisotopes. In particular, the α-particle emitting radioisotope 211At (T1/2 = 7.2 hrs) was hypothesized to be very promising for diabody-directed RAIT. This hypothesis was tested in immunodeficient nude mice bearing established HER2/neu positive MDA-MB-361/DYT2 tumors treated with 211At-SAPS C6.5 diabody (N-succinimidyl N-(4-[211At]astatophenethyl)succinamate-C6.5 diabody) at or below the maximum tolerated dose. A single i.v. injection of 211At-SAPS C6.5 diabody lead to a 30 day delay in tumor growth when a 20 µCi dose was administered and a 57 day delay in tumor growth (60% tumor free after one year) when a 45 µCi dose was employed. Treatment of mice bearing the same tumors with 211At-SAPS T84.66 diabody targeting the carcinoembryonic antigen (CEA) at the same doses led to a delay in tumor growth, but no complete responses, likely due to substantially lower expression of this antigen on the MDA-MB-361/DYT2 tumors. A dose of 20 µCi of 211At-SAPS on an a diabody specific for the Müllerian Inhibiting Substance Type II Receptor which is minimally expressed on this tumor cell line did not impact tumor growth rate, demonstrating specificity. These findings indicate that diabody molecules can be effective agents for targeted radioimmunotherapy of solid tumors using powerful, short-lived

  16. Gene therapy using plasmid DNA-encoded anti-HER2 antibody for cancers that overexpress HER2

    PubMed Central

    Kim, H; Danishmalik, S N; Hwang, H; Sin, J-I; Oh, J; Cho, Y; Lee, H; Jeong, M; Kim, S-H; Hong, H J

    2016-01-01

    Plasmid DNA-encoded antibodies, or DNA-based monoclonal antibodies (dMAbs), are delivered by intramuscular injection and in vivo electroporation (EP) and are effective in virus neutralization, although they have not been evaluated for tumor gene therapy. Here we investigated whether a dMAb was appropriate for tumor gene therapy. We constructed the expression plasmids coding for the heavy or light chain of a parental murine antibody of Herceptin with the antibody genes codon- and RNA-optimized and fused to the Kozak-IgE leader sequence in pVax1. Transfection of the plasmids into human muscle RD cells resulted in functional expression of the antibody, and this exhibited the same in vitro antiproliferative activity as Herceptin. A single intramuscular injection and in vivo EP of the plasmids (100 μg per head) resulted in high and sustained antibody expression in the sera of normal mice and in effective inhibition of tumor growth in nude mice bearing HER2-positive human breast carcinoma BT474 xenografts. The antitumor efficacy of the anti-HER2 dMAb was similar to that of four doses of intravenously injected 10 mg kg−1 Herceptin. The results demonstrate that the dMAb is effective in the treatment of HER2-positive breast cancer, suggesting that this dMAb may be applicable for tumor gene therapy. PMID:27632934

  17. Targeted siRNA delivery by anti-HER2 antibody-modified nanoparticles of mPEG-chitosan diblock copolymer.

    PubMed

    Wang, Yiyi; Liu, Peifeng; Du, Jing; Sun, Ying; Li, Fenghua; Duan, Yourong

    2013-01-01

    This study aims to determine the specificity of anti-human epidermal growth factor receptor antibody (anti-HER2) modified monomethoxy polyethylene glycol-chitosan (mPEG-CS) nanoparticles (anti-HER2/mPEG-CS NPs) in delivering small interfering RNA (siRNA) to the human epidermal growth factor receptor 2 (HER2) positive cancer cells. Physicochemical properties of the siRNA-loaded anti-HER2/mPEG-CS NPs (anti-HER2/mPEG-CS-siRNA NPs), including size, surface charge, siRNA encapsulation efficiency, and in vitro release profile of siRNA from NPs, were characterized by particle size and zeta potential analyzer, and ultraviolet-visible spectrophotometer. MTT assay was used to study the in vitro cytotoxicity of the NPs. Fluorescent microscope and flow cytometer analysis results showed that anti-HER2/mPEG-CS-siRNA NPs had much efficient delivery of siRNA than the siRNA alone, Lipofectamine-siRNA complexes and mPEG-CS-siRNA NPs. These results demonstrated that anti-HER2/mPEG-CS-siRNA NPs had great potential applications as a targeted strategy for siRNA delivery.

  18. 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

  19. Mechanisms of resistance and sensitivity to anti-HER2 therapies in HER2+ breast cancer

    PubMed Central

    de Melo Gagliato, Debora; Leonardo Fontes Jardim, Denis; Marchesi, Mario Sergio Pereira; Hortobagyi, Gabriel N.

    2016-01-01

    Breast Cancer (BC) is a highly prevalent disease. A woman living in the United States has a 12.3% lifetime risk of being diagnosed with breast cancer [1]. It is the most common female cancer and the second most common cause of cancer death in women [2]. Of note, amplification or overexpression of Human Epidermal Receptor 2 (HER2) oncogene is present in approximately 18 to 20% of primary invasive breast cancers, and until personalized therapy became available for this specific BC subtype, the worst rates of Overall Survival (OS) and Recurrence-Free Survival (RFS) were observed in the HER2+ BC cohort, compared to all other types, including triple negative BC (TNBC) [3]. HER2 is a member of the epidermal growth factor receptor (EGFR) family. Other family members include EGFR or HER1, HER3 and HER4. HER2 can form heterodimers with any of the other three receptors, and is considered to be the preferred dimerization partner of the other HER or ErbB receptors [4]. Phosphorylation of tyrosine residues within the cytoplasmic domain is the result of receptor dimerization and culminates into initiation of a variety of signalling pathways involved in cellular proliferation, transcription, motility and apoptosis inhibition [5]. In addition to being an important prognostic factor in women diagnosed with BC, HER2 overexpression also identifies those patients who benefit from treatment with agents that target HER2, such as trastuzumab, pertuzumab, trastuzumab emtansine (T-DM1) and small molecules tyrosine kinase inhibitors of HER2 [6, 11, 127]. In fact, trastuzumab altered the natural history of patients diagnosed with HER2+ BC, both in early and metastatic disease setting, in a major way [8–10]. Nevertheless, there are many women that will eventually develop metastatic disease, despite being treated with anti-HER2 therapy in the early disease setting. Moreover, advanced tumors may reach a point where no anti-HER2 treatment will achieve disease control, including recently

  20. Mechanisms of resistance and sensitivity to anti-HER2 therapies in HER2+ breast cancer.

    PubMed

    de Melo Gagliato, Debora; Jardim, Denis Leonardo Fontes; Marchesi, Mario Sergio Pereira; Hortobagyi, Gabriel N

    2016-09-27

    Breast Cancer (BC) is a highly prevalent disease. A woman living in the United States has a 12.3% lifetime risk of being diagnosed with breast cancer [1]. It is the most common female cancer and the second most common cause of cancer death in women [2]. Of note, amplification or overexpression of Human Epidermal Receptor 2 (HER2) oncogene is present in approximately 18 to 20% of primary invasive breast cancers, and until personalized therapy became available for this specific BC subtype, the worst rates of Overall Survival (OS) and Recurrence-Free Survival (RFS) were observed in the HER2+ BC cohort, compared to all other types, including triple negative BC (TNBC) [3].HER2 is a member of the epidermal growth factor receptor (EGFR) family. Other family members include EGFR or HER1, HER3 and HER4. HER2 can form heterodimers with any of the other three receptors, and is considered to be the preferred dimerization partner of the other HER or ErbB receptors [4]. Phosphorylation of tyrosine residues within the cytoplasmic domain is the result of receptor dimerization and culminates into initiation of a variety of signalling pathways involved in cellular proliferation, transcription, motility and apoptosis inhibition [5].In addition to being an important prognostic factor in women diagnosed with BC, HER2 overexpression also identifies those patients who benefit from treatment with agents that target HER2, such as trastuzumab, pertuzumab, trastuzumab emtansine (T-DM1) and small molecules tyrosine kinase inhibitors of HER2 [6, 11, 127].In fact, trastuzumab altered the natural history of patients diagnosed with HER2+ BC, both in early and metastatic disease setting, in a major way [8-10]. Nevertheless, there are many women that will eventually develop metastatic disease, despite being treated with anti-HER2 therapy in the early disease setting. Moreover, advanced tumors may reach a point where no anti-HER2 treatment will achieve disease control, including recently approved

  1. High HER2 protein levels correlate with increased survival in breast cancer patients treated with anti-HER2 therapy

    PubMed Central

    Aura, Claudia; Garrido-Castro, Ana; Vilaro, Marta; Peg, Vicente; Jimenez, José; Vicario, Rocio; Cecchi, Fabiola; Hoos, William; Burrows, Jon; Hembrough, Todd; Ferreres, Juan Carles; Perez-Garcia, José; Arribas, Joaquin; Cortes, Javier; Scaltriti, Maurizio

    2016-01-01

    Introduction Current methods to determine HER2 (human epidermal growth factor receptor 2) status are affected by reproducibility issues and do not reliably predict benefit from anti-HER2 therapy. Quantitative measurement of HER2 may more accurately identify breast cancer (BC) patients who will respond to anti-HER2 treatments. Methods Using selected reaction monitoring mass spectrometry (SRM-MS), we quantified HER2 protein levels in formalin-fixed, paraffin-embedded (FFPE) tissue samples that had been classified as HER2 0, 1+, 2+ or 3+ by immunohistochemistry (IHC). Receiver operator curve (ROC) analysis was conducted to obtain optimal HER2 protein expression thresholds predictive of HER2 status (by standard IHC or in situ hybridization [ISH]) and of survival benefit after anti-HER2 therapy. Results Absolute HER2 amol/μg levels were significantly correlated with both HER2 IHC and amplification status by ISH (p < 0.0001). A HER2 threshold of 740 amol/μg showed an agreement rate of 94% with IHC and ISH standard HER2 testing (p < 0.0001). Discordant cases (SRM-MS-negative/ISH-positive) showed a characteristic amplification pattern known as double minutes. HER2 levels >2200 amol/μg were significantly associated with longer disease-free survival (DFS) and overall survival (OS) in an adjuvant setting and with longer OS in a metastatic setting. Conclusion Quantitative HER2 measurement by SRM-MS is superior to IHC and ISH in predicting outcome after treatment with anti-HER2 therapy. PMID:26422389

  2. Why man's best friend, the dog, could also benefit from an anti-HER-2 vaccine

    PubMed Central

    Fazekas, Judit; Fürdös, Irene; Singer, Josef; Jensen-Jarolim, Erika

    2016-01-01

    Human epidermal growth factor receptor-2 (HER-2) is a well-established target for anticancer anticancerprecision medicine in humans. A HER-2 homologue with 92% amino acid identity has been described in canine mammary tumors, which whichis termed here as ‘dog epidermal growth factor receptor-2 (DER-2)’, with similar biological implications as those in human breast cancer. Both antigens can principally be immunologically targeted by anti-HER-2 antibodies, such as trastuzumab; however, the in vivo application applicationof humanized antibodies to other species would lead to specific hypersensitivity reactions. Therefore, HER-2 mimotope vaccines that actively induce autologous trastuzumab-like immunoglobulins represent a novel and economic treatment option to overcome species-specific limitations. Thus, the present review proposes the implementation of clinical trials with HER-2 vaccines in canine cancer model modelpatients with spontaneous DER-2 positive mammary gland carcinomas in order to assess their safety and efficacy. This approach would not only pave the way into the veterinary oncology market, but would also similarly generate robust data for human trials and facilitate the testing of novel combinatorial treatments. PMID:27698788

  3. Why man's best friend, the dog, could also benefit from an anti-HER-2 vaccine.

    PubMed

    Fazekas, Judit; Fürdös, Irene; Singer, Josef; Jensen-Jarolim, Erika

    2016-10-01

    Human epidermal growth factor receptor-2 (HER-2) is a well-established target for anticancer anticancerprecision medicine in humans. A HER-2 homologue with 92% amino acid identity has been described in canine mammary tumors, which whichis termed here as 'dog epidermal growth factor receptor-2 (DER-2)', with similar biological implications as those in human breast cancer. Both antigens can principally be immunologically targeted by anti-HER-2 antibodies, such as trastuzumab; however, the in vivo application applicationof humanized antibodies to other species would lead to specific hypersensitivity reactions. Therefore, HER-2 mimotope vaccines that actively induce autologous trastuzumab-like immunoglobulins represent a novel and economic treatment option to overcome species-specific limitations. Thus, the present review proposes the implementation of clinical trials with HER-2 vaccines in canine cancer model modelpatients with spontaneous DER-2 positive mammary gland carcinomas in order to assess their safety and efficacy. This approach would not only pave the way into the veterinary oncology market, but would also similarly generate robust data for human trials and facilitate the testing of novel combinatorial treatments.

  4. New anti-HER2 agents: from second-generation tyrosine kinases inhibitors to bifunctional antibodies.

    PubMed

    Gligorov, Joseph; Richard, Sandrine; Todorovic, Vladimir

    2017-10-03

    HER2-positive breast cancers have benefited since the end of the twentieth century, not only from the improvement of biological knowledge, but also from major technological advances. The latter allowed the synthesis of the first generation of enzymatic inhibitors of the HER receptor family such as lapatinib, but above all, monoclonal antibodies such as trastuzumab or pertuzumab having profoundly modified the management of these cancers. However, despite outstanding progresses, there are still patients who are not cured with these first-generation treatments, and they will need new approaches to improve disease control and impact patients' survival. Understanding the mechanisms of escape to these treatments, more than real resistance, has profoundly changed our pharmacological approaches. They have enabled the development of molecules blocking the signaling pathway downstream of receptors such as mTOR, PI3K inhibitors or molecules interacting with the cellular traffic of the receptor in combination with the first-generation treatments. In addition, new second-generation tyrosine kinase inhibitors have demonstrated increased in-vitro efficacy, but still need to show clinical relevance because of new toxicity profiles. The antibody engineering had also permitted a paradigm evolution of the role of the antibody treatments, particularly with the synthesis of bispecific and trifunctional antibodies, promoting the link between the tumor and the immune system, with the goal to amplify the immune anticancer response. Among the new anti-HER2 agents, second-generation tyrosine kinase inhibitors and bifunctional antibodies are promising approaches that will help to improve disease control and curability of HER2-positive breast cancers.

  5. Twenty years of anti-HER2 therapy-associated cardiotoxicity

    PubMed Central

    Pondé, Noam F; Lambertini, Matteo; de Azambuja, Evandro

    2016-01-01

    Over the past 20 years, the prognosis of HER2-positive breast cancer has been transformed by the development of anti-HER2 targeted therapies. In early clinical trials of trastuzumab (ie, the first anti-HER2 agent to be developed) cardiotoxicity became a major concern. In the first published phase 3 trial of trastuzumab, 27% of patients receiving anthracyclines and trastuzumab experienced cardiac events and 16% suffered from severe congestive heart failure. In subsequent trials conducted in advanced and early settings, the incidence of cardiac events was reduced through changes in chemotherapy regimens, more strict patient selection and close cardiac assessment. However, cardiotoxicity remains a significant problem in clinical practice that is likely to increase as new agents are approved and exposure times increase through improved patients' survival. Though numerous trials have led to improved understanding of many aspects of anti-HER2 therapy-related cardiotoxicity, its underlying physiopathology mechanisms are not well understood. The purpose of this article is to provide an in-depth review on anti-HER2 therapy-related cardiotoxicity, including data on both trastuzumab and the recently developed anti-HER2 targeted agents. PMID:27843627

  6. Synthesis of a Covalent Epirubicin-(C3-amide)-Anti-HER2/neu Immunochemotherapeutic Utilizing a UV-Photoactivated Anthracycline Intermediate

    PubMed Central

    Jones, Toni; Bear, Ryan

    2012-01-01

    Abstract The C3-monoamine on the carbohydrate moiety (daunosamine -NH2-3′) of epirubicin was reacted under anhydrous conditions with succinimidyl 4,4-azipentanoate to create a covalent UV-photoactivated epirubicin-(C3-amide) intermediate with primary amine-reactive properties. A synthetic covalent bond between the UV-photoactivated epirubicin-(C3-amide) intermediate and the ɛ-amine of lysine residues within the amino acid sequence of anti-HER2/neu monoclonal immunoglobulin was subsequently created by exposure to UV light (354 nm) for 15 minutes. Size-separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with immunodetection analysis and chemiluminescent autoradiographic imaging revealed a lack of IgG-IgG polymerization or degradative protein fragmentation of the covalent epirubicin-(C3-amide)-[anti-HER2/neu] immunochemotherapeutic. Retained binding-avidity of epirubicin-(C3-amide)-[anti-HER2/neu] was validated by cell-ELISA utilizing monolayer populations of chemotherapeutic-resistant mammary adenocarcinoma SKBr-3 which highly overexpress membrane-associated HER2/neu complexes. Between epirubicin-equivalent concentrations of 10−10 to 10−6 M the covalent epirubicin-(C3-amide)-[anti-HER2/neu] immunochemotherapeutic consistently evoked levels of cytotoxic anti-neoplastic potency that were highly analogous to chemotherapeutic-equivalent concentrations of epirubicin. Cytotoxic anti-neoplastic potency of epirubicin-(C3-amide)-[anti-HER2/neu] against chemotherapeutic-resistant mammary adenocarcinoma SKBr-3 challenged with epirubicin-(C3-amide)-[anti-HER2/neu] at an epirubicin-equivalent concentration of 10−6 M was 88.5% (e.g., 11.5% residual survival). Between final epirubicin-equivalent concentrations of 10−8 and 10−7 M there was a marked threshold increase in the mean cytotoxic anti-neoplastic activity for epirubicin-(C3-amide)-[anti-HER2/neu] from 9.9% to 66.9% (90.2% to 33.1% residual survival). PMID:22191802

  7. Anti-HER2 immunoliposomes for co-delivery of paclitaxel and rapamycin for breast cancer therapy.

    PubMed

    Eloy, Josimar O; Petrilli, Raquel; Chesca, Deise L; Saggioro, Fabiano P; Lee, Robert J; Marchetti, Juliana Maldonado

    2017-06-01

    Breast cancer is the second leading cause of cancer deaths among women. Paclitaxel (PTX) is used for its treatment, however non-selectivity, rapid systemic clearance and hypersensitivity to the commercially available formulation are major drawbacks. Rapamycin (RAP), an mTOR inhibitor, acts synergistically with PTX, and thus could be used in combination with it. Drug loading into nanocarriers, particularly liposomes, has proven to enhance efficacy and reduce side-effects of chemotherapeutic drugs. Within this context, the functionalization of liposomes with antibodies for overexpressed receptors on tumor surface is a potential strategy to increase specificity and reduce side-effects. Specifically, active targeting of HER2(+) breast cancer cells can be achieved by immunoliposomes consisting of liposomes coated with an anti-HER2 monoclonal antibody, Trastuzumab. Herein, we have synthesized PTX/RAP co-loaded immunoliposomes coated with Trastuzumab, performed physicochemical characterization, and evaluated the formulations for cytotoxicity and uptake in 4T1 (triple negative) and SKBR3 (HER2 positive) cell lines. Furthermore, we aimed to compare the immunoliposomes with liposomes and solution of PTX/RAP in vivo, employing human xenograft HER2-overexpressing tumors in mouse model. The co-loaded immunoliposomes had a mean particle size of 140.3nm, a zeta potential of -9.85mV and drug encapsulation efficiency of 55.87 and 69.51, respectively for PTX and RAP. The functionalization efficiency of Trastuzumab was higher than 70% and the antibody retained HER2 binding activity. Cell studies showed increased cytotoxicity of PTX/RAP for the immunoliposome, compared to the control liposomes in SKBR3 cells, which could be attributed to enhanced uptake mediated through HER2 binding. Furthermore, immunoliposomes were better able to control tumor growth in vivo, with tumor volume averages corresponding to 25.27, 44.38 and 47.78% of tumor volumes of untreated control, PTX/RAP solution

  8. Assessment of the systemic distribution of a bioconjugated anti-Her2 magnetic nanoparticle in a breast cancer model by means of magnetic resonance imaging

    NASA Astrophysics Data System (ADS)

    Huerta-Núñez, L. F. E.; Villanueva-Lopez, G. Cleva; Morales-Guadarrama, A.; Soto, S.; López, J.; Silva, J. G.; Perez-Vielma, N.; Sacristán, E.; Gudiño-Zayas, Marco E.; González, C. A.

    2016-09-01

    The aim of this study was to determine the systemic distribution of magnetic nanoparticles of 100 nm diameter (MNPs) coupled to a specific monoclonal antibody anti-Her2 in an experimental breast cancer (BC) model. The study was performed in two groups of Sprague-Dawley rats: control ( n = 6) and BC chemically induced ( n = 3). Bioconjugated "anti-Her2-MNPs" were intravenously administered, and magnetic resonance imaging (MRI) monitored its systemic distribution at seven times after administration. Non-heme iron presence associated with the location of the bioconjugated anti-Her2-MNPs in splenic, hepatic, cardiac and tumor tissues was detected by Perl's Prussian blue (PPB) stain. Optical density measurements were used to semiquantitatively determine the iron presence in tissues on the basis of a grayscale values integration of T1 and T2 MRI sequence images. The results indicated a delayed systemic distribution of MNPs in cancer compared to healthy conditions with a maximum concentration of MNPs in cancer tissue at 24 h post-infusion.

  9. Anti-HER2/neu peptide-conjugated iron oxide nanoparticles for targeted delivery of paclitaxel to breast cancer cells

    NASA Astrophysics Data System (ADS)

    Mu, Qingxin; Kievit, Forrest M.; Kant, Rajeev J.; Lin, Guanyou; Jeon, Mike; Zhang, Miqin

    2015-10-01

    Nanoparticles (NPs) for targeted therapy are required to have appropriate size, stability, drug loading and release profiles, and efficient targeting ligands. However, many of the existing NPs such as albumin, liposomes, polymers, gold NPs, etc. encounter size limit, toxicity and stability issues when loaded with drugs, fluorophores, and targeting ligands. Furthermore, antibodies are bulky and this can greatly affect the physicochemical properties of the NPs, whereas many small molecule-based targeting ligands lack specificity. Here, we report the utilization of biocompatible, biodegradable, small (~30 nm) and stable iron oxide NPs (IONPs) for targeted delivery of paclitaxel (PTX) to HER2/neu positive breast cancer cells using an anti-HER2/neu peptide (AHNP) targeting ligand. We demonstrate the uniform size and high stability of these NPs in biological medium, their effective tumour targeting in live mice, as well as their efficient cellular targeting and selective killing in human HER2/neu-positive breast cancer cells.Nanoparticles (NPs) for targeted therapy are required to have appropriate size, stability, drug loading and release profiles, and efficient targeting ligands. However, many of the existing NPs such as albumin, liposomes, polymers, gold NPs, etc. encounter size limit, toxicity and stability issues when loaded with drugs, fluorophores, and targeting ligands. Furthermore, antibodies are bulky and this can greatly affect the physicochemical properties of the NPs, whereas many small molecule-based targeting ligands lack specificity. Here, we report the utilization of biocompatible, biodegradable, small (~30 nm) and stable iron oxide NPs (IONPs) for targeted delivery of paclitaxel (PTX) to HER2/neu positive breast cancer cells using an anti-HER2/neu peptide (AHNP) targeting ligand. We demonstrate the uniform size and high stability of these NPs in biological medium, their effective tumour targeting in live mice, as well as their efficient cellular

  10. Seed-specific expression and analysis of recombinant anti-HER2 single-chain variable fragment (scFv-Fc) in Arabidopsis thaliana.

    PubMed

    Dong, Yuanyuan; Li, Jian; Yao, Na; Wang, Dezhong; Liu, Xiuming; Wang, Nan; Li, Xiaowei; Wang, Fawei; Li, Haiyan; Jiang, Chao

    2017-05-01

    Antibodies to human epidermal growth factor receptor 2 (HER2) are a key element of breast cancer therapy; however, they are expensive to produce and their availability is limited. A seed-specific expression system can be used to produce recombinant proteins. We report a seed-specific expression system for the manufacture of anti-HER2 ScFv-Fc in Arabidopsis thaliana, driven by the Phaseolus vulgaris β-phaseolin promoter. Recombinant anti-HER2 ScFv-Fc was successfully and specifically expressed in seeds, and identified by protein analysis. The highest protein accumulation level, with a maximum of 1.1% of total soluble protein, was observed in mature seeds. We also demonstrated the anti-tumor potency of the plant-derived antibody against SK-BR-3 cells. These results suggest that seed-expression systems could contribute to the manufacture of commercial antibodies such as anti-HER2 ScFv-Fc. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. HER2 specific delivery of methotrexate by dendrimer conjugated anti-HER2 mAb

    NASA Astrophysics Data System (ADS)

    Shukla, Rameshwer; Thomas, Thommey P.; Desai, Ankur M.; Kotlyar, Alina; Park, Steve J.; Baker, James R., Jr.

    2008-07-01

    Herceptin, a humanized monoclonal antibody that binds to human growth factor receptor-2 (HER2), was covalently attached to a fifth-generation (G5) polyamidoamine dendrimer containing the cytotoxic drug methotrexate. The specific binding and internalization of this conjugate labeled with FITC was clearly demonstrated in cell lines overexpressing HER2 by flow cytometry as well as confocal microscopic analysis. In addition, binding and uptake of antibody conjugated dendrimers was completely blocked by excess non-conjugated herceptin. The dendrimer conjugate was also shown to inhibit the dihydrofolate reductase with similar activity to methotrexate. Co-localization experiments with lysotracker red indicate that antibody conjugate, although internalized efficiently into cells, has an unusually long residence time in the lysosome. Somewhat lower cytotoxicity of the conjugate in comparison to free methotrexate was attributed to the slow release of methotrexate from the conjugate and its long retention in the lysosomal pocket.

  12. Neural Stem Cells Secreting Anti-HER2 Antibody Improve Survival in a Preclinical Model of HER2 Overexpressing Breast Cancer Brain Metastases.

    PubMed

    Kanojia, Deepak; Balyasnikova, Irina V; Morshed, Ramin A; Frank, Richard T; Yu, Dou; Zhang, Lingjiao; Spencer, Drew A; Kim, Julius W; Han, Yu; Yu, Dihua; Ahmed, Atique U; Aboody, Karen S; Lesniak, Maciej S

    2015-10-01

    The treatment of human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer has been revolutionized by trastuzumab. However, longer survival of these patients now predisposes them to forming HER2 positive brain metastases, as the therapeutic antibodies cannot cross the blood brain barrier. The current oncologic repertoire does not offer a rational, nontoxic targeted therapy for brain metastases. In this study, we used an established human neural stem cell line, HB1.F3 NSCs and generated a stable pool of cells secreting a high amount of functional full-length anti-HER2 antibody, equivalent to trastuzumab. Anti-HER2Ab secreted by the NSCs (HER2Ab-NSCs) specifically binds to HER2 overexpressing human breast cancer cells and inhibits PI3K-Akt signaling. This translates to HER2Ab-NSC inhibition of breast cancer cell growth in vitro. Preclinical in vivo experiments using HER2Ab overexpressing NSCs in a breast cancer brain metastases (BCBM) mouse model demonstrate that intracranial injection of HER2Ab-NSCs significantly improves survival. In effect, these NSCs provide tumor localized production of HER2Ab, minimizing any potential off-target side effects. Our results establish HER2Ab-NSCs as a novel, nontoxic, and rational therapeutic approach for the successful treatment of HER2 overexpressing BCBM, which now warrants further preclinical and clinical investigation.

  13. Anti-HER2 Therapy Beyond Second-Line for HER2-Positive Metastatic Breast Cancer: A Short Review and Recommendations for Several Clinical Scenarios from a Spanish Expert Panel

    PubMed Central

    Martínez-Jañez, Noelia; Chacón, Ignacio; de Juan, Ana; Cruz-Merino, Luis; del Barco, Sònia; Fernández, Isaura; García-Teijido, Paula; Gómez-Bernal, Amalia; Plazaola, Arrate; Ponce, José; Servitja, Sonia; Zamora, Pilar

    2016-01-01

    Summary Background The aim of this project was to provide an expert opinion regarding anti-human epidermal growth factor receptor 2 (HER2) therapy beyond second-line treatment of metastatic breast cancer (mBC). Methods A group of experts discussed specific issues concerning anti-HER2 therapy in late-line settings in mBC. Results Trastuzumab emtansine (T-DM1) or dual HER2 blockade appeared to be good options for HER2-positive mBC after ≥ 2 HER2-targeted therapies. Once an objective response has been achieved with anti-HER2-containing therapy, the anti-HER2 agent can be continued until progression of the disease, unacceptable toxicity or patient decision. mBC treated with ≥ 3 consecutive lines of anti-HER therapy, ≥ 1 being a dual HER2 blockade and with early progression of disease during a fourth or later-line treatment, are clinically resistant to anti-HER therapy. For progression of metastasis in the brain after anti-HER2 therapy, lapatinib and chemotherapy appear to be a good alternative after best local treatment. Conclusions Further clinical trials are needed to provide valuable knowledge about the best treatment options in the later settings of mBC. PMID:27239176

  14. Survival benefit of anti-HER2 therapy after whole-brain radiotherapy in HER2-positive breast cancer patients with brain metastasis.

    PubMed

    Zhang, Qian; Chen, Jian; Yu, Xiaoli; Cai, Gang; Yang, Zhaozhi; Cao, Lu; Hu, Chaosu; Guo, Xiaomao; Sun, Jing; Chen, Jiayi

    2016-09-01

    We aimed to assess the survival benefit of epidermal growth factor receptor 2 (HER2)-positive breast cancer patients with brain metastasis (BM) after whole-brain radiotherapy (WBRT) in combination with systemic treatments, especially anti-HER2 therapy. This retrospective study analyzed the overall survival (OS) of 60 HER2-positive breast cancer patients with BM after WBRT in combination with systemic treatments. Among them, 42 patients received chemotherapy while 18 patients did not receive after WBRT. With regard to anti-HER2 therapy, after WBRT, 17 patients received anti-HER2 treatment without prior adjuvant trastuzumab-based therapy, 7 patients received anti-HER2 treatment with prior adjuvant trastuzumab-based therapy, and 36 patients did not receive further anti-HER2 treatment. All patients were followed up regularly until January 23, 2013. The median OS of patients with BM was 12 months. Patients who received anti-HER2 therapy and chemotherapy after WBRT had significantly better survival compared with patients who did not receive further treatment. Patients who received anti-HER2 treatment after WBRT but did not receive adjuvant trastuzumab-based therapy for early breast cancer had better OS, followed by patients who received anti-HER2 agent both in adjuvant treatment and after WBRT and patients who did not receive anti-HER2 treatment. Multivariate analysis showed that Karnofsky Performance Status, control of extracranial metastases, chemotherapy after WBRT, and anti-HER2 therapy combined with WBRT were all independent predictors for OS. Both chemotherapy and anti-HER2 therapy after WBRT could improve OS. Moreover, patients without prior exposure to adjuvant anti-HER2 treatment may have survival benefit superior to those of patients with prior exposure.

  15. Cancer Cell Targeting Using Folic Acid/Anti-HER2 Antibody Conjugated Fluorescent CdSe/CdS/ZnS-MPA and CdTe-MSA Quantum Dots.

    PubMed

    Singh, Gurpal; Kumar, Manoj; Soni, Udit; Arora, Vikas; Bansal, Vivek; Gupta, Dikshi; Bhat, Madhusudan; Dinda, Amit K; Sapra, Sameer; Singh, Harpal

    2015-12-01

    CdSe/CdS/ZnS and CdTe quantum dots (QDs) were synthesized by successive ion layer adsorption and reaction (SILAR) technique and direct aqueous synthesis respectively using thiol stabilizers. Synthesized CdSe/CdS/ZnS and CdTe QDs stabilized with 3-mercaptopropionic acid (MPA) and mercaptosuccinic acid (MSA) were used as fluorescent labels after conjugation with folic acid (FA) and anti-HER2 antibodies. Photoluminescence quantum yield of folated CdSe/CdS/ZnS-MPA and CdTe-MSA QDs was 59% and 77% than that of non-folated hydrophilic QDs. The folate receptor-mediated delivery of folic acid-conjugated CdTe-MSA and CdSe/CdS/ZnS-MPA QDs showed higher cellular internalization as observed by confocal laser scanning microscopic studies. Folated and non-folated CdTe-MSA QDs were highly toxic and exhibited only 10% cell viability as compared to > 80% cell viability with CdSe/CdS/ZnS-MPA QDs over the concentration ranging from 3.38 to 50 pmoles. Immunohistochemistry (IHC) results of human breast cancer tissue samples showed positive results with anti-HER2 antibody conjugated CdSe/CdS/ZnS-MPA QDs with better sensitivity and specificity as compared to conventional IHC analysis using diaminobenzedene staining.

  16. In vitro HER2 protein-induced affinity dissociation of carbon nanotube-wrapped anti-HER2 aptamers for HER2 protein detection.

    PubMed

    Niazi, Javed H; Verma, Sandeep K; Niazi, Sarfaraj; Qureshi, Anjum

    2015-01-07

    A new in vitro assay was developed to detect human epidermal growth factor receptor 2 (HER2) protein, based on affinity dissociation of carbon nanotube (CNT)-wrapped anti-HER2 ssDNA aptamers. First, we selected an anti-HER2 ssDNA aptamer (H2) using an in vitro serial evolution of ligands by an exponential enrichment (SELEX) process. Then the fluorescently labelled H2 ssDNAs were tightly packed on CNTs that had previously been coupled with magnetic microbeads (MBs), forming MB-CNT-H2 hybrids. The loading capacity of these MB-CNTs heterostructures (2.8 × 10(8)) was determined to be 0.025 to 3.125 μM of H2. HER2 protein-induced H2 dissociation occurred from MB-CNT-H2 hybrids, which was specifically induced by the target HER2 protein, with a dissociation constant (Kd) of 270 nM. The stoichiometric affinity dissociation ratio with respect to H2-to-HER2 protein was shown to be approximately 1 : 1. Our results demonstrated that the developed assay can be an effective approach in detecting native forms of disease biomarkers in free solutions or in biological samples, for accurate diagnosis.

  17. Dual potency anti-HER2/neu and anti-EGFR anthracycline immunoconjugates in chemotherapeutic-resistant mammary carcinoma combined with cyclosporin A and verapamil P-glycoprotein inhibition.

    PubMed

    Coyne, C P; Ross, Matt K; Bailey, John G

    2009-07-01

    Immunoconjugates of epirubicin were synthesized with monoclonal antibodies against the epidermal growth factor receptors, HER2/neu and EGFR, by creating a sulfhydryl-reactive epirubicin intermediate applying heterobifunctional succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), which was introduced at alpha-monoamide groups of the epirubicin carbohydrate moiety. In parallel, N-succinimidyl-S-acetylthioacetate (SATA) was used to incorporate a sulfhydryl group into immunoglobulin at the terminal amine position of -lysine amino acid residues. Eprirubicin-SMCC-SATA-IgG immunoconjugates were produced by reacting epirubicin-SMCC and SATA-IgG at appropriate molar ratios. Epirubicin-(anti-HER2/neu) and epirubicin-(anti-EGFR) had greater potency against chemotherapeutic-resistant SKBr-3 mammary carcinoma than did epirubicin at epirubicin-equivalent concentrations. Epirubicin-(anti-HER2/neu) was more potent than epirubicin-(anti-EGFR), and a synergistic level of antineoplastic activity was detected with an epirubicin immunoconjugate 50/50 combination. Competitive P-glycoprotein inhibition with cyclosporin A or verapamil enhanced the potency of the epirubicin immunoconjugate 50/50 combination. Minor levels of antineoplastic activity were detected only with an immunoglobulin 50/50 combination of anti-HER2/neu and anti-EGFR. The investigations represent a potential strategy for enhancing the selective internalization, intracellular deposition, and antineoplastic potency of chemotherapeutics in multidrug-resistant neoplasias.

  18. The Therapeutic Effect of Anti-HER2/neu Antibody Depends on Both Innate and Adaptive Immunity

    PubMed Central

    Park, SaeGwang; Jiang, Zhujun; Mortenson, Eric D.; Deng, Liufu; Radkevich-Brown, Olga; Yang, Xuanming; Sattar, Husain; Wang, Yang; Brown, Nicholas K.; Greene, Mark; Liu, Yang; Tang, Jie; Wang, Shengdian; Fu, Yang-Xin

    2010-01-01

    SUMMARY Anti-HER2/neu antibody therapy is reported to mediate tumor regression by interrupting oncogenic signals and/or inducing FcR-mediated cytotoxicity. Here, we demonstrate that the mechanisms of tumor regression by this therapy also require the adaptive immune response. Activation of innate immunity and T cells, initiated by antibody treatment, was necessary. Intriguingly, the addition of chemotherapeutic drugs, while capable of enhancing the reduction of tumor burden, could abrogate antibody-initiated immunity leading to decreased resistance to re-challenge or earlier relapse. Increased influx of both innate and adaptive immune cells into the tumor microenvironment by a selected immunotherapy further enhanced subsequent antibody-induced immunity, leading to increased tumor eradication and resistance to re-challenge. Therefore, this study proposes a model and strategy for anti-HER2/neu antibody-mediated tumor clearance. PMID:20708157

  19. HER2 phosphorylation is maintained by a PKB negative feedback loop in response to anti-HER2 herceptin in breast cancer.

    PubMed

    Gijsen, Merel; King, Peter; Perera, Tim; Parker, Peter J; Harris, Adrian L; Larijani, Banafshé; Kong, Anthony

    2010-12-21

    Herceptin (trastuzumab) is used in patients with breast cancer who have HER2 (ErbB2)-positive tumours. However, its mechanisms of action and how acquired resistance to Herceptin occurs are still poorly understood. It was previously thought that the anti-HER2 monoclonal antibody Herceptin inhibits HER2 signalling, but recent studies have shown that Herceptin does not decrease HER2 phosphorylation. Its failure to abolish HER2 phosphorylation may be a key to why acquired resistance inevitably occurs for all responders if Herceptin is given as monotherapy. To date, no studies have explained why Herceptin does not abolish HER2 phosphorylation. The objective of this study was to investigate why Herceptin did not decrease HER2 phosphorylation despite being an anti-HER2 monoclonal antibody. We also investigated the effects of acute and chronic Herceptin treatment on HER3 and PKB phosphorylation in HER2-positive breast cancer cells. Using both Förster resonance energy transfer (FRET) methodology and conventional Western blot, we have found the molecular mechanisms whereby Herceptin fails to abolish HER2 phosphorylation. HER2 phosphorylation is maintained by ligand-mediated activation of EGFR, HER3, and HER4 receptors, resulting in their dimerisation with HER2. The release of HER ligands was mediated by ADAM17 through a PKB negative feedback loop. The feedback loop was activated because of the inhibition of PKB by Herceptin treatment since up-regulation of HER ligands and ADAM17 also occurred when PKB phosphorylation was inhibited by a PKB inhibitor (Akt inhibitor VIII, Akti-1/2). The combination of Herceptin with ADAM17 inhibitors or the panHER inhibitor JNJ-26483327 was able to abrogate the feedback loop and decrease HER2 phosphorylation. Furthermore, the combination of Herceptin with JNJ-26483327 was synergistic in tumour inhibition in a BT474 xenograft model. We have determined that a PKB negative feedback loop links ADAM17 and HER ligands in maintaining HER2

  20. Nonamplification ERBB2 genomic alterations in 5605 cases of recurrent and metastatic breast cancer: An emerging opportunity for anti-HER2 targeted therapies.

    PubMed

    Ross, Jeffrey S; Gay, Laurie M; Wang, Kai; Ali, Siraj M; Chumsri, Saranya; Elvin, Julia A; Bose, Ron; Vergilio, Jo-Anne; Suh, James; Yelensky, Roman; Lipson, Doron; Chmielecki, Juliann; Waintraub, Stanley; Leyland-Jones, Brian; Miller, Vincent A; Stephens, Philip J

    2016-09-01

    Activating, nonamplification ERBB2 mutations (ERBB2mut) are not detected by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH), but are detected by DNA sequencing and may predict clinical responses to human epidermal growth factor receptor (HER2)-targeted therapy. The authors queried 5605 advanced/metastatic breast cancers (mBC) to uncover the frequency of ERBB2mut genomic alterations. Clinical responses to anti-HER2 therapeutics were identified. DNA was extracted from 40 µm of formalin-fixed paraffin-embedded (FFPE) sections. Comprehensive genomic profiling (CGP) was used to evaluate up to 315 genes (592× mean coverage depth). Results were analyzed for base substitutions, short indels, copy number changes, and selected rearrangements. Of 5605 cases, 698 (12.5%) featured ERBB2 alterations, including 596 (10.6%) ERBB2 amplifications (ERBB2amp) and 138 (2.4%) ERBB2mut; 38 cases (0.7%) had co-occurring ERBB2amp and ERBB2mut. ERBB2mut predominantly affected the kinase (124 cases; 90%) or extracellular (15 cases; 11%) domains. Both primary BC (52 cases; 38%) and metastatic site biopsies (86 cases; 62%) were found to harbor ERBB2mut, which were distributed across carcinoma not otherwise specified (NOS) (69 cases; 50%), invasive ductal carcinoma (IDC) (40 cases; 29%), invasive lobular carcinoma (ILC) (27 cases; 20%), and mucinous mBC (2 cases; 1%). Genes commonly coaltered with ERBB2 were tumor protein 53 (TP53) (49%); phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) (42%); cadherin 1, type 1 (CDH1) (37%); MYC (17%); and cyclin D1 protein (CCND1) (16%). CDH1 mutations were enriched in ERBB2mut mBC (P<0.0006) and associated with recurrent mBC. Selected patients with ERBB2mut, without ERBB2amp, who responded to anti-HER2 targeted therapies are presented herein. Within this large series, 1.8% of cases harbored ERBB2mut, which are undetectable by standard-of-care IHC or FISH tests. Metastatic BC driven by ERBB2mut respond to anti-HER2

  1. Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer.

    PubMed

    Chen, Hongwei; Wang, Liya; Yu, Qiqi; Qian, Weiping; Tiwari, Diana; Yi, Hong; Wang, Andrew Y; Huang, Jing; Yang, Lily; Mao, Hui

    2013-01-01

    Antifouling magnetic iron oxide nanoparticles (IONPs) coated with block copolymer poly(ethylene oxide)-block-poly(γ-methacryloxypropyltrimethoxysilane) (PEO-b-PγMPS) were investigated for improving cell targeting by reducing nonspecific uptake. Conjugation of a HER2 antibody, Herceptin®, or a single chain fragment (ScFv) of antibody against epidermal growth factor receptor (ScFvEGFR) to PEO-b-PγMPS-coated IONPs resulted in HER2-targeted or EGFR-targeted IONPs (anti-HER2-IONPs or ScFvEGFR-IONPs). The anti-HER2-IONPs bound specifically to SK-BR-3, a HER2-overexpressing breast cancer cell line, but not to MDA-MB-231, a HER2-underexpressing cell line. On the other hand, the ScFvEGFR-IONPs showed strong reactivity with MDA-MB-231, an EGFR-positive human breast cancer cell line, but not with MDA-MB-453, an EGFR-negative human breast cancer cell line. Transmission electron microscopy revealed internalization of the receptor-targeted nanoparticles by the targeted cancer cells. In addition, both antibody-conjugated and non-antibody-conjugated IONPs showed reduced nonspecific uptake by RAW264.7 mouse macrophages in vitro. The developed IONPs showed a long blood circulation time (serum half-life 11.6 hours) in mice and low accumulation in both the liver and spleen. At 24 hours after systemic administration of ScFvEGFR-IONPs into mice bearing EGFR-positive breast cancer 4T1 mouse mammary tumors, magnetic resonance imaging revealed signal reduction in the tumor as a result of the accumulation of the targeted IONPs.

  2. Synthesis and Characterization of Anti-HER2 Antibody Conjugated CdSe/CdZnS Quantum Dots for Fluorescence Imaging of Breast Cancer Cells.

    PubMed

    Tiwari, Dhermendra K; Tanaka, Shin-Ichi; Inouye, Yasushi; Yoshizawa, Keiko; Watanabe, Tomonobu M; Jin, Takashi

    2009-01-01

    The early detection of HER2 (human epidermal growth factor receptor 2) status in breast cancer patients is very important for the effective implementation of anti-HER2 antibody therapy. Recently, HER2 detections using antibody conjugated quantum dots (QDs) have attracted much attention. QDs are a new class of fluorescent materials that have superior properties such as high brightness, high resistance to photo-bleaching, and multi-colored emission by a single-light source excitation. In this study, we synthesized three types of anti-HER2 antibody conjugated QDs (HER2Ab-QDs) using different coupling agents (EDC/sulfo-NHS, iminothiolane/sulfo-SMCC, and sulfo-SMCC). As water-soluble QDs for the conjugation of antibody, we used glutathione coated CdSe/CdZnS QDs (GSH-QDs) with fluorescence quantum yields of 0.23∼0.39 in aqueous solution. Dispersibility, hydrodynamic size, and apparent molecular weights of the GSH-QDs and HER2Ab-QDs were characterized by using dynamic light scattering, fluorescence correlation spectroscopy, atomic force microscope, and size-exclusion HPLC. Fluorescence imaging of HER2 overexpressing cells (KPL-4 human breast cancer cell line) was performed by using HER2Ab-QDs as fluorescent probes. We found that the HER2Ab-QD prepared by using SMCC coupling with partially reduced antibody is a most effective probe for the detection of HER2 expression in KPL-4 cells. We have also studied the size dependency of HER2Ab-QDs (with green, orange, and red emission) on the fluorescence image of KPL-4 cells.

  3. Synthesis and Characterization of Anti-HER2 Antibody Conjugated CdSe/CdZnS Quantum Dots for Fluorescence Imaging of Breast Cancer Cells

    PubMed Central

    Tiwari, Dhermendra K.; Tanaka, Shin-Ichi; Inouye, Yasushi; Yoshizawa, Keiko; Watanabe, Tomonobu M.; Jin, Takashi

    2009-01-01

    The early detection of HER2 (human epidermal growth factor receptor 2) status in breast cancer patients is very important for the effective implementation of anti-HER2 antibody therapy. Recently, HER2 detections using antibody conjugated quantum dots (QDs) have attracted much attention. QDs are a new class of fluorescent materials that have superior properties such as high brightness, high resistance to photo-bleaching, and multi-colored emission by a single-light source excitation. In this study, we synthesized three types of anti-HER2 antibody conjugated QDs (HER2Ab-QDs) using different coupling agents (EDC/sulfo-NHS, iminothiolane/sulfo-SMCC, and sulfo-SMCC). As water-soluble QDs for the conjugation of antibody, we used glutathione coated CdSe/CdZnS QDs (GSH-QDs) with fluorescence quantum yields of 0.23∼0.39 in aqueous solution. Dispersibility, hydrodynamic size, and apparent molecular weights of the GSH-QDs and HER2Ab-QDs were characterized by using dynamic light scattering, fluorescence correlation spectroscopy, atomic force microscope, and size-exclusion HPLC. Fluorescence imaging of HER2 overexpressing cells (KPL-4 human breast cancer cell line) was performed by using HER2Ab-QDs as fluorescent probes. We found that the HER2Ab-QD prepared by using SMCC coupling with partially reduced antibody is a most effective probe for the detection of HER2 expression in KPL-4 cells. We have also studied the size dependency of HER2Ab-QDs (with green, orange, and red emission) on the fluorescence image of KPL-4 cells. PMID:22291567

  4. Pathologic complete response after preoperative anti-HER2 therapy correlates with alterations in PTEN, FOXO, phosphorylated Stat5, and autophagy protein signaling

    PubMed Central

    2013-01-01

    Background To define protein molecular characteristics of tumor cells prior to, and immediately following, preoperative human epidermal growth factor receptor 2 (HER2)-targeted therapy that correlate with pathologic complete response (pCR) or non response (no pCR) to preoperative HER2-directed therapy and chemotherapy. Methods This open-label, phase II study randomized patients with HER2-positive stage II or III invasive breast cancer to trastuzumab, lapatinib, or both, 2 weeks prior to and during chemotherapy with FEC75 for 4 courses; then paclitaxel 80 mg/m2 weekly for 12 courses, then surgery. Core needle biopsies were collected at baseline and after 2 weeks of anti-HER2 therapy prior to chemotherapy. Data were correlated with pCR, defined as absence of invasive tumor in breast and lymph nodes. Results Of 100 enrolled patients, the analysis population included those who had surgery and received ≥75% chemotherapy (78% [n = 78]). pCRs by arm are: trastuzumab (n = 26), 54% [n = 14]; lapatinib (n = 29), 45% [n = 13]; trastuzumab plus lapatinib (n = 23), 74% [n = 17]). Paired biopsy specimens were available for 49 patients (63%). Tumor cells of patients with pCR in the trastuzumab or lapatinib treatment arms showed nonphosphorylated FOXO, phosphorylated Stat5, and sparse signal-transduction protein network crosstalk representing different patterns of connections with PI3K and autophagy proteins compared with no pCR. Conclusion In this exploratory study, pCR with preoperative anti-HER2 therapy and chemotherapy correlated with the levels and phosphorylation status of specific baseline signal pathway proteins in tumor cells. These data may provide candidate biomarkers to stratify initial treatment and potential combination therapies for future study. Tissue preservation technology introduced here makes this procedure widely feasible. Trial registration ClinicalTrials.gov: NCT00524303 PMID:24304724

  5. Is the skin a sanctuary for breast cancer cells during treatment with anti-HER2 antibodies?

    PubMed

    Graziano, Vincenzo; Scognamiglio, Maria Teresa; Zilli, Marinella; Giampietro, Jamara; Vici, Patrizia; Natoli, Clara; Grassadonia, Antonino

    2015-01-01

    The occurrence of skin metastases is a common event in patients affected by advanced breast cancer, usually associated with systemic disease progression. Here we describe 2 cases of diffuse cutaneous metastases from HER2-overexpressing breast cancer occurring despite a dramatic response in liver and bone, respectively, during treatment with anti-HER2 antibodies Trastuzumab and Pertuzumab. We discuss the reasons for this discrepancy and suggest a possible implication of impaired immune response in the skin. Future research should provide strategies to overcome the induction of immune privilege in the skin in order to avoid discontinuation of effective treatments.

  6. Bio-distribution and toxicity assessment of intravenously injected anti-HER2 antibody conjugated CdSe/ZnS quantum dots in Wistar rats

    PubMed Central

    Tiwari, Dhermendra K; Jin, Takashi; Behari, Jitendra

    2011-01-01

    Anti-HER2 antibody conjugated with quantum dots (anti-HER2ab-QDs) is a very recent fluorescent nanoprobe for HER2+ve breast cancer imaging. In this study we investigated in-vivo toxicity of anti-HER2ab conjugated CdSe/ZnS QDs in Wistar rats. For toxicity evaluation of injected QDs sample, body weight, organ coefficient, complete blood count (CBC), biochemistry panel assay (AST, ALT, ALP, and GGTP), comet assay, reactive oxygen species, histology, and apoptosis were determined. Wistar rat (8–10 weeks old) were randomly divided into 4 treatment groups (n = 6). CBC and biochemistry panel assay showed nonsignificant changes in the anti-HER2ab-QDs treated group but these changes were significant (P < 0.05) in QDs treated group. No tissue damage, inflammation, lesions, and QDs deposition were found in histology and TEM images of the anti-HER2ab-QDs treated group. Apoptosis in liver and kidney was not found in the anti-HER2ab-QDs treated group. Animals treated with nonconjugated QDs showed comet formation and apoptosis. Cadmium deposition was confirmed in the QDs treated group compared with the anti-HER2ab-QDs treated group. The QDs concentration (500 nM) used for this study is suitable for in-vivo imaging. The combine data of this study support the biocompatibility of anti-HER2ab-QDs for breast cancer imaging, suggesting that the antibody coating assists in controlling any possible adverse effect of quantum dots. PMID:21499435

  7. A comparison of anti-HER2 IgA and IgG1 in vivo efficacy is facilitated by high N-glycan sialylation of the IgA.

    PubMed

    Rouwendal, Gerard Ja; van der Lee, Miranda M; Meyer, Saskia; Reiding, Karli R; Schouten, Jan; de Roo, Guy; Egging, David F; Leusen, Jeanette Hw; Boross, Peter; Wuhrer, Manfred; Verheijden, Gijs F; Dokter, Wim H; Timmers, Marco; Ubink, Ruud

    2016-01-01

    Monomeric IgA has been proposed as an alternative antibody format for cancer therapy. Here, we present our studies on the production, purification and functional evaluation of anti-HER2 IgA antibodies as anti-cancer agents in comparison to the anti-HER2 IgG1 trastuzumab. MALDI-TOF MS analysis showed profound differences in glycosylation traits across the IgA isotypes and cell lines used for production, including sialylation and linkage thereof, fucosylation (both core and antennary) and the abundance of high-mannose type species. Increases in sialylation proved to positively correlate with in vivo plasma half-lives. The polymerization propensity of anti-HER2 IgA2m2 could be suppressed by an 18-aa deletion of the heavy chain tailpiece - coinciding with the loss of high-mannose type N-glycan species - as well as by 2 cysteine to serine mutations at positions 320 and 480. The HER2 F(ab')2-mediated anti-proliferative effect of the IgA2m1 and IgA2m2 subtypes was similar to IgG1, whereas the IgA1 isotype displayed considerably lower potency and efficacy. The Fc-mediated induction of antibody-dependent cell-mediated cytotoxicity (ADCC) using human whole blood ADCC assays did not demonstrate such clear differences between the IgA isotypes. However, the potency of the anti-HER2 IgA antibodies in these ADCC assays was found to be significantly lower than that of trastuzumab. In vivo anti-tumor activity of the anti-HER2 IgA antibodies was compared to that of trastuzumab in a BT-474 breast cancer xenograft model. Multiple dosing and sialylation of the IgA antibodies compensated for the short in vivo half-life of native IgA antibodies in mice compared to a single dose of IgG1. In the case of the IgA2m2 antibody, the resulting high plasma exposure levels were sufficient to cause clear tumor stasis comparable to that observed for trastuzumab at much lower plasma exposure levels.

  8. A comparison of anti-HER2 IgA and IgG1 in vivo efficacy is facilitated by high N-glycan sialylation of the IgA

    PubMed Central

    Rouwendal, Gerard JA; van der Lee, Miranda M; Meyer, Saskia; Reiding, Karli R; Schouten, Jan; de Roo, Guy; Egging, David F; Leusen, Jeanette HW; Boross, Peter; Wuhrer, Manfred; Verheijden, Gijs F; Dokter, Wim H; Timmers, Marco; Ubink, Ruud

    2016-01-01

    Monomeric IgA has been proposed as an alternative antibody format for cancer therapy. Here, we present our studies on the production, purification and functional evaluation of anti-HER2 IgA antibodies as anti-cancer agents in comparison to the anti-HER2 IgG1 trastuzumab. MALDI-TOF MS analysis showed profound differences in glycosylation traits across the IgA isotypes and cell lines used for production, including sialylation and linkage thereof, fucosylation (both core and antennary) and the abundance of high-mannose type species. Increases in sialylation proved to positively correlate with in vivo plasma half-lives. The polymerization propensity of anti-HER2 IgA2m2 could be suppressed by an 18-aa deletion of the heavy chain tailpiece - coinciding with the loss of high-mannose type N-glycan species - as well as by 2 cysteine to serine mutations at positions 320 and 480. The HER2 F(ab')2-mediated anti-proliferative effect of the IgA2m1 and IgA2m2 subtypes was similar to IgG1, whereas the IgA1 isotype displayed considerably lower potency and efficacy. The Fc-mediated induction of antibody-dependent cell-mediated cytotoxicity (ADCC) using human whole blood ADCC assays did not demonstrate such clear differences between the IgA isotypes. However, the potency of the anti-HER2 IgA antibodies in these ADCC assays was found to be significantly lower than that of trastuzumab. In vivo anti-tumor activity of the anti-HER2 IgA antibodies was compared to that of trastuzumab in a BT-474 breast cancer xenograft model. Multiple dosing and sialylation of the IgA antibodies compensated for the short in vivo half-life of native IgA antibodies in mice compared to a single dose of IgG1. In the case of the IgA2m2 antibody, the resulting high plasma exposure levels were sufficient to cause clear tumor stasis comparable to that observed for trastuzumab at much lower plasma exposure levels. PMID:26440530

  9. Targeted anti-HER2 cancer therapy in elderly women diagnosed with breast cancer.

    PubMed

    Molina-Garrido, M J; Guillen-Ponce, C; Mora-Rufete, A

    2014-06-01

    Trastuzumab or lapatinib treatment with chemotherapy or hormonotherapy are the gold standard treatments for human epidermal growth factor receptor 2 (HER2)-positive breast cancer (early breast cancer or metastatic breast cancer). Older patients have been largely underrepresented in clinical trials, and few data on trastuzumab or lapatinib efficacy and toxicity have been reported for this subgroup. This article has reviewed the main articles that have analyzed these items.

  10. Giacomo Castelvetro's salads. Anti-HER2 oncogene nutraceuticals since the 17th century?

    PubMed

    Colomer, R; Lupu, R; Papadimitropoulou, A; Vellón, L; Vázquez-Martín, A; Brunet, J; Fernández-Gutiérrez, A; Segura-Carretero, A; Menéndez, J A

    2008-01-01

    We are accumulating evidence to suggest that 17(th) century Renaissance foodways -largely based on the old "Mediterranean dietary traditions"- may provide new nutraceutical management strategies against HER2-positive breast cancer disease in the 21st century. Epidemiological and experimental studies begin to support the notion that "The Sacred Law of Salads" (i.e., "raw vegetables... plenty of generous (olive) oil") -originally proposed in 1614 by Giacomo Castelvetro in its book The Fruit, Herbs & Vegetables of Italy- might be considered the first (unintended) example of customised diets for breast cancer prevention based on individual genetic make-up (i.e., nutraceuticals against human breast carcinomas bearing HER2 oncogene amplification/overexpression). First, the so-called salad vegetables dietary pattern (i.e., a high consumption of raw vegetables and olive oil) appears to exert a protective effect mostly confined to the HER2-positive breast cancer subtype, with no significant influence on the occurrence of HER2-negative breast cancers. Second, all the main olive oil constituents (i.e., the omega-9 monounsaturated fatty acid oleic acid and polyphenolic compounds such as the secoiridoid oleuropein or the lignan 1-[+]-acetoxypinoresinol) dramatically reduce HER2 expression and specifically induce apoptotic cell death in cultured HER2- positive breast cancer cells, with marginal effects against HER2-negative cells. Third, an olive oil-rich diet negatively influences experimental mammary tumorigenesis in rats likewise decreasing HER2 expression levels. If early 1600s Castelvetro's salads can be used as dietary protocols capable to protecting women against biologically aggressive HER2-positive breast cancer subtypes is an intriguing prospect that warrants to be evaluated in human pilot studies in the future. Here, at least, we would like to recognise Giacomo Castelvetro as the father of modern nutritional genomics in oncology.

  11. Distinct Receptor Tyrosine Kinase Subsets Mediate Anti-HER2 Drug Resistance in Breast Cancer.

    PubMed

    Alexander, Peter B; Chen, Rui; Gong, Chang; Yuan, Lifeng; Jasper, Jeff S; Ding, Yi; Markowitz, Geoffrey J; Yang, Pengyuan; Xu, Xin; McDonnell, Donald P; Song, Erwei; Wang, Xiao-Fan

    2017-01-13

    Targeted inhibitors of the human epidermal growth factor receptor 2 (HER2), such as trastuzumab and lapatinib, are among the first examples of molecularly targeted cancer therapy and have proven largely effective for the treatment of HER2-positive breast cancers. However, approximately half of those patients either do not respond to these therapies or develop secondary resistance. Although a few signaling pathways have been implicated, a comprehensive understanding of mechanisms underlying HER2 inhibitor drug resistance is still lacking. To address this critical question, we undertook a concerted approach using patient expression data sets, HER2-positive cell lines, and tumor samples biopsied both before and after trastuzumab treatment. Together, these methods revealed that high expression and activation of a specific subset of receptor tyrosine kinases (RTKs) was strongly associated with poor clinical prognosis and the development of resistance. Mechanistically, these RTKs are capable of maintaining downstream signal transduction to promote tumor growth via the suppression of cellular senescence. Consequently, these findings provide the rationale for the design of therapeutic strategies for overcoming drug resistance in breast cancer via combinational inhibition of the limited number of targets from this specific subset of RTKs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Synthesis, characterization, and biological verification of anti-HER2 indocyanine green-doxorubicin-loaded polyethyleneimine-coated perfluorocarbon double nanoemulsions for targeted photochemotherapy of breast cancer cells.

    PubMed

    Lee, Yu-Hsiang; Ma, Yun-Ting

    2017-05-18

    Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females worldwide. Among various types of breast cancer, the human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer is known to be more aggressive and often resistant to medicinal treatment, leading to an insufficient prognosis and poor susceptibility to chemotherapy and/or hormonal therapy in the current clinic. These circumstances implicate that developing an improved therapeutic strategy rather than persistently changing the anticancer drugs for trying is truly needed to successfully cure this type of breast cancer. In this study, we aimed to fabricate anti-HER2 indocyanine green (ICG)-doxorubicin (DOX)-loaded polyethyleneimine-coated perfluorocarbon double nanoemulsions (HIDPPDNEs) to explore the co-administration of phototherapy and chemotherapy for HER2-overexpressing breast cancer in vitro. The HIDPPDNE was first characterized as a sphere-like nanoparticle with surface charge of -57.1 ± 5.6 mV and size of 340.6 ± 4.5 nm, whereas the DOX release rates for the nanodroplets within 48 h in 4 and 37 °C were obtained by 8.13 ± 2.46% and 19.88 ± 2.75%, respectively. We then examined the target-ability of the nanostructure and found that the adhesion efficiency of the HIDPPDNEs onto HER2+ MDA-MB-453 cells was threefold higher than the nanodroplets without anti-HER2 antibody, indicating that the HIDPPDNEs are the product with HER2 binding specificity. In comparison to freely dissolved ICG, the HIDPPDNEs conferred an enhanced thermal stability to the entrapped ICG, and were able to provide a comparable hyperthermia effect and markedly increased production of singlet oxygen under near infrared irradiation (808 nm; 6 W/cm(2)). Based on the viability analyses, the results showed that the HIDPPDNEs were effective on cell eradication upon near infrared irradiation (808 nm; 6 W/cm(2)), and the resulting cell mortality was even higher

  13. Targeting, bio distributive and tumor growth inhibiting characterization of anti-HER2 affibody coupling to liposomal doxorubicin using BALB/c mice bearing TUBO tumors.

    PubMed

    Akhtari, Javad; Rezayat, Seyed Mahdi; Teymouri, Manouchehr; Alavizadeh, Seyedeh Hoda; Gheybi, Fatemeh; Badiee, Ali; Jaafari, Mahmoud Reza

    2016-05-30

    Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20-30% of breast cancer tumors. In the current investigation, we exploited such a feature and utilized an anti-HER2 affibody (ZHER2:477) in combination with a pegylated liposomal doxorubicin (PLD) for concurrent passive and active targeting of HER2 overexpressing TUBO tumor, using BALB/c mice. It was determined that the affibody coupled liposomes (affisomes) was capable of increasing doxorubicin (Dox) delivery to HER2+ cells (SK-BR-3 and TUBO cells), while transferring drug similarly as low as naïve PLD to HER2- MDA-MB-231 cells. This also resulted in selectively enhance cytotoxicity. The veracity of targeting was further assessed utilizing DiD lipophilic tracer model liposomes via competition assay. An approximated 10 ligand/liposome integration caused Dox delivery at 50% of maximal delivery capacity (Kd). Such integration did not alter Dox release in vitro, while it affected the serum clearance profile. Affibody integration to PLD increased drug concentration in tumor and led to significantly further augmentation of drug in liver and spleen compared to those of PLD. Overall, such differences led to prolonging the mice life spans as compared to PLD. Copyright © 2016. Published by Elsevier B.V.

  14. Anti-Neoplastic Cytotoxicity of Gemcitabine-(C4-amide)-[anti-HER2/neu] in Combination with Griseofulvin against Chemotherapeutic-Resistant Mammary Adenocarcinoma (SKBr-3)

    PubMed Central

    Coyne, CP; Jones, Toni; Bear, Ryan

    2015-01-01

    Introduction Gemcitabine is a pyrimidine nucleoside analog that becomes triphosphorylated and in this form it competitively inhibits cytidine incorporation into DNA strands. Diphosphorylated gemcitabine irreversibly inhibits ribonucleotide reductase thereby preventing deoxyribonucleotide synthesis. Functioning as a potent chemotherapeutic, gemcitabine decreases neoplastic cell proliferation and induces apoptosis which accounts for its effectiveness in the clinical treatment of several leukemia and carcinoma cell types. A brief plasma half-life due to rapid deamination, chemotherapeuticresistance and sequelae restricts gemcitabine utility in clinical oncology. Selective “targeted” gemcitabine delivery represents a molecular strategy for prolonging its plasma half-life and minimizing innocent tissue/organ exposure. Methods A previously described organic chemistry scheme was applied to synthesize a UV-photoactivated gemcitabine intermediate for production of gemcitabine-(C4-amide)-[anti-HER2/neu]. Immunodetection analysis (Western-blot) was applied to detect the presence of any degradative fragmentation or polymerization. Detection of retained binding-avidity for gemcitabine-(C4-amide)-[anti-HER2/neu] was determined by cell-ELISA using populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) that highly over-express the HER2/neu trophic membrane receptor. Anti-neoplastic cytotoxicity of gemcitabine-(C4-amide)-[anti-HER2/neu] and the tubulin/microtubule inhibitor, griseofulvin was established against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3). Related investigations evaluated the potential for gemcitabine-(C4-amide)-[anti-HER2/neu] in dual combination with griseofulvin to evoke increased levels of anti-neoplastic cytotoxicity compared to gemcitabine-(C4-amide)-[anti-HER2/neu]. Results Covalent gemcitabine-(C4-amide)-[anti-HER2/neu] immunochemotherapeutic and griseofulvin exerted anti-neoplastic cytotoxicity against chemotherapeutic

  15. Mutant PIK3CA accelerates HER2-driven transgenic mammary tumors and induces resistance to combinations of anti-HER2 therapies.

    PubMed

    Hanker, Ariella B; Pfefferle, Adam D; Balko, Justin M; Kuba, María Gabriela; Young, Christian D; Sánchez, Violeta; Sutton, Cammie R; Cheng, Hailing; Perou, Charles M; Zhao, Jean J; Cook, Rebecca S; Arteaga, Carlos L

    2013-08-27

    Human epidermal growth factor receptor 2 (HER2; ERBB2) amplification and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations often co-occur in breast cancer. Aberrant activation of the phosphatidylinositol 3-kinase (PI3K) pathway has been shown to correlate with a diminished response to HER2-directed therapies. We generated a mouse model of HER2-overexpressing (HER2(+)), PIK3CA(H1047R)-mutant breast cancer. Mice expressing both human HER2 and mutant PIK3CA in the mammary epithelium developed tumors with shorter latencies compared with mice expressing either oncogene alone. HER2 and mutant PIK3CA also cooperated to promote lung metastases. By microarray analysis, HER2-driven tumors clustered with luminal breast cancers, whereas mutant PIK3CA tumors were associated with claudin-low breast cancers. PIK3CA and HER2(+)/PIK3CA tumors expressed elevated transcripts encoding markers of epithelial-to-mesenchymal transition and stem cells. Cells from HER2(+)/PIK3CA tumors more efficiently formed mammospheres and lung metastases. Finally, HER2(+)/PIK3CA tumors were resistant to trastuzumab alone and in combination with lapatinib or pertuzumab. Both drug resistance and enhanced mammosphere formation were reversed by treatment with a PI3K inhibitor. In sum, PIK3CA(H1047R) accelerates HER2-mediated breast epithelial transformation and metastatic progression, alters the intrinsic phenotype of HER2-overexpressing cancers, and generates resistance to approved combinations of anti-HER2 therapies.

  16. Cancer Cell Targeting Using Folic Acid/Anti-HER2 Antibody Conjugated Fluorescent CdSe/CdS/ZnS-Mercaptopropionic Acid and CdTe-Mercaptosuccinic Acid Quantum Dots.

    PubMed

    Singh, Gurpal; Kumar, Manoj; Soni, Udit; Arora, Vikas; Bansal, Vivek; Gupta, Dikshi; Bhat, Madhusudan; Dinda, Amit K; Sapra, Sameer; Singh, Harpal

    2016-01-01

    CdSe/CdS/ZnS and CdTe quantum dots (QDs) were synthesized by successive ion layer adsorption and reaction (SILAR) technique and direct aqueous synthesis respectively using thiol stabilizers. Synthesized CdSe/CdS/ZnS and CdTe QDs stabilized with 3-mercaptopropionic acid (MPA) and mercaptosuccinic acid (MSA) were used as fluorescent labels after conjugation with folic acid (FA) and anti-HER2 antibodies. Photoluminescence quantum yield of folated CdSe/CdS/ZnS-MPA and CdTe-MSA QDs was 59% and 77% than that of non-folated hydrophilic QDs. The folate receptor-mediated delivery of folic acid-conjugated CdTe-MSA and CdSe/CdS/ZnS-MPA QDs showed higher cellular internalization as observed by confocal laser scanning microscopic studies. Folated and non-folated CdTe-MSA QDs were highly toxic and exhibited only 10% cell viability as compared to > 80% cell viability with CdSe/CdS/ZnS-MPA QDs over the concentration ranging from 3.38 to 50 pmoles. Immunohistochemistry (IHC) results of human breast cancer tissue samples showed positive results with anti-HER2 antibody conjugated CdSe/CdS/ZnS-MPA QDs with better sensitivity and specificity as compared to conventional IHC analysis using diaminobenzedene staining.

  17. A novel inhibitor of fatty acid synthase shows activity against HER2+ breast cancer xenografts and is active in anti-HER2 drug-resistant cell lines

    PubMed Central

    2011-01-01

    Introduction Inhibiting the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of breast carcinoma cells, and this is linked to human epidermal growth factor receptor 2 (HER2) signaling pathways in models of simultaneous expression of FASN and HER2. Methods In a xenograft model of breast carcinoma cells that are FASN+ and HER2+, we have characterised the anticancer activity and the toxicity profile of G28UCM, the lead compound of a novel family of synthetic FASN inhibitors. In vitro, we analysed the cellular and molecular interactions of combining G28UCM with anti-HER drugs. Finally, we tested the cytotoxic ability of G28UCM on breast cancer cells resistant to trastuzumab or lapatinib, that we developed in our laboratory. Results In vivo, G28UCM reduced the size of 5 out of 14 established xenografts. In the responding tumours, we observed inhibition of FASN activity, cleavage of poly-ADPribose polymerase (PARP) and a decrease of p-HER2, p- protein kinase B (AKT) and p-ERK1/2, which were not observed in the nonresponding tumours. In the G28UCM-treated animals, no significant toxicities occurred, and weight loss was not observed. In vitro, G28UCM showed marked synergistic interactions with trastuzumab, lapatinib, erlotinib or gefitinib (but not with cetuximab), which correlated with increases in apoptosis and with decreases in the activation of HER2, extracellular signal-regulated kinase (ERK)1/2 and AKT. In trastuzumab-resistant and in lapatinib-resistant breast cancer cells, in which trastuzumab and lapatinib were not effective, G28UCM retained the anticancer activity observed in the parental cells. Conclusions G28UCM inhibits fatty acid synthase (FASN) activity and the growth of breast carcinoma xenografts in vivo, and is active in cells with acquired resistance to anti-HER2 drugs, which make it a candidate for further pre-clinical development. PMID:22177475

  18. Restoring Lost Anti-HER-2 Th1 Immunity in Breast Cancer: A Crucial Role for Th1 Cytokines in Therapy and Prevention

    PubMed Central

    Nocera, Nadia F.; Lee, M. Catherine; De La Cruz, Lucy M.; Rosemblit, Cinthia; Czerniecki, Brian J.

    2016-01-01

    The ErbB/B2 (HER-2/neu) oncogene family plays a critical role in the development and metastatic spread of several tumor types including breast, ovarian and gastric cancer. In breast cancer, HER-2/neu is expressed in early disease development in a large percentage of DCIS lesions and its expression is associated with an increased risk of invasion and recurrence. Targeting HER-2 with antibodies such as trastuzumab or pertuzumab has improved survival, but patients with more extensive disease may develop resistance to therapy. Interestingly, response to HER-2 targeted therapies correlates with presence of immune response genes in the breast. Th1 cell production of the cytokines interferon gamma (IFNγ) and TNFα can enhance MHC class I expression, PD-L1 expression, augment apoptosis and tumor senescence, and enhances growth inhibition of many anti-breast cancer agents, including anti-estrogens and HER-2 targeted therapies. Recently, we have identified that a loss of anti-HER-2 CD4 Th1 in peripheral blood occurs during breast tumorigenesis and is dramatically diminished, even in Stage I breast cancers. The loss of anti-HER-2 Th1 response is specific and not readily reversed by standard therapies. In fact, this loss of anti-HER-2 Th1 response in peripheral blood correlates with lack of complete response to neoadjuvant therapy and diminished disease-free survival. This defect can be restored with HER-2 vaccinations in both DCIS and IBC. Correcting the anti-HER-2 Th1 response may have significant impact in improving response to HER-2 targeted therapies. Development of immune monitoring systems for anti-HER-2 Th1 to identify patients at risk for recurrence could be critical to improving outcomes, since the anti-HER-2 Th1 response can be restored by vaccination. Correction of the cellular immune response against HER-2 may prevent recurrence in high-risk patients with DCIS and IBC at risk of developing new or recurrent breast cancer. PMID:27766079

  19. Upregulation of ER signaling as an adaptive mechanism of cell survival in HER2-positive breast tumors treated with anti-HER2 therapy

    PubMed Central

    Giuliano, Mario; Hu, Huizhong; Wang, Yen-Chao; Fu, Xiaoyong; Nardone, Agostina; Herrera, Sabrina; Mao, Sufeng; Contreras, Alejandro; Gutierrez, Carolina; Wang, Tao; Hilsenbeck, Susan G.; De Angelis, Carmine; Wang, Nicholas J.; Heiser, Laura M.; Gray, Joe W.; Lopez-Tarruella, Sara; Pavlick, Anne C.; Trivedi, Meghana V.; Chamness, Gary C.; Chang, Jenny C.; Osborne, C. Kent; Rimawi, Mothaffar F.; Schiff, Rachel

    2015-01-01

    Purpose To investigate the direct effect and therapeutic consequences of epidermal growth factor receptor 2 (HER2)-targeting therapy on expression of estrogen receptor (ER) and Bcl2 in preclinical models and clinical tumor samples. Experimental design Archived xenograft tumors from two preclinical models (UACC812 and MCF7/HER2-18) treated with ER and HER2-targeting therapies, and also HER2+ clinical breast cancer specimens collected in a lapatinib neoadjuvant trial (baseline and week 2 post treatment), were used. Expression levels of ER and Bcl2 were evaluated by immunohistochemistry and western blot. The effects of Bcl2 and ER inhibition, by ABT-737 and fulvestrant respectively, were tested in parental versus lapatinib-resistant UACC812 cells in vitro. Results Expression of ER and Bcl2 was significantly increased in xenograft tumors with acquired resistance to anti-HER2 therapy, compared with untreated tumors, in both preclinical models (UACC812: ER p=0.0014; Bcl2 p<0.001. MCF7/HER2-18: ER p=0.0007; Bcl2 p=0.0306). In the neoadjuvant clinical study, lapatinib treatment for two weeks was associated with parallel upregulation of ER and Bcl2 (Spearman’s coefficient: 0.70; p=0.0002). Importantly, 18% of tumors originally ER-negative (ER−) converted to ER+ upon anti-HER2 therapy. In ER−/HER2+ MCF7/HER2-18 xenografts, ER re-expression was primarily observed in tumors responding to potent combination of anti-HER2 drugs. Estrogen deprivation added to this anti-HER2 regimen significantly delayed tumor progression (p=0.018). In the UACC812 cells, fulvestrant, but not ABT-737, was able to completely inhibit anti-HER2-resistant growth (p<0.0001). Conclusion HER2 inhibition can enhance or restore ER expression with parallel Bcl2 upregulation, representing an ER-dependent survival mechanism potentially leading to anti-HER2 resistance. PMID:26015514

  20. Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1.

    PubMed

    Sochaj-Gregorczyk, Alicja M; Ludzia, Patryk; Kozdrowska, Emilia; Jakimowicz, Piotr; Sokolowska-Wedzina, Aleksandra; Otlewski, Jacek

    2017-08-03

    In our previous work we demonstrated that a small protein called affibody can be used for a cytotoxic conjugate development. The anti-HER2 affibody was armed with one moiety of a highly potent auristatin E and specifically killed HER2-positive cancer cells with a nanomolar IC50. The aim of this study was to improve the anti-HER2 affibody conjugate by increasing its size and the number of conjugated auristatin molecules. The affibody was fused to the Fc fragment of IgG1 resulting in a dimeric construct with the molecular weight of 68 kDa, referred to as ZHER2:2891-Fc, ensuring its prolonged half-life in the blood. Due to the presence of four interchain cysteines, the fusion protein could carry four drug molecules. Notably, the in vitro tests of the improved anti-HER2 conjugate revealed that it exhibits the IC50 of 130 pM for the HER2-positive SK-BR-3 cells and 98 nM for the HER2-negative MDA-MB-231 cells. High efficacy and specificity of the auristatin conjugate based on ZHER2:2891-Fc indicate that this construct is suitable for further in vivo evaluation.

  1. Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1

    PubMed Central

    Sochaj-Gregorczyk, Alicja M.; Ludzia, Patryk; Kozdrowska, Emilia; Jakimowicz, Piotr; Sokolowska-Wedzina, Aleksandra; Otlewski, Jacek

    2017-01-01

    In our previous work we demonstrated that a small protein called affibody can be used for a cytotoxic conjugate development. The anti-HER2 affibody was armed with one moiety of a highly potent auristatin E and specifically killed HER2-positive cancer cells with a nanomolar IC50. The aim of this study was to improve the anti-HER2 affibody conjugate by increasing its size and the number of conjugated auristatin molecules. The affibody was fused to the Fc fragment of IgG1 resulting in a dimeric construct with the molecular weight of 68 kDa, referred to as ZHER2:2891-Fc, ensuring its prolonged half-life in the blood. Due to the presence of four interchain cysteines, the fusion protein could carry four drug molecules. Notably, the in vitro tests of the improved anti-HER2 conjugate revealed that it exhibits the IC50 of 130 pM for the HER2-positive SK-BR-3 cells and 98 nM for the HER2-negative MDA-MB-231 cells. High efficacy and specificity of the auristatin conjugate based on ZHER2:2891-Fc indicate that this construct is suitable for further in vivo evaluation. PMID:28771178

  2. In vitro characterization of a bivalent anti-HER-2 affibody with potential for radionuclide-based diagnostics.

    PubMed

    Steffen, Ann-Charlott; Wikman, Maria; Tolmachev, Vladimir; Adams, Gregory P; Nilsson, Fredrik Y; Ståhl, Stefan; Carlsson, Jörgen

    2005-06-01

    The 185 kDa transmembrane glycoprotein human epidermal growth factor receptor 2 (HER-2) (p185/neu, c-ErbB-2) is overexpressed in breast and ovarian cancers. Overexpression in breast cancer correlates with poor patient prognosis, and visualization of HER-2 expression might provide valuable diagnostic information influencing patient management. We have previously described the generation of a new type of affinity ligand, a 58-amino-acid affibody (Z(HER2:4)) with specific binding to HER-2. In order to benefit from avidity effects, we have created a bivalent form of the affibody ligand, (Z(HER2:4))2. The monovalent and bivalent ligands were compared in various assays. The new bivalent affibody has a molecular weight of 15.6 kDa and an apparent affinity (K(D)) against HER-2 of 3 nM. After radioiodination, using the linker molecule N-succinimidyl p-(trimethylstannyl) benzoate (SPMB), in vitro binding assays showed specific binding to HER-2 overexpressing cells. Internalization of 125I was shown after delivery with both the monovalent and the bivalent affibody. The cellular retention of 125I was longer after delivery with the bivalent affibody when compared to delivery with the monovalent affibody. With approximately the same affinity as the monoclonal antibody trastuzumab (Herceptin) but only one tenth of the size, this new bivalent molecule is a promising candidate for radionuclide-based detection of HER-2 expression in tumors. 125I was used in this study as a surrogate marker for the diagnostically relevant radioisotopes 123I for single photon emission computed tomography (SPECT)/gamma-camera imaging and 124I for positron emission tomography (PET).

  3. tabAnti-HER2 (erbB-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO)

    PubMed Central

    Menendez, Javier A; Vazquez-Martin, Alejandro; Garcia-Villalba, Rocio; Carrasco-Pancorbo, Alegria; Oliveras-Ferraros, Cristina; Fernandez-Gutierrez, Alberto; Segura-Carretero, Antonio

    2008-01-01

    Background The effects of the olive oil-rich Mediterranean diet on breast cancer risk might be underestimated when HER2 (ERBB2) oncogene-positive and HER2-negative breast carcinomas are considered together. We here investigated the anti-HER2 effects of phenolic fractions directly extracted from Extra Virgin Olive Oil (EVOO) in cultured human breast cancer cell lines. Methods Solid phase extraction followed by semi-preparative high-performance liquid chromatography (HPLC) was used to isolate phenolic fractions from commercial EVOO. Analytical capillary electrophoresis coupled to mass spectrometry was performed to check for the composition and to confirm the identity of the isolated fractions. EVOO polyphenolic fractions were tested on their tumoricidal ability against HER2-negative and HER2-positive breast cancer in vitro models using MTT, crystal violet staining, and Cell Death ELISA assays. The effects of EVOO polyphenolic fractions on the expression and activation status of HER2 oncoprotein were evaluated using HER2-specific ELISAs and immunoblotting procedures, respectively. Results Among the fractions mainly containing the single phenols hydroxytyrosol and tyrosol, the polyphenol acid elenolic acid, the lignans (+)-pinoresinol and 1-(+)-acetoxypinoresinol, and the secoiridoids deacetoxy oleuropein aglycone, ligstroside aglycone, and oleuropein aglycone, all the major EVOO polyphenols (i.e. secoiridoids and lignans) were found to induce strong tumoricidal effects within a micromolar range by selectively triggering high levels of apoptotic cell death in HER2-overexpressors. Small interfering RNA-induced depletion of HER2 protein and lapatinib-induced blockade of HER2 tyrosine kinase activity both significantly prevented EVOO polyphenols-induced cytotoxicity. EVOO polyphenols drastically depleted HER2 protein and reduced HER2 tyrosine autophosphorylation in a dose- and time-dependent manner. EVOO polyphenols-induced HER2 downregulation occurred regardless the

  4. Synthesis, Characterization, and Biological Evaluation of Anti-HER2 Indocyanine Green-Encapsulated PEG-Coated PLGA Nanoparticles for Targeted Phototherapy of Breast Cancer Cells

    PubMed Central

    Lee, Yu-Hsiang; Lai, Yun-Han

    2016-01-01

    Human epidermal growth factor receptor 2 (HER2)-overexpressed breast cancer is known to be more aggressive and resistant to medicinal treatment and therefore to whom an alternative therapeutics is needed. Indocyanine green (ICG) has been widely exploited in breast cancer phototherapy. However, drawbacks of accelerated degradation and short half-life (2–4 min) in blood seriously hamper its use in the clinic. To overcome these challenges, an anti-HER2 ICG-encapsulated polyethylene glycol-coated poly(lactic-co-glycolic acid) nanoparticles (HIPPNPs) were developed in this study. Through the analyses of degradation rate coefficients of ICG with and without polymeric encapsulation, the photostability of HIPPNP-entrapped ICG significantly enhanced 4 folds (P < 0.05) while its thermal stabilities at 4 and 37°C significantly enhanced 5 and 3 (P < 0.05 for each) folds, respectively, under equal lighting and/or heating treatment for 48 h. The target specificity of HIPPNPs to HER2-positive cells was demonstrated based on a 6-fold (P < 0.05) enhancement of uptake efficiency of HIPPNPs in MDA-MB-453/HER2(+) cells within 4 h as compared with that in MCF7/HER2(-) cells. Moreover, the HIPPNPs with ≤ 25 μM ICG equivalent were nontoxic to cells in the absence of light illumination, and enabled to generate similar amount of singlet oxygen and hyperthermia effect as compared with that used by free ICG upon NIR irradiation. After 808 nm-laser irradiation with intensity of 6 W/cm2 for 5 min, the viability of MDA-MB-453 cells pre-treated by HIPPNPs with ≥ 5 μM ICG equivalent for 4 h significantly reduced as compared with that treated by equal concentration of free ICG (P < 0.05) and > 90% of the cells were eradicated while the dose of HIPPNPs was increased to 25 μM ICG equivalent. In summary, the developed HIPPNPs are anticipated as a feasible tool for use in phototherapy of breast cancer cells with HER2 expression. PMID:27942034

  5. Synthesis, Characterization, and Biological Evaluation of Anti-HER2 Indocyanine Green-Encapsulated PEG-Coated PLGA Nanoparticles for Targeted Phototherapy of Breast Cancer Cells.

    PubMed

    Lee, Yu-Hsiang; Lai, Yun-Han

    2016-01-01

    Human epidermal growth factor receptor 2 (HER2)-overexpressed breast cancer is known to be more aggressive and resistant to medicinal treatment and therefore to whom an alternative therapeutics is needed. Indocyanine green (ICG) has been widely exploited in breast cancer phototherapy. However, drawbacks of accelerated degradation and short half-life (2-4 min) in blood seriously hamper its use in the clinic. To overcome these challenges, an anti-HER2 ICG-encapsulated polyethylene glycol-coated poly(lactic-co-glycolic acid) nanoparticles (HIPPNPs) were developed in this study. Through the analyses of degradation rate coefficients of ICG with and without polymeric encapsulation, the photostability of HIPPNP-entrapped ICG significantly enhanced 4 folds (P < 0.05) while its thermal stabilities at 4 and 37°C significantly enhanced 5 and 3 (P < 0.05 for each) folds, respectively, under equal lighting and/or heating treatment for 48 h. The target specificity of HIPPNPs to HER2-positive cells was demonstrated based on a 6-fold (P < 0.05) enhancement of uptake efficiency of HIPPNPs in MDA-MB-453/HER2(+) cells within 4 h as compared with that in MCF7/HER2(-) cells. Moreover, the HIPPNPs with ≤ 25 μM ICG equivalent were nontoxic to cells in the absence of light illumination, and enabled to generate similar amount of singlet oxygen and hyperthermia effect as compared with that used by free ICG upon NIR irradiation. After 808 nm-laser irradiation with intensity of 6 W/cm2 for 5 min, the viability of MDA-MB-453 cells pre-treated by HIPPNPs with ≥ 5 μM ICG equivalent for 4 h significantly reduced as compared with that treated by equal concentration of free ICG (P < 0.05) and > 90% of the cells were eradicated while the dose of HIPPNPs was increased to 25 μM ICG equivalent. In summary, the developed HIPPNPs are anticipated as a feasible tool for use in phototherapy of breast cancer cells with HER2 expression.

  6. Monoclonal Antibody That Defines Human Myoepithelium

    NASA Astrophysics Data System (ADS)

    Dairkee, Shahnaz Hashmi; Blayney, Carlene; Smith, Helene S.; Hackett, Adeline J.

    1985-11-01

    We have isolated a mouse monoclonal antibody that, upon immunohistochemical localization in frozen sections, displays specificity for human myoepithelial cells in the resting mammary gland, sweat glands, and salivary glands. Furthermore, this antibody was strongly and homogeneously reactive with frozen sections of 3 of 60 breast carcinoma specimens. Using immunolocalization techniques in conjunction with polyacrylamide gel electrophoresis, we have determined that the reactivity of this monoclonal antibody is directed toward a 51,000-dalton keratin polypeptide. The potential uses of this antibody in the prognosis of human mammary carcinoma and in understanding the role of the myoepithelium in development and differentiation are discussed.

  7. Dual Fatty Acid Synthase and HER2 Signaling Blockade Shows Marked Antitumor Activity against Breast Cancer Models Resistant to Anti-HER2 Drugs

    PubMed Central

    Blancafort, Adriana; Giró-Perafita, Ariadna; Oliveras, Glòria; Palomeras, Sònia; Turrado, Carlos; Campuzano, Òscar; Carrión-Salip, Dolors; Massaguer, Anna; Brugada, Ramon; Palafox, Marta; Gómez-Miragaya, Jorge; González-Suárez, Eva; Puig, Teresa

    2015-01-01

    Blocking the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of HER2-positive breast carcinoma cells. The hypothesis is that blocking FASN, in combination with anti-HER2 signaling agents, would be an effective antitumor strategy in preclinical HER2+ breast cancer models of trastuzumab and lapatinib resistance. We developed and molecularly characterized in vitro HER2+ models of resistance to trastuzumab (SKTR), lapatinib (SKLR) and both (SKLTR). The cellular interactions of combining anti-FASN polyphenolic compounds (EGCG and the synthetic G28UCM) with anti-HER2 signaling drugs (trastuzumab plus pertuzumab and temsirolimus) were analyzed. Tumor growth inhibition after treatment with EGCG, pertuzumab, temsirolimus or the combination was evaluated in two in vivo orthoxenopatients: one derived from a HER2+ patient and another from a patient who relapsed on trastuzumab and lapatinib-based therapy. SKTR, SKLR and SKLTR showed hyperactivation of EGFR and p-ERK1/2 and PI3KCA mutations. Dual-resistant cells (SKLTR) also showed hyperactivation of HER4 and recovered levels of p-AKT compared with mono-resistant cells. mTOR, p-mTOR and FASN expression remained stable in SKTR, SKLR and SKLTR. In vitro, anti-FASN compounds plus pertuzumab showed synergistic interactions in lapatinib- and dual- resistant cells and improved the results of pertuzumab plus trastuzumab co-treatment. FASN inhibitors combined with temsirolimus displayed the strongest synergistic interactions in resistant cells. In vivo, both orthoxenopatients showed strong response to the antitumor activity of the combination of EGCG with pertuzumab or temsirolimus, without signs of toxicity. We showed that the simultaneous blockade of FASN and HER2 pathways is effective in cells and in breast cancer models refractory to anti-HER2 therapies. PMID:26107737

  8. Dual fatty acid synthase and HER2 signaling blockade shows marked antitumor activity against breast cancer models resistant to anti-HER2 drugs.

    PubMed

    Blancafort, Adriana; Giró-Perafita, Ariadna; Oliveras, Glòria; Palomeras, Sònia; Turrado, Carlos; Campuzano, Òscar; Carrión-Salip, Dolors; Massaguer, Anna; Brugada, Ramon; Palafox, Marta; Gómez-Miragaya, Jorge; González-Suárez, Eva; Puig, Teresa

    2015-01-01

    Blocking the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of HER2-positive breast carcinoma cells. The hypothesis is that blocking FASN, in combination with anti-HER2 signaling agents, would be an effective antitumor strategy in preclinical HER2+ breast cancer models of trastuzumab and lapatinib resistance. We developed and molecularly characterized in vitro HER2+ models of resistance to trastuzumab (SKTR), lapatinib (SKLR) and both (SKLTR). The cellular interactions of combining anti-FASN polyphenolic compounds (EGCG and the synthetic G28UCM) with anti-HER2 signaling drugs (trastuzumab plus pertuzumab and temsirolimus) were analyzed. Tumor growth inhibition after treatment with EGCG, pertuzumab, temsirolimus or the combination was evaluated in two in vivo orthoxenopatients: one derived from a HER2+ patient and another from a patient who relapsed on trastuzumab and lapatinib-based therapy. SKTR, SKLR and SKLTR showed hyperactivation of EGFR and p-ERK1/2 and PI3KCA mutations. Dual-resistant cells (SKLTR) also showed hyperactivation of HER4 and recovered levels of p-AKT compared with mono-resistant cells. mTOR, p-mTOR and FASN expression remained stable in SKTR, SKLR and SKLTR. In vitro, anti-FASN compounds plus pertuzumab showed synergistic interactions in lapatinib- and dual- resistant cells and improved the results of pertuzumab plus trastuzumab co-treatment. FASN inhibitors combined with temsirolimus displayed the strongest synergistic interactions in resistant cells. In vivo, both orthoxenopatients showed strong response to the antitumor activity of the combination of EGCG with pertuzumab or temsirolimus, without signs of toxicity. We showed that the simultaneous blockade of FASN and HER2 pathways is effective in cells and in breast cancer models refractory to anti-HER2 therapies.

  9. Comparison between Internalizing Anti-HER2 mAbs and Non-Internalizing Anti-CEA mAbs in Alpha-Radioimmunotherapy of Small Volume Peritoneal Carcinomatosis Using 212Pb

    PubMed Central

    Busson, Muriel; Garambois, Véronique; Jarlier, Marta; Charalambatou, Paraskevi; Pèlegrin, André; Paillas, Salomé; Chouin, Nicolas; Quenet, François; Maquaire, Patrick; Torgue, Julien; Navarro-Teulon, Isabelle; Pouget, Jean-Pierre

    2013-01-01

    Background and Purpose We assessed the contribution of antibody internalization in the efficacy and toxicity of intraperitoneal α-radioimmunotherapy (RIT) of small volume carcinomatosis using 212Pb-labeled monoclonal antibodies (mAbs) that target HER2 (internalizing) or CEA (non-internalizing) receptors. Materials and Methods Athymic nude mice bearing 2–3 mm intraperitoneal tumor xenografts were intraperitoneally injected with similar activities (370, 740 and 1480 kBq; 37 MBq/mg) of 212Pb-labeled 35A7 (anti-CEA), trastuzumab (anti-HER2) or PX (non-specific) mAbs, or with equivalent amounts of unlabeled mAbs, or with NaCl. Tumor volume was monitored by bioluminescence and survival was reported. Hematologic toxicity and body weight were assessed. Biodistribution of 212Pb-labeled mAbs and absorbed dose-effect relationships using MIRD formalism were established. Results Transient hematological toxicity, as revealed by white blood cells and platelets numbering, was reported in mice treated with the highest activities of 212Pb-labeled mAbs. The median survival (MS) was significantly higher in mice injected with 1.48 MBq of 212Pb-35A7 (non-internalizing mAbs) (MS = 94 days) than in animals treated with the same activity of 212Pb-PX mAbs or with NaCl (MS = 18 days). MS was even not reached after 130 days when follow-up was discontinued in mice treated with 1.48 MBq of 212Pb-trastuzumab. The later efficacy was unexpected since final absorbed dose resulting from injection of 1.48 MBq, was higher for 212Pb-35A7 (35.5 Gy) than for 212Pb-trastuzumab (27.6 Gy). These results also highlight the lack of absorbed dose-effect relationship when mean absorbed dose was calculated using MIRD formalism and the requirement to perform small-scale dosimetry. Conclusions These data indicate that it might be an advantage of using internalizing anti-HER2 compared with non-internalizing anti-CEA 212Pb-labeled mAbs in the therapy of small volume xenograft tumors. They support clinical

  10. Use of Human Hybridoma Technology To Isolate Human Monoclonal Antibodies.

    PubMed

    Smith, Scott A; Crowe, James E

    2015-02-01

    The human hybridoma technique offers an important approach for isolation of human monoclonal antibodies. A diversity of approaches can be used with varying success. Recent technical advances in expanding the starting number of human antigen-specific B cells, improving fusion efficiency, and isolating new myeloma partners and new cell cloning methods have enabled the development of protocols that make the isolation of human monoclonal antibodies from blood samples feasible. Undoubtedly, additional innovations that could improve efficiency are possible.

  11. Synthesis, preclinical validation, dosimetry, and toxicity of 68Ga-NOTA-anti-HER2 Nanobodies for iPET imaging of HER2 receptor expression in cancer.

    PubMed

    Xavier, Catarina; Vaneycken, Ilse; D'huyvetter, Matthias; Heemskerk, Johannes; Keyaerts, Marleen; Vincke, Cécile; Devoogdt, Nick; Muyldermans, Serge; Lahoutte, Tony; Caveliers, Vicky

    2013-05-01

    .0218 mSv/MBq, which would yield a radiation dose of 4 mSv to a patient after injection of 185 MBq of (68)Ga-NOTA-2Rs15d. In the toxicity study, no adverse effects were observed after injection of a 10 mg/kg dose of NOTA-2Rs15d. A new anti-HER2 PET tracer, (68)Ga-NOTA-2Rs15d, was synthesized via a rapid procedure under mild conditions. Preclinical validation showed high-specific-contrast imaging of HER2-positive tumors with no observed toxicity. (68)Ga-NOTA-2Rs15d is ready for first-in-human clinical trials.

  12. Preclinical Evaluation of 18F-Labeled Anti-HER2 Nanobody Conjugates for Imaging HER2 Receptor Expression by ImmunoPET

    PubMed Central

    Vaidyanathan, Ganesan; McDougald, Darryl; Choi, Jaeyeon; Koumarianou, Eftychia; Weitzel, Douglas; Osada, Takuya; Lyerly, H. Kim; Zalutsky, Michael R.

    2016-01-01

    The human growth factor receptor type 2 (HER2) is overexpressed in breast as well as other types of cancer. ImmunoPET, a noninvasive imaging procedure that could assess HER2 status in both primary and metastatic lesions simultaneously, could be a valuable tool for optimizing application of HER2-targeted therapies in individual patients. Herein, we have evaluated the tumor targeting potential of the 5F7 anti-HER2 Nanobody (single-domain antibody fragment; ~13 kDa) after 18F labeling by two methods. Methods The 5F7 Nanobody was labeled with 18F using the novel residualizing label N-succinimidyl 3-((4-(4-18F-fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate (18F-SFBTMGMB; 18F-RL-I) and also via the most commonly utilized 18F protein labeling prosthetic agent, N-succinimidyl 3-18F-fluorobenzoate (18F-SFB). For comparison, 5F7 Nanobody was also labeled using the residualizing radioiodination agent N-succinimidyl 4-guanidinomethyl-3-125I-iodobenzoate (125I-SGMIB). Paired label (18F/125I) internalization assays and biodistribution studies were performed on HER2-expressing BT474M1 breast carcinoma cells and in mice with BT474M1 subcutaneous xenografts, respectively. Micro positron emission tomography/computed tomography (microPET/CT) imaging of 5F7 Nanobody labeled using 18F-RL-I also was performed. Results Internalization assays indicated that intracellularly retained radioactivity for 18F-RL-I-5F7 was similar to that for co-incubated 125I-SGMIB-5F7, while that for 18F-SFB-5F7 was lower than co-incubated 125I-SGMIB-5F7 and decreased with time. BT474M1 tumor uptake of 18F-RL-I-5F7 was 28.97 ± 3.88 %ID/g at 1 h and 36.28 ± 14.10 %ID/g at 2 h, reduced by >90% trastuzumab blocking, indicating HER2-specificity of uptake, and also 26–28% higher (P < 0.05) than that of 18F-SFB-5F7. At 2 h, the tumor-to-blood ratio for 18F-RL-I-5F7 (47.4 ± 13.1) was significantly higher (P < 0.05) than for 18F-SFB-5F7 (25.4 ± 10.3); however, kidney uptake was 28–36

  13. Preclinical Evaluation of 18F-Labeled Anti-HER2 Nanobody Conjugates for Imaging HER2 Receptor Expression by Immuno-PET.

    PubMed

    Vaidyanathan, Ganesan; McDougald, Darryl; Choi, Jaeyeon; Koumarianou, Eftychia; Weitzel, Douglas; Osada, Takuya; Lyerly, H Kim; Zalutsky, Michael R

    2016-06-01

    The human growth factor receptor type 2 (HER2) is overexpressed in breast as well as other types of cancer. Immuno-PET, a noninvasive imaging procedure that could assess HER2 status in both primary and metastatic lesions simultaneously, could be a valuable tool for optimizing application of HER2-targeted therapies in individual patients. Herein, we have evaluated the tumor-targeting potential of the 5F7 anti-HER2 Nanobody (single-domain antibody fragment; ∼13 kDa) after (18)F labeling by 2 methods. The 5F7 Nanobody was labeled with (18)F using the novel residualizing label N-succinimidyl 3-((4-(4-(18)F-fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ((18)F-SFBTMGMB; (18)F-RL-I) and also via the most commonly used (18)F protein-labeling prosthetic agent N-succinimidyl 3-(18)F-fluorobenzoate ((18)F-SFB). For comparison, 5F7 Nanobody was also labeled using the residualizing radioiodination agent N-succinimidyl 4-guanidinomethyl-3-(125)I-iodobenzoate ((125)I-SGMIB). Paired-label ((18)F/(125)I) internalization assays and biodistribution studies were performed on HER2-expressing BT474M1 breast carcinoma cells and in mice with BT474M1 subcutaneous xenografts, respectively. Small-animal PET/CT imaging of 5F7 Nanobody labeled using (18)F-RL-I also was performed. Internalization assays indicated that intracellularly retained radioactivity for (18)F-RL-I-5F7 was similar to that for coincubated (125)I-SGMIB-5F7, whereas that for (18)F-SFB-5F7 was lower than coincubated (125)I-SGMIB-5F7 and decreased with time. BT474M1 tumor uptake of (18)F-RL-I-5F7 was 28.97 ± 3.88 percentage injected dose per gram of tissue (%ID/g) at 1 h and 36.28 ± 14.10 %ID/g at 2 h, reduced by more than 90% on blocking with trastuzumab, indicating HER2 specificity of uptake, and was also 26%-28% higher (P < 0.05) than that of (18)F-SFB-5F7. At 2 h, the tumor-to-blood ratio for (18)F-RL-I-5F7 (47.4 ± 13.1) was significantly higher (P < 0.05) than for (18)F-SFB-5F7 (25.4 ± 10

  14. Production of Monoclonal Antibody against Human Nestin.

    PubMed

    Hadavi, Reza; Zarnani, Amir Hassan; Ahmadvand, Negah; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Sadeghi, Mohammad-Reza; Soltanghoraee, Haleh; Akhondi, Mohammad Mehdi; Tarahomi, Majid; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2010-04-01

    We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140-250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays.

  15. Production of Monoclonal Antibody against Human Nestin

    PubMed Central

    Hadavi, Reza; Zarnani, Amir Hassan; Ahmadvand, Negah; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Sadeghi, Mohammad-Reza; Soltanghoraee, Haleh; Akhondi, Mohammad Mehdi; Tarahomi, Majid; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2010-01-01

    We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140–250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays. PMID:23407796

  16. Improved drug delivery and therapeutic efficacy of PEgylated liposomal doxorubicin by targeting anti-HER2 peptide in murine breast tumor model.

    PubMed

    Zahmatkeshan, Masoumeh; Gheybi, Fatemeh; Rezayat, Seyed Mahdi; Jaafari, Mahmoud Reza

    2016-04-30

    Targeted cancer therapy is a powerful therapeutic strategy to management of cancer. HER2 as an anticancer target has long been studied. Its overexpression plays an important role in the pathogenesis and progressiveness of breast and other cancers. To establish efficient and reliable drug delivery to HER2-overexpressing cells, the authors of this study have developed anti-HER2 (ErbB2) peptide-liposomal formulations of doxorubicin (DOX) by an engineered breast tumor-targeting peptide ligand, AHNP, Anti-HER2/neu peptide, (FCDGFYACYADV) with three glycine amino acids as spacer before its original sequencing. Towards this goal, PEGylated liposome doxorubicin (PLD) bearing different ligand densities of AHNP was prepared and characterized for their size, zeta potential and peptide conjugation. The AHNP functionalization and density effects on breast tumor cell uptake, selective cytotoxicity, prevention of tumor growth and the tissue biodistribution of encapsulated DOX were studied in mice bearing TUBO breast cancer tumor model. The findings demonstrated that increasing the ligand density of AHNP increases cytotoxicity and cell-uptake in SKBR3 and TUBO cells which overexpress HER2 but not in MDA-MB-231with low HER2 expression profile. The anticancer activity was also superior for targeted liposomal DOX with more AHNP densities. Overall, the results showed that optimum AHNP density functionalization of PLD can significantly improve selectivity and the therapeutic index of liposomal DOX in the treatment of HER2 positive breast cancer and merits further investigation. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. ctDNA dynamics: a novel indicator to track resistance in metastatic breast cancer treated with anti-HER2 therapy

    PubMed Central

    Guan, Yanfang; Yang, Ling; Xia, Xuefeng; Chen, Shanshan; Li, Qiao; Guan, Xiuwen; Yi, Zongbi; Qian, Haili; Yi, Xin; Xu, Binghe

    2016-01-01

    Background Most studies utilizing circulating tumor DNA (ctDNA) to monitor disease interrogated only one or a few genes and failed to develop workable criteria to inform clinical practice. We evaluated the feasibility of detecting resistance to anti-HER2 therapy by serial gene-panel ctDNA sequencing. Results Primary therapeutic resistance was identified in 6 out of 14 patients with events of progressive disease. For this subset comparison of pre- and post-treatment ctDNA assay results revealed that HER2 amplification concurred with disease progression (4/6, 66.7%). Mutations in TP53 (3/6, 50.0%) and genes implicated in the PI3K/mTOR pathway (3/6, 50.0%) were also dominant markers of resistance. Together, resistance to HER2 blockade should be indicated during treatment if any of the following situations applies: 1) recurrence or persistence of HER2 amplification in the blood; 2) emergence or ≥20% increase in the fraction of mutations in any of these resistance-related genes including TP53/PIK3CA/MTOR/PTEN. Compared with CT scans, dynamic ctDNA profiling utilizing pre-defined criteria was sensitive in identifying drug resistance (sensitivity 85.7%, specificity 55.0%), with a concordance rate up to 82.1%. Besides, the ctDNA criteria had a discriminating role in the prognosis of HER2-positive metastatic breast cancer. Methods 52 plasma samples were prospectively collected from 18 patients with HER2-positive metastatic breast cancer who were treated with an oral anti-HER1/HER2 tyrosine kinase inhibitor (ClinicalTrials.gov NCT01937689). ctDNA was assayed by gene-panel target-capture next-generation sequencing. Conclusions Longitudinal gene-panel ctDNA sequencing could be exploited to determine resistance and guide the precise administration of anti-HER2 targeted therapy in the metastatic setting. PMID:27602761

  18. Application of the 2013 ASCO/CAP guideline and the SISH technique for HER2 testing of breast cancer selects more patients for anti-HER2 treatment.

    PubMed

    Polónia, António; Leitão, Dina; Schmitt, Fernando

    2016-04-01

    The aim of this study is to assess the impact of changes of the 2013 ASCO/CAP guideline on the results of HER2 testing in breast cancer. A series of 916 primary invasive breast cancer cases, assessed as HER2 2+ by IHC in part using the 2007 and in part the 2013 ASCO/CAP criteria, was evaluated for HER2 amplification status by SISH and classified according to both 2007 and 2013 ASCO/CAP ISH guideline criteria. We observed a significant increase of HER2-positive cases (12.4 to 16.8%) and a decrease of HER2-equivocal cases (3.6 to 0.7%). Of the cases studied, 52.1% fulfilled both criteria of HER2/CEP17 ratio and average HER2 copy number per cell to be classified as HER2-positive. Reclassification of the cases from before the introduction of the new ASCO/CAP guideline with the 2013 ISH criteria resulted in an increase of cases with a HER2-positive status (12.4 to 14.2%) and in a decrease of HER2-equivocal cases (3.6 to 1.6%). The 2013 ASCO/CAP guideline selects more patients for anti-HER2 targeted therapy, mostly based on the modifications of criteria to evaluate ISH-HER2.

  19. Evaluation of anti-HER2 scFv-conjugated PLGA-PEG nanoparticles on 3D tumor spheroids of BT474 and HCT116 cancer cells

    NASA Astrophysics Data System (ADS)

    Thuy Duong Le, Thi; Pham, Thu Hong; Nghia Nguyen, Trong; Giang Ngo, Thi Hong; Nhung Hoang, Thi My; Huan Le, Quang

    2016-06-01

    Three-dimensional culture cells (spheroids) are one of the multicellular culture models that can be applied to anticancer chemotherapeutic development. Multicellular spheroids more closely mimic in vivo tumor-like patterns of physiologic environment and morphology. In previous research, we designed docetaxel-loaded pegylated poly(D, L-lactide-co-glycolide) nanoparticles conjugated with anti-HER2 single chain antibodies (scFv-Doc-PLGA-PEG) and evaluated them in 2D cell culture. In this study, we continuously evaluate the cellular uptake and cytotoxic effect of scFv-Doc-PLGA-PEG on a 3D tumor spheroid model of BT474 (HER2-overexpressing) and HCT116 (HER2-underexpressing) cancer cells. The results showed that the nanoparticle formulation conjugated with scFv had a significant internalization effect on the spheroids of HER2-overexpressing cancer cells as compared to the spheroids of HER2-underexpressing cancer cells. Therefore, cytotoxic effects of targeted nanoparticles decreased the size and increased necrotic score of HER2-overexpressing tumor spheroids. Thus, these scFv-Doc-PLGA-PEG nanoparticles have potential for active targeting for HER2-overexpressing cancer therapy. In addition, BT474 and HCT116 spheroids can be used as a tumor model for evaluation of targeting therapies.

  20. Monoclonal antibodies against methionyl recombinant human prolactin.

    PubMed

    Paris, N; Robert, R; Mercier, L

    1993-02-01

    Hybridoma cell lines producing monoclonal antibody (Mab) against recombinant human prolactin (rhPrl) were established from fusion between X63-Ag8 myeloma cells and Balb/c mice splenocytes. Four Mabs numbered I to IV were selected by ELISA, purified and characterized. All these Mabs were of the Ig1 kappa isotype and able to recognize oxidized as well as reduced rhPrl. As shown by a competitive inhibition assay, Mab IV did not compete with any of the three others. Moreover, both rhPrl and hPrl extracted from human pituitaries, were recognized equally by this Mab. Properties displayed by Mab IV make it very attractive for the evaluation of prolactin levels by sandwich immunoassays.

  1. Recombinant genetic libraries and human monoclonal antibodies.

    PubMed

    Adams, Jarrett J; Nelson, Bryce; Sidhu, Sachdev S

    2014-01-01

    In order to comprehensively manipulate the human proteome we require a vast repertoire of pharmacological reagents. To address these needs we have developed repertoires of synthetic antibodies by phage display, where diversified oligonucleotides are used to modify the complementarity-determining regions (CDRs) of a human antigen-binding fragment (Fab) scaffold. As diversity is produced outside the confines of the mammalian immune system, synthetic antibody libraries allow us to bypass several limitations of hybridoma technology while improving the experimental parameters under which pharmacological reagents are produced. Here we describe the methodologies used to produce synthetic antibody libraries from a single human framework with diversity restricted to four CDRs. These synthetic repertoires can be extremely functional as they produce highly selective, high affinity Fabs to the majority of soluble human antigens. Finally we describe selection methodologies that allow us to overcome immuno-dominance in our selections to target a variety of epitopes per antigen. Together these methodologies allow us to produce human monoclonal antibodies to manipulate the human proteome.

  2. Orientation and density control of bispecific anti-HER2 antibody on functionalized carbon nanotubes for amplifying effective binding reactivity to cancer cells

    NASA Astrophysics Data System (ADS)

    Kim, Hye-In; Hwang, Dobeen; Jeon, Su-Ji; Lee, Sangyeop; Park, Jung Hyun; Yim, Dabin; Yang, Jin-Kyoung; Kang, Homan; Choo, Jaebum; Lee, Yoon-Sik; Chung, Junho; Kim, Jong-Ho

    2015-03-01

    Nanomaterial bioconjugates have gained unabated interest in the field of sensing, imaging and therapy. As a conjugation process significantly affects the biological functions of proteins, it is crucial to attach them to nanomaterials with control over their orientation and the nanomaterial-to-protein ratio in order to amplify the binding efficiency of nanomaterial bioconjugates to targets. Here, we describe a targeting nanomaterial platform utilizing carbon nanotubes functionalized with a cotinine-modified dextran polymer and a bispecific anti-HER2 × cotinine tandem antibody. This new approach provides an effective control over antibody orientation and density on the surface of carbon nanotubes through site-specific binding between the anti-cotinine domain of the bispecific tandem antibody and the cotinine group of the functionalized carbon nanotubes. The developed synthetic carbon nanotube/bispecific tandem antibody conjugates (denoted as SNAs) show an effective binding affinity against HER2 that is three orders of magnitude higher than that of the carbon nanotubes bearing a randomly conjugated tandem antibody prepared by carbodiimide chemistry. As the density of a tandem antibody on SNAs increases, their effective binding affinity to HER2 increases as well. SNAs exhibit strong resonance Raman signals for signal transduction, and are successfully applied to the selective detection of HER2-overexpressing cancer cells.Nanomaterial bioconjugates have gained unabated interest in the field of sensing, imaging and therapy. As a conjugation process significantly affects the biological functions of proteins, it is crucial to attach them to nanomaterials with control over their orientation and the nanomaterial-to-protein ratio in order to amplify the binding efficiency of nanomaterial bioconjugates to targets. Here, we describe a targeting nanomaterial platform utilizing carbon nanotubes functionalized with a cotinine-modified dextran polymer and a bispecific anti-HER2

  3. First FDA approval of dual anti-HER2 regimen: pertuzumab in combination with trastuzumab and docetaxel for HER2-positive metastatic breast cancer.

    PubMed

    Blumenthal, Gideon M; Scher, Nancy S; Cortazar, Patricia; Chattopadhyay, Somesh; Tang, Shenghui; Song, Pengfei; Liu, Qi; Ringgold, Kimberly; Pilaro, Anne M; Tilley, Amy; King, Kathryn E; Graham, Laurie; Rellahan, Barbara L; Weinberg, Wendy C; Chi, Bo; Thomas, Colleen; Hughes, Patricia; Ibrahim, Amna; Justice, Robert; Pazdur, Richard

    2013-09-15

    On June 8, 2012, the U.S. Food and Drug Administration (FDA) approved pertuzumab (Perjeta, Genentech) for use in combination with trastuzumab (Herceptin, Genentech) and docetaxel for the treatment of patients with HER2-positive metastatic breast cancer (MBC) who have not received prior anti-HER2 therapy or chemotherapy for metastatic disease. Approval was based on the results of a randomized, double-blind, placebo-controlled trial conducted in 808 patients with HER2-positive MBC. Patients were randomized (1:1) to receive pertuzumab (n = 402) or placebo (n = 406) in combination with trastuzumab and docetaxel. The primary endpoint was progression-free survival (PFS) and a key secondary endpoint was overall survival (OS). A statistically significant improvement in PFS (difference in medians of 6.1 months) was observed in patients receiving pertuzumab [HR, 0.62; 95% confidence interval (CI), 0.51-0.75; P < 0.0001]. A planned interim analysis suggested an improvement in OS (HR, 0.64; 95% CI, 0.47-0.88; P = 0.0053) but the HR and P value did not cross the stopping boundary. Common adverse reactions (>30%) observed in patients on the pertuzumab arm included diarrhea, alopecia, neutropenia, nausea, fatigue, rash, and peripheral neuropathy. No additive cardiac toxicity was observed. Significant manufacturing issues were identified during the review. On the basis of substantial evidence of efficacy for pertuzumab in MBC and the compelling public health need, FDA did not delay availability to patients pending final resolution of all manufacturing concerns. Therefore, FDA approved pertuzumab but limited its approval to lots not affected by manufacturing problems. The applicant agreed to multiple manufacturing and testing postmarketing commitments under third-party oversight to resolve manufacturing issues. ©2013 AACR.

  4. Monoclonal antibodies to human urinary thrombopoietin

    SciTech Connect

    McDonald, T.P.; Clift, R.; Cottrell, M.

    1986-01-01

    Monoclonal antibodies (MA) to a thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) were obtained from hybridomas derived from the fusion of P3 x 63/Ag 8 cells and spleen cells from TSF-immunized BALB/c mice. Media from several hybrid cultures were tested in a microantibody detection technique that measured the binding of MA to a /sup 125/I-purified TSF preparation from human embryonic kidney (HEK) cells. Hybridized cells were injected into pristane-primed mice and the antibodies produced in the ascites fluid were also shown to bind the /sup 125/I-TSF. Compared to the results of normal mouse serum, ascites fluid containing MA was shown to bind the unlabeled TSF from HEK cells. The TSF activity was significantly reduced in the supernatant fluid after precipitating the TSF-anti-TSF immune complex by a second antibody when tested in an immunothrombocythemic mouse assay. After SDS-PAGE, the precipitate from this TSF-Ma conjugate showed that the antiserum bound a single 32,000 mol wt component, indicating the monospecificity of the MA. MA directed toward human TSF will allow studies that were not previously possible.

  5. A monoclonal antibody against human MUDENG protein.

    PubMed

    Wagley, Yadav; Choi, Jun-Ha; Wickramanayake, Dimuthu Dhammika; Choi, Geun-Yeol; Kim, Chang-Kyu; Kim, Tae-Hyoung; Oh, Jae-Wook

    2013-08-01

    MUDENG (mu-2-related death-inducing gene, MuD) encodes a predicted ∼54-kDa protein in humans, considered to be involved in trafficking proteins from endosomes toward other membranous compartments as well as in inducing cell death. Here we report on the generation of a mouse monoclonal antibody (MAb) against the middle domain of human (h) MuD. This IgG sub 1 MAb, named M3H9, recognizes residues 244-326 in the middle domain of the MuD protein. Thus, the MuD proteins expressed in an astroglioma cell line and primary astrocytes can be detected by the M3H9 MAb. We showed that M3H9 MAb can be useful in enzyme-linked immunosorbent assay (ELISA) and immunoblot experiments. In addition, M3H9 MAb can detect the expression of the MuD protein in formalin-fixed, paraffin-embedded mouse ovary and uterus tissues. These results indicate that the MuD MAb M3H9 could be useful as a new biomarker of hereditary spastic paraplegia and other related diseases.

  6. Preclinical evaluation of anti-HER2 Affibody molecules site-specifically labeled with 111In using a maleimido derivative of NODAGA.

    PubMed

    Altai, Mohamed; Perols, Anna; Karlström, Amelie Eriksson; Sandström, Mattias; Boschetti, Frederic; Orlova, Anna; Tolmachev, Vladimir

    2012-05-01

    Affibody molecules have demonstrated potential for radionuclide molecular imaging. The aim of this study was to synthesize and evaluate a maleimido derivative of the 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) for site-specific labeling of anti-HER2 Affibody molecule. The maleimidoethylmonoamide NODAGA (MMA-NODAGA) was synthesized and conjugated to Z(HER2:2395) Affibody molecule having a C-terminal cysteine. Labeling efficiency, binding specificity to and cell internalization by HER2-expressing cells of [(111)In-MMA-NODAGA-Cys(61)]-Z(HER2:2395) were studied. Biodistribution of [(111)In-MMA-NODAGA-Cys(61)]-Z(HER2:2395) and [(111)In-MMA-DOTA-Cys(61)]-Z(HER2:2395) was compared in mice. The affinity of [MMA-NODAGA-Cys(61)]-Z(HER2:2395) binding to HER2 was 67 pM. The (111)In-labeling yield was 99.6%±0.5% after 30 min at 60°C. [(111)In-MMA-NODAGA-Cys(61)]-Z(HER2:2395) bound specifically to HER2-expressing cells in vitro and in vivo. Tumor uptake of [(111)In-MMA-NODAGA-Cys(61)]-Z(HER2:2395) in mice bearing DU-145 xenografts (4.7%±0.8% ID/g) was lower than uptake of [(111)In-MMA-DOTA-Cys(61)]-Z(HER2:2395) (7.5%±1.6% ID/g). However, tumor-to-organ ratios were higher for [(111)In-MMA-NODAGA-Cys(61)]-Z(HER2:2395) due to higher clearance rate from normal tissues. MMA-NODAGA is a promising chelator for site-specific labeling of targeting proteins containing unpaired cysteine. Appreciable influence of chelators on targeting properties of Affibody molecules was demonstrated. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Addition of anti-estrogen therapy to anti-HER2 dendritic cell vaccination improves regional nodal immune response and pathologic complete response rate in patients with ER(pos)/HER2(pos) early breast cancer.

    PubMed

    Lowenfeld, Lea; Zaheer, Salman; Oechsle, Crystal; Fracol, Megan; Datta, Jashodeep; Xu, Shuwen; Fitzpatrick, Elizabeth; Roses, Robert E; Fisher, Carla S; McDonald, Elizabeth S; Zhang, Paul J; DeMichele, Angela; Mick, Rosemarie; Koski, Gary K; Czerniecki, Brian J

    2017-01-01

    HER2-directed therapies are less effective in patients with ER(pos) compared to ER(neg) breast cancer, possibly reflecting bidirectional activation between HER2 and estrogen signaling pathways. We investigated dual blockade using anti-HER2 vaccination and anti-estrogen therapy in HER2(pos)/ER(pos) early breast cancer patients. In pre-clinical studies of HER2(pos) breast cancer cell lines, ER(pos) cells were partially resistant to CD4(+) Th1 cytokine-induced metabolic suppression compared with ER(neg) cells. The addition of anti-estrogen treatment significantly enhanced cytokine sensitivity in ER(pos), but not ER(neg), cell lines. In two pooled phase-I clinical trials, patients with HER2(pos) early breast cancer were treated with neoadjuvant anti-HER2 dendritic cell vaccination; HER2(pos)/ER(pos) patients were treated with or without concurrent anti-estrogen therapy. The anti-HER2 Th1 immune response measured in the peripheral blood significantly increased following vaccination, but was similar across the three treatment groups (ER(neg) vaccination alone, ER(pos) vaccination alone, ER(pos) vaccination + anti-estrogen therapy). In the sentinel lymph nodes, however, the anti-HER2 Th1 immune response was significantly higher in ER(pos) patients treated with combination anti-HER2 vaccination plus anti-estrogen therapy compared to those treated with anti-HER2 vaccination alone. Similar rates of pathologic complete response (pCR) were observed in vaccinated ER(neg) patients and vaccinated ER(pos) patients treated with concurrent anti-estrogen therapy (31.4% vs. 28.6%); both were significantly higher than the pCR rate in vaccinated ER(pos) patients who did not receive anti-estrogen therapy (4.0%, p = 0.03). Since pCR portends long-term favorable outcomes, these results support additional clinical investigations using HER2-directed vaccines in combination with anti-estrogen treatments for ER(pos)/HER2(pos) DCIS and invasive breast cancer.

  8. Conjugation of Monoclonal Antibodies to Super Paramagnetic Iron Oxide Nanoparticles for Detection of her2/neu Antigen on Breast Cancer Cell Lines.

    PubMed

    Shamsipour, Fereshteh; Zarnani, Amir Hassan; Ghods, Roya; Chamankhah, Mahmood; Forouzesh, Flora; Vafaei, Sedigheh; Bayat, Ali Ahmad; Akhondi, Mohammad Mehdi; Ali Oghabian, Mohammad; Jeddi-Tehrani, Mahmood

    2009-04-01

    Conjugation of monoclonal antibodies to super paramagnetic nanoparticles is an effective method for cancer diagnosis and treatment. In this study the humanized anti her2/neu monoclonal antibody- Herceptin- was conjugated to super paramagnetic iron oxide (SPIO) nanoparticles using EDC method. The concentration of the conjugated antibodies was measured by Bradford assay. The antibody-nanoparticle conjugates were incubated with SKBR-3 and T47D human breast carcinoma cell lines and the presence of the conjugates on cell surface was confirmed by Prussian blue iron staining method. Conjugation of Herceptin to SPIO resulted in a precipitate-free conjugate containing 20µg antibody/mg SPIO. Prussian blue iron-staining of cells showed successful binding of the conjugates to the cell surfaces. Conjugation of monoclonal antibodies to SPIO may be a useful method for detection of tumor cells, especially by MRI techniques.

  9. Localisation of malignant glioma by a radiolabelled human monoclonal antibody.

    PubMed Central

    Phillips, J; Alderson, T; Sikora, K; Watson, J

    1983-01-01

    Human monoclonal antibodies were produced by fusing intratumoral lymphocytes from patients with malignant gliomas with a human myeloma line. One antibody was selected for further study after screening for binding activity to glioma cell lines. The patient from whom it was derived developed recurrent glioma. 1 mg of antibody was purified, radiolabelled with 131I, and administered intravenously. The distribution of antibody was determined in the blood, CSF and tumour cyst fluid and compared with that of a control human monoclonal immunoglobulin. Antibody localisation in the tumour was observed and confirmed by external scintiscanning. Images PMID:6101173

  10. Therapeutic monoclonal antibodies in human breast milk: a case study.

    PubMed

    Ross, Elle; Robinson, Steven E; Amato, Carol; McMillan, Colette; Westcott, Jay; Wolf, Tiffany; Robinson, William A

    2014-04-01

    Recently, therapeutic monoclonal antibodies have been introduced for the treatment of advanced melanoma and other diseases. It remains unclear whether these drugs can be safely administered to women who are breast feeding because of the potential hazardous side effects for nursing infants. One such therapy for metastatic melanoma is ipilimumab, a human monoclonal antibody that blocks cytotoxic T-lymphocyte-antigen-4, and is the preferred treatment for patients with metastatic melanoma when other molecular therapies are not viable. This study measured ipilimumab levels in the breast milk of a patient undergoing treatment that were enough to raise concerns for a nursing infant exposed to ipilimumab.

  11. [Production of human monoclonal antibody reactive with gastrointestinal carcinoma].

    PubMed

    Soyama, N; Ohyanagi, H; Saitoh, Y

    1990-12-01

    Lymphocytes obtained from regional lymph nodes and spleen in the patients with gastrointestinal carcinoma were fused with the human B lymphoblastoid cell line GC01 and human hybridomas producing human monoclonal antibody (MoAb) were derived. Human MoAb No. 235 (IgM) derived from spleen cell of a gastric cancer patient reacted with adenocarcinoma of stomach, colon, and pancreas in the new immunohistochemical assay, modified direct immunoperoxidase method, and reacted with KATO III cells in cultured cell lines. The antigenic determinant of this antibody was suspected to be protein moiety after enzyme treatment. The competitive binding inhibition assay indicated that its epitope was different from anti-CEA monoclonal antibodies (KM10, A10, B9, AH3, JA4) and KM01. These findings suggested the possible use of human MoAb No. 235 for clinical application of targeting cancer chemotherapy in the future.

  12. Immunohistochemical identification of cytotoxic lymphocytes using human perforin monoclonal antibody.

    PubMed Central

    Hameed, A.; Olsen, K. J.; Cheng, L.; Fox, W. M.; Hruban, R. H.; Podack, E. R.

    1992-01-01

    Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforin in cardiac allograft rejection was observed. The detection and quantitative analysis of perforin-associated cytotoxic cells by the human anti-perforin monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue. Images Figure 1 PMID:1374586

  13. Comparative evaluation of synthetic anti-HER2 Affibody molecules site-specifically labelled with 111In using N-terminal DOTA, NOTA and NODAGA chelators in mice bearing prostate cancer xenografts.

    PubMed

    Malmberg, Jennie; Perols, Anna; Varasteh, Zohreh; Altai, Mohamed; Braun, Alexis; Sandström, Mattias; Garske, Ulrike; Tolmachev, Vladimir; Orlova, Anna; Karlström, Amelie Eriksson

    2012-03-01

    In disseminated prostate cancer, expression of human epidermal growth factor receptor type 2 (HER2) is one of the pathways to androgen independence. Radionuclide molecular imaging of HER2 expression in disseminated prostate cancer might identify patients for HER2-targeted therapy. Affibody molecules are small (7 kDa) targeting proteins with high potential as tracers for radionuclide imaging. The goal of this study was to develop an optimal Affibody-based tracer for visualization of HER2 expression in prostate cancer. A synthetic variant of the anti-HER2 Z(HER2:342) Affibody molecule, Z(HER2:S1), was N-terminally conjugated with the chelators DOTA, NOTA and NODAGA. The conjugated proteins were biophysically characterized by electrospray ionization mass spectroscopy (ESI-MS), circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR)-based biosensor analysis. After labelling with (111)In, the biodistribution was assessed in normal mice and the two most promising conjugates were further evaluated for tumour targeting in mice bearing DU-145 prostate cancer xenografts. The HER2-binding equilibrium dissociation constants were 130, 140 and 90 pM for DOTA-Z(HER2:S1), NOTA-Z(HER2:S1) and NODAGA-Z(HER2:S1), respectively. A comparative study of (111)In-labelled DOTA-Z(HER2:S1), NOTA-Z(HER2:S1) and NODAGA-Z(HER2:S1) in normal mice demonstrated a substantial influence of the chelators on the biodistribution properties of the conjugates. (111)In-NODAGA-Z(HER2:S1) had the most rapid clearance from blood and healthy tissues. (111)In-NOTA-Z(HER2:S1) showed high hepatic uptake and was excluded from further evaluation. (111)In-DOTA-Z(HER2:S1) and (111)In-NODAGA-Z(HER2:S1) demonstrated specific uptake in DU-145 prostate cancer xenografts in nude mice. The tumour uptake of (111)In-NODAGA-Z(HER2:S1), 5.6 ± 0.4%ID/g, was significantly lower than the uptake of (111)In-DOTA-Z(HER2:S1), 7.4 ± 0.5%ID/g, presumably because of lower bioavailability due to more rapid clearance

  14. Monoclonal Antibodies Against Human Cardiac Troponin I for Immunoassays II.

    PubMed

    Lee, Gregory; Liu, Suefay

    2015-06-01

    Human cardiac troponin I (cTnI) is one of the most specific biomarkers for detection of acute myocardial infarction (AMI). To formulate immunoassay kits for rapid immunodiagnosis of AMI, monoclonal antibodies with high affinity and specificity were generated against cTnI and subsequently tested through a series of experiments. C57BL/6 mice were immunized with cTnI as the immunogen and cell fusions with myeloma cells of BALB/c origin were performed to generate hybridomas. The supernatants of the hybridoma cell culture were routinely screened for antibody secretions against intact cTnI and synthetic peptides from the N-terminal half of cTnI (amino acid residues N1-30, N24-40, N59-79, and N80-95). Monoclonal antibodies specific to different epitope regions were then determined and selected, according to their respective affinity and specificity, for formulation of enzyme immunoassay kits. The results of this study found that most of the selected antibodies revealed comparable binding affinity to cTnI and to the corresponding synthetic peptides. Optimal sandwich enzyme immunoassays with high sensitivity could be achieved through proper combinations of the epitope-distinct monoclonal antibodies in different capture-detection pairs; signal enhancements were frequently observed when a mixture of epitope-distinct anti-cTnI monoclonal antibodies was used for coating. This indicates that a combination of epitope-distinct anti-cTnI monoclonal antibodies recognizing the N-terminal half of cTnI yield reliable detection and greater sensitivity for cTnI in AMI patients.

  15. Discovery of a potent, selective, and orally active human epidermal growth factor receptor-2 sheddase inhibitor for the treatment of cancer.

    PubMed

    Yao, Wenqing; Zhuo, Jincong; Burns, David M; Xu, Meizhong; Zhang, Colin; Li, Yun-Long; Qian, Ding-Quan; He, Chunhong; Weng, Lingkai; Shi, Eric; Lin, Qiyan; Agrios, Costas; Burn, Timothy C; Caulder, Eian; Covington, Maryanne B; Fridman, Jordan S; Friedman, Steven; Katiyar, Kamna; Hollis, Gregory; Li, Yanlong; Liu, Changnian; Liu, Xiangdong; Marando, Cindy A; Newton, Robert; Pan, Max; Scherle, Peggy; Taylor, Nancy; Vaddi, Kris; Wasserman, Zelda R; Wynn, Richard; Yeleswaram, Swamy; Jalluri, Ravi; Bower, Michael; Zhou, Bing-Bing; Metcalf, Brian

    2007-02-22

    The design, synthesis, evaluation, and identification of a novel class of (6S,7S)-N-hydroxy-6-carboxamide-5-azaspiro[2.5]octane-7-carboxamides as the first potent and selective inhibitors of human epidermal growth factor receptor-2 (HER-2) sheddase is described. Several compounds were identified that possess excellent pharmacodynamic and pharmacokinetic properties and were shown to decrease tumor size, cleaved HER-2 extracellular domain plasma levels, and potentiate the effects of the humanized anti-HER-2 monoclonal antibody (trastuzumab) in vivo in a HER-2 overexpressing cancer murine xenograft model.

  16. Generation of human monoclonal antibodies reactive with cellular antigens.

    PubMed Central

    Cote, R J; Morrissey, D M; Houghton, A N; Beattie, E J; Oettgen, H F; Old, L J

    1983-01-01

    Human lymphocytes from lymph node, peripheral blood, spleen, and tumor specimens have been fused with the LICR-LON-HMy2 (LICR-2) or SKO-007 human cell lines or the NS-1 mouse myeloma line. Over 75 fusions with the three myeloma-lymphoblastoid lines have been performed. Several factors appeared to improve the fusion outcome, including maintenance of the myeloma-lymphoblastoid lines in logarithmic phase growth at greater than or equal to 95% viability, a delay of 24 hr in the introduction of aminopterin to the fused cells, and preselection of the fetal calf serum used in the medium. For a given number of lymphocytes, fusions with NS-1 produced 5-20 times more clones than fusions with LICR-2 or SKO-007, and LICR-2 produced 4 times as many clones as SKO-007. The percentage of clones secreting human immunoglobulin, the range of immunoglobulin production, and the proportion of IgM, IgA, and IgG secretors were comparable for clones derived from the three myeloma-lymphoblastoid lines. Stable Ig-secreting clones were isolated with approximately equal frequency from LICR-2 and NS-1 fusions. A number of stable clones producing human monoclonal antibodies reacting with cell-surface, cytoplasmic, or nuclear antigens have been isolated from tumor-bearing patients and normal individuals. A surface antigenic system present on normal and malignant cells has been defined with a human monoclonal antibody derived from a patient with breast cancer. Techniques for producing human monoclonal antibody now appear to be sufficiently advanced to initiate a serological dissection of the humoral immune response to cancer. Images PMID:6572959

  17. Initial Characterization of Monoclonal Antibodies against Human Monocytes

    NASA Astrophysics Data System (ADS)

    Ugolini, Valentina; Nunez, Gabriel; Smith, R. Graham; Stastny, Peter; Capra, J. Donald

    1980-11-01

    Three monoclonal antibodies against human monocytes have been produced by somatic cell fusion. Extensive specificity analysis suggests that these antibodies react with most if not all human peripheral blood monocytes and not with highly purified T or B cells. Initial chemical characterization of the monocyte antigen recognized by two of these antibodies is presented. The molecule is a single polypeptide chain with an apparent molecular weight of 200,000. These reagents should prove useful in the clinical definition of disorders of monocyte differentiation, in studies of monocyte function, and in the elucidation of the genetics and structure of monocyte cell surface antigens.

  18. Limiting the protein corona: A successful strategy for in vivo active targeting of anti-HER2 nanobody-functionalized nanostars.

    PubMed

    D'Hollander, Antoine; Jans, Hilde; Velde, Greetje Vande; Verstraete, Charlotte; Massa, Sam; Devoogdt, Nick; Stakenborg, Tim; Muyldermans, Serge; Lagae, Liesbet; Himmelreich, Uwe

    2017-04-01

    Gold nanoparticles hold great promise as anti-cancer theranostic agents against cancer by actively targeting the tumor cells. As this potential has been supported numerously during in vitro experiments, the effective application is hampered by our limited understanding and control of the interactions within complex in vivo biological systems. When these nanoparticles are exposed to a biological environment, their surfaces become covered with proteins and biomolecules, referred to as the protein corona, reducing the active targeting capabilities. We demonstrate a chemical strategy to overcome this issue by reducing the protein corona's thickness by blocking the active groups of the self-assembled monolayer on gold nanostars. An optimal blocking agent, 2-mercapto ethanol, has been selected based on charge and length of the carbon chain. By using a nanobody as a biological ligand of the human epidermal growth factor 2 receptor (HER2), the active targeting is demonstrated in vitro and in vivo in an experimental tumor model by using darkfield microscopy and photoacoustic imaging. In this study, we have established gold nanostars as a conceivable theranostic agent with a specificity for HER2-positive tumors.

  19. Determining true HER2 gene status in breast cancers with polysomy by using alternative chromosome 17 reference genes: implications for anti-HER2 targeted therapy.

    PubMed

    Tse, Chun Hing; Hwang, Harry C; Goldstein, Lynn C; Kandalaft, Patricia L; Wiley, Jesse C; Kussick, Steven J; Gown, Allen M

    2011-11-01

    The ratio of human epidermal growth factor receptor 2 (HER2) to CEP17 by fluorescent in situ hybridization (FISH) with the centromeric probe CEP17 is used to determine HER2 gene status in breast cancer. Increases in CEP17 copy number have been interpreted as representing polysomy 17. However, pangenomic studies have demonstrated that polysomy 17 is rare. This study tests the hypothesis that the use of alternative chromosome 17 reference genes might more accurately assess true HER2 gene status. In all, 171 patients with breast cancer who had HER2 FISH that had increased mean CEP17 copy numbers (> 2.6) were selected for additional chromosome 17 studies that used probes for Smith-Magenis syndrome (SMS), retinoic acid receptor alpha (RARA), and tumor protein p53 (TP53) genes. A eusomic copy number exhibited in one or more of these loci was used to calculate a revised HER2-to-chromosome-17 ratio by using the eusomic gene locus as the reference. Of 132 cases classified as nonamplified on the basis of their HER2:CEP17 ratios, 58 (43.9%) were scored as amplified by using alternative chromosome 17 reference gene probes, and 13 (92.9%) of 14 cases scored as equivocal were reclassified as amplified. Among the cases with mean HER2 copy number of 4 to 6, 41 (47.7%) of 86 had their HER2 gene status upgraded from nonamplified to amplified, and four (4.7%) of 86 were upgraded from equivocal to amplified. Our results support the findings of recent pangenomic studies that true polysomy 17 is uncommon. Additional FISH studies that use probes to the SMS, RARA, and TP53 genes are an effective way to determine the true HER2 amplification status in patients with polysomy 17 and they have important potential implications for guiding HER2-targeted therapy in breast cancer.

  20. [Neutralizing Monoclonal and Chimeric Antibodies to Human IFN-γ].

    PubMed

    Larina, M V; Aliev, T K; Solopova, O N; Pozdnyakova, L P; Korobova, S V; Yakimov, S A; Sveshnikov, P G; Dolgikh, D A; Kirpichnikov, M P

    2015-01-01

    Autoiminune disorders are chronic diseases characterized by abnormal immune response directed against self-antigens that leads to tissue damage and violation of its normal functioning. Such diseases often result in disability or even death of patients. Nowadays a number of monoclonal antibodies to pro-inflammatory cytokines and their receptors are successfully used for the targeted treatment of autoimmune diseases. One of the perspective targets in autoimmune disease therapy is interferon gamma, a key cytokine in Th1 cells differentiation, activation of macrophages, and inflammation. In the present work, 5 monoclonal antibodies to human IFN-γ were obtained. For the development of potential therapeutic agent, we have performed neutralizing activity and affinity analysis of the antibodies. Based on the data obtained, the monoclonal antibody F1 was selected. This antibody has a dissociation constant 1.7 x 10(-9) M and IC90 = 8.9 ± 2.0 nM measured upon antibody inhibition of the IFN-γ-induced HLA-DR expression on the surface of U937 cells. We have constructed a bicistronic vector for the production of recombinant chimeric Fab fragment F1 chim in E. coli cells. The recombinant chimeric Fab fragment Fl chim neutralizes IFN-γ activity in vitro and has a dissociation constant 1.8 x 10(-9) M.

  1. The Use of Monoclonal Antibodies in Human Prion Disease

    NASA Astrophysics Data System (ADS)

    Bodemer, Walter

    Detection of PrP and its pathological isoform(s) is the key to understanding the etiology and pathogenesis of transmissible spongiform encephalopathy. There is ample evidence that PrP isoforms constitute a major component of an unknown and perhaps unconventional infectious agent. An etiological relationship between human and zoonotic transmissible spongiform encephalopathies may be revealed with monoclonal antibodies. Knowledge of the conformational transition rendering a nonpathogenic, almost ubiquitous cellular protein into a pathogenic one is crucial to defining pathomechanisms. The stepwise or even continuous formation of pathogenic molecules can be monitored. Any improvement in the early diagnosis could help to conceive new therapeutic measures which are not currently available. Determination of PrP isoforms in tissue, cells, or body fluids may be of prognostic value. Many experimental approaches in molecular medicine and molecular biology of the prion protein already rely on monoclonal antibodies. Recombinant antibodies such as the single-chain Fv may soon replace traditional hybridoma techniques. Binding affinity can easily be manipulated by a number of techniques, including in vitro mutagenesis - a step which could never be carried out using the traditional hybridoma technology. Monoclonal antibodies are and will remain an essential support for ongoing research on the prion protein in general and on the unconventional infectious prions.

  2. Sperm-immobilizing monoclonal antibody to human seminal plasma antigens.

    PubMed Central

    Shigeta, M; Watanabe, T; Maruyama, S; Koyama, K; Isojima, S

    1980-01-01

    Rat spleen cells immunized to human azoospermic semen (a mixture of seminal plasma components) and mouse myeloma cells (P3/X63 Ag8U1; P3U1) (Marguilies et al., 1976) were successfully fused with polyethylene glycol (PEG 1500) and 19 of 89 fused cell cultures were found to produce sperm-immobilizing antibody. The cells that produced antibody indicating the highest sperm-immobilizing activity were distributed into wells for further recloning and 10 clones producing sperm-immobilizing antibody were established. The clone (1C4) producing the highest antibody titre was found to produce a large amount of IgG in culture supernatants and to contain a mixture of rat and mouse chromosomes. It was proved by immunodiffusion test that the monoclonal antibody was produced to the human seminal plasma antigen No. 7 which is common to human milk protein. Using this hybridoma which produced a large amount of monoclonal sperm-immobilizing antibody, a new method could be developed for purifying human seminal plasma antigen by immunoaffinity chromatography with bound antibody from the hybridoma. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6783353

  3. Efficient generation of human IgA monoclonal antibodies.

    PubMed

    Lorin, Valérie; Mouquet, Hugo

    2015-07-01

    Immunoglobulin A (IgA) is the most abundant antibody isotype produced in humans. IgA antibodies primarily ensure immune protection of mucosal surfaces against invading pathogens, but also circulate and are present in large quantities in blood. IgAs are heterogeneous at a molecular level, with two IgA subtypes and the capacity to form multimers by interacting with the joining (J) chain. Here, we have developed an efficient strategy to rapidly generate human IgA1 and IgA2 monoclonal antibodies in their monomeric and dimeric forms. Recombinant monomeric and dimeric IgA1/IgA2 counterparts of a prototypical IgG1 monoclonal antibody, 10-1074, targeting the HIV-1 envelope protein, were produced in large amounts after expression cloning and transient transfection of 293-F cells. 10-1074 IgAs were FPLC-purified using a novel affinity-based resin engrafted with anti-IgA chimeric Fabs, followed by a monomers/multimers separation using size exclusion-based FPLC. ELISA binding experiments confirmed that the artificial IgA class switching of 10-1074 did not alter its antigen recognition. In summary, our technical approach allows the very efficient production of various forms of purified recombinant human IgA molecules, which are precious tools in dissecting IgA B-cell responses in physiological and pathophysiological conditions, and studying the biology, function and therapeutic potential of IgAs.

  4. Current status of cancer immunodetection with radiolabeled human monoclonal antibodies.

    PubMed

    De Jager, R; Abdel-Nabi, H; Serafini, A; Pecking, A; Klein, J L; Hanna, M G

    1993-04-01

    The use of radiolabeled murine monoclonal antibodies (MoAbs) for cancer immunodetection has been limited by the development of human antimouse antibodies (HAMA). Human monoclonal antibodies do not elicit a significant human antihuman (HAHA) response. The generation and production of human monoclonal antibodies met with technical difficulties that resulted in delaying their clinical testing. Human monoclonal antibodies of all isotypes have been obtained. Most were immunoglobulin (Ig) M directed against intracellular antigens. Two antibodies, 16.88 (IgM) and 88BV59 (IgG3k), recognize different epitopes on a tumor-associated antigen, CTA 16.88, homologous to cytokeratins 8, 18, and 19. CTA 16.88 is expressed by most epithelial-derived tumors including carcinomas of the colon, pancreas, breast, ovary, and lung. The in vivo targeting by these antibodies is related to their localization in nonnecrotic areas of tumors. Repeated administration of 16.88 over 5 weeks to a cumulative dose of 1,000 mg did not elicit a HAHA response. Two of 53 patients developed a low titer of HAHA 1 to 3 months after a single administration of 88BV59. Planar imaging of colorectal cancer with Iodine-131 (131I)-16.88 was positive in two studies in 9 of 12 and 16 of 20 patients preselected by immunohistochemistry. Tumors less than 2 cm in diameter are usually not detected. The lack of immunogenicity and long tumor residence time (average = 17 days) makes 16.88 a good candidate for therapy. Radioimmunlymphoscintigraphy with indium-111 (111In)-LiLo-16.88 administered by an intramammary route was used in the presurgical staging of primary breast cancer. The negative predictive value of lymph node metastases for tumors less than 3 cm was 90.5%. Planar and single photon emission computed tomography imaging of colorectal carcinoma with technetium-99m (99mTc) 88BV59 was compared with computed tomography (CT) scan in 36 surgical patients. The antibody scan was more sensitive than the CT scan in detecting

  5. A human monoclonal antibody that binds serotype A botulinum neurotoxin.

    PubMed

    Adekar, Sharad P; Jones, R Mark; Elias, M D; Al-Saleem, Fetweh H; Root, Michael J; Simpson, Lance L; Dessain, Scott K

    2008-02-01

    Monoclonal antibodies have demonstrated significant potential as therapeutics for botulinum neurotoxin exposures. We previously described a hybridoma method for cloning native human antibodies that uses a murine myeloma cell line that ectopically expresses the human telomerase catalytic subunit gene (hTERT) and the murine interleukin-6 gene (mIL-6). Here we describe a heterohybridoma cell line that ectopically expresses mIL-6 and hTERT and has improved stability of hTERT expression. We fused this cell line to human peripheral blood B cells from a subject who had received the botulinum toxoid vaccine, cloning a high-affinity antibody (13A) specific for serotype A botulinum neurotoxin (BoNT/A). The 13A antibody is an affinity-matured, post-germinal center IgG(1) lambda antibody that has partial neutralization activity in vivo. 13A binds an epitope on BoNT/A that overlaps the binding epitope of an IgG antibody previously shown to fully neutralize a lethal dose of BoNT/A in vivo. The 13A antibody may be useful for diagnostic testing or for incorporation into an oligoclonal therapeutic to counteract BoNT/A exposure.

  6. Human liver catalase: cloning, expression and characterization of monoclonal antibodies.

    PubMed

    Jin, Li Hua; Kim, Dae Won; Eum, Won Sik; Yoon, Chang Sik; Jang, Sang Ho; Choi, Hee Soon; Choi, Soo Hyun; Kim, Young Hoon; Kim, So Young; Jung, Mi Ryoung; Kang, Tae-Cheon; Won, Moo Ho; Lee, Hyeon Yong; Kang, Jung Hoon; Kwon, Oh-Shin; Cho, Sung-Woo; Lee, Kil Soo; Park, Jinseu; Choi, Soo Young

    2003-06-30

    We isolated a cDNA encoding liver catalase from a human liver cDNA library. The cDNA had a high degree of sequence similarity to the corresponding enzyme from other sources. It was expressed in E. coli using the pET15b vector. The protein produced was enzymatically active after purification, and its kinetic parameters closely resembled those of other mammalian catalases. Monoclonal antibodies were generated against the purified catalase; six antibodies recognizing different epitopes were obtained, one of which inhibited the enzyme. The cross reactions of the antibodies with brain catalases from human and other mammalian tissues were investigated, and all the immunoreactive bands obtained on Western blots had molecular masses of about 58 kDa. Similarly fractionated extracts of several mammalian cell lines all gave a single band of molecular mass 58 kDa. These results indicate that mammalian livers and human cell lines contain only one major type of immunologically reactive catalase, even though some of catalases have been previously reported to differ in certain properties.

  7. Production of human anti-HLA monoclonal antibodies

    SciTech Connect

    Walker, M.C.; Mercier, F.; Roger, J.; Varin, M.

    1986-03-01

    Only 40% of the several hundred anti-HLA murine monoclonal antibodies (MAbs) that have been made detect HLA-A,B,C or DR specificities previously defined by human alloantisera, the range of recognized specificities is very narrow, and few of the MAbs have proven useful as tissue typing reagents. In hopes of obtaining HLA typing reagents, the authors are developing a protocol for the production of human anti-HLA MAbs from HLA-antigen (Ag) immunized peripheral blood B cells of volunteering renal patients, immunized to one or more HLA Ags through therapeutic blood transfusions. A simple enrichment of the donor B cells has not been sufficient for anti-HLA MAb production, the authors are currently delineating the conditions necessary for increasing the number of HLA-specific donor B cells by in vitro stimulation with cells expressing the HLA Ag to which the B cell donor is immunized. For the production of MAbs, the stimulated B cells are transformed with Epstein-Barr virus and subsequently fused with KR-4 lymphoblastoid cells. Hybridomas are selected by HAT and Ouabain. Supernatants are screened for anti-HLA activity against lymphocyte targets expressing the original immunizing HLA Ag by complement mediated /sup 51/Cr release assay. Antibody specificity is determined by the complement-dependent microcytotoxicity test used for HLA typing.

  8. The influence of Bz-DOTA and CHX-A''-DTPA on the biodistribution of ABD-fused anti-HER2 Affibody molecules: implications for (114m)In-mediated targeting therapy.

    PubMed

    Tolmachev, Vladimir; Wållberg, Helena; Andersson, Karl; Wennborg, Anders; Lundqvist, Hans; Orlova, Anna

    2009-09-01

    Affibody molecules represent a novel class of high-affinity agents for radionuclide tumour targeting. Fusion of the Affibody molecules with an albumin-binding domain (ABD) enables modification of the blood kinetics of the Affibody molecules and reduction of the renal dose. (177)Lu-CHX-A''-DTPA-ABD-(Z(HER2:342))(2), an anti-HER2 Affibody molecule-ABD fusion protein has earlier demonstrated promising results in treatment of HER2-expressing micro-xenografts in mice. The use of the in vivo generator (114m)In/(114)In as a label for ABD-fused Affibody molecules would create preconditions for efficient treatment of both micrometastases (due to conversion and Auger electrons of (114m)In) and bulky tumours (due to high-energy beta particles from the daughter nuclide (114)In). The goal of this study was to investigate if different chelators influence the biodistribution of ABD-(Z(HER2:342))(2) and to find an optimal chelator for attachment of (114m)In to the Affibody molecule-ABD fusion protein. Isothiocyanate derivatives of Bz-DOTA and CHX-A''-DTPA were coupled to ABD-(Z(HER2:342))(2). The cellular processing of both conjugates was studied in vitro. The influence of chelators on the biodistribution was investigated in mice using double isotope ((114m)In and (111)In) labelling. The apparent affinity of CHX-A''-DTPA-ABD-(Z(HER2:342))(2) and Bz-DOTA-ABD-(Z(HER2:342))(2) to the extracellular domain of HER2 was similar, 13.5 and 15.0 pM, respectively. It was found that both conjugates were internalized by SKOV-3 cells. The use of CHX-A''-DTPA provided better cellular retention of the radioactivity, better tumour accumulation of radioactivity and better tumour to organ dose ratios than Bz-DOTA-ABD-(Z(HER2:342))(2). CHX-A''-DTPA is more suitable for (114m)In labelling of Affibody molecule-ABD fusion proteins for radionuclide therapy.

  9. Neutralizing human monoclonal antibodies prevent Zika virus infection in macaques.

    PubMed

    Magnani, Diogo M; Rogers, Thomas F; Beutler, Nathan; Ricciardi, Michael J; Bailey, Varian K; Gonzalez-Nieto, Lucas; Briney, Bryan; Sok, Devin; Le, Khoa; Strubel, Alexander; Gutman, Martin J; Pedreño-Lopez, Núria; Grubaugh, Nathan D; Silveira, Cassia G T; Maxwell, Helen S; Domingues, Aline; Martins, Mauricio A; Lee, David E; Okwuazi, Erica E; Jean, Sherrie; Strobert, Elizabeth A; Chahroudi, Ann; Silvestri, Guido; Vanderford, Thomas H; Kallas, Esper G; Desrosiers, Ronald C; Bonaldo, Myrna C; Whitehead, Stephen S; Burton, Dennis R; Watkins, David I

    2017-10-04

    Therapies to prevent maternal Zika virus (ZIKV) infection and its subsequent fetal developmental complications are urgently required. We isolated three potent ZIKV-neutralizing monoclonal antibodies (nmAbs) from the plasmablasts of a ZIKV-infected patient-SMZAb1, SMZAb2, and SMZAb5-directed against two different domains of the virus. We engineered these nmAbs with Fc LALA mutations that abrogate Fcγ receptor binding, thus eliminating potential therapy-mediated antibody-dependent enhancement. We administered a cocktail of these three nmAbs to nonhuman primates 1 day before challenge with ZIKV and demonstrated that the nmAbs completely prevented viremia in serum after challenge. Given that numerous antibodies have exceptional safety profiles in humans, the cocktail described here could be rapidly developed to protect uninfected pregnant women and their fetuses. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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

    PubMed

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

    1985-12-01

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

  11. Anti-Neoplastic Cytotoxicity of Gemcitabine-(C4-amide)-[anti-EGFR] in Dual-combination with Epirubicin-(C3-amide)-[anti-HER2/neu] against Chemotherapeutic-Resistant Mammary Adenocarcinoma (SKBr-3) and the Complementary Effect of Mebendazole

    PubMed Central

    Coyne, CP; Jones, Toni; Bear, Ryan

    2015-01-01

    Aims Delineate the feasibility of simultaneous, dual selective “targeted” chemotherapeutic delivery and determine if this molecular strategy can promote higher levels anti-neoplastic cytotoxicity than if only one covalent immunochemotherapeutic is selectively “targeted” for delivery at a single membrane associated receptor over-expressed by chemotherapeutic-resistant mammary adenocarcinoma. Methodology Gemcitabine and epirubicin were covalently bond to anti-EGFR and anti-HER2/neu utilizing a rapid multi-phase synthetic organic chemistry reaction scheme. Determination that 96% or greater gemcitabine or epirubicin content was covalently bond to immunoglobulin fractions following size separation by micro-scale column chromatography was established by methanol precipitation analysis. Residual binding-avidity of gemcitabine-(C4-amide)-[anti-EG-FR] applied in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu] was determined by cell-ELIZA utilizing chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) populations. Lack of fragmentation or polymerization was validated by SDS-PAGE/immunodetection/chemiluminescent autoradiography. Anti-neoplastic cytotoxic potency was determined by vitality stain analysis of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) monolayers known to uniquely over-express EGFR (2 × 105/cell) and HER2/neu (1 × 106/cell) receptor complexes. The covalent immunochemotherapeutics gemcitabine-(C4-amide)-[anti-EGFR] and epirubicin-(C3-amide)-[anti-HER2/neu] were applied simultaneously in dual-combination to determine their capacity to collectively evoke elevated levels of anti-neoplastic cytotoxicity. Lastly, the tubulin/microtubule inhibitor mebendazole evaluated to determine if it’s potential to complemented the anti-neoplastic cytotoxic properties of gemcitabine-(C4-amide)-[anti-EGFR] in dual-combination with epirubicin-(C3-amide)-[anti-HER2/neu]. Results Dual-combination of gemcitabine-(C4-amide)-[anti-EGFR] with

  12. Monoclonal antibodies to human butyrylcholinesterase reactive with butyrylcholinesterase in animal plasma.

    PubMed

    Peng, Hong; Brimijoin, Stephen; Hrabovska, Anna; Krejci, Eric; Blake, Thomas A; Johnson, Rudolph C; Masson, Patrick; Lockridge, Oksana

    2016-01-05

    Five mouse anti-human butyrylcholinesterase (BChE) monoclonal antibodies bind tightly to native human BChE with nanomolar dissociation constants. Pairing analysis in the Octet system identified the monoclonal antibodies that bind to overlapping and independent epitopes on human BChE. The nucleotide and amino acid sequences of 4 monoclonal antibodies are deposited in GenBank. Our goal was to determine which of the 5 monoclonal antibodies recognize BChE in the plasma of animals. Binding of monoclonal antibodies 11D8, B2 18-5, B2 12-1, mAb2 and 3E8 to BChE in animal plasma was measured using antibody immobilized on Pansorbin cells and on Dynabeads Protein G. A third method visualized binding by the shift of BChE activity bands on nondenaturing gels stained for BChE activity. Gels were counterstained for carboxylesterase activity. The three methods agreed that B2 18-5 and mAb2 have broad species specificity, but the other monoclonal antibodies interacted only with human BChE, the exception being 3E8, which also bound chicken BChE. B2 18-5 and mAb2 recognized BChE in human, rhesus monkey, horse, cat, and tiger plasma. A weak response was found with rabbit BChE. Monoclonal mAb2, but not B2 18-5, bound pig and bovine BChE. Gels stained for carboxylesterase activity confirmed that plasma from humans, monkey, pig, chicken, and cow does not contain carboxylesterase, but plasma from horse, cat, tiger, rabbit, guinea pig, mouse, and rat has carboxylesterase. Rabbit plasma carboxylesterase hydrolyzes butyrylthiocholine. In conclusion monoclonal antibodies B2 18-5 and mAb2 can be used to immuno extract BChE from the plasma of humans, monkey and other animals.

  13. Monoclonal antibodies to human butyrylcholinesterase reactive with butyrylcholinesterase in animal plasma

    PubMed Central

    Peng, Hong; Brimijoin, Stephen; Hrabovska, Anna; Krejci, Eric; Blake, Thomas A.; Johnson, Rudolph C.; Masson, Patrick; Lockridge, Oksana

    2016-01-01

    Five mouse anti-human butyrylcholinesterase (BChE) monoclonal antibodies bind tightly to native human BChE with nanomolar dissociation constants. Pairing analysis in the Octet system identified the monoclonal antibodies that bind to overlapping and independent epitopes on human BChE. The nucleotide and amino acid sequences of 4 monoclonal antibodies are deposited in GenBank. Our goal was to determine which of the 5 monoclonal antibodies recognize BChE in the plasma of animals. Binding of monoclonal antibodies 11D8, B2 18-5, B2 12-1, mAb2 and 3E8 to BChE in animal plasma was measured using antibody immobilized on Pansorbin cells and on Dynabeads Protein G. A third method visualized binding by the shift of BChE activity bands on nondenaturing gels stained for BChE activity. Gels were counterstained for carboxylesterase activity. The three methods agreed that B2 18-5 and mAb2 have broad species specificity, but the other monoclonal antibodies interacted only with human BChE, the exception being 3E8, which also bound chicken BChE. B2 18-5 and mAb2 recognized BChE in human, rhesus monkey, horse, cat, and tiger plasma. A weak response was found with rabbit BChE. Monoclonal mAb2, but not B2 18-5, bound pig and bovine BChE. Gels stained for carboxylesterase activity confirmed that plasma from humans, monkey, pig, chicken, and cow does not contain carboxylesterase, but plasma from horse, cat, tiger, rabbit, guinea pig, mouse, and rat has carboxylesterase. Rabbit plasma carboxylesterase hydrolyzes butyrylthiocholine. In conclusion monoclonal antibodies B2 18-5 and mAb2 can be used to immunoextract BChE from the plasma of humans, monkey and other animals. PMID:26585590

  14. Human Monoclonal Antibodies Broadly Neutralizing against Influenza B Virus

    PubMed Central

    Yasugi, Mayo; Kubota-Koketsu, Ritsuko; Yamashita, Akifumi; Kawashita, Norihito; Du, Anariwa; Sasaki, Tadahiro; Nishimura, Mitsuhiro; Misaki, Ryo; Kuhara, Motoki; Boonsathorn, Naphatsawan; Fujiyama, Kazuhito; Okuno, Yoshinobu; Nakaya, Takaaki; Ikuta, Kazuyoshi

    2013-01-01

    Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus. PMID:23408886

  15. In vitro inhibition of Cryptosporidium parvum infection by human monoclonal antibodies.

    PubMed Central

    Elliot, B C; Wisnewski, A V; Johnson, J; Fenwick-Smith, D; Wiest, P; Hamer, D; Kresina, T; Flanigan, T P

    1997-01-01

    Cryptosporidium parvum infection of the small epithelial intestine causes unremitting diarrhea and malabsorption that can lead to chronic and sometimes fatal illness in patients with AIDS. The illness may be ameliorated by passive oral immunoglobulin therapy. The objective of this study was to produce anti-Cryptosporidium human monoclonal antibodies for evaluation as potential therapy. All human monoclonal cell lines that produced C. parvum antibodies were originally generated from the peripheral blood lymphocytes of a human immunodeficiency virus-seronegative woman. She had recovered from C. parvum infection and had a high specific antibody titer. Hybridization of these lymphocytes with a tumor cell line was accomplished by hypo-osmolar electrofusion. Twelve clones were identified by enzyme-linked immunosorbent assay (ELISA) as secreting anti-Cryptosporidium antibodies after the initial hybridization. From the 12 positive clones, two high antibody-secreting clones, 17A and 17B, were maintained in long-term culture. A second hybridization produced two other human monoclonal cell lines, EC5 and BB2. Human monoclonal antibody from the first two cell lines bound to C. parvum sporozoites and oocysts by immunofluorescence. The ability of human monoclonal antibodies to inhibit C. parvum infection in vitro was assessed by using a human enterocyte cell line, HT29.74. The antibodies of the four different human hybridomas inhibited infection by 35 to 68% (P < 0.05) compared to a control irrelevant human monoclonal antibody derived in a similar fashion. Human monoclonal antibodies are candidate molecules for immunotherapy of C. parvum infection. PMID:9284173

  16. Belimumab: anti-BLyS human monoclonal antibody, anti-BLyS monoclonal antibody, BmAb, human monoclonal antibody to B-lymphocyte stimulator.

    PubMed

    2008-01-01

    Belimumab is a fully human monoclonal antibody that specifically recognizes and inhibits the biological activity of B-lymphocyte stimulator, or BLyS. Belimumab is in phase III trials for the treatment of systemic lupus erythematosus (SLE) and has completed a phase II trial in rheumatoid arthritis (RA); the product may also have potential in the treatment of other autoimmune disorders. In May 2001, Cambridge Antibody Technology (now MedImmune) completed its discovery programme and Human Genome Sciences identified belimumab as a candidate for clinical development. More than 1000 distinct human antibodies specific to BLyS were characterized by the collaboration.B-lymphocyte stimulator is a naturally occurring protein discovered by Human Genome Sciences that stimulates B-lymphocytes to develop into mature B cells. Laboratory studies have indicated that higher than normal levels of B-lymphocyte stimulator may contribute to the pathogenesis of autoimmune diseases, such as SLE and RA. Human Genome Sciences (HGS) and Cambridge Antibody Technology signed a collaborative agreement in August 1999 to study the B-lymphocyte stimulator as a human protein target. HGS is also developing other BLyS products. In March 2000, HGS and Cambridge Antibody Technology expanded their agreement into a 10-year collaboration and product development alliance, providing Human Genome Sciences with the right to use the antibody technology of Cambridge Antibody Technology to fully develop human antibodies for therapeutic and diagnostic purposes. Cambridge Antibody Technology will receive royalty payments on product sales from HGS, as well as the development and milestone payments it has already received. Belimumab will be manufactured in Human Genome Sciences' manufacturing facility, located in Rockville, MD, USA. HGS holds commercial rights to the drug. In July 2005, GlaxoSmithKline (GSK) exercised its co-development and co-promotion option to belimumab. In an agreement made in June 1996, HGS had

  17. The Use of Humanized Monoclonal Antibodies for the Prevention of Respiratory Syncytial Virus Infection

    PubMed Central

    Arcuri, Santo; Galletti, Silvia; Faldella, Giacomo

    2013-01-01

    Monoclonal antibodies are widely used both in infants and in adults for several indications. Humanized monoclonal antibodies (palivizumab) have been used for many years for the prevention of respiratory syncytial virus infection in pediatric populations (preterm infants, infants with chronic lung disease or congenital heart disease) at high risk of severe and potentially lethal course of the infection. This drug was reported to be safe, well tolerated and effective to decrease the hospitalization rate and mortality in these groups of infants by several clinical trials. In the present paper we report the development and the current use of monoclonal antibodies for prophylaxis against respiratory syncytial virus. PMID:23840240

  18. A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells

    NASA Astrophysics Data System (ADS)

    Colcher, D.; Horan Hand, P.; Nuti, M.; Schlom, J.

    1981-05-01

    Splenic lymphocytes of mice, immunized with membrane-enriched fractions of metastatic human mammary carcinoma tissues, were fused with the NS-1 non-immunoglobulin-secreting murine myeloma cell line. This resulted in the generation of hybridoma cultures secreting immunoglobulins reactive in solid-phase radioimmunoassays with extracts of metastatic mammary carcinoma cells from involved livers, but not with extracts of apparently normal human liver. As a result of further screening of immunoglobulin reactivities and double cloning of cultures, 11 monoclonal antibodies were chosen that demonstrated reactivities with human mammary tumor cells and not with apparently normal human tissues. These monoclonal antibodies could be placed into at least five major groups on the basis of their differential binding to the surface of various live human mammary tumor cells in culture, to extracts of mammary tumor tissues, or to tissue sections of mammary tumor cells studied by the immunoperoxidase technique. Whereas a spectrum of reactivities to mammary tumors was observed with the 11 monoclonal antibodies, no reactivity was observed to apparently normal cells of the following human tissues: breast, lymph node, lung, skin, testis, kidney, thymus, bone marrow, spleen, uterus, thyroid, intestine, liver, bladder, tonsils, stomach, prostate, and salivary gland. Several of the antibodies also demonstrated a ``pancarcinoma'' reactivity, showing binding to selected non-breast carcinomas. None of the monoclonal antibodies showed binding to purified ferritin or carcinoembryonic antigen. Monoclonal antibodies of all five major groups, however, demonstrated binding to human metastatic mammary carcinoma cells both in axillary lymph nodes and at distal sites.

  19. Characterization of monoclonal antibodies against human apolipoprotein E.

    PubMed Central

    Milne, R W; Douste-Blazy, P; Marcel, Y L; Retegui, L

    1981-01-01

    From a single cell fusion, five stable hybridomas secreting antiapolipoprotein E (apo E) were obtained. The immunoglobulin (Ig)G subclasses containing the respective monoclonal antibodies were isolated and were used as the antibody component in a solid-phase radioimmunoassay. The binding of 125I-apo E to the insolubilized antibody was inhibited by unlabeled apo E but not by unlabeled apoproteins A-I, A-II, C-II, and C-III, or by low density lipoprotein immunodepleted of endogenous apo E. Competition curves were obtained with lipoprotein subfractions that had the same shape as those obtained with purified apo E. Apo E levels in normal and hyperlipidemic plasma were well correlated when measured by the five monoclonal antibodies and polyclonal anti-apo E, although differences in absolute values were observed. In normal subjects 34, 10, 20, and 36% of apo E was recovered in the very low density lipoprotein, low density lipoprotein, high density lipoprotein, and the d greater than 1.21-gl/ml fractions, respectively, whereas these values were 34, 7, 12, and 47%, respectively, in type III patients. All antibodies indicated the same subfraction distribution of apo E. The monoclonal antibodies reacted with all of the isomorphs of apo E. One of the antibodies could be clearly distinguished by its reactivity with chemically modified very low density lipoprotein. Images PMID:6788802

  20. Polyspecificity of monoclonal lupus autoantibodies produced by human-human hybridomas.

    PubMed

    Shoenfeld, Y; Rauch, J; Massicotte, H; Datta, S K; André-Schwartz, J; Stollar, B D; Schwartz, R S

    1983-02-24

    We studied the serologic properties of monoclonal autoantibodies that were produced by hybridomas derived from lymphocytes of patients with systemic lupus erythematosus. The hybridomas were made by fusion of a human lymphoblastoid cell line, GM 4672 (derived from a patient with multiple myeloma), with peripheral-blood or splenic lymphocytes from six patients with lupus. Thirty monoclonal autoantibodies, selected for their ability to react with denatured DNA, were analyzed. Eighteen of them reacted with three or more additional polynucleotides, including native DNA, left-handed double-helical DNA (Z-DNA), poly(l), and poly(dT). Ten reacted both with nucleic acids and the phospholipid cardiolipin. The multiple binding reactions of the monoclonal autoantibodies may be explained by the presence of appropriately spaced phosphodiester groups in both the polynucleotides and the phospholipid. The sharing of antigenic groups by polymers of different natures may contribute to the apparent diversity of serologic reactions in systemic lupus erythematosus. These findings suggest that DNA itself need not be the immunogenic stimulus for autoantibody formation in this disease.

  1. 77 FR 5036 - Prospective Grant of Exclusive License: The Development of Human Anti-Mesothelin Monoclonal...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-01

    ... the treatment of pancreatic cancer, ovarian cancer, lung cancer, mesothelioma, and stomach/gastric... the treatment of mesothelin-expressing cancers, including mesothelioma, lung cancer, stomach/gastric... Human Anti-Mesothelin Monoclonal Antibodies for the Treatment of Human Cancers AGENCY: National...

  2. Characterization of pathogenic human monoclonal autoantibodies against GM-CSF

    PubMed Central

    Wang, Yanni; Thomson, Christy A.; Allan, Lenka L.; Jackson, Linda M.; Olson, Melanie; Hercus, Timothy R.; Nero, Tracy L.; Turner, Amanda; Parker, Michael W.; Lopez, Angel L.; Waddell, Thomas K.; Anderson, Gary P.; Hamilton, John A.; Schrader, John W.

    2013-01-01

    The origin of pathogenic autoantibodies remains unknown. Idiopathic pulmonary alveolar proteinosis is caused by autoantibodies against granulocyte–macrophage colony-stimulating factor (GM-CSF). We generated 19 monoclonal autoantibodies against GM-CSF from six patients with idiopathic pulmonary alveolar proteinosis. The autoantibodies used multiple V genes, excluding preferred V-gene use as an etiology, and targeted at least four nonoverlapping epitopes on GM-CSF, suggesting that GM-CSF is driving the autoantibodies and not a B-cell epitope on a pathogen cross-reacting with GM-CSF. The number of somatic mutations in the autoantibodies suggests that the memory B cells have been helped by T cells and re-entered germinal centers. All autoantibodies neutralized GM-CSF bioactivity, with general correlations to affinity and off-rate. The binding of certain autoantibodies was changed by point mutations in GM-CSF that reduced binding to the GM-CSF receptor. Those monoclonal autoantibodies that potently neutralize GM-CSF may be useful in treating inflammatory disease, such as rheumatoid arthritis and multiple sclerosis, cancer, and pain. PMID:23620516

  3. SPAM-8, a mouse-human heteromyeloma fusion partner in the production of human monoclonal antibodies. Establishment of a human monoclonal antibody against cytomegalovirus.

    PubMed

    Gustafsson, B; Jondal, M; Sundqvist, V A

    1991-01-01

    A heteromyeloma (mouse x human) cell line (SPAM-8) was produced by fusing mouse myeloma cells (SP2/0) with human peripheral blood lymphocytes. The cells were sensitive to aminopterin and resistant to ouabain. The cells showed a doubling time of about 19 hours and a cloning efficiency of 0.8 cells/well (to obtain growth in 50% of wells seeded) using mouse thymocytes as feeder cells. The number of chromosomes was about 86 and 1% of the total DNA was of human origin. Fusion of SPAM-8 cells with lymphocytes prepared from human spleens resulted in approximately one hybridoma per 10(5) seeded lymphocytes. A trioma (human x [mouse x human]) cell line was established by fusing cells of an Epstein-Barr virus-transformed B cell line with SPAM-8 cells. The trioma cells produced antibodies (IgG1, K) against cytomegalovirus, in a concentration of 7 micrograms/ml in spent medium, over a period of six months of continuous culture. The results obtained indicate that the heteromyeloma SPAM-8 may be used as a fusion partner in the production of human monoclonal antibodies.

  4. 77 FR 9678 - Prospective Grant of Exclusive License: The Development of Human Anti-CD22 Monoclonal Antibodies...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-17

    ... and m972 (SMB-002) monoclonal antibodies as therapies for the treatment of B cell cancers and... preferentially expressed on certain types of cancer cells, and is involved in the modulation of the immune system... Human Anti-CD22 Monoclonal Antibodies for the Treatment of Human Cancers and Autoimmune Disease AGENCY...

  5. A human-mouse hybridoma producing monoclonal antibody against human sperm coating antigen.

    PubMed Central

    Kyurkchiev, S D; Shigeta, M; Koyama, K; Isojima, S

    1986-01-01

    Since anti-sperm antibodies were first discovered in the sera of women, the relationship of these antibodies to sterility has been studied by many investigators. In order to determine the antigens of spermatozoa responsible for raising antibodies to spermatozoa in humans, many studies have been carried out by purifying human spermatozoa cell membrane and seminal plasma components. Since it was found that the purification was difficult by physiochemical procedures, the immunoaffinity chromatography bound monoclonal antibody (Mab) to spermatozoa antigens was attempted for this purpose. The establishment of hybridomas producing Mabs to human seminal plasma and human spermatozoa was reported by Shigeta et al. (1980), Isojima, Koyoma & Fujiwara (1982), Lee et al. (1982) and Isahakia & Alexander (1984). The ordinary approaches to obtain the Mabs consisted of xenogenic immunization with human semen and cell fusion of immunized spleen cells with mouse myeloma cells. However, the antigenic epitopes of human spermatozoa, which induced antibody production, are xenogenic for the mouse, and therefore there is a possibility that there is a difference in recognized antigenic epitopes in humans as isotypic and in mice as xenogenic. In order to study these antigenic epitopes, which correspond to antibodies against spermatozoa in women, the establishment of human-mouse hybridomas, which produced anti-semen antibodies as produced in sterile women, became essential. In these studies, we used recently developed cell fusion techniques to fuse immunized human peripheral lymphocytes with mouse myeloma cells. PMID:3456978

  6. Monoclonal antibodies directed against human Rh antigens in tests with the red cells of nonhuman primates.

    PubMed

    Socha, W W; Ruffie, J

    1990-01-01

    Monoclonal antibodies against Rh related antigens on human red cells often crossreact with the red cells of the highest subhuman primate species. Depending on specificity of antibody, the species tested, and technique used, these reactions can be either species-specific or type specific. In tests with chimpanzee red cells, some of the latter type reactions have specificities related to the R antigen of the R-C-E-F blood group system of chimpanzee; specificities of some others seem to be unrelated to any known chimpanzee blood groups. Monoclonal anti-D reagents that give uniformly positive reactions with human D-positive (common and rare types) red cells, display wide individual differences in tests with chimpanzee blood. This indicates that there are minute structural variations of antibody molecules from one monoclonal anti-D antibodies apparently have no bearing on recognition of the D combining site on the human red cells, but come into play when in contact with chimpanzee rbcs. Some of the monoclonal antibodies directed against Rh and LW molecules are distinguished by unusually strong reactions with the red cells of the Old World monkeys (macaques and baboons), which is in contrast with negative or weak reactions of the same antibodies with the red cells of anthropoid apes and human bloods. One may recall, that polyclonal anti-Rh sera do not react with the blood of rhesus monkeys, the phenomenon that was the source of controversy surrounding the discovery of the rhesus factor of the human blood.

  7. Monoclonal antibodies to human plasma low-density lipoproteins. I. Enhanced binding of 125I-labeled low-density lipoproteins by combined use of two monoclonal antibodies.

    PubMed

    Mao, S J; Patton, J G; Badimon, J J; Kottke, B A; Alley, M C; Cardin, A D

    1983-11-01

    Four monoclonal antibodies (IgG2b) to human plasma low-density lipoproteins (LDL) have been characterized. The binding affinities of each monoclonal antibody to 125I-labeled LDL were moderately high, ranging from 10(8) to 10(10) L/mol at 4 degrees C, but were reduced by at least 50-70% at 37 degrees C. The maximum binding of each monoclonal antibody was unique, ranging from 20 to 95% of total 125I-labeled LDL, suggesting that LDL particles were immunochemically heterogeneous. One antibody, LP-34, had both high and low binding affinities to LDL. Another, LP-47, exhibited high affinity for isolated LDL, yet reacted poorly with native LDL in plasma, indicating that the conformation of isolated LDL differs from that of native LDL in plasma. Unlike polyclonal serum antibodies, a mixture of four monoclonal antibodies failed to precipitate LDL, but did show a drastic increase in binding to LDL. We found that only two of our monoclonal antibodies were necessary for such synergistic enhancement. We propose that one of the monoclonal antibodies may serve as a catalytic reagent, and discuss the clinical significance of this finding.

  8. Production of Human Monoclonal Rheumatoid Factor Secreting Hybridomas Derived from Rheumatoid Synovial Cells

    DTIC Science & Technology

    1989-01-01

    antisera to human [gM and human IgG heavy chains , kappa and lambda lightTable 2: Rheumatoid synowal cell tRF subelass speclicity profiles chains , and...antisera to whole mouse Ig including light chains .(ELISA)frompantie MKin Table 1& AD7 RF was a human 1gM k monoclonal antibody without Well IgGI gG2

  9. Human monoclonal rheumatoid factors derived from the polyclonal repertoire of rheumatoid synovial tissue: production and characterization.

    PubMed Central

    Randen, I; Thompson, K M; Natvig, J B; Førre, O; Waalen, K

    1989-01-01

    A panel of 20 human monoclonal antibodies was produced from cells derived from the synovial tissue of two rheumatoid arthritis patients and one polyarticular juvenile rheumatoid arthritis patient. Fourteen IgM monoclonal antibodies were classical rheumatoid factors (RFs) with specificities restricted to IgG and included twelve kappa and two lambda proteins. Two of them were pan-specific for IgG and reacted with all four human subclasses. Nine showed the Ga specificity, i.e. they reacted with IgG1, IgG2 and IgG4 subclass proteins. Two showed the 'new Ga-related' specificity, i.e. they reacted with IgG1, IgG2 and IgG4 subclass proteins, and also with the Ge3m(st) allotype proteins. One RF demonstrated an anti-G3m(u) anti-allotypic specificity. Five IgM(lambda) monoclonal antibodies and one IgM(kappa) monoclonal antibody demonstrated polyreactivity against various antigens including DNA, human thyroglobulin, human serum albumin and tetanus toxoid. PMID:2805416

  10. Monoclonal antibody specific for human colon fibroblast-derived T-PA

    SciTech Connect

    Schaumann, J.P.; Olander, J.V.; Harakas, N.K.; Feder, J

    1989-05-23

    This patent describes a murine-derived hybridoma cell line capable of producing monoclonal antibody against human colon fibroblast-derived tissue plasminogen activator and the cell line selected from the group consisting of cell lines 63-4 (ATCC HB 9155), 54-2 (ATCC HB 9157) or 79-7 (ATCC HB 9156).

  11. Accessing of recombinant human monoclonal antibodies from patient libraries by eukaryotic ribosome display.

    PubMed

    Tang, Jie; Wang, Lin; Markiv, Anatoliy; Jeffs, Simon A; Dreja, Hanna; McKnight, Áine; He, Mingyue; Kang, Angray S

    2012-01-01

    What are effective antibodies and when do they arise to prevent or delay disease onset during a natural infection or in the course of vaccination? To address these questions at a molecular level requires longitudinal studies, capturing and analyzing the antibody repertoire at regular intervals following exposure or sero-conversion. Such studies require a method that allows the rapid generation and evaluation of monoclonal antibodies from relatively small volumes of blood. Here we describe an approach for rapidly generating human monoclonal antibodies in vitro by directly screening single-chain antibody repertories derived from donor peripheral blood mononuclear cells using ribosome display. Two single-chain antibody libraries were constructed using RNA extracted from peripheral blood mononuclear cells of two HIV-1 long-term non-progressor donors (K530 and M325). Both libraries were subjected to a single round of in vitro ribosome display for enrichment of human monoclonal antibodies against recombinant gp120(K530), derived from virus isolated from donor K530. This study has validated a novel, in vitro method for the rapid generation of human monoclonal antibodies. An antibody library could be constructed from as little as 3 μg of total RNA, the equivalent of 3-5 mL of human blood.

  12. Harnessing the immune system's arsenal: producing human monoclonal antibodies for therapeutics and investigating immune responses

    PubMed Central

    Sullivan, Meghan; Kaur, Kaval; Pauli, Noel

    2011-01-01

    Monoclonal antibody technology has undergone rapid and innovative reinvention over the last 30 years. Application of these technologies to human samples revealed valuable therapeutic and experimental insights. These technologies, each with their own benefits and flaws, have proven indispensable for immunological research and in our fight to provide new treatments and improved vaccines for infectious disease. PMID:21876728

  13. Human Monoclonal Antibodies Targeting Glypican-2 in Neuroblastoma | NCI Technology Transfer Center | TTC

    Cancer.gov

    Researchers at the National Cancer Institute’s Laboratory of Molecular Biology (NCI LMB) have developed and isolated several single domain monoclonal human antibodies against GPC2. NCI seeks parties interested in licensing or co-developing GPC2 antibodies and/or conjugates.

  14. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    SciTech Connect

    Holers, V.M.; Kotzin, B.L.

    1985-09-01

    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases.

  15. Monoclonal antibodies to human apolipoproteins: application to the study of high density lipoprotein subpopulations.

    PubMed

    Bustos, P; Ulloa, N; Calvo, C; Muller, D; Durán, D; Martínez, J; Salazar, L; Quiroga, A

    2000-09-01

    We produced, selected and cloned hybridomas that secrete monoclonal antibodies against human apolipoprotein (apo) A-I. All of the antibodies corresponded to the IgG(1) subclass and were named 1C11, 2B4, 2C10, 7C5, 8A4 and 8A5. The antibodies were characterized by their reactivity with whole lipoproteins, apolipoproteins, synthetic peptides and fragments generated by cleavage of the apo A-I. Three of the monoclonal antibodies studied (2B4, 2C10 and 7C5) were similarly inhibited by an amino-terminal peptide (amino acid sequence 1-20) of apo A-I, whereas antibodies 1C11, 8A4 and 8A5 had no reaction. Other results show that monoclonal antibody 1C11 recognizes an epitope located between amino acids 135-148. We evaluated the monoclonal antibody 8A4 against different HDL subpopulations by competitive displacement analysis and it showed a similar reactivity with the HDL particles: LpA-I and LpA-I:A-II. This antibody was used to standardize a sandwich ELISA to quantitate LpA-I in plasma. We conclude that these monoclonal antibodies are relevant for the study of apo A-I epitope expression and for quantitating apo A-I containing lipoparticles.

  16. Specific killing of human melanoma cells with an efficient 10B-compound on monoclonal antibodies

    SciTech Connect

    Komura, A.; Tokuhisa, T.; Nakagawa, T.; Sasase, A.; Ichihashi, M.; Ferrone, S.; Mishima, Y. )

    1989-07-01

    We previously established methods which have enabled us to target a sufficient number of 10B atoms on human melanoma cells to destroy them by thermal neutron irradiation. Monoclonal antibodies were here used as vector of 10B atoms on the target cell. Thermal neutrons require at least 10(9) 10B atoms to destroy the cell. In order to accumulate an adequate number of 10B atoms on target cells, our first approach was to make an effective compound that contains 12 atoms of 10B in a molecule. The second step was to conjugate the compound with an avidin molecule (10B12-avidin). One molecule of the 10B12-avidin carries about 30 atoms of 10B. This 10B12-avidin can be specifically targeted on human melanoma cells by biotinated monoclonal antibodies specific for the cells. Furthermore, the number of 10B atoms on target cells can be augmented by a hapten-antihapten monoclonal antibody system. The cultured human melanoma cells treated with these methods were damaged by thermal neutron irradiation. This is the first study that indicates thermal neutrons do injure target cells boronated by monoclonal antibodies.

  17. Identification of human plasma cells with a lamprey monoclonal antibody

    PubMed Central

    Yu, Cuiling; Liu, Yanling; Chan, Justin Tze Ho; Tong, Jiefei; Li, Zhihua; Shi, Mengyao; Davani, Dariush; Parsons, Marion; Khan, Srijit; Zhan, Wei; Kyu, Shuya; Grunebaum, Eyal; Campisi, Paolo; Propst, Evan J.; Jaye, David L.; Trudel, Suzanne; Moran, Michael F.; Ostrowski, Mario; Herrin, Brantley R.; Lee, F. Eun-Hyung; Sanz, Ignacio; Cooper, Max D.; Ehrhardt, Götz R.A.

    2016-01-01

    Ab-producing plasma cells (PCs) serve as key participants in countering pathogenic challenges as well as being contributors to autoimmune and malignant disorders. Thus far, only a limited number of PC–specific markers have been identified. The characterization of the unique variable lymphocyte receptor (VLR) Abs that are made by evolutionarily distant jawless vertebrates prompted us to investigate whether VLR Abs could detect novel PC antigens that have not been recognized by conventional Abs. Here, we describe a monoclonal lamprey Ab, VLRB MM3, that was raised against primary multiple myeloma cells. VLRB MM3 recognizes a unique epitope of the CD38 ectoenzyme that is present on plasmablasts and PCs from healthy individuals and on most, but not all, multiple myelomas. Binding by the VLRB MM3 Ab coincides with CD38 dimerization and NAD glycohydrolase activity. Our data demonstrate that the lamprey VLRB MM3 Ab is a unique reagent for the identification of plasmablasts and PCs, with potential applications in the diagnosis and therapeutic intervention of PC or autoimmune disorders. PMID:27152361

  18. Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells.

    PubMed

    Nogales-Gadea, Gisela; Saxena, Abhishek; Hoffmann, Carolin; Hounjet, Judith; Coenen, Daniëlle; Molenaar, Peter; Losen, Mario; Martinez-Martinez, Pilar

    2015-06-05

    Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

  19. Monoclonal antibody therapy directed against human acute myeloid leukemia stem cells.

    PubMed

    Majeti, R

    2011-03-03

    Accumulating evidence indicates that many human cancers are organized as a cellular hierarchy initiated and maintained by self-renewing cancer stem cells. This cancer stem cell model has been most conclusively established for human acute myeloid leukemia (AML), although controversies still exist regarding the identity of human AML stem cells (leukemia stem cell (LSC)). A major implication of this model is that, in order to eradicate the cancer and cure the patient, the cancer stem cells must be eliminated. Monoclonal antibodies have emerged as effective targeted therapies for the treatment of a number of human malignancies and, given their target antigen specificity and generally minimal toxicity, are well positioned as cancer stem cell-targeting therapies. One strategy for the development of monoclonal antibodies targeting human AML stem cells involves first identifying cell surface antigens preferentially expressed on AML LSC compared with normal hematopoietic stem cells. In recent years, a number of such antigens have been identified, including CD123, CD44, CLL-1, CD96, CD47, CD32, and CD25. Moreover, monoclonal antibodies targeting CD44, CD123, and CD47 have demonstrated efficacy against AML LSC in xenotransplantation models. Hopefully, these antibodies will ultimately prove to be effective in the treatment of human AML.

  20. 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

  1. Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

    NASA Astrophysics Data System (ADS)

    Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

    1995-07-01

    Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

  2. Development of a PBPK model for monoclonal antibodies and simulation of human and mice PBPK of a radiolabelled monoclonal antibody.

    PubMed

    Heiskanen, Tomi; Heiskanen, Tomas; Kairemo, Kalevi

    2009-01-01

    Physiology based pharmacokinetic (PBPK) modeling and simulation is a useful method for prediction of biodistribution of both macromolecules and small molecules. It can enhance our understanding of the underlying mechanisms of biodistribution and hence may help in rational design of macromolecules used as diagnostic and therapeutic agents. In this review we discuss PBPK modeling and simulation of a radiolabelled Monoclonal Antibody ((111)In-DOTA-hAFP31 IgG) ("MAB") in mice without tumor and in a human with tumor. This study is part of Xemet Co.'s effort to develop a more accurate and reliable PBPK model and simulation platform, which is applicable both for small molecules and macromolecules. The simulated results were fitted to experimental time series data by varying parameters which were not fixed a priori. It was demonstrated that the PBPK model describes the main features of the pharmacokinetics of the studied systems. It was also shown that simulation can be used for evaluating the parameters of the system and scaling up the pharmacokinetics of MAB from mice to man. We identified several areas of improvement and further development needed to improve the accuracy of PBPK simulation for MAB and other macromolecules. It was concluded that the transvascular permeabilities are the most important parameters and more research is needed to enable prediction of permeabilities from molecular characteristics of macromolecules. It would also be necessary to understand better and describe with a more detailed model the microstructure of the tumor and to measure or predict the antigen concentration in tumor. Non-specific, non-saturable binding in other organs/tissues should be understood better and the kinetic constants of the binding should be measured experimentally. Although the metabolism and clearance were neglected in this study they need to be included in more detailed studies. Also the intracellular trafficking of macromolecules, which was not included in this study

  3. The Cloning and Expression of Human Monoclonal Antibodies: Implications for Allergen Immunotherapy.

    PubMed

    James, Louisa K

    2016-02-01

    Allergic responses are dependent on the highly specific effector functions of IgE antibodies. Conversely, antibodies that block the activity of IgE can mediate tolerance to allergen. Technologies that harness the unparalleled specificity of antibody responses have revolutionized the way that we diagnose and treat human disease. This area of research continues to advance at a rapid pace and has had a significant impact on our understanding of allergic disease. This review will present an overview of humoral responses and provide an up-to-date summary of technologies used in the generation of human monoclonal antibodies. The impact that monoclonal antibodies have on allergic disease will be discussed, with a particular focus on allergen immunotherapy, which remains the only form of treatment that can modulate the underlying immune mechanisms and induce long-term clinical tolerance.

  4. Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

    DOEpatents

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

    1984-11-29

    The present invention provides monoclonal antibodies specific to and distinguishing between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype. 4 figs.

  5. Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

    DOEpatents

    Jensen, Ronald H.; Vanderlaan, Martin; Bigbee, William L.; Stanker, Larry H.; Branscomb, Elbert W.; Grabske, Robert J.

    1988-01-01

    The present invention provides monoclonal antibodies specific to and distinguish between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype.

  6. The human thymus microenvironment: heterogeneity detected by monoclonal anti-epithelial cell antibodies.

    PubMed Central

    de Maagd, R A; MacKenzie, W A; Schuurman, H J; Ritter, M A; Price, K M; Broekhuizen, R; Kater, L

    1985-01-01

    Monoclonal antibodies were raised against human thymus stromal cells and their specificity for the epithelial component of thymus stroma assessed by double immunofluorescence using anti-keratin antibodies to identify epithelium. Our monoclonal antibodies identify six distinct patterns of epithelial cell antigen expression within the thymus: pan epithelial (antibody IP1); cortex (MR3 and MR6); cortical/medullary junction (IP2); subcapsule and subpopulation of medulla (MR10/MR14); Hassall's corpuscles and adjacent subpopulation of medulla (IP3); Hassall's corpuscles only (MR13/IP4). This heterogeneity of antigen expression suggests that many different epithelial microenvironments exist within the human thymus. Images Figure 1 Figure 1 Cont Figure 2 PMID:3884494

  7. [Anti Shiga-like toxin II(SLT-II) humanized monoclonal antibody].

    PubMed

    Matsumoto, Yoh-ichi

    2002-03-01

    Anti-Shiga-Like Toxin II(SLT-II) Humanized Monoclonal Antibody(TMA-15) was constructed from Mouse Monoclonal Antibody(MuVTm1.1) recognizing the same antigen using recombinant and CDR grafting technology. TMA-15 had almost the same affinity to SLT-II as MuVTm1.1 and showed the good protective activity of mice challenged either with SLT-II or with SLT-II secreting Shiga-like Toxin producing E. coli(STEC). TMA-15 showed no acute toxicity to monkeys and no cross-reactivity to human tissues in pre-clinical safety studies. From the preliminary results of Phase 1 clinical trial using healthy adult volunteers, doses up to planned maximum dose were well tolerated and TMA-15 showed long half life in blood almost comparable to gamma globulin preparations. Therefore, TMA-15 is expected to show clinical efficacy in coming clinical trial using pediatric STEC patients.

  8. Accumulation characteristics of human colon carcinomas after monoclonal antibody ex vivo perfusion.

    PubMed Central

    Löhde, E.; Schwarzendahl, P.; Schlicker, H.; Abri, O.; Kalthoff, H.; Matzku, S.; Epenetos, A. A.; Kraas, E.

    1990-01-01

    Human colon carcinomas were operatively resected and the tumour-bearing segments interposed into an oxygenised ex vivo perfusion system. Pressure, flow, temperature, pH and metabolic parameters were controlled. Over a period of 45 min the 131I-labelled monoclonal antibody AUA1 was administered and its distribution in the tumour tissue analysed scintigraphically. The accumulated activity was determined in different tissues. The results showed that the AUA1 uptake increased with the degree of histological tumour differentiation. The main tumour:non-tumour ratio reached 0.8 in poorly, 4.1 in moderately and 5.9 in highly differentiated adenocarcinomas. Introducing the oxygenised erythrocyte-enriched perfusion media significantly increased the viability of the colon tissue. The ex vivo perfusion system will help to analyse factors determining monoclonal antibody accumulation in human colon carcinomas. Images Figure 3 PMID:2383474

  9. New monoclonal-antibody two-site solid-phase immunoradiometric assay for human thyrotropin evaluated

    SciTech Connect

    Pekary, A.E.; Hershman, J.M.

    1984-07-01

    The authors compared results with a commercial solid-phase two-site immunoradiometric assay kit for human thyrotropin in which monoclonal antibodies are used with those by our radioimmunoassay, which is optimized for measurement of low concentrations of thyrotropin. In the immunoradiometric assay a specific antibody to the beta subunit of human thyrotropin is immobilized on a polystyrene bead, and a radiolabeled monoclonal antibody directed against the alpha subunit provides a measure of bead-immobilized hormone. The mean thyrotropin concentrations in 70 euthyroid serum samples were similar in the two assays. Values for hypothyroid patients were clearly higher in both assays than values for euthyroid individuals. In commercial assays the major source of error in measurement of thyrotropin response to thyroliberin in terms of the increment over the basal concentration of thyrotropin has been systematic errors in the measurement of those basal concentrations. With the present assay, however, basal values are obtained with good precision and accuracy.

  10. Humanized Monoclonal Antibody That Passively Protects Mice against Systemic and Intranasal Ricin Toxin Challenge

    PubMed Central

    Sully, Erin K.; Bohorova, Natasha; Bohorov, Ognian; Kim, Do; Pauly, Michael H.; Whaley, Kevin J.

    2016-01-01

    PB10 is a murine monoclonal antibody against an immunodominant epitope on ricin toxin's enzymatic subunit. Here, we characterize a fully humanized version of PB10 IgG1 (hPB10) and demonstrate that it has potent in vitro and in vivo toxin-neutralizing activities. We also report the minimum serum concentrations of hPB10 required to protect mice against 10 times the 50% lethal dose of ricin when delivered by injection and inhalation. PMID:27466351

  11. Human-monoclonal-antibody therapy protects nonhuman primates against advanced Lassa fever.

    PubMed

    Mire, Chad E; Cross, Robert W; Geisbert, Joan B; Borisevich, Viktoriya; Agans, Krystle N; Deer, Daniel J; Heinrich, Megan L; Rowland, Megan M; Goba, Augustine; Momoh, Mambu; Boisen, Mathew L; Grant, Donald S; Fullah, Mohamed; Khan, Sheik Humarr; Fenton, Karla A; Robinson, James E; Branco, Luis M; Garry, Robert F; Geisbert, Thomas W

    2017-09-04

    There are no approved treatments for Lassa fever, which is endemic to the same regions of West Africa that were recently devastated by Ebola. Here we show that a combination of human monoclonal antibodies that cross-react with the glycoproteins of all four clades of Lassa virus is able to rescue 100% of cynomolgus macaques when treatment is initiated at advanced stages of disease, including up to 8 d after challenge.

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

    PubMed

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

    2012-12-01

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

  13. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type

    NASA Technical Reports Server (NTRS)

    He, X. M.; Ruker, F.; Casale, E.; Carter, D. C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 degrees. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  14. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type

    NASA Technical Reports Server (NTRS)

    He, X. M.; Ruker, F.; Casale, E.; Carter, D. C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 degrees. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  15. Human Neutralizing Monoclonal Antibody Inhibition of Middle East Respiratory Syndrome Coronavirus Replication in the Common Marmoset.

    PubMed

    Chen, Zhe; Bao, Linlin; Chen, Cong; Zou, Tingting; Xue, Ying; Li, Fengdi; Lv, Qi; Gu, Songzhi; Gao, Xiaopan; Cui, Sheng; Wang, Jianmin; Qin, Chuan; Jin, Qi

    2017-06-15

    Middle East respiratory syndrome coronavirus (MERS-CoV) infection in humans is highly lethal, with a fatality rate of 35%. New prophylactic and therapeutic strategies to combat human infections are urgently needed. We isolated a fully human neutralizing antibody, MCA1, from a human survivor. The antibody recognizes the receptor-binding domain of MERS-CoV S glycoprotein and interferes with the interaction between viral S and the human cellular receptor human dipeptidyl peptidase 4 (DPP4). To our knowledge, this study is the first to report a human neutralizing monoclonal antibody that completely inhibits MERS-CoV replication in common marmosets. Monotherapy with MCA1 represents a potential alternative treatment for human infections with MERS-CoV worthy of evaluation in clinical settings. © Crown copyright 2017.

  16. Human Monoclonal Antibodies as a Countermeasure Against Botulinum Toxins

    DTIC Science & Technology

    2012-11-30

    producing wells were determined by  FRET   analysis.  Those wells were consolidated and supernatants tested for BoNT/E first by  FRET , and reconfirmed by...believed to express well when humanized. The A ) Intact G10 m ass 147500 150000 152500 155000 % 0 100 080212_004 148 (13.087) M1 [Ev-18813,It44] (Gs

  17. Monoclonal antibodies against human BAP31 for immunocytochemistry.

    PubMed

    Song, Chaojun; Wang, Fuli; Xu, Zhuwei; Hu, Jintao; Tao, Haiqiang; Yang, Angang; Yang, Kun; Jin, Boquan

    2009-06-01

    Human BAP31 is a 28 kDa polytopic integral protein of the ER and part of a large BAP hetero-oligomeric complex that includes the related BAP29 protein and connections to actomyosin. BAP31 interacts with mIgD, cellubrevin, major histocompatibility complex class I, and BCL-2/BCL-X(L), and plays an important role in regulating the egress of these proteins and in apoptosis. Northern blot analyses have revealed BAP31 RNA transcripts in many tissues, including thymus, spleen, brain, kidney, testis, liver, and lung. However, prominent BAP31 protein expression analyzed by immunohistochemistry is restricted to a minority of cells in normal human tissue. Further studies should be made to verify the expression profiles of BAP31 in the protein level. Production of high affinity MAbs suitable for immunohistochemical staining has lagged. Here we generate a set of MAbs that could be used in Western blot, immunoprecipitation, and immunocytochemistry, providing a new powerful tool for investigation of expression profile of BAP31 protein and furthers the study of BAP31 functions.

  18. Development and characterization of monoclonal antibody against human IL-37b.

    PubMed

    Gao, Yu-Chi; Jia, Yan; Xiao, De-Qian; Wang, Xin; Dai, You-Chao; Yu, Shi-Yan; Chen, Chen; Zhuang, Ze-Gang; Fu, Xiao-Xia; Zhang, Jun-Ai; Zheng, Bi-Ying; Chen, Zhi-Hong; Zhong, Ji-Xin; Chen, Zhang-Quan; Xu, Jun-Fa

    2017-04-01

    IL-37 has been described as a natural inhibitor of immune responses. Monoclonal antibody (mAb) against human IL-37b with high affinity and specificity can serve as a molecular probe to detect IL-37 and study IL-37 functions, mechanisms and related signal pathways in inflammatory diseases. However, there are very few such mAbs against human IL-37 commercially available so far. In the current study, monoclonal antibodies against human IL-37b were developed by fusing splenocytes from immunized mouse with SP2/0 myeloma cells and polyethylene glycol. Then the antibodies were screened with prokaryotic expressed human IL-37b protein and eukaryotic expressed human IL-37b protein subsequently. Western blot and flow cytometry analysis revealed that selected mAb clons were able to recognize human IL-37 with high specificity. And more importantly, the IL-37b mAbs were fluorescently labeled and can be directly used in flow cytometry and immunohistochemistry. In conclusion, the current study developed new mAbs against human IL-37b, which are applicable in flow cytometry and immunohistochemistry.

  19. Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies

    SciTech Connect

    Britton, W.J.; Hellqvist, L.; Basten, A.; Raison, R.L.

    1985-12-01

    Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenicbands. These two bands, of M/sub tau/ 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast the two other bands of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae.

  20. Development of monoclonal antibodies against parathyroid hormone: genetic control of the immune response to human PTH

    SciTech Connect

    Nussbaum, S.R.; Lin, C.S.; Potts, J.T. Jr.; Rosenthal, A.S.; Rosenblatt, M.

    1985-01-01

    Seventeen monocloanl antibodies against the aminoterminal portion of parathyroid hormone (PTH) were generated by using BALB/c mouse for immunization fully biologically active synthetic human PTH-(1-34) and bovine PTH-(1-84) as immunogens, monoclonal antibody methods, and a solid-phase screening assay. Isotypic analysis of these monoclonal antibodies was performed using affinity purified goat antimouse immunoglobulins specific for IgG heavy chains and ..mu..(IgM). All antibodies were IgM as evidenced by 40 times greater than background activity when 25,000 cpm of /sup 125/I-labelled goat anti-mouse IgM was used as second antibody in a radioimmunoassay.

  1. Imaging of bone tumors using a monoclonal antibody raised against human osteosarcoma

    SciTech Connect

    Armitage, N.C.; Perkins, A.C.; Pimm, M.V.; Wastie, M.; Hopkins, J.S.; Dowling, F.; Baldwin, R.W.; Hardcastle, J.D.

    1986-07-01

    The radiolabeled monoclonal antibody 791T/36 raised against a human osteosarcoma was injected into 20 patients with known or suspected bone tumors. Gamma camera images were acquired at 48 or 72 hours after injection, and assessed for antibody localization. Positive images were obtained in all five osteosarcomas and four other primary malignant sarcomas. Two of the four other primary bone tumors gave positive images. Three patients with trauma had negative images as did one patient with Paget's disease. Two patients with suppurative disease gave positive images. The antibody localized in the majority of malignant sarcomas tested. In one tumor where tissue was available, a tumor:non-tumor ratio of 2.8:1 was measured. Repeat imaging was performed in five patients. Immunoscintigraphy using the monoclonal antibody 791T/36 has shown tumor localization in patients with bone and soft tissue sarcomas.

  2. Protein polymorphism of human IL-18 identified by monoclonal antibodies.

    PubMed

    Seya, T; Matsumoto, M; Shiratori, I; Fukumori, Y; Toyoshima, K

    2001-11-01

    Six mAbs were raised against human "functionally inactive" recombinant IL-18, ELISA for determination of "functionally inactive" forms of IL-18 were established using two of these mAbs (#21 and #132), and inactive species of IL-18 protein were examined with human blood plasma and macrophages (Mp). In 6-day GM-CSF-treated monocytes, namely Mp, the mAb #21 recognized the IL-18 proform (24 kDa) and a 48 kDa dimer by immunoblotting. In contrast, only the 24 kDa species was detected as a relatively faint band with a commercial mAb against "active" IL-18. No IL-18 species was detected in premature monocytes. Thus, the dimeric IL-18 was produced in Mp and detectable with the mAb we established. In blood plasma of normal subjects and patients, the #21-recognizable IL-18 was also detected by ELISA, the levels of which were not consistent with those obtained with the commercially available kit for determination of "functionally active" IL-18. We designated the former as type 2 and the latter as type 1. Strikingly, IL-18 type 1 was detected in all volunteers while type 2 was detected in approximately 30% of healthy subjects, and the levels of type 2 were high (10-100 ng/ml) compared to those of type 1 (0.02-0.55 ng/ml) in their blood plasma. In patients with atopic dermatitis, the mean value of type 1 was high (200 ng/ml) compared to those of normal subjects (0.122 ng/ml) and patients with lung cancer (0.113 ng/ml). Production of high type 1 may be associated with an immunomodulatory state in atopic dermatitis. The levels and frequencies of IL-18 type 2 were not significantly changed among these populations. Hence, large amounts of type 2 species are produced in monocyte-Mp differentiation, and their levels and frequencies are unchanged in blood plasma irrespective of the levels of type 1.

  3. Characterization of Two Human Monoclonal Antibodies Neutralizing Influenza A H7N9 Viruses

    PubMed Central

    Wang, Jianmin; Chen, Zhe; Bao, Linlin; Zhang, Weijia; Xue, Ying; Pang, XingHuo; Zhang, Xi

    2015-01-01

    H7N9 was a cause of significant global health concern due to its severe infection and approximately 35% mortality in humans. By screening a Fab antibody phage library derived from patients who recovered from H7N9 infections, we characterized two human monoclonal antibodies (HuMAbs), HNIgGD5 and HNIgGH8. The epitope of these two antibodies was dependent on two residues in the receptor binding site at positions V186 and L226 of the hemagglutinin glycoprotein. Both antibodies possessed high neutralizing activity. PMID:26063436

  4. Systematic Review of the Side Effects Associated With Anti-HER2-Targeted Therapies Used in the Treatment of Breast Cancer, on Behalf of the EORTC Quality of Life Group.

    PubMed

    Sodergren, Samantha C; Copson, Ellen; White, Alice; Efficace, Fabio; Sprangers, Mirjam; Fitzsimmons, Deborah; Bottomley, Andrew; Johnson, Colin D

    2016-06-01

    Targeted therapies (TTs), notably trastuzumab, have improved outcomes for breast cancer characterised by overexpression of human epidermal growth factor receptors including HER2. Compared with chemotherapy treatments, TTs are more specific in their targets and are delivered over longer periods of time, thus presenting different side-effect profiles. The objective of this paper is to systematically review and describe the side effects associated with TTs used in the adjuvant and metastatic settings for HER2+ breast cancer. The MEDLINE, EMBASE, CINAHL, Web of Science and Cochrane Library databases were searched from January 2007 to March 2015 to identify clinical trials and prospective studies reporting toxicities associated with TTs (mainly trastuzumab and lapatinib) used without other therapies in the treatment of HER2-positive breast cancer. Two independent reviewers selected papers based on their titles and abstracts. All papers selected by either reviewer were included. A third reviewer extracted and tabulated the relevant data using a data extraction form. We identified 5478 papers, of which 299 were reviewed and 18 trials identified involving 6980 patients. A total of 66 side effects were identified, including 46 "patient-based" symptoms and 20 "medically defined" outcomes. Side effects were more common for patients treated with therapies other than trastuzumab or with dual-HER2 regimens and for patients with metastatic disease. Diarrhoea and skin rash were the most prevalent symptoms, experienced by 29 % and 22 % of patients overall, respectively. There were 119 (2 %) cardiac events reported, and these were not exclusive to trastuzumab-treated patients. The majority of side effects (n = 52) were experienced by 1 % or less of patients and were predominantly of grade 1/2 toxicity. This systematic review provides a detailed analysis of side effects of HER2+ therapies in a large number of patients included in trials, enabling an accurate estimate of

  5. Production and Characterization of a Murine Monoclonal Antibody Against Human Ferritin

    PubMed Central

    Bayat, Ali Ahmad; Yeganeh, Omid; Ghods, Roya; Zarnani, Amir Hassan; Ardekani, Reza Bahjati; Mahmoudi, Ahmad Reza; Mahmoudian, Jafar; Haghighat-Noutash, Farzaneh; Jeddi-Tehrani, Mahmood

    2013-01-01

    Background Ferritin is an iron storage protein, which plays a key role in iron metabolism. Measurement of ferritin level in serum is one of the most useful indicators of iron status and also a sensitive measurement of iron deficiency. Monoclonal antibodies may be useful as a tool in various aspects of ferritin investigations. In this paper, the production of a murine monoclonal antibody (mAb) against human ferritin was reported. Methods Balb/c mice were immunized with purified human ferritin and splenocytes of hyper immunized mice were fused with Sp2/0 myeloma cells. After four times of cloning by limiting dilution, a positive hybridoma (clone: 2F9-C9) was selected by ELISA using human ferritin. Anti-ferritin mAb was purified from culture supernatants by affinity chromatography. Results Determination of the antibody affinity for ferritin by ELISA revealed a relatively high affinity (2.34×109 M -1) and the isotype was determined to be IgG2a. The anti-ferritin mAb 2F9-C9 reacted with 79.4% of Hela cells in flow cytometry. The antibody detected a band of 20 kDa in K562 cells, murine and human liver lysates, purified ferritin in Western blot and also ferritin in human serum. Conclusion This mAb can specifically recognize ferritin and may serve as a component of ferritin diagnostic kit if other requirements of the kit are met. PMID:24285995

  6. A Fully Human Inhibitory Monoclonal Antibody to the Wnt Receptor RYK

    PubMed Central

    Parish, Clare L.; Takano, Elena A.; Fox, Stephen; Layton, Daniel; Nice, Edouard; Stacker, Steven A.

    2013-01-01

    RYK is an unusual member of the receptor tyrosine kinase (RTK) family that is classified as a putative pseudokinase. RYK regulates fundamental biological processes including cell differentiation, migration and target selection, axon outgrowth and pathfinding by transducing signals across the plasma membrane in response to the high affinity binding of Wnt family ligands to its extracellular Wnt inhibitory factor (WIF) domain. Here we report the generation and initial characterization of a fully human inhibitory monoclonal antibody to the human RYK WIF domain. From a naïve human single chain fragment variable (scFv) phage display library, we identified anti-RYK WIF domain–specific scFvs then screened for those that could compete with Wnt3a for binding. Production of a fully human IgG1κ from an inhibitory scFv yielded a monoclonal antibody that inhibits Wnt5a-responsive RYK function in a neurite outgrowth assay. This antibody will have immediate applications for modulating RYK function in a range of settings including development and adult homeostasis, with significant potential for therapeutic use in human pathologies. PMID:24058687

  7. Monoclonal antibodies to human interferon-gamma: production, affinity purification and radioimmunoassay.

    PubMed Central

    Novick, D; Eshhar, Z; Fischer, D G; Friedlander, J; Rubinstein, M

    1983-01-01

    Human interferon-gamma (IFN-gamma) purified to electrophoretic homogeneity by a cation exchange h.p.l.c., was used for the development of monoclonal antibodies. Following immunization, spleen lymphocytes of two mice showing the highest binding and neutralizing titers were isolated, fused with NSO mouse myeloma cells and cloned. The screening of hybridomas was based on precipitation of the immune complexes with a second antibody and recovery of the biological activity of IFN-gamma from the precipitate. Twenty nine independent hybridomas secreting antibodies specific to IFN-gamma were obtained. Twelve out of these 29 hybridomas produced antibodies that neutralized the antiviral activity of pure as well as crude IFN-gamma. Moreover, IFN-gamma obtained by various induction procedures was neutralized as well, indicating that these various IFN-gamma subtypes are immunologically cross-reactive. Immune precipitation of partially purified 125I-labelled IFN-gamma by several monoclonal antibodies revealed two protein bands of 26,000 and 21,000 daltons. Immunoaffinity chromatography of IFN-gamma gave a 50-fold purification to a specific activity > or = 4 x 10(7) units/mg. Two of the monoclonal antibodies were found suitable for a sensitive and rapid double antibody solid-phase radioimmunoassay, allowing the detection of IFN-gamma at concentrations of at least 4 ng/ml (150 units/ml) within 8 h. Images Fig. 1. Fig. 2. PMID:11892806

  8. Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity

    SciTech Connect

    Qian Weizhu; Wang Ling; Li Bohua; Wang Hao; Hou Sheng; Hong Xueyu; Zhang Dapeng; Guo Yajun

    2008-03-07

    Despite the widespread clinical use of CD34 antibodies for the purification of human hematopoietic stem/progenitor cells, all the current anti-human CD34 monoclonal antibodies (mAbs) are murine, which have the potential to elicit human antimouse antibody (HAMA) immune response. In the present study, we developed three new mouse anti-human CD34 mAbs which, respectively, belonged to class I, class II and class III CD34 epitope antibodies. In an attempt to reduce the immunogenicity of these three murine mAbs, their chimeric antibodies, which consisted of mouse antibody variable regions fused genetically to human antibody constant regions, were constructed and characterized. The anti-CD34 chimeric antibodies were shown to possess affinity and specificity similar to that of their respective parental murine antibodies. Due to the potentially better safety profiles, these chimeric antibodies might become alternatives to mouse anti-CD34 antibodies routinely used for clinical application.

  9. Expression of CR2/EBV receptors on human thymocytes detected by monoclonal antibodies.

    PubMed

    Tsoukas, C D; Lambris, J D

    1988-08-01

    The biologic effects of the third component of complement, C3, are mediated via receptors which specifically bind the enzymatic degradation products resulting from the cleavage of C3. One of the products, C3d, has been associated with binding to the second complement receptor CR2 (CD21). This receptor, which is identical to the receptor for Epstein-Barr virus (EBV), has been primarily found on cells of the B lineage, but not on mature T cells or other cells of erythroid or myeloid lineages. In the present investigation, we report the presence of CR2 on human thymocytes. Indirect immunofluorescence analysis employing monoclonal anti-CR2 antibodies revealed a range of thymocyte reactivity from 15% to 63% in thirteen experiments using cells of different donors. Reactivity was always greater with the monoclonal anti-CR2 (CD21) antibody HB-5 than with two other antibodies which recognize distinct epitopes on the CR2 molecule. Two-color immunofluorescence analysis indicated that the brightest of the HB-5-stained thymocytes also reacted with the monoclonal anti-CD1 antibody T6 (immature thymocyte marker) while some of the duller HB-5-staining cells reacted with the monoclonal anti-CD3 antibody Leu-4 (mature thymocyte marker). Immunoprecipitation of CR2 on thymocytes with antibody HB-5 and polyacrylamide gel electrophoretic analysis revealed a protein of 145 kDa molecular mass which is consistent with the size of CR2 found on B lymphocytes. These findings raise several questions regarding the biologic role of CR2-EBV receptor on cells of the T lineage.

  10. Preparation of Recombinant Human Monoclonal Antibody Fab Fragments Specific for Entamoeba histolytica

    PubMed Central

    Tachibana, Hiroshi; Cheng, Xun-Jia; Watanabe, Katsuomi; Takekoshi, Masataka; Maeda, Fumiko; Aotsuka, Satoshi; Kaneda, Yoshimasa; Takeuchi, Tsutomu; Ihara, Seiji

    1999-01-01

    Genes coding for human antibody Fab fragments specific for Entamoeba histolytica were cloned and expressed in Escherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A)+ RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase PCR. The amplified DNA fragments were ligated with a plasmid vector and were introduced into Escherichia coli. Three thousand colonies were screened for the production of antibodies to E. histolytica HM-1:IMSS by an indirect fluorescence-antibody (IFA) test. Lysates from five Escherichia coli clones were positive. Analysis of the DNA sequences of the five clones showed that three of the five heavy-chain sequences and four of the five light-chain sequences differed from each other. When the reactivities of the Escherichia coli lysates to nine reference strains of E. histolytica were examined by the IFA test, three Fab fragments with different DNA sequences were found to react with all nine strains and another Fab fragment was found to react with seven strains. None of the four human monoclonal antibody Fab fragments reacted with Entamoeba dispar reference strains or with other enteric protozoan parasites. These results indicate that the bacterial expression system reported here is effective for the production of human monoclonal antibodies specific for E. histolytica. The recombinant human monoclonal antibody Fab fragments may be applicable for distinguishing E. histolytica from E. dispar and for use in the serodiagnosis of amebiasis. PMID:10225840

  11. Neutralization of diverse human immunodeficiency virus type 1 variants by an anti-V3 human monoclonal antibody.

    PubMed Central

    Gorny, M K; Conley, A J; Karwowska, S; Buchbinder, A; Xu, J Y; Emini, E A; Koenig, S; Zolla-Pazner, S

    1992-01-01

    The third variable region (V3) of the HIV-1 gp120 envelope glycoprotein is thought to induce potent neutralizing antibodies which are generally defined as type specific and reactive with individual viral isolates. In contrast, the CD4-binding domain is thought to induce neutralizing antibodies that are group specific and capable of neutralizing all isolates of HIV-1. However, in this study, we used a panel of human monoclonal antibodies to these regions of gp120 which displays specificities and neutralizing activities that challenge these tenets. In particular, we used a human monoclonal antibody to the V3 domain with exceptionally potent and broad neutralizing activity against many diverse HIV-1 isolates. The anti-CD4-binding domain antibodies, on the other hand, showed a more restricted pattern of activity. PMID:1433529

  12. Development of broad-spectrum human monoclonal antibodies for rabies post-exposure prophylaxis.

    PubMed

    De Benedictis, Paola; Minola, Andrea; Rota Nodari, Elena; Aiello, Roberta; Zecchin, Barbara; Salomoni, Angela; Foglierini, Mathilde; Agatic, Gloria; Vanzetta, Fabrizia; Lavenir, Rachel; Lepelletier, Anthony; Bentley, Emma; Weiss, Robin; Cattoli, Giovanni; Capua, Ilaria; Sallusto, Federica; Wright, Edward; Lanzavecchia, Antonio; Bourhy, Hervé; Corti, Davide

    2016-04-01

    Currently available rabies post-exposure prophylaxis (PEP) for use in humans includes equine or human rabies immunoglobulins (RIG). The replacement of RIG with an equally or more potent and safer product is strongly encouraged due to the high costs and limited availability of existing RIG. In this study, we identified two broadly neutralizing human monoclonal antibodies that represent a valid and affordable alternative to RIG in rabies PEP. Memory B cells from four selected vaccinated donors were immortalized and monoclonal antibodies were tested for neutralizing activity and epitope specificity. Two antibodies, identified as RVC20 and RVC58 (binding to antigenic site I and III, respectively), were selected for their potency and broad-spectrum reactivity. In vitro, RVC20 and RVC58 were able to neutralize all 35 rabies virus (RABV) and 25 non-RABV lyssaviruses. They showed higher potency and breath compared to antibodies under clinical development (namely CR57, CR4098, and RAB1) and commercially available human RIG. In vivo, the RVC20-RVC58 cocktail protected Syrian hamsters from a lethal RABV challenge and did not affect the endogenous hamster post-vaccination antibody response. © 2016 Humabs BioMed SA Published under the terms of the CC BY 4.0 license.

  13. Monoclonal Antibodies Detect a Spectrin-Like Protein in Normal and Dystrophic Human Skeletal Muscle

    NASA Astrophysics Data System (ADS)

    Appleyard, S. T.; Dunn, M. J.; Dubowitz, V.; Scott, M. L.; Pittman, S. J.; Shotton, D. M.

    1984-02-01

    Spectrin is the major protein of the erythrocyte membrane skeleton, which is bound to the cytoplasmic surface of the membrane's lipid bilayer and is responsible for cell shape and membrane elasticity. Inability to identify spectrin in other cell types led to the assumption that this protein was unique to erythrocytes. However, spectrin-like proteins have been demonstrated recently in a variety of cell types, including skeletal and cardiac muscle, in several species. We used monoclonal antibodies against human erythrocyte spectrin subunits in an immunocytochemical study to detect related proteins in normal and diseased human skeletal muscle. Six of seven monoclonal antibodies against β -spectrin determinants were bound at the cytoplasmic surface of muscle fiber plasma membranes, whereas none of six monoclonal antibodies against α -spectrin determinants was bound. Muscle fibers of patients with neuromuscular diseases showed similar distribution and specificity of antibody binding to those of normal subjects, but the intensity of binding was increased. In contrast, probable regenerating fibers in muscle of patients with muscular dystrophies showed reduced binding of antibodies, but reduced binding was not seen in fetal muscle fibers nor in those of a patient with a myotubular myopathy. We conclude that human skeletal muscle fibers possess a spectrin-related protein associated with their plasma membrane that shows extensive β -chain similarities to erythrocyte spectrin but differs significantly with respect to the α -subunit. Its function may be associated with the maintenance of membrane and myofibril integrity during contraction, and the increased antibody binding in diseased muscle may reflect a structural rearrangement of spectrin or a compensatory increase in spectrin abundance in response to increased stress on these systems.

  14. Human Monoclonal Antibodies to Pf 155, a Major Antigen of Malaria Parasite Plasmodium falciparum

    NASA Astrophysics Data System (ADS)

    Udomsangpetch, Rachanee; Lundgren, Katarina; Berzins, Klavs; Wahlin, Birgitta; Perlmann, Hedvig; Troye-Blomberg, Marita; Carlsson, Jan; Wahlgren, Mats; Perlmann, Peter; Bjorkman, Anders

    1986-01-01

    Pf 155, a protein of the human malaria parasite Plasmodium falciparum, is strongly immunogenic in humans and is believed to be a prime candidate for the preparation of a vaccine. Human monoclonal antibodies to Pf 155 were obtained by cloning B cells that had been prepared from an immune donor and transformed with Epstein-Barr virus. When examined by indirect immunofluorescence, these antibodies stained the surface of infected erythrocytes, free merozoites, segmented schizonts, and gametocytes. They bound to a major polypeptide with a relative molecular weight of 155K and to two minor ones (135K and 120K), all having high affinity for human glycophorin. The antibodies strongly inhibited merozoite reinvasion in vitro, suggesting that they might be appropriate reagents for therapeutic administration in vivo.

  15. Human Monoclonal Antibodies Against a Plethora of Viral Pathogens From Single Combinatorial Libraries

    NASA Astrophysics Data System (ADS)

    Williamson, R. Anthony; Burioni, Roberto; Sanna, Pietro P.; Partridge, Lynda J.; Barbas, Carlos F., III; Burton, Dennis R.

    1993-05-01

    Conventional antibody generation usually requires active immunization with antigen immediately prior to the preparation procedure. Combinatorial antibody library technology offers the possibility of cloning a range of antibody specificities at a single point in time and then accessing these specificities at will. Here we show that human monoclonal antibody Fab fragments against a plethora of infectious agents can be readily derived from a single library. Further examination of a number of libraries shows that whenever antibody against a pathogen can be detected in the serum of the donor, then specific antibodies can be derived from the corresponding library. We describe the generation of human Fab fragments against herpes simplex virus types 1 and 2, human cytomegalovirus, varicella zoster virus, rubella, human immunodeficiency virus type 1, and respiratory syncytial virus. The antibodies are shown to be highly specific and a number are effective in neutralizing virus in vitro.

  16. Targeting Breast Cancer With Anti-HER2/neu Diabodies

    DTIC Science & Technology

    2002-07-01

    single-chain Fv fragments,- 15. NUMBER OF PAGES radioimmunotherapy, radioimmunoimaging, radioimmunoguided surgery, yttrium-90, 7 iodine-131, astatine -211... astatine -21 1 (211At) conjugated to an engineered antibody-based molecule. This study (described in detail below) demonstrated the efficacy of...profile. While we will continue our work to evaluate astatine -21 1, we feel that yttrium- 90 based therapy has demonstrated sufficient efficacy to justify

  17. A plant-derived human monoclonal antibody induces an anti-carbohydrate immune response in rabbits.

    PubMed

    Jin, Chunsheng; Altmann, Friedrich; Strasser, Richard; Mach, Lukas; Schähs, Matthias; Kunert, Renate; Rademacher, Thomas; Glössl, Josef; Steinkellner, Herta

    2008-03-01

    A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic N-glycosylation. A major concern is the presence of beta 1,2-xylose and core alpha 1,3-fucose residues on complex N-glycans as these nonmammalian N-glycan residues may provoke unwanted side effects in humans. In this study we have investigated the potential antigenicity of plant-type N-glycans attached to a human monoclonal antibody (2G12). Using glyco-engineered plant lines as expression hosts, four 2G12 glycoforms differing in the presence/absence of beta 1,2-xylose and core alpha 1,3-fucose were generated. Systemic immunization of rabbits with a xylose and fucose carrying 2G12 glycoform resulted in a humoral immune response to both N-glycan epitopes. Furthermore, IgE immunoblotting with sera derived from allergic patients revealed binding to plant-produced 2G12 carrying core alpha 1,3 fucosylated N-glycan structures. Our results provide evidence for the adverse potential of nonmammalian N-glycan modifications present on monoclonal antibodies produced in plants. This emphasizes the need for the use of glyco-engineered plants lacking any potentially antigenic N-glycan structures for the production of plant-derived recombinant proteins intended for parenteral human application.

  18. Characterization of four monoclonal antibodies to recombinant human tartrate-resistant acid phosphatase.

    PubMed

    Miyazaki, Takashi; Matsunaga, Toshiyuki; Miyazaki, Shuichi; Hokari, Shigeru; Komoda, Tsugikazu

    2002-06-01

    In this study we produced a recombinant human Tartrate-resistant acid phosphatase (TRAP) enzyme from baculovirus-infected insect cells, generated four monoclonal antibodies (MAbs) 15A4, 13B9, 1C6 and 3G7, to the enzyme, and characterized these antibodies. In the human serum and lung specimen, all four antibodies appeared to have a high specificity for native TRAP enzyme in western blot analysis, immunohistochemical analysis and enzyme immunoassay. These antibodies may react with respective conformational determinants, therefore, they may be useful for detection of active TRAP. Only one of the antibodies, 15A4 also reacted with a denatured epitope, therefore, it is suitable for western blot analysis, enzyme immunoassay and for immunohistochemistry in the rat. Taken together, having characterized properties of four monoclonal antibodies against recombinant human TRAP enzyme may be useful for development of TRAP specific immunoassays in pathology and hematology of the bone. They will certainly be of use for the study of biosynthesis, regulation and function of the TRAP enzyme.

  19. Unique glycoprotein-proteoglycan complex defined by monoclonal antibody on human melanoma cells.

    PubMed Central

    Bumol, T F; Reisfeld, R A

    1982-01-01

    A monoclonal antibody, 9.2.27, with a high specificity for human melanoma cell surfaces has been utilized for biosynthetic studies in M21 human melanoma cells to define a unique antigenic complex consisting of a 250-kilodalton N-linked glycoprotein and a high molecular weight proteoglycan component larger than 400 kilodaltons. The 250-kilodalton glycoprotein has endoglycosidase H-sensitive precursors and shows a lower apparent molecular weight after treatment with neuraminidase. The biosynthesis of the proteoglycan component is inhibited by exposure of M21 cells to the monovalent ionophore monensin, this component can be labeled biosynthetically with 35SO4, is sensitive to beta-elimination in dilute base, and is degraded by both chondroitinase AC and ABC lyases, suggesting that it is a chondroitin sulfate proteoglycan. These data demonstrate that the antigenic determinant recognized by monoclonal antibody 9.2.27 is located on a glycoprotein-proteoglycan complex which may have unique implications for the interaction of glycoconjugates at the human melanoma tumor cell surface. Images PMID:6175965

  20. Generation and characterization of human B lymphocyte stimulator blocking monoclonal antibody.

    PubMed

    Zhuang, Weiliang; Zhang, Jianjun; Pei, Lili; Fang, Shuping; Liu, Honghao; Wang, Ruixue; Su, Yunpeng

    2016-09-01

    The cytokine, B lymphocyte stimulator (Blys) is essential for activation and proliferation of B cells and is involved in the pathogenesis of B-cell mediated autoimmune diseases. Based on its essential activity, Blys may be a potential therapeutic target for human autoimmune diseases. In this article, we have described the development of a novel humanized anti-Blys antibody, NMB04, that binds with high affinity and specificity to both soluble and membrane bound Blys. This monoclonal antibody has the potential to block Blys binding to all its three receptors, TACI, BCMA and BR-3. Further in vivo studies revealed that NMB04 possessed more potent inhibitory activity against human Blys as compared to an existing antibody, Belimumab. Therefore, NMB04 may have potential as a therapeutic candidate targeting autoimmune diseases.

  1. Cell surface antigens of human trophoblast: definition of an apparently unique system with a monoclonal antibody.

    PubMed Central

    Mueller, U W; Hawes, C S; Jones, W R

    1986-01-01

    An epitope with apparent specificity for the surface of human syncytiotrophoblast was defined by a murine monoclonal antibody, FDO46B (IgG1, kappa). The epitope was predominantly expressed during the first trimester of pregnancy. Binding was detected on frozen tissue sections and on cultured trophoblast by the immunoperoxidase technique. It was also detected on the surface of a small percentage (less than 10%) of cultured choriocarcinoma cells (JEG-3). A panel of human tissues was negative, as were normal and malignant human lymphocytes. The antigen bearing the FDO46B epitope was still expressed by trophoblast after culture in the presence of tunicamycin, indicating that it is possibly protein in nature. This antigen may have potential utility as a target for a contraceptive vaccine. Images Figure 1 Figure 2 Figure 3 PMID:2428734

  2. A human monoclonal antibody specific to placental alkaline phosphatase, a marker of ovarian cancer

    PubMed Central

    Ravenni, Niccolò; Weber, Marcel; Neri, Dario

    2014-01-01

    Placental alkaline phosphatase (PLAP) is a promising ovarian cancer biomarker. Here, we describe the isolation, affinity-maturation and characterization of two fully human monoclonal antibodies (termed B10 and D9) able to bind to human PLAP with a dissociation constant (Kd) of 10 and 30 nM, respectively. The ability of B10 and D9 antibodies to recognize the native antigen was confirmed by Biacore analysis, FACS and immunofluorescence studies using ovarian cancer cell lines and freshly-frozen human tissues. A quantitative biodistribution study in nude mice revealed that the B10 antibody preferentially localizes to A431 tumors, following intravenous administration. Anti-PLAP antibodies may serve as a modular building blocks for the development of targeted therapeutic products, armed with cytotoxic drugs, radionuclides or cytokines as payloads. PMID:24247025

  3. Generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune B cells

    PubMed Central

    Funaro, Ada; Gribaudo, Giorgio; Luganini, Anna; Ortolan, Erika; Lo Buono, Nicola; Vicenzi, Elisa; Cassetta, Luca; Landolfo, Santo; Buick, Richard; Falciola, Luca; Murphy, Marianne; Garotta, Gianni; Malavasi, Fabio

    2008-01-01

    Background Human monoclonal antibodies (mAbs) generated as a result of the immune response are likely to be the most effective therapeutic antibodies, particularly in the case of infectious diseases against which the immune response is protective. Human cytomegalovirus (HCMV) is an ubiquitous opportunistic virus that is the most serious pathogenic agent in transplant patients. The available therapeutic armamentarium (e.g. HCMV hyperimmune globulins or antivirals) is associated with severe side effects and the emergence of drug-resistant strains; therefore, neutralizing human mAb may be a decisive alternative in the prevention of primary and re-activated HCMV infections in these patients. Results The purpose of this study was to generate neutralizing mAb against HCMV from the immunological repertoire of immune donors. To this aim, we designed an efficient technology relying on two discrete and sequential steps: first, human B-lymphocytes are stimulated with TLR9-agonists and IL-2; second, after both additives are removed, the cells are infected with EBV. Using this strategy we obtained 29 clones secreting IgG neutralizing the HCMV infectivity; four among these were further characterized. All of the mAbs neutralize the infection in different combinations of HCMV strains and target cells, with a potency ~20 fold higher than that of the HCMV hyperimmune globulins, currently used in transplant recipients. Recombinant human monoclonal IgG1 suitable as a prophylactic or therapeutic tool in clinical applications has been generated. Conclusion The technology described has proven to be more reproducible, efficient and rapid than previously reported techniques, and can be adopted at low overall costs by any cell biology laboratory for the development of fully human mAbs for immunotherapeutic uses. PMID:19014469

  4. Monoclonal antibodies to human colorectal tumor-associated antigens: improved elicitation and subclass restriction.

    PubMed

    Morgan, A C; Woodhouse, C S; Knost, J A; Abrams, P G; Clarke, G C; Arthur, L O; McIntyre, R; Ochs, J J; Foon, K A; Oldham, R K

    1984-01-01

    Monoclonal antibodies to tumor-associated antigens (TAA) of human colorectal cancer were elicited using immunosorbents of lectins combined with peripheral protein extracts of xenografted colon adenocarcinoma. This method of immunization was compared with whole cells from surgical specimens and to crude membranes from xenografted tumors. The immunosorbent immunogens were superior to the other immunogens in three ways: (1) the number of hybrids reactive with colon tumor cells or extracts, but not with lymphoid cells or extracts, (2) the number of stable hybrids after cloning, and (3) the number of hybridoma clones reactive with tissue sections of colon tumors, but not normal colonic mucosa. In addition, lectin immunosorbents elicited primarily IgG antibodies, especially IgG3, with almost 50% of the clones of interest reacting to seemingly less immunogenic glycoproteins. The improved elicitation of monoclonal antibodies to TAA by the use of lectin immunosorbents and peripheral protein extracts has considerable potential for generating reagents useful in diagnosis and therapy of human tumors.

  5. Therapeutic effect of a radiolabeled monoclonal antibody on human ovarian cancer xenograft in nude mice

    SciTech Connect

    Manetta, A.; Satyaswaroop, P.G.; Hamilton, T.; Ozols, R.; Mortel, R.

    1989-03-01

    The therapeutic value of 131I-OC125, a radiolabeled monoclonal antibody directed against a human ovarian tumor associated antigen CA125, was examined in an ascites forming intraperitoneal human ovarian carcinoma nude mouse model. Nude mice were injected intraperitoneally with NIH:OVCAR3 cells. Twenty-one days after tumor transplantation, groups of animals were injected intraperitoneally as follows: Group 1 with 200 microCi of 131I-OC125 (n = 20), Group 2 with 200 microCi of 131I (n = 17), Group 3 with 200 microCi of 131I-IgG (n = 21), Group 4 with 60 micrograms of OC125 (n = 18), and Group 5 was left untreated (n = 21). Survival of the tumor-bearing animals was used as the endpoint of the experiment. Mean survivals were found to be 52 +/- 18 days for the 131I-OC125 group, 53 +/- 16 days for the 131I-IgG group, 49 +/- 13 days for the 131I group, 47 +/- 24 days for the OC125 group, and 47 +/- 15 days for the untreated control. These results would indicate no therapeutic advantage of 131I-OC125 over controls in this animal model. However, other approaches using single as well as multiple radiolabeled monoclonal antibodies need to be tested in this model in order to definitely establish the efficacy of this treatment modality.

  6. Production and characterisation of monoclonal antibodies against RAI3 and its expression in human breast cancer

    PubMed Central

    2009-01-01

    Background RAI3 is an orphan G-protein coupled receptor (GPCR) that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens. Methods We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA), western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147) and normal breast tissues (n = 44) using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50) as well as lymph node metastases (n = 3) for RAI3 mRNA expression. Results The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. Conclusion We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic analysis of RAI3

  7. Biochemical and pharmacological characterization of human c-Met neutralizing monoclonal antibody CE-355621

    PubMed Central

    Michaud, Neil R.; Jani, Jitesh P.; Hillerman, Stephen; Tsaparikos, Konstantinos E.; Barbacci-Tobin, Elsa G.; Knauth, Elisabeth; Putz Jr., Henry; Campbell, Mary; Karam, George A.; Chrunyk, Boris; Gebhard, David F.; Green, Larry L.; Xu, Jinghai J.; Dunn, Margaret C.; Coskran, Tim M.; Lapointe, Jean-Martin; Cohen, Bruce D.; Coleman, Kevin G.; Bedian, Vahe; Vincent, Patrick; Kajiji, Shama; Steyn, Stefan J.; Borzillo, Gary V.; Los, Gerrit

    2012-01-01

    The c-Met proto-oncogene is a multifunctional receptor tyrosine kinase that is stimulated by its ligand, hepatocyte growth factor (HGF), to induce cell growth, motility and morphogenesis. Dysregulation of c-Met function, through mutational activation or overexpression, has been observed in many types of cancer and is thought to contribute to tumor growth and metastasis by affecting mitogenesis, invasion, and angiogenesis. We identified human monoclonal antibodies that bind to the extracellular domain of c-Met and inhibit tumor growth by interfering with ligand-dependent c-Met activation. We identified antibodies representing four independent epitope classes that inhibited both ligand binding and ligand-dependent activation of c-Met in A549 cells. In cells, the antibodies antagonized c-Met function by blocking receptor activation and by subsequently inducing downregulation of the receptor, translating to phenotypic effects in soft agar growth and tubular morphogenesis assays. Further characterization of the antibodies in vivo revealed significant inhibition of c-Met activity (≥ 80% lasting for 72–96 h) in excised tumors corresponded to tumor growth inhibition in multiple xenograft tumor models. Several of the antibodies identified inhibited the growth of tumors engineered to overexpress human HGF and human c-Met (S114 NIH 3T3) when grown subcutaneously in athymic mice. Furthermore, lead candidate antibody CE-355621 inhibited the growth of U87MG human glioblastoma and GTL-16 gastric xenografts by up to 98%. The findings support published pre-clinical and clinical data indicating that targeting c-Met with human monoclonal antibodies is a promising therapeutic approach for the treatment of cancer. PMID:23007574

  8. Biochemical and pharmacological characterization of human c-Met neutralizing monoclonal antibody CE-355621.

    PubMed

    Michaud, Neil R; Jani, Jitesh P; Hillerman, Stephen; Tsaparikos, Konstantinos E; Barbacci-Tobin, Elsa G; Knauth, Elisabeth; Putz, Henry; Campbell, Mary; Karam, George A; Chrunyk, Boris; Gebhard, David F; Green, Larry L; Xu, Jinghai J; Dunn, Margaret C; Coskran, Tim M; Lapointe, Jean-Martin; Cohen, Bruce D; Coleman, Kevin G; Bedian, Vahe; Vincent, Patrick; Kajiji, Shama; Steyn, Stefan J; Borzillo, Gary V; Los, Gerrit

    2012-01-01

    The c-Met proto-oncogene is a multifunctional receptor tyrosine kinase that is stimulated by its ligand, hepatocyte growth factor (HGF), to induce cell growth, motility and morphogenesis. Dysregulation of c-Met function, through mutational activation or overexpression, has been observed in many types of cancer and is thought to contribute to tumor growth and metastasis by affecting mitogenesis, invasion, and angiogenesis. We identified human monoclonal antibodies that bind to the extracellular domain of c-Met and inhibit tumor growth by interfering with ligand-dependent c-Met activation. We identified antibodies representing four independent epitope classes that inhibited both ligand binding and ligand-dependent activation of c-Met in A549 cells. In cells, the antibodies antagonized c-Met function by blocking receptor activation and by subsequently inducing downregulation of the receptor, translating to phenotypic effects in soft agar growth and tubular morphogenesis assays. Further characterization of the antibodies in vivo revealed significant inhibition of c-Met activity (≥ 80% lasting for 72-96 h) in excised tumors corresponded to tumor growth inhibition in multiple xenograft tumor models. Several of the antibodies identified inhibited the growth of tumors engineered to overexpress human HGF and human c-Met (S114 NIH 3T3) when grown subcutaneously in athymic mice. Furthermore, lead candidate antibody CE-355621 inhibited the growth of U87MG human glioblastoma and GTL-16 gastric xenografts by up to 98%. The findings support published pre-clinical and clinical data indicating that targeting c-Met with human monoclonal antibodies is a promising therapeutic approach for the treatment of cancer.

  9. Characterization of a Novel Monoclonal Antibody against Human Mitochondrial Ferritin and Its Immunohistochemical Application in Human and Monkey Substantia Nigra

    PubMed Central

    Yang, Mingchun; Yang, Hongkuan; Guan, Hongpeng; Kato, Tomoko; Mukaisho, Kenichi; Sugihara, Hiroyuki; Ogasawara, Kazumasa; Terada, Tomohiro; Tooyama, Ikuo

    2017-01-01

    Mitochondrial ferritin (FtMt) is a novel iron storage protein with high homology to H-ferritin. Unlike the ubiquitously expressed H- and L-ferritin, FtMt is expressed in specific tissues such as the testis, heart, and brain. The function of FtMt is not fully understood; however, evidence suggests that it has a neuroprotective role in neurodegenerative diseases. We have previously reported that FtMt is expressed in catecholaminergic neurons of the monkey brainstem. To explore FtMt expression in human dopaminergic neurons, we designed a novel monoclonal antibody, C65-2, directed against human FtMt. Here, we report the properties of our C65-2 antibody. Western blots analysis and immunoabsorption tests demonstrated that the C65-2 antibody specifically recognized FtMt with no cross-reactivity to H-ferritin. Immunohistochemistry showed that the C65-2 antibody detected FtMt in neurons of the substantia nigra pars compacta (SNc) in humans and monkeys. We confirmed that FtMt is expressed in dopaminergic neurons of the human SNc. Our results suggest that FtMt is involved in various physiological and pathological mechanisms in human dopaminergic neurons, and the C65-2 monoclonal antibody promises to be a useful tool for determining the localization and biological functions of FtMt in the brain. PMID:28386150

  10. Acetylcholinesterase of human erythrocytes and neuromuscular junctions: homologies revealed by monoclonal antibodies.

    PubMed Central

    Fambrough, D M; Engel, A G; Rosenberry, T L

    1982-01-01

    Human erythrocyte acetylcholinesterase was used to immunize mice, and hybridomas were generated by fusion of mouse spleen cells with cells of the Sp 2/0 myeloma cell line. Five independently derived hybridoma clones produced antibodies that bound to purified erythrocyte acetylcholinesterase. All of these antibodies crossreacted with human and monkey neuromuscular junctions; immunocytochemical staining patterns corresponded to the distribution of junctional acetylcholinesterase. The monoclonal antibodies fell into at least four categories based on differences in crossreactivity with neuromuscular acetylcholinesterase of rabbit, dog, calf, and guinea pig, and competition tests indicated that the antibodies defined five different antigenic sites on the acetylcholinesterase molecule. It is concluded that there is a high level of homology between the acetylcholinesterases of erythrocytes and neuromuscular junctions. Images PMID:6175961

  11. Isolation and characterization of broad and ultrapotent human monoclonal antibodies with therapeutic activity against chikungunya virus

    PubMed Central

    Smith, Scott A.; Silva, Laurie A.; Fox, Julie M.; Flyak, Andrew; Kose, Nurgun; Sapparapu, Gopal; Khomadiak, Solomiia; Ashbrook, Alison W.; Kahle, Kristen M.; Fong, Rachel H.; Swayne, Sherri; Doranz, Benjamin J.; McGee, Charles E.; Heise, Mark T.; Pal, Pankaj; Brien, James D.; Austin, S. Kyle; Diamond, Michael S.; Dermody, Terence S.; Crowe, James E.

    2015-01-01

    SUMMARY Chikungunya virus (CHIKV) is a mosquito-transmitted RNA virus that causes acute febrile infection associated with polyarthralgia in humans. Mechanisms of protective immunity against CHIKV are poorly understood, and no effective therapeutics or vaccines are available. We isolated and characterized human monoclonal antibodies (mAbs) that neutralize CHIKV infectivity. Among the 30 mAbs isolated, 13 had broad and ultrapotent neutralizing activity (IC50 < 10 ng/mL), and all of these mapped to domain A of the E2 envelope protein. Potent inhibitory mAbs blocked post-attachment steps required for CHIKV membrane fusion, and several were protective in a lethal challenge model in immunocompromised mice, even when administered at late time points after infection. These highly protective mAbs could be considered for prevention or treatment of CHIKV infection, and their epitope location in domain A of E2 could be targeted for rational structure-based vaccine development. PMID:26159721

  12. [Study of the antigenic structure of human immunoglobulin lambda-chain using monoclonal antibodies].

    PubMed

    Arsen'eva, E L; Bogacheva, G T; Solomon, A; Weiss, D; Ibragimov, A R; Rokhlin, O V

    1990-01-01

    Nine monoclonals against human Ig lambda chains were produced, 4 antibodies react with C-domain, 5--with V-domain of the lambda chain. Anti-C lambda domain antibodies recognize not less than 3 epitopes and one of them is expressed only on the isolated chain. Anti-V lambda antibodies bind both isolated lambda chain and intact IgG, IgM, IgA. Four epitopes are expressed by few lambda Bence Jones proteins of the III subgroup, the immunogen possessing the same isotype. The 4 mentioned epitopes represent private idiotypic determinants. The epitope 3E10 is characteristic of 50% Bence Jones proteins of the II and III V lambda-subgroups thus representing a common idiotypic determinant. Using anti-V lambda antibodies germ line variability of V lambda III proteins was analysed and the similarity of antigenic structure of normal and myeloma human Ig lambda chains was demonstrated.

  13. Human monoclonal antibodies as candidate therapeutics against emerging viruses and HIV-1.

    PubMed

    Zhu, Zhongyu; Prabakaran, Ponraj; Chen, Weizao; Broder, Christopher C; Gong, Rui; Dimitrov, Dimiter S

    2013-04-01

    More than 40 monoclonal antibodies (mAbs) have been approved for a number of disease indications with only one of these (Synagis) - for a viral disease, and not for therapy but for prevention. However, in the last decade novel potent mAbs have been discovered and characterized with potential as therapeutics against viruses of major importance for public health and biosecurity including Hendra virus (HeV), Nipah virus (NiV), severe acute respiratory syndrome coronavirus (SARS-CoV), Ebola virus (EBOV), West Nile virus (WNV), influenza virus (IFV) and human immunodeficiency virus type 1 (HIV-1). Here, we review such mAbs with an emphasis on antibodies of human origin, and highlight recent results as well as technologies and mechanisms related to their potential as therapeutics.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2010-10-13

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

  16. New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells.

    PubMed

    O'Brien, Carmel M; Chy, Hun S; Zhou, Qi; Blumenfeld, Shiri; Lambshead, Jack W; Liu, Xiaodong; Kie, Joshua; Capaldo, Bianca D; Chung, Tung-Liang; Adams, Timothy E; Phan, Tram; Bentley, John D; McKinstry, William J; Oliva, Karen; McMurrick, Paul J; Wang, Yu-Chieh; Rossello, Fernando J; Lindeman, Geoffrey J; Chen, Di; Jarde, Thierry; Clark, Amander T; Abud, Helen E; Visvader, Jane E; Nefzger, Christian M; Polo, Jose M; Loring, Jeanne F; Laslett, Andrew L

    2017-03-01

    The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. Stem Cells 2017;35:626-640.

  17. Maturation of human B lymphocytes--studies with a panel of monoclonal antibodies against membrane antigens.

    PubMed Central

    Zola, H; McNamara, P J; Moore, H A; Smart, I J; Brooks, D A; Beckman, I G; Bradley, J

    1983-01-01

    The expression of six different membrane markers by cells of the human B lymphocyte lineage has been studied, using monoclonal antibodies. B cells representing various stages of differentiation/maturation have been examined, using normal cells, leukaemia cells, and continuous cell lines. The expression of the six markers has been compared with maturation stages defined by immunoglobulin expression. The HLA/beta 2-microglobulin complex is present throughout the B cell lineage, whilst the Ia (p28,33) marker is present from the earliest stage that can be attributed to the B lineage, but is lost during plasma cell differentiation. A marker detected by monoclonal antibody FMC 1 is present only on mature B lymphocytes, being absent from pre-B cells or plasma cells. FMC 7 detects an antigen found on a relatively mature subpopulation, whereas FMC 8 detects early as well as mature B cells. FMC 3 expression is found on a proportion of cells at any maturation stage, suggesting that expression of this marker is controlled by factors unrelated to maturation. Images Fig. 1 PMID:6191892

  18. Structural and functional characterization of a human IgG monoclonal antiphospholipid antibody.

    PubMed

    Prinz, Nadine; Häuser, Friederike; Lorenz, Mareike; Lackner, Karl J; von Landenberg, Philipp

    2011-01-01

    Antiphospholipid antibodies (aPL) are likely involved in the pathogenesis of the antiphospholipid syndrome (APS). This study analyzes the structural and functional characteristics of a human monoclonal aPL (HL7G) from the IgG2 subtype with λ light chains generated from a patient with primary APS and recurrent cerebral microemboli. DNA encoding the variable region of heavy and light chains of the antibody was sequenced, analyzed, and compared to HL5B a previously described monoclonal aPL from the same patient. Both antibodies are derived from the same germline genes. HL7G had similar but more extensive somatic mutations in the CDR1 and 2 regions than HL5B, indicating that both antibodies are closely related and derived by a T cell-dependent antigen driven process. In ELISA assays HL7G bound to cardiolipin and several other phospholipid antigens in the absence of protein cofactors. Different from HL5B this aPL bound to β2-glycoprotein I (β2GPII). This suggests that reactivity of aPL against β2GPI is determined by only few specific amino acid exchanges. HL7G was able to induce tissue factor (TF) as one of the procoagulant effects of aPL. Our data suggest that the binding specificity of aPL is only of limited value to predict the biological effect and the pathophysiological impact of the antibodies. Copyright © 2010 Elsevier GmbH. All rights reserved.

  19. Unveiling a glycation hot spot in a recombinant humanized monoclonal antibody.

    PubMed

    Zhang, Boyan; Yang, Yi; Yuk, Inn; Pai, Roger; McKay, Patrick; Eigenbrot, Charles; Dennis, Mark; Katta, Viswanatham; Francissen, Kathleen Champion

    2008-04-01

    Biotechnological companies and regulatory agencies are pursuing the complete characterization of protein therapeutics in every detail as a means to mitigate risks of product quality related safety issues. During the characterization of a recombinant humanized monoclonal antibody (referred to as rhuMAb), electrospray mass spectrometric analysis suggested that the light chain was highly glycated. The glycated and unglycated materials, separated using boronate affinity chromatography, were fully characterized using tryptic peptide mapping and tandem mass spectrometry. Using an automatic SEQUEST search of the single protein database for this antibody and extensive manual investigations of the mass spectra of the matched peptides, multiple tentative glycation sites in the light and heavy chains were observed in the highly glycated (>53%) samples. A predominant glycation site was identified and confirmed to be lysine 49 on the light chain, by performing extensive sequence analysis on an isolated glycated peptide utilizing Edman degradation analysis and MALDI-TOF/TOF mass spectrometry. Sequence alignments of rhuMAb with 12 other recombinant monoclonal antibodies and computer modeling of the Fab part of rhuMAb suggest that the unusually high level of glycation of lysine residue 49, which is located adjacent to the second complementarity-determining region (CDR2) in the light chain, is due to a spatial proximity effect in catalyzing the Amadori rearrangement by aspartic acid residue 31 in the CDR1 on the light chain.

  20. [Pharmacokinetics of injection of iodine-131 labelling MEI-TUO-XI monoclonal antibody in human body].

    PubMed

    Li, Yunchun; Tan, Tianzhi; Mo, Tingshu; Lu, Wusheng; Deng, Houfu; Yang, Xiaochuan; Li, Xiao

    2007-08-01

    To study pharmacokinetics of injection of iodine-131 labelling MEI-TUO-XI monoclonal antibody (hepatoma monoclonal antibody HAb18 F(ab')2) in vivo. 24 cases of primary hepatocelluar carcinoma (PHC) were equally divided into the low dose group, middle dose group and high dose group. After the relevant injection was administrated into the hepatic artery of each case, intravenous blood and urine samples were separately collected at different time for determination of the radioactive count ratio (min(-1)). The proportion of 131I-HAb18 F(ab')2 in serum of each blood sample was determined, and the radioactive count ratio (min(-1)) of druggery for each blood sample was revised according to the proportion. The pharmacokinetic parameters were calculated using DAS ver 1.0 (Drug And Statistics for Windows) program. The component of urine radiomaterial was determined and the percentages of urine radioactivity in administration dosage were calculated. The catabolism of the injection with time accorded with dynamics two-compartment model. The catabolism product was mainly free-131I and was excreted via kidney; the urine radioactivity was 47.70%-51.16% of administration dosage during 120 h after administration of drug. Therefore, the pharmacokinetics of the injection can satisfy the clinical demands. The drug dose recommended for clinical use was 27.75 MBq of the injection for each kg of human body.

  1. Identification of a Monoclonal Antibody Against Pneumococcal Pilus 1 Ancillary Protein Impairing Bacterial Adhesion to Human Epithelial Cells.

    PubMed

    Amerighi, Fulvia; Valeri, Maria; Donnarumma, Danilo; Maccari, Silvia; Moschioni, Monica; Taddei, Annarita; Lapazio, Lucia; Pansegrau, Werner; Buccato, Scilla; De Angelis, Gabriella; Ruggiero, Paolo; Masignani, Vega; Soriani, Marco; Pezzicoli, Alfredo

    2016-02-15

    The adhesion of Streptococcus pneumoniae is a key step during colonization of human respiratory tract mucosae. Here we demonstrate that pneumococcal type I pilus significantly increases the adhesiveness of poorly adhering highly capsulated strains in vitro. Interestingly, preincubation of bacteria with antibodies against the major pilus backbone subunit (RrgB) or the adhesin component (RrgA) impaired pneumococcal association to human epithelial cells. Screening for anti-RrgA monoclonal antibodies specifically affecting the adhesive capacity of S. pneumoniae led to the identification of the monoclonal 11B9/61 antibody, which greatly reduced pilus-dependent cell contact. Proteomic-based epitope mapping of 11B9/61 monoclonal antibody revealed a well-exposed epitope on the D2 domain of RrgA as the target of this functional antibody. The data presented here confirm the importance of pilus I for S. pneumoniae pathogenesis and the potential use of antipilus antibodies to prevent bacterial colonization.

  2. Development and characterization of new monoclonal antibodies against human recombinant CA XII.

    PubMed

    Dekaminaviciute, Dovile; Lasickiene, Rita; Parkkila, Seppo; Jogaite, Vaida; Matuliene, Jurgita; Matulis, Daumantas; Zvirbliene, Aurelija

    2014-01-01

    Carbonic anhydrases (CAs) are enzymes that catalyse the reversible hydration of CO2 to bicarbonate. CA XII is considered a potential biomarker of tumor cells and a promising target for specific therapies. The aim of the current study was to develop new monoclonal antibodies (MAbs) against human recombinant CA XII and evaluate their diagnostic potential. An extracellular catalytic domain of human CA XII was expressed in E. coli and used as an immunogen. Seven stable hybridoma cell lines producing high-affinity IgG antibodies against human CA XII were generated. The majority of MAbs were highly specific to CA XII and did not cross-react with human recombinant CA I, CA II, CA VII, and CA XIII. In order to demonstrate the diagnostic value of the MAbs, they were employed for the immunohistochemistry analysis of CA XII expression in tissues. Two MAbs (15A4 and 4A6) demonstrated a strong and specific immunostaining of CA XII in human tissue specimens. Flow cytometry analysis of 5 human tumor cell lines with the MAb 15A4 revealed its immunoreactivity with cellular CA XII. In conclusion, the MAbs raised against recombinant catalytic domain of CA XII recognize cellular CA XII and represent a promising diagnostic tool for the immunodetection of CA XII-expressing cells.

  3. Human immunoglobulin allotypes: previously unrecognized determinants and alleles defined with monoclonal antibodies.

    PubMed Central

    Zelaschi, D; Newby, C; Parsons, M; van West, B; Cavalli-Sforza, L L; Herzenberg, L A; Herzenberg, L A

    1983-01-01

    The highly polymorphic system of serologically defined genetic markers on human IgG heavy chains (Gm allotypes) is second only to the HLA complex in terms of the large number of determinants, alleles, and haplotypes that can be used for analyses of disease associations and other genetic studies. However, present typing methods are based on the use of anti-Gm antisera that are derived mainly from fortuitously immunized human donors, often requiring processing before use, and must be used in a hemagglutination-inhibition assay that cannot be used in typing for isoallotypic determinants (currently termed "non-markers"). In studies presented here, we describe an allotyping system that utilizes monoclonal antibodies in a "sandwich" modification of the solid-phase radioimmunoassay, which is capable of reliable quantitative typing of allotypic, isoallotypic, and isotypic immunoglobulin determinants. We show that these highly reproducible, easily disseminated, and essentially inexhaustible reagents can be used for rapid, sensitive, and quantitative Gm typing. Using this system we define two previously unrecognized Gm determinants, one of which, found to date only in Caucasians, is different from all known Gm markers and thus defines previously unrecognized alleles and haplotypes. The other determinant co-segregates with the conventional G3m(b1) marker but is distinct from that marker on serological grounds. The successful preparation of mouse monoclonal antibodies that detect human Gm allotypic differences and the development of an assay system capable of typing isoallotypic as well as allotypic determinants opens the way to further dissection and application of this rich genetic system. PMID:6190180

  4. Detection of Rh D-associated epitopes in human and animal tissues using human monoclonal anti-D antibodies.

    PubMed

    Thorpe, S J

    1989-12-01

    The distribution of Rh D-associated epitopes in human and animal tissues has been assessed by immunochemical techniques using six human monoclonal anti-D antibodies. The IgM antibody MAD-2 reacted predominantly with endothelial cells lining blood vessels and with animal leucocytes and human leucocytes from both Rh D-positive and Rh D-negative donors. Immunoblotting revealed reactivity with a 55 kDa tissue component which was most prominent in homogenates of heart, lung and spleen. In contrast, the IgG antibodies UCHD4 and FOG-1 stained smooth muscle, and UCHD4 in addition weakly stained cardiac and skeletal muscle and the glomeruli of kidney. Immunoblotting with UCHD4 revealed reactivity with a 27 kDa tissue component which was most prominent in heart homogenates. The results show that the six monoclonal anti-D antibodies recognize at least four different epitopes and that Rh D-associated epitopes may occur in non-erythroid cell types.

  5. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

    PubMed Central

    Yin, Yongjun; Ren, Xiaodi; Smith, Craig; Guo, Qianxu; Malabunga, Maria; Guernah, Ilhem; Zhang, Yiwei; Shen, Juqun; Sun, Haijun; Chehab, Nabil; Loizos, Nick; Ludwig, Dale L.; Ornitz, David M.

    2016-01-01

    ABSTRACT Activating mutations in fibroblast growth factor receptor 3 (FGFR3) have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9), a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC) specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11) with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3. PMID:27056048

  6. Human monoclonal antibodies that recognize conserved epitopes in the core-lipid A region of lipopolysaccharides.

    PubMed Central

    Pollack, M; Raubitschek, A A; Larrick, J W

    1987-01-01

    Epstein-Barr virus (EBV)-transformed human B lymphocytes were fused with a murine-human heteromyeloma to produce stable hybrid cell lines that secreted human monoclonal antibodies (mAbs) of the IgM class that recognized conserved epitopes in the core-lipid A region of lipopolysaccharides (LPS). Three of the mAbs reacted with epitopes on the lipid A moiety, while a fourth recognized a determinant in the core oligosaccharide. The lipid A-specific mAbs cross-reacted with heterologous rough LPS and with lipid As released by acid hydrolysis of different intact (smooth) LPS. Carbohydrate groups in the O-side chain and core oligosaccharide of isolated, smooth LPS restricted antibody access to antigenic sites on lipid A. Yet, one lipid A-reactive mAb recognized its epitope on the surfaces of a variety of intact bacteria. These findings confirm the presence of highly conserved epitopes in the core-lipid A complex and prove the existence of human B cell clones with the potential for secreting high avidity IgM antibodies that react with these widely shared determinants. Such human mAbs might provide protective activity against disease caused by diverse gram-negative bacteria. Images PMID:2437155

  7. Expression of POTE protein in human testis detected by novel monoclonal antibodies

    SciTech Connect

    Ise, Tomoko; Das, Sudipto; Nagata, Satoshi; Maeda, Hiroshi; Lee, Yoomi; Onda, Masanori; Anver, Miriam R.; Pastan, Ira

    2008-01-25

    The POTE gene family is composed of 13 highly homologous paralogs preferentially expressed in prostate, ovary, testis, and placenta. We produced 10 monoclonal antibodies (MAbs) against three representative POTE paralogs: POTE-21, POTE-2{gamma}C, and POTE-22. One reacted with all three paralogs, six MAbs reacted with POTE-2{gamma}C and POTE-22, and three MAbs were specific to POTE-21. Epitopes of all 10 MAbs were located in the cysteine-rich repeats (CRRs) motifs located at the N-terminus of each POTE paralog. Testing the reactivity of each MAb with 12 different CRRs revealed slight differences among the antigenic determinants, which accounts for differences in cross-reactivity. Using MAbs HP8 and PG5 we were able to detect a POTE-actin fusion protein in human testis by immunoprecipitation followed by Western blotting. By immunohistochemistry we demonstrated that the POTE protein is expressed in primary spermatocytes, implying a role in spermatogenesis.

  8. Expression of POTE protein in human testis detected by novel monoclonal antibodies.

    PubMed

    Ise, Tomoko; Das, Sudipto; Nagata, Satoshi; Maeda, Hiroshi; Lee, Yoomi; Onda, Masanori; Anver, Miriam R; Bera, Tapan K; Pastan, Ira

    2008-01-25

    The POTE gene family is composed of 13 highly homologous paralogs preferentially expressed in prostate, ovary, testis, and placenta. We produced 10 monoclonal antibodies (MAbs) against three representative POTE paralogs: POTE-21, POTE-2gammaC, and POTE-22. One reacted with all three paralogs, six MAbs reacted with POTE-2gammaC and POTE-22, and three MAbs were specific to POTE-21. Epitopes of all 10 MAbs were located in the cysteine-rich repeats (CRRs) motifs located at the N-terminus of each POTE paralog. Testing the reactivity of each MAb with 12 different CRRs revealed slight differences among the antigenic determinants, which accounts for differences in cross-reactivity. Using MAbs HP8 and PG5 we were able to detect a POTE-actin fusion protein in human testis by immunoprecipitation followed by Western blotting. By immunohistochemistry we demonstrated that the POTE protein is expressed in primary spermatocytes, implying a role in spermatogenesis.

  9. Expression of POTE protein in human testis detected by novel monoclonal antibodies

    PubMed Central

    Ise, Tomoko; Das, Sudipto; Nagata, Satoshi; Maeda, Hiroshi; Lee, Yoomi; Onda, Masanori; Anver, Miriam R.; Bera, Tapan K.; Pastan, Ira

    2008-01-01

    The POTE gene family is composed of 13 highly homologous paralogs preferentially expressed in prostate, ovary, testis and placenta. We produced 10 monoclonal antibodies (MAbs) against 3 representative POTE paralogs: POTE-21, POTE-2γC and POTE-22. One reacted with all 3 paralogs, 6 MAbs reacted with POTE-2γC and POTE-22, and 3 MAbs were specific to POTE-21. Epitopes of all 10 MAbs were located in the cysteine-rich repeats (CRRs) motifs located at the N-terminus of each POTE paralog. Testing the reactivity of each MAb with 12 different CRRs revealed slight differences among the antigenic determinants, which accounts for differences in cross-reactivity. Using MAbs HP8 and PG5 we were able to detect a POTE-actin fusion protein in human testis by immunoprecipitation followed by Western blotting. By immunohistochemistry we demonstrated that the POTE protein is expressed in primary spermatocytes, implying a role in spermatogenesis. PMID:17996727

  10. Potent human monoclonal antibodies against SARS CoV, Nipah and Hendra viruses.

    PubMed

    Prabakaran, Ponraj; Zhu, Zhongyu; Xiao, Xiaodong; Biragyn, Arya; Dimitrov, Antony S; Broder, Christopher C; Dimitrov, Dimiter S

    2009-03-01

    Recently, several potently neutralizing fully human monoclonal antibodies (hmAbs) targeting the severe acute respiratory syndrome-associated coronavirus (SARS CoV) S glycoprotein, and the G glycoprotein of the paramyxoviruses Hendra virus (HeV) and Nipah virus (NiV) have been discovered [corrected]. To examine, compare and contrast the functional characteristics of hmAbs with the potential for prophylaxis and treatment of diseases caused by SARS CoV, HeV and NiV. A review of relevant literature. Structural, functional and biochemical analyses [corrected] have provided insights into the molecular mechanisms of receptor recognition and antibody neutralization, and suggested that these antibodies alone or in combination could fight the viruses' heterogeneity and mutability, which is a major problem in the development of effective therapeutic agents against viruses, including therapeutic antibodies.

  11. Binding and regulation of cellular functions by monoclonal antibodies against human tumor necrosis factor receptors

    PubMed Central

    1990-01-01

    The present study was undertaken to further characterize the interaction of monoclonal antibodies (mAbs) against tumor necrosis factor (TNF) receptors with different targets, and to assess their ability to influence TNF effects on U937 and human endothelial cell (HEC) functions. Actions of recombinant TNF-alpha on U937 and HEC were effectively inhibited by Htr-5 and Utr-1, and to a greater extent by a combination of both mAbs. These observations indicate that TNF interaction with antigenically different components of membrane receptors (p55 and p75) represents a crucial step in transduction of signals for TNF toxicity against U937 and TNF activation of HEC functions. PMID:2172437

  12. Phage display-based strategies for cloning and optimization of monoclonal antibodies directed against human pathogens.

    PubMed

    Clementi, Nicola; Mancini, Nicasio; Solforosi, Laura; Castelli, Matteo; Clementi, Massimo; Burioni, Roberto

    2012-01-01

    In the last two decades, several phage display-selected monoclonal antibodies (mAbs) have been described in the literature and a few of them have managed to reach the clinics. Among these, the anti-respiratory syncytial virus (RSV) Palivizumab, a phage-display optimized mAb, is the only marketed mAb directed against microbial pathogens. Palivizumab is a clear example of the importance of choosing the most appropriate strategy when selecting or optimizing an anti-infectious mAb. From this perspective, the extreme versatility of phage-display technology makes it a useful tool when setting up different strategies for the selection of mAbs directed against human pathogens, especially when their possible clinical use is considered. In this paper, we review the principal phage display strategies used to select anti-infectious mAbs, with particular attention focused on those used against hypervariable pathogens, such as HCV and influenza viruses.

  13. Monoclonal antibodies to the cell surface and a soluble form of the human nerve growth factor receptor

    SciTech Connect

    Clagett-Dame, M.; Chung, C.; Chao, M.V.; DiStefano, P.S. )

    1990-12-01

    Monoclonal antibodies (designated IIIG5, VIID1, VIIIC8, and XIF1) have been produced that bind to the human nerve growth factor receptor (NGF-R) as well as to a soluble, truncated form of the receptor (NGF-Rt). The antibodies were generated against partially purified NGF-Rt from the conditioned medium of E9b cells, a transfected mouse fibroblast cell line (Ltk-) that expresses large numbers of the low affinity form of the human NGF-R on its cell surface. Hybridomas were screened by radiometric immunosorbent assay (RISA) and by immunoprecipitation of solubilized cell surface receptor covalently cross-linked to {sup 125}I-NGF. Four positive lines were cloned by limiting dilution and were found to secrete monoclonal antibodies of the IgGl,k subclass. All monoclonal antibodies bound to both NGF-R and NGF-Rt. Two monoclonal antibodies (VIID1, XIF1) immunoblotted the NGF-R from E9b cell preparations resolved on non-reducing sodium dodecyl sulfate (SDS)-polyacrylamide gels. The antibodies immunoprecipitated NGF-R from both E9b cells and from SH-SY5Y human neuroblastoma cells. The monoclonal antibodies bound to monkey (rhesis and cynomolgus) NGF-Rt, but did not cross-react with NGF-R from chick or rat. Results of antibody competition studies demonstrated that three antibodies bound to a similar or overlapping epitope on the NGF-Rt and one monoclonal antibody (IIIG5) recognized a distinct receptor epitope. Antibodies that bound to different sites on the receptor were used to develop a sensitive 2-site RISA. The 2-site RISA can be used to rapidly quantitate NGF-R and NGF-Rt in large numbers of biological samples in the absence of added {sup 125}I-labeled NGF.

  14. Development and characterization of a monoclonal antibody to human embryonal carcinoma

    SciTech Connect

    Khazaeli, M.B.; Beierwaltes, W.H.; Pitt, G.S.; Kabza, G.A.; Rogers, K.J.; LoBuglio, A.F.

    1987-06-01

    A monoclonal anti-testicular carcinoma antibody was obtained via the somatic cell fusion technique by immunization of BALB/c mice with freshly prepared single cell suspension from a patient with testicular embryonal carcinoma with choriocarcinoma components. The hybridoma supernates were screened against the testicular carcinoma cells used in the immunization as well as normal mononuclear white blood cells isolated from the same patient. An antibody (5F9) was selected which bound to fresh tumor cells from two patients with embryonal testicular carcinoma and failed to bind to fresh tumor cells from 24 patients (2 seminoma, 2 melanoma, 3 neck, 2 esophageal, 1 ovarian, 3 colon, 1 prostate, 2 breast, 1 liposarcoma, 3 endometrial, 1 kidney, 1 adrenal, 1 larynx and 1 bladder tumors) or cell suspensions prepared from normal liver, lung, spleen, ovary, testes, kidney, red blood cells or white blood cells. The antibody was tested for its binding to several well established cancer cell lines, and was found to bind to the BeWo human choriocarcinoma and two human embryonal carcinoma cell lines. The antibody did not react with 22 other cell lines or with hCG. The antibody was labeled with /sup 131/I and injected into nude mice bearing BeWo tumors and evaluated for tumor localization by performing whole body scans with a gamma camera 5 days later. Six mice injected with the antibody showed positive tumor localization without the need for background subtraction while six mice injected with MOPC-21, a murine myeloma immunoglobulin, demonstrated much less tumor localization. Tissue distribution studies performed after scanning showed specific tumor localization (8:1 tumor: muscle) for the monoclonal antibody and no specific localization for MOPC-21.

  15. Novel monoclonal antibody against beta 1 integrin enhances cisplatin efficacy in human lung adenocarcinoma cells.

    PubMed

    Kim, Min-Young; Cho, Woon-Dong; Hong, Kwon Pyo; Choi, Da Bin; Hong, Jeong Won; Kim, Soseul; Moon, Yoo Ri; Son, Seung-Myoung; Lee, Ok-Jun; Lee, Ho-Chang; Song, Hyung Geun

    2016-05-01

    The use of anti-beta 1 integrin monoclonal antibody in lung cancer treatment has proven beneficial. Here, we developed a novel monoclonal antibody (mAb), called P5, by immunizing mice with human peripheral blood mononuclear cells (PBMC). Its anti-tumor effect is now being tested, in a clinical phase III trial, in combinatorial treatments with various chemical drugs. To confirm that P5 indeed binds to beta 1 integrin, cell lysates were immunoprecipitated with commercial anti-beta 1 integrin mAb (TS2/16) and immunoblotted against P5 to reveal a 140 kDa molecular weight band, as expected. Immunoprecipitation with P5 followed by LC/MS protein sequence analysis further verified P5 antigen to be beta 1 integrin. Cisplatin treatment upregulated cell surface expression of beta 1 integrin in A549 cells, while causing inhibition of cell growth. When cells were co-treated with different concentrations of P5 mAb, the cisplatin-mediated inhibitory effect was enhanced in a dose-dependent manner. Our findings show that a combinatorial treatment of P5 mAb and cisplatin in A549 cells resulted in a 30% increase in apoptosis, compared to baseline, and significantly more when compared to either the cisplatin or P5 alone group. The entire peptide sequences in CDR from variable region of Ig heavy and light chain gene for P5 mAb are also disclosed. Together, these results provide evidence of the beneficial effect of P5 mAb in combinatorial treatment of human lung adenocarcinoma.

  16. Monoclonal antibody GB3, a new probe for the study of human basement membranes and hemidesmosomes

    SciTech Connect

    Verrando, P.; Pisani, A.; Serieys, N.; Ortonne, J.P. ); Hsi, Baeli; Yeh, Changjing )

    1987-05-01

    A monoclonal antibody, GB3, has been raised against human amnion. Not only does GB3 bind to amniotic basement membrane, but it also recognizes an antigenic structure expressed by epidermal as well as by some other human basement membranes. This antigen is synthesized (and excreted) by cultured normal human epidermal keratinocytes. It is expressed to a lesser extent by the A431 epidermoid carcinoma cell line, but is not expressed by the SV40 virus-transformed SVK14 keratinocyte cell line. In ultrastructural studies, this antigen was located in the epidermal basement membrane, both in the lamina densa and in the lamina lucida, associated with hemidesmosomes. It was identified as a protein by in vitro proteolytic cleavage studies. The radio-immunoprecipitates from cultured human keratinocytes, analyzed by SDS-PAGE, showed that GB3 recognized five polypeptides of 93.5, 125, 130, 146 and 150 kD under reducing conditions. The tissue distribution of the antigen and the molecular weights (MWs) of its constitutive polypeptides suggest that it is different from other known components of basement membranes. It may provide a biochemical marker for hemidesmosomes. Furthermore, GB3 represents an interesting and original clinical probe, since the antigenic structure recognized by GB3 is lacking in Junctional Epidermolysis Bullosa, a lethal genodermatosis in which a dermo-epidermal splitting occurs at the level of lamina lucida.

  17. Rheumatoid factor interference in immunogenicity assays for human monoclonal antibody therapeutics.

    PubMed

    Tatarewicz, Suzanna; Miller, Jill M; Swanson, Steven J; Moxness, Michael S

    2010-05-31

    Rheumatoid factors (RFs) are endogenous human antibodies that bind to human gamma globulins. RFs demonstrate preferential binding to aggregated gamma globulins and are involved in the clearing mechanism of immune complexes. Immunoassays designed to measure human anti-human antibodies (HAHA) after administration of monoclonal antibody therapeutics are thus vulnerable to interference from RFs. When using a sensitive electrochemiluminescent (ECL) bridging immunoassay, samples from subjects with rheumatoid arthritis demonstrated much higher baseline reactivity than healthy subjects. Interference was found to be dependent on the aggregation state of the therapeutic antibody that had been conjugated with the detection reagent (ruthenium). Size exclusion high performance liquid chromatography (SE-HPLC) demonstrated that of the total integrated peaks, as little as 0.55% high molecular weight aggregates (>600kDa) were sufficient to cause increased reactivity. Stability studies of the ruthenium and biotin conjugated therapeutic antibody indicated that storage time, temperature and buffer formulation were critical in maintaining the integrity of the reagents. Through careful SE-HPLC monitoring we were able to choose appropriate storage and buffer conditions which led to a reduction in the false reactivity rate in therapeutic-naïve serum from a rheumatoid arthritis population.

  18. Unique glycoprotein antigen defined by monoclonal antibody on human neurobiastoma cells

    SciTech Connect

    Mujoo, K.; Spiro, R.C.; Reisfeld, R.A.

    1986-05-01

    The authors have characterized a new target antigen on the surface of human neuroblastoma cells and defined it with a monoclonal antibody (Mab) 5G3. This antibody is of IgG2a type and has an association constant of 8 x 10/sup 9/ M/sup -1/. In ELISA assays, Mab 5G3 reacted with human neuroblastoma as well as melanoma, squamous lung, skin carcinoma, and osteogenic sarcoma. Immunocytochemical analysis of frozen tissue sections revealed strong reactivity with all neuroblastoma tissues and marginal reactivity with melanoma and glioma tissues. There was no reactivity with fetal or normal tissues with the exception of cerebellum. The antigen recognized by Mab 5G3 is a glycoprotein of 200 and 215 kDa expressed on the SK-N-AS neuroblastoma cells. The antigen appears to contain N-linked carbohydrates based on treatment of human neuroblastoma cells with tunicamycin before and after intrinsic radiolabeling followed by indirect immunoprecipitation. The pulse-chase biosynthetic studies followed by indirect immunoprecipitation and SDS-PAGE indicated the precursor/product relationship between 200 and 215 kDa molecules. The 200 kDa component is endoglycosidase H-sensitive, whereas 215 kDa molecule is Endo-H resistant. The 215 kDa component is also sulfated, sialylated, and phosphorylated at serine residues. Preliminary data suggests that Mab, aside from identifying a unique target antigen on human neuroblastoma cells, may be suited as a targeting device for chemotherapeutic drugs.

  19. Efficient generation of a monoclonal antibody against the human C-type lectin receptor DCIR by targeting murine dendritic cells

    PubMed Central

    Heidkamp, Gordon F.; Neubert, Kirsten; Haertel, Eric; Nimmerjahn, Falk; Nussenzweig, Michel C.; Dudziak, Diana

    2010-01-01

    1. Summary Dendritic cells (DCs) are very important for the generation of long lasting immune responses against pathogens or the induction of anti-tumor responses. Targeting antigen to dendritic cells via monoclonal antibodies specific for DC cell surface receptors such as DEC205 was shown to elicit potent cellular and humoral immune responses in vivo. Therefore we investigated whether this novel strategy might also be useful for the generation of new monoclonal antibodies against molecules of choice. We show, that by targeting the extracellular domain of the human C-type lectin receptor ClecSF6/DCIR/LLIR (hDCIR) to DEC205 on DCs in vivo, we were able to generate highly specific monoclonal antibodies against hDCIR. PMID:20566350

  20. Immunohistochemical Examination of Novel Rat Monoclonal Antibodies against Mouse and Human Podoplanin

    PubMed Central

    Kaji, Chiaki; Tsujimoto, Yuta; Kato Kaneko, Mika; Kato, Yukinari; Sawa, Yoshihiko

    2012-01-01

    This study aims to develop new monoclonal antibodies (mAbs) against mouse and human podoplanin. Rats were immunized with synthetic peptides, corresponding to amino acids 38–51 of mouse podoplanin or human podoplanin which is 100% homologous to the same site of monkey podoplanin; anti-mouse podoplanin mAb PMab-1 (IgG2a) and anti-human mAb NZ-1.2 (IgG2a) were established. In immunocytochemistry, the mouse melanoma B16-F10 and mouse podoplanin (mPDPN)-expressed CHO transfectant were stained by PMab-1; human lymphatic endothelial cells (LEC) and human podoplanin (hPDPN)-expressed squamous cell carcinoma HSC3 transfectant, were stained by NZ-1.2. Western-blot analysis detected an about 40-kDa protein in CHO-mPDPN and B16-F10 by PMab-1, and in HSC3-hPDPN and LEC by NZ-1.2. In frozen sections, PMab-1 reacted with mouse kidney, pulmonary alveoli, pulmonary pleura, and salivary gland myoepithelial cells while NZ-1.2 reacted to the human salivary gland myoepithelial cells. The immunostaining of paraffin-embedded sections also showed the reaction of PMab-1 or NZ-1.2 to the mouse or monkey kidney glomerulus, pulmonary alveoli, and lung lymphatic vessels. These results indicate that the two novel rat mAbs to the mouse and human/monkey podoplanin are useful for Western-blot and immunostaining of somatic tissues on paraffin-embedded sections as well as frozen sections. PMID:23012488

  1. Human anti-murine immune response following administration of radiolabeled monoclonal antibodies

    SciTech Connect

    Reynolds, J.C.; Carrasquillo, J.C.; Larson, S.M.

    1985-05-01

    The author's purpose is to measure circulating anti-murine immunoglobulin antibodies (HAMA) in patients who previously received radiolabeled monoclonal antibodies (MoAb) for tumor imaging and therapy. Because the presence of HAMA may negate further use of MoAb in patients, it is important to determine the frequency and rate of HAMA development. Patients received radiolabeled MoAb Fab 96.5 (IgG2a), Fab 48.7 (IgG1), T101 (IgG2a), B72.3 (IgG1), 9.2.27 (IgG2a) and 791T/36 (IgG2b). HAMA was measured by incubating I-125 labeled 96.5, 48.7 or B72.3 with serum and isolating human IgG with Staphyloccocal protein A cells by centrifugation. The assays were capable of detecting HAMA concentrations which bound 20 ng/ml of monoclonal antibody. 12 of 37 patients who received IgG developed HAMA within 4 months of a single injection. For one patient this occurred as early as 1 week post injection. 2 of 18 patients who received Fab developed HAMA. One of these patients received multiple injections of MoAb. 2 of 3 patients who received IgG2B were positive for HAMA. There was no apparent difference in the positive HAMA when antibody or fragment was given SubQ or IV. The authors conclude that the use of IgG MoAb are more likely to lead to the development of antimurine immunoglobulin antibodies.

  2. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment

    PubMed Central

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region. PMID:25692880

  3. Sequence similarities among kappa IIIb chains of monoclonal human IgM kappa autoantibodies

    PubMed Central

    1984-01-01

    Light chains of the serologically and chemically defined V region sub- subgroup kappa IIIb are preferentially associated with several types of human IgM kappa (monoclonal) autoantibodies and are remarkably homologous in primary structure, as evidenced by partial amino acid sequence data. To establish the extent of homology among such proteins, we have determined the complete variable region (V) sequence of the light chains of four monoclonal IgM kappa autoantibodies, of which two (GAR and GOT) are rheumatoid factors (RFs), the third (SON) has anti- apo beta lipoprotein specificity, and the fourth (PIE) binds specifically to intermediate filaments. The region encoded by the V kappa segment gene (positions 1-95) in all four light (L) chains is virtually identical in sequence, differing by only one residue in the FR3 of protein SON and in the first CDR of protein GOT. Further, the CDR3 of kappa chain SON contains an additional residue (prolyl) located at the carboxyl-terminus of the V segment. The region encoded by the J gene (positions 96-108) is identical after position 96 for the two RFs GAR and GOT (J kappa 2), but different in proteins SON (J kappa 4) and PIE (J kappa 1). The amino acid residue at position 96, located in CDR3 at the site of combinatoriaL joining of the V kappa and J kappa gene segments and involved as a contacting residue in the hapten binding site, is different in all four light chains. These results demonstrate the extensive homology in sequence among light chains of IgM kappa autoantibodies and indicate that a particular V kappa germ line gene, kappa IIIb, is expressed as a phylogenetic response to certain self antigens or as part of a selection process by which these autoimmune responses are regulated. PMID:6432934

  4. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

    PubMed

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

  5. Production and Characterization of Monoclonal Antibodies against Human Nuclear Protein FAM76B

    PubMed Central

    Zheng, Xiaojing; Li, Yanqing; Zhao, Junli; Wang, Dongyang; Xia, Haibin; Mao, Qinwen

    2016-01-01

    Human FAM76B (hFAM76B) is a 39 kDa protein that contains homopolymeric histidine tracts, a targeting signal for nuclear speckles. FAM76B is highly conserved among different species, suggesting that it may play an important physiological role in normal cellular functions. However, a lack of appropriate tools has hampered study of this potentially important protein. To facilitate research into the biological function(s) of FAM76B, murine monoclonal antibodies (MAbs) against hFAM76B were generated by using purified, prokaryotically expressed hFAM76B protein. Six strains of MAbs specific for hFAM76B were obtained and characterized. The specificity of MAbs was validated by using FAM76B-/- HEK 293 cell line. Double immunofluorescence followed by laser confocal microscopy confirmed the nuclear speckle localization of hFAM76B, and the specific domains recognized by different MAbs were further elucidated by Western blot. Due to the high conservation of protein sequences between mouse and human FAM76B, MAbs against hFAM76B were shown to react with mouse FAM76B (mFAM76B) specifically. Lastly, FAM76B was found to be expressed in the normal tissues of most human organs, though to different extents. The MAbs produced in this study should provide a useful tool for investigating the biological function(s) of FAM76B. PMID:27018871

  6. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits.

    PubMed

    Robinson, James E; Hastie, Kathryn M; Cross, Robert W; Yenni, Rachael E; Elliott, Deborah H; Rouelle, Julie A; Kannadka, Chandrika B; Smira, Ashley A; Garry, Courtney E; Bradley, Benjamin T; Yu, Haini; Shaffer, Jeffrey G; Boisen, Matt L; Hartnett, Jessica N; Zandonatti, Michelle A; Rowland, Megan M; Heinrich, Megan L; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C; Andersen, Kristian G; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J; Fonnie, Richard; Jalloh, Simbirie C; Kargbo, Brima; Vandi, Mohamed A; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A; Okokhere, Peter O; Follarin, Onikepe A; Schieffelin, John S; Pitts, Kelly R; Geisbert, Joan B; Kulakoski, Peter C; Wilson, Russell B; Happi, Christian T; Sabeti, Pardis C; Gevao, Sahr M; Khan, S Humarr; Grant, Donald S; Geisbert, Thomas W; Saphire, Erica Ollmann; Branco, Luis M; Garry, Robert F

    2016-05-10

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design.

  7. Human monoclonal antibodies to West Nile virus identify epitopes on the prM protein.

    PubMed

    Calvert, Amanda E; Kalantarov, Gavreel F; Chang, Gwong-Jen J; Trakht, Ilya; Blair, Carol D; Roehrig, John T

    2011-02-05

    Hybridoma cell lines (2E8, 8G8 and 5G12) producing fully human monoclonal antibodies (hMAbs) specific for the pre-membrane (prM) protein of West Nile virus (WNV) were prepared using a human fusion partner cell line, MFP-2, and human peripheral blood lymphocytes from a blood donor diagnosed with WNV fever in 2004. Using site-directed mutagenesis of a WNV-like particle (VLP) we identified 4 amino acid residues in the prM protein unique to WNV and important in the binding of these hMAbs to the VLP. Residues V19 and L33 are important epitopes for the binding of all three hMAbs. Mutations at residue, T20 and T24 affected the binding of hMAbs, 8G8 and 5G12 only. These hMAbs did not significantly protect AG129 interferon-deficient mice or Swiss Webster outbred mice from WNV infection.

  8. Efficacy of broadly neutralizing monoclonal antibody PG16 in HIV-infected humanized mice.

    PubMed

    Stoddart, Cheryl A; Galkina, Sofiya A; Joshi, Pheroze; Kosikova, Galina; Long, Brian R; Maidji, Ekaterina; Moreno, Mary E; Rivera, Jose M; Sanford, Ukina R; Sloan, Barbara; Cieplak, Witold; Wrin, Terri; Chan-Hui, Po-Ying

    2014-08-01

    Highly potent broadly neutralizing human monoclonal antibodies hold promise for HIV prophylaxis and treatment. We used the SCID-hu Thy/Liv and BLT humanized mouse models to study the efficacy of these antibodies, primarily PG16, against HIV-1 clades A, B, and C. PG16 targets a conserved epitope in the V1/V2 region of gp120 common to 70-80% of HIV-1 isolates from multiple clades and has extremely potent in vitro activity against HIVJR-CSF. PG16 was highly efficacious in SCID-hu mice as a single intraperitoneal administration the day before inoculation of R5-tropic HIV directly into their Thy/Liv implants and demonstrated even greater efficacy if PG16 administration was continued after Thy/Liv implant HIV inoculation. However, PG16 as monotherapy had no activity in humanized mice with established R5-tropic HIV infection. These results provide evidence of tissue penetration of the antibodies, which could aid in their ability to prevent infection if virus crosses the mucosal barrier.

  9. Monoclonal antibodies to two epitopes of beta-human chorionic gonadotropin for the treatment of cancer.

    PubMed

    Iversen, Patrick L; Mourich, Dan V; Moulton, Hong M

    2003-04-01

    Human chorionic gonadotropin (hCG) is expressed by many histological types of cancer and may play an important role in tumor maintenance and progression. Vaccination of patients with the therapeutic peptide Avicine (CTP37; AVI BioPharma Inc/SuperGen Inc), that contains 37 amino acids from the carboxyl terminus (CTP37; AVI BioPharma Inc/SuperGen Inc) of hCG, can result in two distinct antibody responses to separate epitopes within the peptide. Colorectal cancer patients who develop both anti-hCG responses show a significant improvement in median survival time. These observations provide a compelling rationale for the development of two human monoclonal antibodies (mAbs), one for each of the epitopes within the 37 amino acid peptide region of hCG. Two such human mAbs, both exhibiting a high degree of specificity and affinity have been prepared using XenoMouse technology. These mAbs may prove useful in multiple clinical settings for the treatment of various cancers. Treatment options may include passive immunotherapy with both mAbs, mixed passive supplement to active specific immunotherapy with Avicine and conjugation of the mAbs with radioisotopes or cytotoxic drugs. The requirement for dual mAb therapy is consistent with current trends in the development of complex, non-toxic therapies for cancer.

  10. Human monoclonal antibodies to West Nile virus identify epitopes on the prM protein

    SciTech Connect

    Calvert, Amanda E.; Kalantarov, Gavreel F.; Chang, Gwong-Jen J.; Trakht, Ilya; Blair, Carol D.; Roehrig, John T.

    2011-02-05

    Hybridoma cell lines (2E8, 8G8 and 5G12) producing fully human monoclonal antibodies (hMAbs) specific for the pre-membrane (prM) protein of West Nile virus (WNV) were prepared using a human fusion partner cell line, MFP-2, and human peripheral blood lymphocytes from a blood donor diagnosed with WNV fever in 2004. Using site-directed mutagenesis of a WNV-like particle (VLP) we identified 4 amino acid residues in the prM protein unique to WNV and important in the binding of these hMAbs to the VLP. Residues V19 and L33 are important epitopes for the binding of all three hMAbs. Mutations at residue, T20 and T24 affected the binding of hMAbs, 8G8 and 5G12 only. These hMAbs did not significantly protect AG129 interferon-deficient mice or Swiss Webster outbred mice from WNV infection.

  11. Distribution of cardiac myosin isozymes in human conduction system. Immunohistochemical study using monoclonal antibodies.

    PubMed Central

    Kuro-o, M; Tsuchimochi, H; Ueda, S; Takaku, F; Yazaki, Y

    1986-01-01

    To determine the presence and distribution of cardiac myosin isozymes in the human conduction system, we performed an immunohistochemical study using monoclonal antibodies CMA19 and HMC14, which are specific for myosin heavy chains of human atrial type (alpha-type) and ventricular type (beta-type), respectively. Serial frozen sections of human hearts were obtained from autopsy samples and examined by indirect immunofluorescence. Alpha-type was found in all myofibers of sinus node and atrio-ventricular node, and in 55.2 +/- 10.2% (mean +/- SD, n = 5) of the myofibers of ventricular conduction tissue, which consists of the bundle of His, bundle branches, and the Purkinje network. In contrast, beta-type was found in all myofibers of the atrio-ventricular node and ventricular conduction tissue, whereas almost all myofibers of the sinus node were unlabeled by HMC14. Although the number of ventricular myofibers labeled by CMA19 was small, the labeled myofibers were more numerous in the subepicardial region than in the subendocardial region. These findings show that the gene coding for alpha-type is expressed predominantly in specialized myocardium compared with the adjacent ordinary working myocardium. Images PMID:3511096

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

    SciTech Connect

    Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki; Oshita, Masatoshi; Ideno, Shoji; Yunoki, Mikihiro; Kuhara, Motoki; Yamamoto, Naomasa; Okuno, Yoshinobu; Ikuta, Kazuyoshi

    2009-09-11

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

  13. Production and characterization of a panel of monoclonal antibodies against native human cellular prion protein.

    PubMed

    Jones, Michael; McLoughlin, Victoria; Connolly, John G; Farquhar, Christine F; MacGregor, Ian R; Head, Mark W

    2009-02-01

    The human prion diseases, such as variant Creutzfeldt-Jakob disease (vCJD), are characterized by the conversion of the normal cellular prion protein (PrP(C)) into an abnormal disease associated form (PrP(Sc)). Monoclonal antibodies (MAbs) that recognize these different PrP isoforms are valuable reagents both in the diagnosis of these diseases and in prion disease research in general but we know of no attempts to raise MAbs against native human PrP(C). We immunized prion protein gene ablated (PrP(-/-)) mice with native human PrP(C) purified from platelets (pHuPrP) generating a predominantly IgG isotype anti-pHuPrP polyclonal antibody response in all mice. Following fusion of splenocytes from the immunized mice with SP2/0 myeloma cells, we were able to identify single cell clone and cryopreserve 14 stable hybridoma cell lines producing MAbs that reacted with pHuPrP. The properties of these MAbs (such as isotype, binding to native/denatured pHuPrP, and HuPrP epitopes recognized) are described. Furthermore, several of these MAbs showed a selectivity in their ability to immunoprecipitate disease associated PrP(Sc) and its corresponding protease resistant core (PrP(res)).

  14. Production, characterization, and applications of two novel monoclonal antibodies against human interleukin-28A.

    PubMed

    Li, M; Chen, Y; Huang, T; Liu, Y; He, S

    2006-12-01

    Interleukin-28A (IL-28A) is a novel cytokine discovered in recent years and has been shown to have antiviral activity. In this study, IL-28A complementary DNA was inserted into prokaryotic expression vector pET-44 Ek/LIC. The Nus-S-His-tagged IL-28A fusion protein was expressed in Escherichia coli BL21 (DE3) in the soluble fraction. The fusion protein was purified by S-protein agarose affinity chromatography, and the fusion tag was removed from recombinant IL-28A by cleavage with thrombin. To prepare specific monoclonal antibody against human IL-28A, BALB/c mice were immunized with IL-28A, and hybridoma cell lines were obtained by fusing mouse spleen cells with myeloma NS-1 cells. Two strains of hybridoma cells, which produced the anti-human IL-28A antibodies 1B9 and 4B5 were obtained. They are IgM isotype and working in western blot analysis and enzyme-linked immunosorbent assay. In the present study, it was shown for the first time that human umbilical vein endothelial cells treated with interferon-alpha and poly(I:C) express IL-28A protein assessed by flow cytometry and immunofluorescent staining techniques. Immunohistochemistry showed that macrophage-like cells in colon and lung tissue and alveolar epithelial cells in lung tissue contain IL-28A, indicating a novel mechanism for both cell types to carry out their antivirus or antitumor functions.

  15. First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers.

    PubMed

    Stevens, Misty W; Henry, Ralph L; Owens, S Michael; Schutz, Ralph; Gentry, W Brooks

    2014-01-01

    This first-in-human study examined the safety and pharmacokinetics of ch-mAb7F9, an anti-methamphetamine monoclonal antibody, in healthy volunteers. Single, escalating doses of ch-mAb7F9 over the range of 0.2 to 20 mg/kg were administered to 42 subjects who were followed for 147 d. Safety was measured by physical examinations, adverse events, vital signs, electrocardiograms, and clinical laboratory testing. Serum ch-mAb7F9 concentration and immunogenicity analyses were performed. There were no serious adverse reactions or discontinuations from the study due to adverse events. No trends emerged in the frequency, relatedness, or severity of adverse events with increased dose or between active and placebo treated subjects. Ch-mAb7F9 displayed expected IgG pharmacokinetic parameters, including a half-life of 17-19 d in the 3 highest dose groups and volume of distribution of 5-6 L, suggesting the antibody is confined primarily to the vascular compartment. Four (12.5%) of the 32 subjects receiving ch-mAb7F9 were confirmed to have developed a human anti-chimeric antibody response by the end of the study; however, this response did not appear to be dose related. Overall, no apparent safety or tolerability concerns were identified; a maximum tolerated dose was not reached in this Phase 1 study. Ch-mAb7F9 therefore appears safe for human administration.

  16. Binding properties of monoclonal antibodies recognizing external epitopes of the human MDR1 P-glycoprotein.

    PubMed

    Schinkel, A H; Arceci, R J; Smit, J J; Wagenaar, E; Baas, F; Dollé, M; Tsuruo, T; Mechetner, E B; Roninson, I B; Borst, P

    1993-09-30

    Monoclonal antibodies (MAbs) recognizing external epitopes of the human MDR1 P-glycoprotein have been used both for the detection of multidrug-resistant cells and as specific inhibitors of P-glycoprotein-mediated multidrug resistance. Using a panel of recently developed transfected or transgenic cell lines containing variants of the human MDR1 and MDR3 P-glycoproteins, we have compared the specificity and binding properties of the previously isolated MAbs MRK16, HYB-241, UIC2 and 4E3, and of the newly isolated MAb 7G4. The removal of 1, 2 or all 3 of the N-glycosylation sites present in the first extracellular loop of MDR1 P-glycoprotein did not significantly affect the binding of these MAbs. In contrast, 20 amino acid deletion in the first extracellular loop of MDR1 P-glycoprotein completely abolished binding of UIC2, whereas the binding of all other MAbs was hardly affected. None of the MAbs tested bound detectably to cell lines containing a high level of the human MDR3 P-glycoprotein. The differences in the binding specificity between UIC2 and the other tested antibodies parallel the reported functional differences in the ability of these antibodies to inhibit P-glycoprotein-mediated drug efflux.

  17. Efficacy of Broadly Neutralizing Monoclonal Antibody PG16 in HIV-infected Humanized Mice

    PubMed Central

    Stoddart, Cheryl A.; Galkina, Sofiya A.; Joshi, Pheroze; Kosikova, Galina; Long, Brian R.; Maidji, Ekaterina; Moreno, Mary E.; Rivera, Jose M.; Sanford, Ukina R.; Sloan, Barbara; Cieplak, Witold; Wrin, Terri; Chan-Hui, Po-Ying

    2014-01-01

    Highly potent broadly neutralizing human monoclonal antibodies hold promise for HIV prophylaxis and treatment. We used the SCID-hu Thy/Liv and BLT humanized mouse models to study the efficacy of these antibodies, primarily PG16, against HIV-1 clade A, B, and C. PG16 targets a conserved epitope in the V1/V2 region of gp120 common to 70–80% of HIV-1 isolates from multiple clades and has extremely potent in vitro activity against HIVJR-CSF. PG16 was highly efficacious in SCID-hu mice as a single intraperitoneal administration the day before inoculation of R5-tropic HIV-1 directly into their Thy/Liv implants and demonstrated even greater efficacy if PG16 administration was continued after Thy/Liv implant HIV-1 infection. However, PG16 as monotherapy had no activity in humanized mice with established R5-tropic HIV-1 infection. These results provide evidence of tissue penetration of the antibodies, which could aid in their ability to prevent infection if virus crosses the mucosal barrier. PMID:24971704

  18. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

    PubMed Central

    Robinson, James E.; Hastie, Kathryn M.; Cross, Robert W.; Yenni, Rachael E.; Elliott, Deborah H.; Rouelle, Julie A.; Kannadka, Chandrika B.; Smira, Ashley A.; Garry, Courtney E.; Bradley, Benjamin T.; Yu, Haini; Shaffer, Jeffrey G.; Boisen, Matt L.; Hartnett, Jessica N.; Zandonatti, Michelle A.; Rowland, Megan M.; Heinrich, Megan L.; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C.; Andersen, Kristian G.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J.; Fonnie, Richard; Jalloh, Simbirie C.; Kargbo, Brima; Vandi, Mohamed A.; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A.; Okokhere, Peter O.; Follarin, Onikepe A.; Schieffelin, John S.; Pitts, Kelly R.; Geisbert, Joan B.; Kulakoski, Peter C.; Wilson, Russell B.; Happi, Christian T.; Sabeti, Pardis C.; Gevao, Sahr M.; Khan, S. Humarr; Grant, Donald S.; Geisbert, Thomas W.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  19. Novel monoclonal antibodies broadly reactive to human recombinant sapovirus-like particles.

    PubMed

    Kitamoto, Noritoshi; Oka, Tomoichiro; Katayama, Kazuhiko; Li, Tian-Cheng; Takeda, Naokazu; Kato, Yoji; Miyoshi, Tatsuya; Tanaka, Tomoyuki

    2012-11-01

    Sapovirus (SaV), a member of the family Caliciviridae, is an important cause of acute epidemic gastroenteritis in humans. Human SaV is genetically and antigenically diverse and can be classified into four genogroups (GI, GII, GIV, and GV) and 16 genotypes (7 GI [GI.1-7], 7 GII, [GII.1-7], 1 GIV and 1 GV), based on capsid sequence similarities. Monoclonal antibodies (MAbs) are powerful tools for examining viruses and proteins. PAI myeloma cells were fused with spleen cells from mice immunized with a single type of recombinant human SaV virus-like particles (VLPs) (GI.1, GI.5, GI.6, GII.3, GIV, or GV). Sixty-five hybrid clones producing MAbs were obtained. Twenty-four MAbs were characterized by ELISA, according to their cross-reactivity to each VLP (GI.1, GI.5, GI.6, GII.2, GII.3, GII.4, GII.7, GIV, and GV). The MAbs were classified by this method into: (i) MAbs broadly cross-reactive to all GI, GII, GIV and GV strains; (ii) those reactive in a genogroup-specific; and (iii) those reactive in a genotype-specific manner. Further analysis of three broadly cross-reactive MAbs with a competitive ELISA demonstrated that at least two different common epitopes are located on the capsid protein of human SaVs in the four genogroups. The MAbs generated and characterized in this study will be useful tools for further study of the antigenic and structural topography of the human SaV virion and for developing new diagnostic assays for human SaV.

  20. Fully-human Monoclonal Antibodies Against Human EphrinB2 and EphB4 | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cacner Institute's Nanobiology Program is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize fully-human monoclonal antibodies against human EphrinB2 and EphB4.

  1. Radial-immunodiffusion assay of human apolipoprotein A-I with use of two monoclonal antibodies combined.

    PubMed

    Marcovina, S; Di Cola, G; Catapano, A L

    1986-12-01

    We produced and characterized several monoclonal antibodies directed toward human plasma apolipoprotein A-I. Two of them, A-I-12 and A-I-57, individually precipitated purified or native high-density lipoprotein in agarose gel by double immunodiffusion. Because radial immunodiffusion performed with a single monoclonal antibody gave faint and diffuse rings of precipitation, we developed and optimized working conditions for using these two monoclonal antibodies combined to determine apolipoprotein A-I in human plasma. This combination gave easy-to-measure, clear, sharp rings, and linear and parallel standard curves for HDL3 (the primary standard) and a reference serum (the secondary standard). Moreover, no pretreatment of samples with dissociating agents or detergents is necessary. The assay was complete after overnight incubation, as compared with two to three days when polyclonal antisera were used. Apolipoprotein A-I concentrations as measured in 128 normolipidemic subjects and in 72 patients with various lipid disorders by the radial immunodiffusion technique with monoclonal antibodies (x) compared well (r = 0.882; y = 1.029x-0.036) with those measured by radial immunodiffusion with polyclonal antisera (y).

  2. Generation of Human Antigen-Specific Monoclonal IgM Antibodies Using Vaccinated “Human Immune System” Mice

    PubMed Central

    van Geelen, Caroline M. M.; Noerder, Miriam; Huntington, Nicholas D.; Lim, Annick; Yasuda, Etsuko; Diehl, Sean A.; Scheeren, Ferenc A.; Ott, Michael; Weijer, Kees; Wedemeyer, Heiner; Di Santo, James P.; Beaumont, Tim; Guzman, Carlos A.; Spits, Hergen

    2010-01-01

    Background Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the ‘humanization’ of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique. Methodology/Principal Findings After transplantation with CD34+CD38− human hematopoietic progenitor cells, BALB/c Rag2−/−IL-2Rγc−/− mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. “Human Immune System” mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19+CD27+ B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively. Conclusion/Significance This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens. PMID:20957227

  3. Monoclonal antibodies reactive with human breast or ovarian carcinoma: In vivo applications

    SciTech Connect

    Thor, A.D.; Edgerton, S.M. )

    1989-10-01

    Monoclonal antibodies (MoAbs) are unique and useful bioprobes that allow in vivo targeting of membrane-associated or circulating antigens. Most of the clinical trials to date have used low dosages of radiolabeled MoAb given in a single dose. Newer studies have included antibody fragments, repeated injections, intraperitoneal (IP) administration, and other labels such as 90Y. Clinical MoAb trials are often arduous, expensive, and time-consuming to perform. Before human use, animal studies and extensive MoAb characterization are required. The production of pharmaceutical grade, radiolabeled MoAb is technically difficult and costly. Clinical trials require administrative and patient consent as well as extensive written protocols. These studies necessitate interdepartmental and intradepartmental cooperation and coordination. Furthermore, the use of in vivo radiolabeled probes impacts many levels of health care providers from janitorial, nursing, and technical staff to laboratories and physicians. Simple blood tests or disposal of body excretions may concern nursing or technical staff with the possibility of radiation exposure. The responsibility for study design, personnel involvement, and prospective use in patients without a definitive cancer diagnosis ultimately rests with the physician. While many issues have been addressed, additional clinical trials, consideration of safety issues, and standardization between institutions will be necessary before the use of radiolabeled MoAb for diagnosis, management, or therapy of human tumors becomes routine. Continued cooperation and funding should ensure its achievement. 136 references.

  4. [Monoclonal antibodies of the ICO series against differentiation antigens of human lymphocytes].

    PubMed

    Baryshnikov, A Iu

    1990-08-01

    The principal characteristics of monoclonal antibodies (MCA) ICO have been presented. The MCA ICO panel includes MCA against differentiating antigens of T- and B-lymphocytes, myelomonocytes, human leukemia-associated antigens. The following MCA have been described: MCA ICO-87 against common T-cell antigen CD7, ICO-33 and ICO-80 against common T-cell antigen CD5, MCA ICO-10 against Thy-1 antigen of early thymocytes, ICO-44 against CD1c antigen of cortical thymocytes, MCA ICO-90 against CD3 antigen of mature T-lymphocytes, MCA ICO-86 against CD4 antigen of T-helper/inductor cells, MCA ICO-31 against CD8 antigen of T-suppressor/cytotoxic cells, MCA ICO-1 against nonpolymorphic antigens of HLA II class, MCA ICO-12 against CD22 antigen of B-lymphocytes, MCA ICO-30 against mu-chain of human IgGM, MCA ICO-66 against CD37 antigen of B-lymphocytes, MCA ICO-88 against antigen of activated T- and B-cells, MCA ICO-35 against lymphoblasts, MCA ICO-88 against CD38 antigen of thymocytes and activated cells.

  5. Tracking the emerging human pathogen Pseudallescheria boydii by using highly specific monoclonal antibodies.

    PubMed

    Thornton, Christopher R

    2009-05-01

    Pseudallescheria boydii has long been known to cause white grain mycetoma in immunocompetent humans, but it has recently emerged as an opportunistic pathogen of humans, causing potentially fatal invasive infections in immunocompromised individuals and evacuees of natural disasters, such as tsunamis and hurricanes. The diagnosis of P. boydii is problematic since it exhibits morphological characteristics similar to those of other hyaline fungi that cause infectious diseases, such as Aspergillus fumigatus and Scedosporium prolificans. This paper describes the development of immunoglobulin M (IgM) and IgG1 kappa-light chain monoclonal antibodies (MAbs) specific to P. boydii and certain closely related fungi. The MAbs bind to an immunodominant carbohydrate epitope on an extracellular 120-kDa antigen present in the spore and hyphal cell walls of P. boydii and Scedosporium apiospermum. The MAbs do not react with S. prolificans, Scedosporium dehoogii, or a large number of clinically relevant fungi, including A. fumigatus, Candida albicans, Cryptococcus neoformans, Fusarium solani, and Rhizopus oryzae. The MAbs were used in immunofluorescence and double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to accurately differentiate P. boydii from other infectious fungi and to track the pathogen in environmental samples. Specificity of the DAS-ELISA was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of environmental isolates.

  6. Simulation of monoclonal antibody pharmacokinetics in humans using a minimal physiologically based model.

    PubMed

    Li, Linzhong; Gardner, Iain; Dostalek, Miroslav; Jamei, Masoud

    2014-09-01

    Compared to small chemical molecules, monoclonal antibodies and Fc-containing derivatives (mAbs) have unique pharmacokinetic behaviour characterised by relatively poor cellular permeability, minimal renal filtration, binding to FcRn, target-mediated drug disposition, and disposition via lymph. A minimal physiologically based pharmacokinetic (PBPK) model to describe the pharmacokinetics of mAbs in humans was developed. Within the model, the body is divided into three physiological compartments; plasma, a single tissue compartment and lymph. The tissue compartment is further sub-divided into vascular, endothelial and interstitial spaces. The model simultaneously describes the levels of endogenous IgG and exogenous mAbs in each compartment and sub-compartment and, in particular, considers the competition of these two species for FcRn binding in the endothelial space. A Monte-Carlo sampling approach is used to simulate the concentrations of endogenous IgG and mAb in a human population. Existing targeted-mediated drug disposition (TMDD) models are coupled with the minimal PBPK model to provide a general platform for simulating the pharmacokinetics of therapeutic antibodies using primarily pre-clinical data inputs. The feasibility of utilising pre-clinical data to parameterise the model and to simulate the pharmacokinetics of adalimumab and an anti-ALK1 antibody (PF-03446962) in a population of individuals was investigated and results were compared to published clinical data.

  7. LpMab-23: A Cancer-Specific Monoclonal Antibody Against Human Podoplanin.

    PubMed

    Yamada, Shinji; Ogasawara, Satoshi; Kaneko, Mika K; Kato, Yukinari

    2017-04-07

    Human podoplanin (hPDPN), the ligand of C-type lectin-like receptor-2, is involved in cancer metastasis. Until now, many monoclonal antibodies (mAbs) have been established against hPDPN. However, it is still difficult to develop a cancer-specific mAb (CasMab) against hPDPN because the protein sequence of hPDPN expressed in cancer cells is the same as that in normal cells. Herein, we report LpMab-23 of the mouse IgG1 subclass, a novel CasMab against hPDPN. In an immunohistochemical analysis, LpMab-23 reacted with tumor cells of human oral cancer, but did not react with normal cells such as lymphatic endothelial cells (LECs). In contrast, LpMab-17, another anti-hPDPN mAb, reacted with both tumor cells and LECs. Furthermore, flow cytometric analysis revealed that LpMab-23 reacted with hPDPN-expressing cancer cell lines (LN319, RERF-LC-AI/hPDPN, Y-MESO-14/hPDPN, and HSC3/hPDPN) but showed little reaction with normal cells (LECs and HEK-293T), although another anti-hPDPN mAb, LpMab-7, reacted with both hPDPN-expressing cancer cells and normal cells, indicating that LpMab-23 is a CasMab against hPDPN.

  8. Epitope mapping of 10 monoclonal antibodies against the pig analogue of human membrane cofactor protein (MCP)

    PubMed Central

    PéRez De La Lastra, J M; Van Den Berg, C W; Bullido, R; Almazán, F; Domínguez, J; Llanes, D; Morgan, B P

    1999-01-01

    Pig membrane cofactor protein (MCP; CD46) is a 50 000–60 000 MW glycoprotein that is expressed on a wide variety of cells, including erythrocytes. Pig MCP has cofactor activity for factor I-mediated cleavage of C3b and is an efficient regulator of the classical and alternative pathway of human and pig complement. A panel of 10 monoclonal antibodies (mAbs) was collected from two different laboratories; all of these mAbs were raised against pig leucocytes and all recognized the same complex banding pattern on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) of erythrocyte membranes. All were shown to be reactive with pig MCP and were divided into four groups of mutually competitive antibodies based on competition studies for membrane-bound MCP and for soluble MCP, the latter by surface plasmon resonance (SPR) analysis. The antigenic properties of membrane-bound and soluble MCP were similar, although some interesting differences were revealed. None of the 10 mAbs were cross-reactive with human MCP and only one showed cross-reactivity with leucocytes from a panel of large mammals – a weak cross-reactivity with a subset of dog leucocytes. All antibodies in one of the epitope groups and some in a second epitope group were able to block the functional activity of pig MCP, as measured by inhibition of MCP-catalysed C3 degradation by factor I. PMID:10233756

  9. Purification of human seminal plasma no. 7 antigen by immunoaffinity chromatography on bound monoclonal antibody.

    PubMed Central

    Isojima, S; Koyama, K; Fujiwara, N

    1982-01-01

    Human seminal plasma (HSP) No. 7 antigen was purified by immunoaffinity chromatography on bound 1C4 monoclonal antibody (Moab) (Shigeta et al., 1980b). The pooled HSP protein was applied to a CNBr-activated Sepharose 4B column of bound 1C4 Moab gamma globulin and the antibody bound fraction (fr) eluted was further purified by rechromatography in the same way. The purified antigen in the antibody bound fr obtained by rechromatography gave a single band on SDS-PAGE in a position corresponding to a molecular weight of 15,000 daltons. This preparation was 196.2 times more effective than the original HSP protein in neutralizing the sperm immobilizing activity of 1C4 Moab. The purified HSP No. 7 antigen contained iron, but was different from lactoferrin and transferrin. It did not show any enzymatic activities, such as those of acid phosphatase, LDH or trypsin inhibitor, and shared antigenicity with human milk protein. It was present in seminal plasma as a molecule with a higher molecular weight but seemed to be cleaved to a monomer of 15,000 daltons during purification procedures. This antigen is present on spermatozoa as sperm-coating antigen and the corresponding antibody can immobilize spermatozoa with complement. Images Fig. 3 PMID:7127911

  10. Early short-term treatment with neutralizing human monoclonal antibodies halts SHIV infection in infant macaques.

    PubMed

    Hessell, Ann J; Jaworski, J Pablo; Epson, Erin; Matsuda, Kenta; Pandey, Shilpi; Kahl, Christoph; Reed, Jason; Sutton, William F; Hammond, Katherine B; Cheever, Tracy A; Barnette, Philip T; Legasse, Alfred W; Planer, Shannon; Stanton, Jeffrey J; Pegu, Amarendra; Chen, Xuejun; Wang, Keyun; Siess, Don; Burke, David; Park, Byung S; Axthelm, Michael K; Lewis, Anne; Hirsch, Vanessa M; Graham, Barney S; Mascola, John R; Sacha, Jonah B; Haigwood, Nancy L

    2016-04-01

    Prevention of mother-to-child transmission (MTCT) of HIV remains a major objective where antenatal care is not readily accessible. We tested HIV-1-specific human neutralizing monoclonal antibodies (NmAbs) as a post-exposure therapy in an infant macaque model for intrapartum MTCT. One-month-old rhesus macaques were inoculated orally with the simian-human immunodeficiency virus SHIVSF162P3. On days 1, 4, 7 and 10 after virus exposure, we injected animals subcutaneously with NmAbs and quantified systemic distribution of NmAbs in multiple tissues within 24 h after antibody administration. Replicating virus was found in multiple tissues by day 1 in animals that were not treated. All NmAb-treated macaques were free of virus in blood and tissues at 6 months after exposure. We detected no anti-SHIV T cell responses in blood or tissues at necropsy, and no virus emerged after CD8(+) T cell depletion. These results suggest that early passive immunotherapy can eliminate early viral foci and thereby prevent the establishment of viral reservoirs.

  11. Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein

    PubMed Central

    Tang, Aimin; Callahan, Cheryl; Pristatsky, Pavlo; Swoyer, Ryan; Cejas, Pedro; Nahas, Debbie; Galli, Jennifer; Cosmi, Scott; DiStefano, Daniel; Hoang, Van M.; Bett, Andrew; Casimiro, Danilo

    2016-01-01

    Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F) glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab) is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs) against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction. PMID:27258388

  12. Development of humanized rabbit monoclonal antibodies against vascular endothelial growth factor receptor 2 with potential antitumor effects.

    PubMed

    Yu, Yanlan; Lee, Pierre; Ke, Yaohuang; Zhang, Yongke; Chen, Jungang; Dai, Jihong; Li, Mingzhen; Zhu, Weimin; Yu, Guo-Liang

    2013-07-05

    Vascular endothelial growth factor-A (VEGF-A) plays a critical role in physiologic and pathologic angiogenesis through its receptors especially through VEGFR2. The lack of cross-reactivity of monoclonal antibodies with human VEGFR2/mouse Flk-1 is a major obstacle in preclinical developments. In this study, using a unique hybridoma technique, we generated a panel of 30 neutralization anti-VEGFR2 rabbit monoclonal antibodies (RabMAbs) either blocking VEGF/VEGFR2 interaction or inhibiting VEGF-stimulated VEGFR2 tyrosine kinase phosphorylation. Among 18 RabMAbs with human/mouse VEGFR2 cross-reactivity, we humanized one lead candidate RabMAb by Mutational Lineage Guided (MLG) method and further demonstrated its potent inhibition of tumor growth in xenograft mouse model. Our study suggests that RabMAbs are highly relevant for therapeutic applications.

  13. Monoclonal Antibodies to the Human Insulin Receptor that Activate Glucose Transport but not Insulin Receptor Kinase Activity

    NASA Astrophysics Data System (ADS)

    Forsayeth, John R.; Caro, Jose F.; Sinha, Madhur K.; Maddux, Betty A.; Goldfine, Ira D.

    1987-05-01

    Three mouse monoclonal antibodies were produced that reacted with the α subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.

  14. A monoclonal IgM protein with antibody-like activity for human albumin.

    PubMed

    Hauptman, S; Tomasi, T B

    1974-03-01

    The serum of a patient (L'ec) with an IgM lambda monoclonal protein was noted to bind albumin on immunoelectrophoresis. Analytical ultracentrifugation of the L'ec serum demonstrated 23S and 12S peaks, but no 4S (albumin) boundary. Immunologically identical 20S and 9S IgM proteins were isolated from the serum and the addition in vitro of either the patient's albumin or albumin isolated from normal serum was shown to reconstitute the 23S and 12S boundaries. The binding of high molecular weight IgM to albumin was demonstated by Sephadex G200 chromatography with (125)I-labeled albumin and isolated IgM. Immunoelectrophoresis of the L'ec IgM developed with aggregated albumin (reverse immunoelectrophoresis) also demonstrated the binding of albumin to IgM. That all of the patient's IgM complexed with albumin was shown by affinity chromatography employing an aggregated albumin-immunoadsorbent column. Binding was shown to be of the noncovalent type by polyacrylamide gel electrophoresis in 8 M urea. With hot trypsin proteolysis, Fabmu and Fcmu5 fragments were isolated, and monomer albumin was shown to complex only with the Fabmu fragment by both analytical ultracentrifugation and molecular sieve chromatogaphy employing (125)I-labeled Fab fragments. 1 mol of Fabmu fragment bound 1 mol of monomer albumin. Polymers of human albumin, produced by heat aggregation, precipitated with the isolated L'ec protein on gel diffusion analysis and, when coated on sheep red blood cells, gave a hemagglutination titer greater than 1 million with the whole L'ec serum. 50 additional monoclonal IgM, 33 IgA, and 80 IgG sera failed to show precipitation or hemagglutination with aggregated albumin. Native monomer albumin inhibited precipitation only at high concentrations (> 50 mg/ml); dimer albumin or fragments of albumin produced by trypsin digestion inhibited at low concentrations (0.4 mg/ml). No reactivity occurred with the albumin of five other mammalian species, including bovine. The L

  15. A neutralizing human monoclonal antibody protects African Green monkeys from Hendra virus challenge

    PubMed Central

    Bossart, Katharine N.; Geisbert, Thomas W.; Feldmann, Heinz; Zhu, Zhongyu; Feldmann, Friederike; Geisbert, Joan B.; Yan, Lianying; Feng, Yan-Ru; Brining, Doug; Scott, Dana; Wang, Yanping; Dimitrov, Antony S.; Callison, Julie; Chan, Yee-Peng; Hickey, Andrew C.; Dimitrov, Dimiter S.; Broder, Christopher C.; Rockx, Barry

    2012-01-01

    Hendra virus (HeV) is a recently emerged zoonotic paramyxovirus that can cause a severe and often fatal disease in horses and humans. HeV is categorized as a biosafety level 4 agent, which has made the development of animal models and testing of potential therapeutics and vaccines challenging. Infection of African Green monkeys (AGMs) with HeV was recently demonstrated and disease mirrored fatal HeV infection in humans, manifesting as a multisystemic vasculitis with widespread virus replication in vascular tissues and severe pathologic manifestations in the lung, spleen and brain. Here, we demonstrate that m102.4, a potent HeV neutralizing human monoclonal antibody (hmAb), can protect AGMs from disease post infection (p.i.) with HeV. Fourteen AGMs were challenged intratracheally with a lethal dose of HeV and twelve subjects were infused twice with a 100 mg dose of m102.4 beginning at either 10 hr, 24 hr or 72 hr p.i. and again approximately 48 hrs later. The presence of viral RNA, infectious virus and HeV-specific immune responses demonstrated that all subjects were infected following challenge. All twelve AGMs that received m102.4 survived infection; whereas the untreated control subjects succumbed to disease on day 8 p.i.. Animals in the 72 hr treatment group exhibited neurological signs of disease but all animals started to recover by day 16 p.i.. These results represent successful post-exposure in vivo efficacy by an investigational drug against HeV and highlight the potential impact a hmAb can have on human disease. PMID:22013123

  16. Idiotypic and subgroup analysis of human monoclonal rheumatoid factors. Implications for structural and genetic basis of autoantibodies in humans.

    PubMed Central

    Silverman, G J; Goldfien, R D; Chen, P; Mageed, R A; Jefferis, R; Goñi, F; Frangione, B; Fong, S; Carson, D A

    1988-01-01

    Rheumatoid factors (RFs) in humans have been studied intensively because of their association with autoimmune and lymphoproliferative diseases. Many human IgM-RFs express cross-reactive idiotypes (CRIs) and have homologous light chains, some of which are encoded by a single V kappa gene, termed V kappa 325. However, although antibody activity generally requires the interaction between heavy and light chain variable regions, much less is known about structural relationships among RF heavy chains. To delineate further the structural and genetic basis of RF autoantibody synthesis, we generated "sequence-dependent" reagents specific for the human heavy and kappa light chain subgroups, and used them to analyze a panel of 27 monoclonal RFs. In addition, these proteins were tested for the expression of a heavy chain-associated CRI (G6), and a light chain-associated CRI (17.109). The results showed that most 17.109-reactive RFs contain heavy chains of the VHI subgroup, which bear the G6 idiotypic marker. However, among the 14 17.109-reactive RFs, two have heavy chains of the VHII subgroup, and another two contain heavy chains of the VHIII subgroup. Previously, we have shown that 17.109 is a phenotypic marker of the human V kappa 325 gene. Accordingly, these results demonstrate that the same human V kappa gene can combine with several VH genes from different VH gene subgroups to generate RF activity. Images PMID:3136191

  17. Recombinant human monoclonal antibodies to human cytomegalovirus glycoprotein B neutralize virus in a complement-dependent manner.

    PubMed

    Ohta, Akane; Fujita, Ayano; Murayama, Tsugiya; Iba, Yoshitaka; Kurosawa, Yoshikazu; Yoshikawa, Tetsushi; Asano, Yoshizo

    2009-11-01

    Human antibodies specific for HCMV are currently considered as potential anti-HCMV therapeutic agents. In this study, we used a combinatorial human antibody library to isolate and characterize complete human monoclonal antibodies that effectively neutralize HCMV in a complement-dependent manner. One hundred and six clones were isolated in two independent screens using HCMV virions and recombinant glycoprotein B, gB654, as antigens. All of the clones recognized the same molecule gB and were classified into 14 groups based on the amino acid sequence of the V(H) region. Seven representative clones from these 14 groups had a strong gB654 binding affinity by surface plasmon resonance (SPR). A pairwise binding competition analysis suggested that there were three groups based on differences in the gB recognition sites. Although Fab fragments of the seven groups showed strong affinity for gB, none of the Fab fragments neutralized HCMV infectivity in vitro. In contrast, complete human IgG(1) antibodies of at least three groups neutralized HCMV in a complement-dependent manner. These data suggest that potent therapeutic antibodies can be obtained from a human antibody library, including most of the functional antibodies that mediate humoral immunity to the selected pathogen.

  18. Crystallization of the Fab from a human monoclonal antibody against gp 41 of human immunodeficiency virus type I

    NASA Technical Reports Server (NTRS)

    Casale, Elena; He, Xiao-Min; Snyder, Robert S.; Carter, Daniel C.; Wenisch, Elisabeth; Jungbauer, Alois; Tauer, Christa; Ruker, Florian; Righetti, Pier Giorgio

    1990-01-01

    A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-1) has been crystallized in both intact and Fab forms. Crystals of the intact antibody grow as tetragonal-like prisms too small for conventional X-ray analysis. However, the Fab portion of the antibody produces suitable platelike crystals which belong to the space group P2(1)2(1)2(1) with unit cell constants of a = 66.5 A, b = 74.3 A, and c = 105.3 A. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.

  19. Crystallization of the Fab from a human monoclonal antibody against gp 41 of human immunodeficiency virus type I

    NASA Technical Reports Server (NTRS)

    Casale, Elena; He, Xiao-Min; Snyder, Robert S.; Carter, Daniel C.; Wenisch, Elisabeth; Jungbauer, Alois; Tauer, Christa; Ruker, Florian; Righetti, Pier Giorgio

    1990-01-01

    A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-1) has been crystallized in both intact and Fab forms. Crystals of the intact antibody grow as tetragonal-like prisms too small for conventional X-ray analysis. However, the Fab portion of the antibody produces suitable platelike crystals which belong to the space group P2(1)2(1)2(1) with unit cell constants of a = 66.5 A, b = 74.3 A, and c = 105.3 A. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.

  20. Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: KD values, binding pairs, and amino acid sequences.

    PubMed

    Peng, Hong; Brimijoin, Stephen; Hrabovska, Anna; Targosova, Katarina; Krejci, Eric; Blake, Thomas A; Johnson, Rudolph C; Masson, Patrick; Lockridge, Oksana

    2015-10-05

    Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger of nerve agents and organophosphorus pesticides. Mass spectrometry methods detect stable nerve agent adducts on the active site serine of HuBChE. The first step in sample preparation is immunopurification of HuBChE from plasma. Our goal was to identify monoclonal antibodies that could be used to immunopurify HuBChE on Dynabeads Protein G. Mouse anti-HuBChE monoclonal antibodies were obtained in the form of ascites fluid, dead hybridoma cells stored frozen at -80 °C for 30 years, or recently frozen hybridoma cells. RNA from 4 hybridoma cell lines was amplified by PCR for determination of their nucleotide and amino acid sequences. Full-length light and heavy chains were expressed, and the antibodies purified from culture medium. A fifth monoclonal was purchased. The 5 monoclonal antibodies were compared for ability to capture HuBChE from human plasma on Dynabeads Protein G. In addition, they were evaluated for binding affinity by Biacore and ELISA. Epitope mapping by pairing analysis was performed on the Octet Red96 instrument. The 5 monoclonal antibodies, B2 12-1, B2 18-5, 3E8, mAb2, and 11D8, had similar KD values of 10(-9) M for HuBChE. Monoclonal B2 18-5 outperformed the others in the Dynabeads Protein G assay where it captured 97% of the HuBChE in 0.5 ml plasma. Pairing analysis showed that 3E8 and B2 12-1 share the same epitope, 11D8 and B2 18-5 share the same epitope, but mAb2 and B2 12-1 or mAb2 and 3E8 bind to different epitopes on HuBChE. B2 18-5 was selected for establishment of a stable CHO cell line for production of mouse anti-HuBChE monoclonal.

  1. Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: KD values, binding pairs, and amino acid sequences

    PubMed Central

    Peng, Hong; Brimijoin, Stephen; Hrabovska, Anna; Targosova, Katarina; Krejci, Eric; Blake, Thomas A.; Johnson, Rudolph C.; Masson, Patrick; Lockridge, Oksana

    2016-01-01

    Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger of nerve agents and organophosphorus pesticides. Mass spectrometry methods detect stable nerve agent adducts on the active site serine of HuBChE. The first step in sample preparation is immunopurification of HuBChE from plasma. Our goal was to identify monoclonal antibodies that could be used to immunopurify HuBChE on Dynabeads Protein G. Mouse anti-HuBChE monoclonal antibodies were obtained in the form of ascites fluid, dead hybridoma cells stored frozen at −80°C for 30 years, or recently frozen hybridoma cells. RNA from 4 hybridoma cell lines was amplified by PCR for determination of their nucleotide and amino acid sequences. Full-length light and heavy chains were expressed, and the antibodies purified from culture medium. A fifth monoclonal was purchased. The 5 monoclonal antibodies were compared for ability to capture HuBChE from human plasma on Dynabeads Protein G. In addition, they were evaluated for binding affinity by Biacore and ELISA. Epitope mapping by pairing analysis was performed on the Octet Red96 instrument. The 5 monoclonal antibodies, B2 12-1, B2 18-5, 3E8, mAb2, and 11D8, had similar KD values of 10−9 M for HuBChE. Monoclonal B2 18-5 outperformed the others in the Dynabeads Protein G assay where it captured 97% of the HuBChE in 0.5 ml plasma. Pairing analysis showed that 3E8 and B2 12-1 share the same epitope, 11D8 and B2 18-5 share the same epitope, but mAb2 and B2 12-1 or mAb2 and 3E8 bind to different epitopes on HuBChE. B2 18-5 was selected for establishment of a stable CHO cell line for production of mouse anti-HuBChE monoclonal. PMID:26343001

  2. Selective Blockade of Human Natural Killer Cells by a Monoclonal Antibody

    NASA Astrophysics Data System (ADS)

    Newman, Walter

    1982-06-01

    A murine monoclonal antibody, 13.1, which blocks human natural killer (NK) cell-mediated lysis, has been developed. Hybridoma 13.1 was derived by fusion of NS-1 cells with spleen cells from mice immunized with an enriched population of NK cells. Supernatants of growing hybridomas were screened for their ability to block NK cell-mediated lysis of K562 targets. Antibody 13.1 is an IgG1 with a single light chain type and it does not fix complement. The 13.1 antigen is expressed on all peripheral blood mononuclear cells, with an antigen density approximately 1/30th that of HLA antigen heavy chain. Pretreatment and washing experiments revealed that inhibition of cytotoxicity occurred at the effector cell level only. Significant blocking was achieved with nanogram quantities of antibody and was not due to toxic effects on NK cells. Likewise, controls with other antibodies of the same subclass demonstrated that blocking was not a consequence of mere binding to NK cells. When a panel of 17 NK cell-susceptible targets was tested, the lysis of only 5 of these was blocked, namely K562, HL-60, KG-1, Daudi, and HEL, a human erythroleukemic cell line. The lysis of 12 human B cell and T cell line targets was not inhibited. In addition to the demonstration that the 13.1 antigen is a crucial cell surface structure involved in NK lysis, a heterogeneity of target cell recognition has been revealed that argues for the proposition that individual NK cells have multiple recognitive capabilities.

  3. Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy

    PubMed Central

    Hart, Felix; Danielczyk, Antje; Goletz, Steffen

    2017-01-01

    IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich) and hematological (CD20) cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated. PMID:28952521

  4. Identification of a Human Monoclonal Antibody To Replace Equine Diphtheria Antitoxin for Treatment of Diphtheria Intoxication

    PubMed Central

    Sevigny, Leila M.; Booth, Brian J.; Rowley, Kirk J.; Leav, Brett A.; Cheslock, Peter S.; Garrity, Kerry A.; Sloan, Susan E.; Thomas, William; Babcock, Gregory J.

    2013-01-01

    Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (hMAbs) directly from antibody-secreting cells in the circulation of immunized human volunteers. We isolated a panel of diverse hMAbs that recognized diphtheria toxoid, as well as a variety of recombinant protein fragments of diphtheria toxin. Forty-five unique hMAbs were tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays with a 50% effective concentration of 0.65 ng/ml for the lead candidate hMAb, 315C4. In addition, 25 μg of 315C4 completely protected guinea pigs from intoxication in an in vivo lethality model, yielding an estimated relative potency of 64 IU/mg. In comparison, 1.6 IU of DAT was necessary for full protection from morbidity and mortality in this model. We further established that our lead candidate hMAb binds to the receptor-binding domain of diphtheria toxin and physically blocks the toxin from binding to the putative receptor, heparin-binding epidermal growth factor-like growth factor. The discovery of a specific and potent human neutralizing antibody against diphtheria toxin holds promise as a potential therapeutic. PMID:23940209

  5. Identification of a human monoclonal antibody to replace equine diphtheria antitoxin for treatment of diphtheria intoxication.

    PubMed

    Sevigny, Leila M; Booth, Brian J; Rowley, Kirk J; Leav, Brett A; Cheslock, Peter S; Garrity, Kerry A; Sloan, Susan E; Thomas, William; Babcock, Gregory J; Wang, Yang

    2013-11-01

    Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (hMAbs) directly from antibody-secreting cells in the circulation of immunized human volunteers. We isolated a panel of diverse hMAbs that recognized diphtheria toxoid, as well as a variety of recombinant protein fragments of diphtheria toxin. Forty-five unique hMAbs were tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays with a 50% effective concentration of 0.65 ng/ml for the lead candidate hMAb, 315C4. In addition, 25 μg of 315C4 completely protected guinea pigs from intoxication in an in vivo lethality model, yielding an estimated relative potency of 64 IU/mg. In comparison, 1.6 IU of DAT was necessary for full protection from morbidity and mortality in this model. We further established that our lead candidate hMAb binds to the receptor-binding domain of diphtheria toxin and physically blocks the toxin from binding to the putative receptor, heparin-binding epidermal growth factor-like growth factor. The discovery of a specific and potent human neutralizing antibody against diphtheria toxin holds promise as a potential therapeutic.

  6. Targeting human vasohibin-2 by a neutralizing monoclonal antibody for anti-cancer treatment.

    PubMed

    Koyanagi, Takahiro; Suzuki, Yasuhiro; Komori, Kazuki; Saga, Yasushi; Matsubara, Shigeki; Fujiwara, Hiroyuki; Sato, Yasufumi

    2016-12-29

    There are 2 members of the vasohibin (VASH) family, VASH1 and VASH2. VASH1 is expressed mainly in endothelial cells to inhibit angiogenesis, whereas VASH2 is expressed mainly in cancer cells to stimulate tumor growth. The aim of the present study was to establish neutralizing monoclonal antibody (mAb) against human VASH2 and apply it as an anti-cancer treatment. We previously raised mAbs against several synthetic peptides of hVASH1, and found that one of them exhibited neutralizing activity against hVASH1. Because of the similarity in the amino acid sequences between VASH1 and VASH2, we hypothesized that they shared the bioactive center. When we mutated 4 amino acids within the region, the mutant VASH2 lost its pro-angiogenic activity. We therefore raised mAb against a synthetic peptide overlapping the mutated amino acids of hVASH2, and isolated one clone (1760) that almost completely inhibited the stimulatory effect of hVASH2 on the migration of and tube formation by ECs. When we used this clone 1760 antibody for cancer treatment, the peritoneal injection of it inhibited both tumor growth and angiogenesis in a mouse xenograft model of human cancer cells. In terms of anti-tumor activity, 25 mg/kg of clone 1760 was equivalent to 5 mg/kg of bevacizmab. From these results, we propose the targeting of human VASH2 with neutralizing mAb as a new strategy for cancer treatment. This article is protected by copyright. All rights reserved.

  7. Monoclonal Antibodies.

    ERIC Educational Resources Information Center

    Killington, R. A.; Powell, K. L.

    1984-01-01

    Monoclonal antibodies have provided an exciting addition to the "armory" of the molecular biologist and immunologist. This article discusses briefly the concept of, techniques available for, production of, and possible uses of monoclonal antibodies. (Author)

  8. Monoclonal Antibodies.

    ERIC Educational Resources Information Center

    Killington, R. A.; Powell, K. L.

    1984-01-01

    Monoclonal antibodies have provided an exciting addition to the "armory" of the molecular biologist and immunologist. This article discusses briefly the concept of, techniques available for, production of, and possible uses of monoclonal antibodies. (Author)

  9. Production and characterization of domain-specific monoclonal antibodies against human ECM1.

    PubMed

    Li, Ya; Li, Yanqing; Zhao, Junli; Wang, Dongyang; Mao, Qinwen; Xia, Haibin

    2016-05-01

    Human extracellular matrix protein-1 (hECM1), a secreted glycoprotein, is widely expressed in different tissues and organs. ECM1 has been implicated in multiple biological functions, which are potentially mediated by the interaction of different ECM1 domains with its ligands. However, the exact biological functions of ECM1 have not been elucidated yet, and the functional study of ECM1 has been partially hampered by the lack of sensitive and specific antibodies, especially those targeting different ECM1 domains. In this study, six strains of monoclonal antibody (MAb) against hECM1 were generated using purified, prokaryotically-expressed hECM1 as an immunogen. The MAbs were shown to be highly sensitive and specific, and suitable for western blot, immunoprecipitation assays and immunohistochemistry. Furthermore, the particular ECM1 domains recognized by different MAbs were identified. Lastly, the MAbs were found to have neutralizing activities, inhibiting the proliferation, migration and metastasis of MDA-MB-231 cells. In conclusion, the domain-specific anti-ECM1 MAbs produced in this study should provide a useful tool for investigating ECM1's biological functions, and cellular pathways in which it is involved.

  10. Applications of monoclonal antibodies and recombinant cytokines for the treatment of human colorectal and other carcinomas

    SciTech Connect

    Greiner, J.W.; Smalley, R.V.; Borden, E.C.; Martin, E.W.; Guadagni, F.; Roselli, M.; Schlom, J. )

    1991-01-01

    Monoclonal antibodies (MAbs) which recognize a human tumor antigen, termed tumor-associated glycoprotein-72 (TAG-72), have successfully been used to localize primary as well as metastatic colorectal tumor lesions in patients. The localization of the anti-TAG-72 MAbs has also been exploited intraoperatively using a hand-held gamma probe. That procedure, termed radioimmunoguided surgery (RIGS), has identified occult tumors which were not detected using standard external imaging techniques. In another clinical trial, interferon-gamma (IFN-gamma) was administered intraperitoneally to patients diagnosed with either gastrointestinal or ovarian carcinoma with secondary ascites. Analysis of the tumor cells isolated from the malignant ascites revealed a substantial increase in TAG-72 expression on the surface of tumor cells isolated from seven of eight patients. The results provide evidence that the combination of an anti-carcinoma MAb with the administration of a cytokine, such as IFN-gamma, may be an effective approach for the detection and subsequent treatment, of colorectal carcinoma. 15 references.

  11. Efficacy of monoclonal antibody against human recombinant tumor necrosis factor in E. coli-challenged swine.

    PubMed Central

    Jesmok, G.; Lindsey, C.; Duerr, M.; Fournel, M.; Emerson, T.

    1992-01-01

    Monoclonal antibody against human tumor necrosis factor alpha (TNF MAb) prevents death induced by intravenous gram-negative bacteria or lipopolysaccharide (LPS) in primates. Although these studies have demonstrated that TNF plays a prominent role in the development of lethal septic shock, exploration of dose-response relationships and possible mechanisms of protection have been limited. We addressed these questions in a series of experiments conducted in E. coli-challenged pigs. First, we determined that TNF MAb neutralized the cytotoxic activity found in septic pig plasma and in culture media from pig monocytes incubated with LPS. Second, we demonstrated that pretreatment with TNF MAb promotes survival, in a dose-dependent fashion, in an otherwise lethal E. coli bacteremic pig model. The results of the survival study highly correlate (r = 0.96, P < 0.01) the presence of TNF in the circulation with mortality. In an additional series of physiologic monitoring experiments designed to delineate possible mechanisms of protection, the authors demonstrate that TNF MAb pretreatment abrogates the prolonged leukopenia, thrombocytopenia, and microvascular leakiness resulting from intravenous bacterial challenge and maintains arterial blood pressure while diminishing pulmonary edema. These findings may provide a mechanism whereby neutralization of TNF systemically affords protection against the lethal sequelae of bacteremia. PMID:1443053

  12. Synthetic human monoclonal antibodies toward staphylococcal enterotoxin B (SEB) protective against toxic shock syndrome.

    PubMed

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

    2012-07-20

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

  13. Highly potent anti-human GPVI monoclonal antibodies derived from GPVI knockout mouse immunization.

    PubMed

    Matsumoto, Yutaka; Takizawa, Hisao; Gong, Xiaoqi; Le, Sang; Lockyer, Simon; Okuyama, Keiji; Tanaka, Michinori; Yoshitake, Masuhiro; Tandon, Narendra N; Kambayashi, Junichi

    2007-01-01

    Recent progress in the understanding of thrombus formation has suggested an important role for glycoprotein (GP) VI in this process. To clarify the exact role in detail, it is necessary to use specific, high affinity inhibitory antibodies. However, possibly due to the conserved structure of GPVI among species, it has been difficult to obtain potent antibodies. In this study, we developed highly potent anti-human GPVI monoclonal antibodies using GPVI knockout mice for immunization. Fab fragments of these antibodies, named OM1 and OM2, potently inhibit collagen-induced platelet aggregation. The IC(50) values for OM1 and OM2 are 0.6+/-0.05 and 1.7+/-0.5 microg/mL, respectively, showing potency greater than, or equal to that of abciximab (1.7+/-0.3 microg/mL), an anti-GPIIb/IIIa antibody. Fab fragments of OM1 and OM2 also potently inhibit collagen-induced ATP release, thromboxane A(2) formation, and platelet adhesion to immobilized collagen under static and flow conditions. Interestingly, platelet aggregation induced with collagen-related peptide was potently inhibited by OM2 but not OM1, indicating that OM1 recognizes an epitope that is different from collagen-related peptide-binding site on GPVI. These results suggest that OM1 and OM2 may be useful tools to understand the role of GPVI in thrombus formation. Furthermore, these antibodies have the potential to be developed as a new class of therapeutic tool.

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

    PubMed Central

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

    2012-01-01

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

  15. Diagnostic assays with monoclonal antibodies for the human serum parvovirus-like virus (SPLV).

    PubMed Central

    Cohen, B. J.; Mortimer, P. P.; Pereira, M. S.

    1983-01-01

    Monoclonal antibodies to the serum parvovirus-like virus (SPLV) were prepared by the hybridoma technique. They provided an antibody reagent which was used to develop solid phase antibody-capture assays for anti-SPLV IgM and IgG and for SPLV antigen. These assays were more sensitive than those based on human convalescent antibody as a reagent, and were more economical in the use of SPLV antigen. Their use enabled the serological responses to SPLV to be studied more fully and their sensitivity revealed the extent of SPLV infection. SPLV antigen was detected in four patients by both counter-immuno electrophoresis (CIE) and radioimmunoassay (RIA) and in two others by RIA alone. Parvovirus particles were seen in all six by electron microscopy. The anti-SPLV IgM response was measured in patients infected by SPLV. It was strong 5-18 days after the onset of illness, then declined and was only detectable in trace amounts after 6 months. Anti-SPLV IgG was also formed early, and persisted for at least 6 months. In a survey of 310 blood donors anti-SPLV was detected in 134 (43%) by CIE, but in 190 (61%) by IgG antibody capture RIA. Images Plate 1 PMID:6886408

  16. Early short-term treatment with neutralizing human monoclonal antibodies halts SHIV infection in newborn macaques

    PubMed Central

    Hessell, Ann J.; Jaworski, J. Pablo; Epson, Erin; Matsuda, Kenta; Pandey, Shilpi; Kahl, Christoph; Reed, Jason; Sutton, William F.; Hammond, Katherine B.; Cheever, Tracy A.; Barnette, Philip T.; Legasse, Alfred W.; Planer, Shannon; Stanton, Jeffrey J.; Pegu, Amarendra; Chen, Xuejun; Wang, Keyun; Siess, Don; Burke, David; Park, Byung S.; Axthelm, Michael K.; Lewis, Anne; Hirsch, Vanessa M.; Graham, Barney S.; Mascola, John R.; Sacha, Jonah B.; Haigwood, Nancy L.

    2016-01-01

    Prevention of mother to child transmission (MTCT) of HIV remains a major objective where antenatal care is not readily accessible. We tested anti-HIV-1 human neutralizing monoclonal antibodies (NmAb) as post-exposure therapy in an infant macaque model for intrapartum MTCT. One-month-old rhesus macaques were inoculated orally with SHIVSF162P3. On days 1, 4, 7, and 10 after virus exposure, we injected animals subcutaneously with NmAbs and quantified systemic distribution of NmAbs in multiple tissues within 24 h following administration. Replicating virus was found in multiple tissues by day 1 in animals without treatment. All NmAb-treated macaques were free of virus in blood and tissues at 6 months post-exposure. We detected no anti-SHIV T cell responses in blood or tissues at necropsy, and no virus emerged following CD8+ T cell depletion. These results suggest early passive immunotherapy can eliminate early viral foci and thereby prevent the establishment of viral reservoirs. PMID:26998834

  17. A novel human B-lymphocyte antigen shared with lymphoid dendritic cells: characterization by monoclonal antibody.

    PubMed Central

    Ishii, Y; Takami, T; Kokai, Y; Yuasa, H; Fujimoto, J; Takei, T; Kikuchi, K

    1985-01-01

    A novel cell-surface antigen (L25) expressed on human B cells was identified using a B cell-reactive monoclonal antibody (TB1-4D5). This L25 antigen was expressed on most B-lineage cells but not other cell types including thymocytes, T cells, granulocytes and monocytes. Thus, L25 existed on the majority of normal B cells present in the blood and lymphoid tissues, on cultured cell lines derived from normal and malignant B cells, and on neoplastic cells isolated from patients with B cell-derived malignancies. Though L25 was persistently expressed on B cells until 7 days after their activation with pokeweed mitogen (PWM), neither normal nor neoplastic plasma cells expressed L25. Moreover, L25 was present on cultured as well as freshly isolated leukaemic cells with common acute lymphatic leukaemia (CALL) antigen, which have been thought to correspond to the early B-cell ontogeny. Besides pan-B cell reactivity of TB1-4D5 antibody, it apparently cross-reacted with so-called dendritic or interdigitating cells located in the thymic-dependent areas of peripheral lymphoid organs, which have been presumably ascribed to those associated with accessory-cell function. Functional studies showed that anti-L25 (TB1-4D5) antibody had inhibitory effect on induction of immunoglobulin synthesis by PWM-stimulated B cells. Images Fig. 2 PMID:3907905

  18. Generation of Rat Monoclonal Antibodies Against Human Pancreatic Ductal Adenocarcinoma Cells.

    PubMed

    Higashi, Kiyoshi; Fujii, Nobuaki; Kushida, Masahiko; Yamada, Keita; Suzuki, Noriyuki; Saito, Koichi; Tachibana, Taro

    2016-06-01

    Pancreatic ductal adenocarcinoma is an aggressive tumor with a poor prognosis. Biomarkers that can detect the tumor in its early stages when it may be amenable to curative resection might improve prognosis. To discover novel markers expressed in primary pancreatic cancer, we generated a panel of monoclonal antibodies against pancreatic ductal adenocarcinoma cell line BxPC3 using a rat medial iliac lymph node method. The antigen recognized by 1B5A5 was expressed on the cell surface and secreted into the conditioned medium of BxPC3 cells, and characterized as glycoproteins with molecular mass between 60 and 90 kDa. A wide range of molecular weights of 1B5A5 antigen in several pancreatic cancer cell lines were observed. Immunohistochemistry using a human multiple organ tumor tissue array showed an enhanced expression of 1B5A5 antigen in pancreas, lung, stomach, breast, urinary bladder, colon, and cervix uteri cancers. Immunoprecipitation followed by proteomic analyses identified CEACAM6 as a 1B5A5 antigen. In addition, western blot analysis results indicated that the 1B5A5 epitope is located within an amino-terminal domain of CEACAM6. These results raised the possibility that our approach could lead to discovery of novel biomarkers for the early stage of cancers in a relatively short period of time.

  19. Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

    PubMed

    Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

    2016-04-22

    High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies.

  20. Characterization and Epitope Mapping of Human Monoclonal Antibodies to PDC-E2, the Immunodominant Autoantigen of Primary Biliary Cirrhosis

    PubMed Central

    Leung, Patrick S. C.; Krams, Sheri; Munoz, Santiago; Surh, Charles P.; Ansari, Aftab; Kenny, Thomas; Robbins, Dick L.; Fung, John; Starzl, Thomas E.; Maddrey, Willis; Coppel, Ross L.; Gershwin, M. Eric

    2010-01-01

    Further to define the epitopes of PDC-E2, the major autoantigen in primary biliary cirrhosis (PBC), we have developed and characterized five human monoclonal antibodies. These antibodies were derived by fusing a regional hepatic lymph node from a patient with PBC with the mouse human heterohybrid cell line F3B6. Previous studies of epitope mapping ofPDC-E2 have relied on whole sera and have suggested that the immunodominant epitope lies within the inner lipoyl domain of the molecule. However, selective absorption studies using whole sera and a series of overlapping recombinant peptides of PDC-E2 have suggested that the epitope may also include a large conformational component. Moreover, several laboratories have suggested that autoantibodies against the 2-oxo acids dehydrogenase autoantigens are cross-reactive. The five Inonoclonal antibodies generated included three IgG2a and two IgM antibodies and were studied for antigen specificity using recombinant PDC-E2, recombinant BCKD-E2, histone, dsDNA, IgG (Fe), collagen and a recombinant irrelevant liver specific control, the F alloantigen. The antibodies were also used to probe blots of human, bovine, mouse and rat mitochondria. Finally, fine specificity was studied by selective ELISA and absorption against overlapping expressing fragments of PDC-E2. All five monoclonals, but none of the other mitochondrial auto antigens were specific for PDC-E2. In fact, although affinity purified antibodies to PDC-E2 from patients with PBC cross-reacted with protein X, the human monoclonals did not, suggesting that protein X contains an epitope distinct from that found on PDC-E2. Additionally, all three IgG2 monoclonals recognized distinct epitopes within the inner lipoyl domain of PDC-E2. PMID:1283300

  1. Human biodistribution of sup 111 In-labeled B72. 3 monoclonal antibody

    SciTech Connect

    Harwood, S.J.; Carroll, R.G.; Webster, W.B.; Zangara, L.M.; Laven, D.L.; Morrissey, M.A.; Sinni, B.J. )

    1990-02-01

    The murine IgG1 monoclonal antibody B72.3 reacts with human colorectal, breast, lung, pancreatic, gastric, and ovarian tumors. Human biodistribution studies using intact {sup 131}I-B72.3 have been reported by Carrasquillo et al.. We have performed similar studies on five patients using i.v. infusion of 20 mg of intact {sup 111}In-B72.3 (Cytogen Corp.). Serum clearance is similar with a t1/2 of 64.2 h (range, 44-80) for {sup 111}In-B72.3 and 65 h (range, 32-106) for {sup 131}I-B72.3. However, organ biodistribution is markedly different. For {sup 131}I-B72.3, hepatic and splenic clearance mirrors blood pool clearance. For {sup 111}In-B72.3, there is rapid uptake in tumor, liver, spleen, kidney, lumbar spine, and testes by 2-6 h with no significant clearance over the next 9 days. For {sup 111}In-B72.3, quantitative analysis of liver (from biopsy specimens), spleen, kidney, and lumbar spine (from scintiphoto regions of interest after background subtraction and attenuation correction) shows the following peak organ biodistributions in percentage infused dose: liver, 32%; spleen, 3.9%; kidneys, 3.5%; and lumbar vertebral bodies (marrow sample), 2.7%. For both {sup 111}In-B72.3 and {sup 131}I-B72.3, the principal route of excretion from the body is urinary with excretion rate of {sup 131}I faster than {sup 111}In. The marked differences between {sup 111}In-B72.3 and {sup 131}I-B72.3 biodistribution and clearance strongly influence the dosimetry, immunodetection, and immunotherapeutic potentials of B72.3 MoAb.

  2. Characterization of a monoclonal antibody as the first specific inhibitor of human NTP diphosphohydrolase-3

    PubMed Central

    Munkonda, Mercedes N.; Pelletier, Julie; Ivanenkov, Vasily V.; Fausther, Michel; Tremblay, Alain; Künzli, Beat; Kirley, Terence L.; Sévigny, Jean

    2016-01-01

    The study and therapeutic modulation of purinergic signaling is hindered by a lack of specific inhibitors for NTP diphosphohydrolases (NTPDases), which are the terminating enzymes for these processes. In addition, little is known of the NTPDase protein structural elements that affect enzymatic activity and which could be used as targets for inhibitor design. In the present study, we report the first inhibitory monoclonal antibodies specific for an NTPDase, namely human NTPDase3 (EC 3.6.1.5), as assessed by ELISA, western blotting, flow cytometry, immunohistochemistry and inhibition assays. Antibody recognition of NTPDase3 is greatly attenuated by denaturation with SDS, and abolished by reducing agents, indicating the significance of the native conformation and the disulfide bonds for epitope recognition. Using site-directed chemical cleavage, the SDS-resistant parts of the epitope were located in two fragments of the C-terminal lobe of NTPDase3 (i.e. Leu220–Cys347 and Cys347–Pro485), which are both required for antibody binding. Additional site-directed mutagenesis revealed the importance of Ser297 and the fifth disulfide bond (Cys399–Cys422) for antibody binding, indicating that the discontinuous inhibitory epitope is located on the extracellular C-terminal lobe of NTPDase3. These antibodies inhibit recombinant NTPDase3 by 60–90%, depending on the conditions. More importantly, they also efficiently inhibit the NTPDase3 expressed in insulin secreting human pancreatic islet cells in situ. Because insulin secretion is modulated by extracellular ATP and purinergic receptors, this finding suggests the potential application of these inhibitory antibodies for the study and control of insulin secretion. PMID:19120451

  3. Detection of acrolein-derived cyclic DNA adducts in human cells by monoclonal antibodies.

    PubMed

    Pan, Jishen; Awoyemi, Bisola; Xuan, Zhuoli; Vohra, Priya; Wang, Hsiang-Tsui; Dyba, Marcin; Greenspan, Emily; Fu, Ying; Creswell, Karen; Zhang, Lihua; Berry, Deborah; Tang, Moon-Shong; Chung, Fung-Lung

    2012-12-17

    Acrolein (Acr) is a ubiquitous environmental pollutant found in cigarette smoke and automobile exhaust. It can also be produced endogenously by oxidation of polyunsaturated fatty acids. The Acr-derived 1,N(2)-propanodeoxyguanosine (Acr-dG) adducts in DNA are mutagenic lesions that are potentially involved in human cancers. In this study, monoclonal antibodies were raised against Acr-dG adducts and characterized using ELISA. They showed strong reactivity and specificity toward Acr-dG, weaker reactivity toward crotonaldehyde- and trans-4-hydroxy-2-nonenal-derived 1,N(2)-propanodeoxyguanosines, and weak or no reactivity toward 1,N(6)-ethenodeoxyadenosine and 8-oxo-deoxyguanosine. Using these antibodies, we developed assays to detect Acr-dG in vivo: first, a simple and quick FACS-based assay for detecting these adducts directly in cells; second, a highly sensitive direct ELISA assay for measuring Acr-dG in cells and tissues using only 1 μg of DNA without DNA digestion and sample enrichment; and third, a competitive ELISA for better quantitative measurement of Acr-dG levels in DNA samples. The assays were validated using Acr-treated HT29 cell DNA samples or calf thymus DNA, and the results were confirmed by LC-MS/MS-MRM. An immunohistochemical assay was also developed to detect and visualize Acr-dG in HT29 cells as well as in human oral cells. These antibody-based methods provide useful tools for the studies of Acr-dG as a cancer biomarker and of the molecular mechanisms by which cells respond to Acr-dG as a ubiquitous DNA lesion.

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

    PubMed

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

    2017-02-24

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

  5. Human cancer detection and immunotherapy with conjugated and non-conjugated monoclonal antibodies.

    PubMed

    Bodey, B; Siegel, S E; Kaiser, H E

    1996-01-01

    Classical therapeutic modalities such as surgery, radiation and chemotherapy not only fail to cure the majority of neoplastic disease, but their employment also leads to severe and debilitating side effects. The severe cancer related morbidity is often associated with the use of radiation and chemotherapy, making them less than ideal forms of therapy. Entirely new approaches to cancer therapy that are tumor cell directed, and specifically lethal to malignant cells and less toxic to normal tissues are being observed and developed, adhering to the old prayer "Destroy the diseased tissues, preserve the normal." Following the initial advances of Ehrlich, immunotherapy as a fourth modality of cancer therapy has already been developed and proven to be quite effective. Unfortunately, the cancer cell population is not a static entity, but rather a continually changing one. Considerable variations have been determined between individual malignant cells. Our strong belief is that it is necessary for present-day clinical oncologists to become aware of the existence of immunotherapy and learn how to employ it in order to improve the efficacy and decrease the side effects of modern cancer therapy. The development of hybridoma technology and the advances in monoclonal antibody (MoAB) production have revitalized the concept concerning the existence of cancer cell-targeted, specific "magic bullets". In addition, a variety of different agents (e.g. toxins, radionuclides, chemotherapeutic drugs) have been conjugated to mouse and human MoABs for selective delivery to cancer cells. Preclinical observations in athymic, nude mice using xenografted human cancers and mouse, anti-human MoABs were more than impressive and have lead to several clinical trials. Strategies for the employment of MoABs for cancer immunotherapy include: a) Immune reaction directed destruction of cancer cells; b) Interference with the growth and differentiation of malignant cells; c) Antigen epitope directed

  6. Preparation of Monoclonal Antibodies and a Simple Myeloperoxidase-Immunosorbent Assay for Detecting Human Myeloperoxidase.

    PubMed

    Bian, Zhi-Ping; Li, Xiong-Zhi; Wu, Heng-Fang; Xu, Jin-Dan; Gu, Chun-Rong; Chen, Xiang-Jian; Yang, Di

    2016-04-01

    Myeloperoxidase (MPO), a leukocyte hemoprotein released from neutrophils, is thought to be a potential participant in plaque formation and plaque rupture. Therefore, MPO is regarded as an early marker predicting the risk for atherosclerosis, especially for coronary artery disease and acute coronary syndrome. We generated hybridoma clones 1E3 and 3E8 secreting monoclonal antibodies (mAbs) specific to human MPO. BALB/c mice were immunized with MPO protein purified from human neutrophils. Splenocytes from these mice were fused with the mouse myeloma cell line SP2/0. Based on isotyping of the mAbs, both clones 1E3 and 3E8 were referred to the IgG1 subclass. The specificities of 1E3 and 3E8 were assessed by enzyme-linked immunosorbent assay (ELISA), and only 3E8 was confirmed by western blot. We developed a simple MPO-immunosorbent assay (MPO-ISA) on microplate based on both the immune activity and peroxidase activity of MPO. The mAb secreted by clone 3E8 was chosen as coating antibody to capture the plasma MPO without interfering with the peroxidase activity of MPO. Then, tetramethylbenzidine substrate was added to the microwell directly, catalyzed by captured MPO, and a colored product was formed. The simple MPO-ISA test has a sensitivity of 3.68 ng/mL. The linear concentration of MPO-ISA for commercial MPO standard ranged to 250 ng/mL. The average recovery rate is 101.02%. The imprecision within-day was <10% at three different MPO levels. The imprecision between-day was <10% at low and middle MPO levels and varied to 14.61% at the high MPO level. We found that the established MPO-ISA can detect the plasma MPO from human and cavy, but not from mouse and rat. Compared with the commercial human MPO ELISA assay, the MPO-ISA can be used to detect the natural human MPO protein, but not recombinant MPO polypeptides. The generated mAbs and MPO-ISA test may be useful tools to assess risk for inflammation and cardiac events.

  7. Production of monoclonal antibody recognizing a subpopulation of human B lymphocytes producing B cell growth factor (BCGF)

    SciTech Connect

    Witzel, N.; Ambrus, J.L. Jr.; Jurgensen, C.H.; Mostowski, H.; Fauci, A.S.

    1986-03-05

    Several laboratories have recently reported production of B cell growth factors (BCGF) by a variety of human B cell lines. The authors have described production of BCGF by clones of B cell lymphoma lines and by normal Staphylococcus aureus Cowan I-activated B cells. To further evaluate whether BCGF production was the function of particular subpopulations of B cells, they immunized mice with the BCGF producing line Namalva and then developed a panel of hybridomas. Monoclonal antibodies were screened for binding to Namalva and the absence of binding to a non-BCGF producing B cell line. One monoclonal antibody, NN4, which met these criteria was also noted by fluorescence-activated cell sorter analysis to stain clones from a B cell lymphoma line producing BCGF but not clones from the same line which do not produce BCGF. The monoclonal antibody did not stain T cells or monocytes but stained 1-5% of normal human B lymphocytes from peripheral blood or tonsils. Binding of /sup 125/I-labeled NN4 to NN4/sup +/ B cells was not affected by the presence of BCGF. Therefore, NN4 recognizes a unique antigen present on B cell lines which produces BCGF; studies are in progress to determine the significance of this antigen on normal B cells.

  8. Triggering of monoclonal human lymphoma B cells with antibodies to IgM heavy chains: differences of response obtained with monoclonal as compared to polyclonal antibodies.

    PubMed Central

    Godal, T; Ruud, E; Heikkilä, R; Funderud, S; Michaelsen, T; Jefferis, R; Ling, N R; Hildrum, K

    1983-01-01

    A comparative study of human B lymphoma cells activation by monoclonal (murine hybridoma) antibodies to mu heavy chains (Ma-mu) as compared to polyclonal (rabbit) antibodies to mu heavy chains (Ra-mu) has been carried out. Early events related to calmodulin activation such as 86Rb influx and changes in cell volume at 4 h could be induced by Ma-mu. One antibody (AF6) approached Ra-mu with regard to the strength of response obtained. However, Ma-mus including AF6 were deficient in inducing DNA synthesis under conditions where this was achieved with Ra-mu. Studies in one lymphoma, where stimulation of re-expressed surface IgM could be studied, revealed that Ma-mu was deficient in stimulating re-expressed sIgM. These findings raise questions with regard to polyclonal antibody to surface Ig as a model for B cell triggering by antigen and suggest that antigen-induced B cell triggering may be more complex than indicated by previous studies with polyclonal antibody. PMID:6418424

  9. A murine monoclonal antibody that binds N-terminal extracellular segment of human protease-activated receptor-4.

    PubMed

    Sangawa, Takeshi; Nogi, Terukazu; Takagi, Junichi

    2008-10-01

    Abstract A monoclonal antibody that recognizes native G protein coupled receptors (GPCR) is generally difficult to obtain. Protease-activated receptor-4 (PAR4) is a GPCR that plays an important role in platelet activation as a low-affinity thrombin receptor. By immunizing peptide corresponding to the N-terminal segment of human PAR4, we obtained a monoclonal antibody that recognizes cell surface expressed PAR4. Epitope mapping using a series of artificial fusion proteins that carry PAR4-derived peptide revealed that the recognition motif is fully contained within the 6-residue portion adjacent to the thrombin cleavage site. The antibody blocked PAR4 peptide cleavage by thrombin, suggesting its utility in the functional study of PAR4 signaling.

  10. Immunodiagnosis of human cysticercosis (Taenia solium) with antigens purified by monoclonal antibodies.

    PubMed Central

    Nascimento, E; Tavares, C A; Lopes, J D

    1987-01-01

    Monoclonal antibodies were generated from mice immunized with scolex protein antigen of Cysticercus cellulosae. Three monoclonal antibodies specific for cysticercal antigens, which did not show any cross-reactivity with Taenia solium or Taenia saginata antigens, were selected. Each monoclonal antibody coupled to Sepharose could purify one antigen, which appeared as a single band on polyacrylamide gel electrophoresis. When antigens purified by monoclonal antibodies were used to detect antibody in serum samples taken from patients with cysticercosis, taeniasis, and other parasitic infections in an enzyme-linked immunosorbent assay, cross-reactivity was observed until a serum dilution of 1:128 was reached. Since serum samples from unexposed subjects showed positive reactions until a dilution of 1:64 was reached, we chose a discriminative dilution (1:128) above which no cross-reaction was observed. The percent positive serum samples from cysticercosis patients was 100% by the enzyme-linked immunosorbent assay with any of the antigens purified by monoclonal antibodies. Images PMID:3611310

  11. Sensitive radioimmunoassay of. cap alpha. human atrial natriuretic polypeptide using monoclonal antibody recognizing human form ring structure

    SciTech Connect

    Naomi, S.; Umeda, T.; Iwaoka, T.; Miura, F.; Ohno, M.; Sasaki, M.; Oishi, S.; Sato, T.; Takatsu, K.

    1988-01-01

    A monoclonal antibody (C351) against ..cap alpha.. human atrial natriuretic polypeptide (..cap alpha..hANP) recognizing human form ring structure was established and applied to a radioimmunoassay of plasma ..cap alpha..hANP. The minimum detectable amount in terms of 10 % radioligand displacement relative to zero dose were 0.28 fmol/tube, corresponding to 0.7 fmol/ml in plasma after extraction using Sep-Pak C18 cartridges. When the mean plasma levels at recumbent position in fasted morning were compared in 10 young (<30 years) and 10 elderly (greater than or equal to50 years) healthy subjects taking normal sodium diet, it was slightly higher in the latter. After i.v. infusion of hypertonic saline at a rate of 0.24 ml/kg/min for 20 min in 6 normal subjects (26 to 35 years), it was increased from 4.1 +/- 0.4 to 5.9 +/- 0.7 fmol/ml (p<0.01). In 6 patients with essential hypertension (34 to 57 years), it was elevated with high salt intake. From these results, the radioimmunoassay of plasma IR-..cap alpha..hANP using MAb C351 seems to be quite suitable to detect rather small changes at low plasma concentrations and to investigate a physiological importance of ..cap alpha..hANP in man.

  12. Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-γ Using Human-Bovine Interferon-γ Chimeras

    PubMed Central

    Zuber, Bartek; Rudström, Karin; Ehrnfelt, Cecilia

    2016-01-01

    Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-γ. Based on the mAbs' (n = 12) ability to simultaneously bind hIFN-γ in ELISA, 2 epitope clusters with 5 mAbs in each were defined; 2 mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-γ, epitopes were identified using 7 h/bIFN-γ chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-γ residues. Chimeras had a N-terminal peptide tag enabling the analysis of mAb recognition of chimeras in ELISA. The 2 mAb clusters mapped to region A and E, respectively; the epitopes of several mAbs also involved additional regions. MAbs in cluster A neutralized, to various degrees, IFN-γ-mediated activation of human cells, in line with the involvement of region A in the IFN-γ receptor interaction. MAbs mapping to region E displayed a stronger neutralizing capacity although this region has not been directly implicated in the receptor interaction. The results corroborate earlier studies and provide a detailed picture of the link between the epitope specificity and neutralizing capacity of mAbs. They further demonstrate the general use of peptide-tagged chimeric proteins as a powerful and straightforward method for efficient mapping of conformational epitopes. PMID:27336613

  13. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type 1

    NASA Technical Reports Server (NTRS)

    He, Xiao M.; Rueker, Florian; Casale, Elena; Carter, Daniel C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 deg. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  14. Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type 1

    NASA Technical Reports Server (NTRS)

    He, Xiao M.; Rueker, Florian; Casale, Elena; Carter, Daniel C.

    1992-01-01

    The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 deg. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

  15. Polymorphic expression of a human superficial bladder tumor antigen defined by mouse monoclonal antibodies.

    PubMed Central

    Fradet, Y; Islam, N; Boucher, L; Parent-Vaugeois, C; Tardif, M

    1987-01-01

    Three mouse monoclonal antibodies (mAbs), which define a highly restricted antigen, were obtained by simultaneous immunizations with superficial papillary bladder tumor cells and mouse polyclonal serum against normal urothelium. The antigen was detected by the avidin/biotin/peroxidase method in 30/44 superficial bladder tumors (68%) but in only 4/27 infiltrating urothelial cancers (with much less intensity). No normal adult or fetal tissues tested expressed the antigen, including normal urothelium from 40 individuals, 13 of whom had a bladder tumor positive for the antigen. Only 1 of 45 nonbladder tumors showed some reactivity with one of the three mAbs. Serological tests on a large panel of human cancer cell lines and normal cultured cells were negative. The antigen is highly stable and well preserved on paraffin-embedded tissues. Electrophoretic transfer blot experiments with fresh tumor extracts showed that all three mAbs react with a determinant on a component of 300,000 Mr (pI 9.5) and 62,000 Mr (pI 6.5). The antigen shows polymorphic expression at the cellular level on tissue sections and also at a molecular level on immunoblots where the two bands are differentially detected on extracts of a series of tumors but are not visualized on normal urothelium extracts. The characteristics of this antigenic system suggest that it may provide some insights about the biology of bladder cancer. Specific detection of the antigen on 70% of superficial bladder tumors with normal cytology may be useful for their diagnosis and follow-up. Images PMID:3313389

  16. Serologic analysis of human rotavirus serotypes P1A and P2 by using monoclonal antibodies.

    PubMed Central

    Padilla-Noriega, L; Werner-Eckert, R; Mackow, E R; Gorziglia, M; Larralde, G; Taniguchi, K; Greenberg, H B

    1993-01-01

    Three human rotavirus (HRV) VP4 serotypes and one subtype have been described on the basis of a fourfold or an eightfold-or-greater difference in neutralization titer when tested with hyperimmune antisera to recombinant VP4 or VP8* (serotypes P1A, P1B, P2, and P3). To start to analyze the antigenic basis underlying serotype specificity, we produced a library of 13 VP4-specific neutralizing monoclonal antibodies (NMAbs) to two HRVs, the serotype P1A strain Wa and the serotype P2 strain ST3, and characterized the reactivity of these NMAbs with a panel of serotypically diverse HRV strains by neutralization assay and enzyme-linked immunosorbent assay (ELISA). We characterized the serotypic specificity of the NMAbs by using a fourfold or an eightfold-or-greater difference in titer against the homologous (i.e., immunogen) and heterologous strains as a criterion for serotype. Some ST3-derived NMAbs reacted specifically with serotype P2 HRVs by ELISA and/or neutralization assay, while some Wa-derived NMAbs reacted specifically by ELISA and/or neutralization assay with some or all serotype P1A HRVs. Other Wa- and ST3-derived NMAbs reacted with some or all serotype P1A and P2 HRV strains by neutralization assay and ELISA. Most NMAbs did not react with serotype P1B or P3 strains. In previous studies, three distinct operationally defined epitopes have been identified on VP4 by examining the reactivity patterns of selected antigenic variants of HRV strain KU. At least one of the NMAbs described here recognizes an epitope unrelated to these previously identified epitopes, since it neutralized both KU and its variants. Images PMID:7681440

  17. N-terminal or signal peptide sequence engineering prevents truncation of human monoclonal antibody light chains.

    PubMed

    Gibson, S J; Bond, N J; Milne, S; Lewis, A; Sheriff, A; Pettman, G; Pradhan, R; Higazi, D R; Hatton, D

    2017-03-28

    Monoclonal antibodies (mAbs) contain short N-terminal signal peptides on each individual polypeptide that comprises the mature antibody, targeting them for export from the cell in which they are produced. The signal peptide is cleaved from each heavy chain (Hc) and light chain (Lc) polypeptide after translocation to the ER and prior to secretion. This process is generally highly efficient, producing a high proportion of correctly cleaved Hc and Lc polypeptides. However, mis-cleavage of the signal peptide can occur, resulting in truncation or elongation at the N-terminus of the Hc or Lc. This is undesirable for antibody manufacturing as it can impact efficacy and can result in product heterogeneity. Here, we describe a truncated variant of the Lc that was detected during a routine developability assessment of the recombinant human IgG1 MEDI8490 in Chinese hamster ovary cells. We found that the truncation of the Lc was caused due to the use of the murine Hc signal peptide together with a lambda Lc containing an SYE amino acid motif at the N-terminus. This truncation was not caused by mis-processing of the mRNA encoding the Lc and was not dependent on expression platform (transient or stable), the scale of the fed-batch culture or clonal lineage. We further show that using alternative signal peptides or engineering the Lc SYE N-terminal motif prevented the truncation and that this strategy will improve Lc homogeneity of other SYE lambda Lc-containing mAbs. This article is protected by copyright. All rights reserved.

  18. Affinity Maturation to Improve Human Monoclonal Antibody Neutralization Potency and Breadth against Hepatitis C Virus*

    PubMed Central

    Wang, Yong; Keck, Zhen-yong; Saha, Anasuya; Xia, Jinming; Conrad, Fraser; Lou, Jianlong; Eckart, Michael; Marks, James D.; Foung, Steven K. H.

    2011-01-01

    A potent neutralizing antibody to a conserved hepatitis C virus (HCV) epitope might overcome its extreme variability, allowing immunotherapy. The human monoclonal antibody HC-1 recognizes a conformational epitope on the HCV E2 glycoprotein. Previous studies showed that HC-1 neutralizes most HCV genotypes but has modest potency. To improve neutralization, we affinity-matured HC-1 by constructing a library of yeast-displayed HC-1 single chain Fv (scFv) mutants, using for selection an E2 antigen from one of the poorly neutralized HCVpp. We developed an approach by parallel mutagenesis of the heavy chain variable (VH) and κ-chain variable (Vk) genes separately, then combining the optimized VH and Vk mutants. This resulted in the generation of HC-1-related scFv variants exhibiting improved affinities. The best scFv variant had a 92-fold improved affinity. After conversion to IgG1, some of the antibodies exhibited a 30-fold improvement in neutralization activity. Both surface plasmon resonance and solution kinetic exclusion analysis showed that the increase in affinity was largely due to a lowering of the dissociation rate constant, Koff. Neutralization against a panel of HCV pseudoparticles and infectious 2a HCV virus improved with the affinity-matured IgG1 antibodies. Interestingly, some of these antibodies neutralized a viral isolate that was not neutralized by wild-type HC-1. Moreover, propagating 2a HCVcc under the selective pressure of WT HC-1 or affinity-matured HC-1 antibodies yielded no viral escape mutants and, with the affinity-matured IgG1, needed 100-fold less antibody to achieve complete virus elimination. Taken together, these findings suggest that affinity-matured HC-1 antibodies are excellent candidates for therapeutic development. PMID:22002064

  19. In vivo Evaluation of Human Embryonic Stem Cells Isolated by 57-C11 Monoclonal Antibody

    PubMed Central

    Kim, Won-Tae; Lee, Hyun Min; Kim, Min Kyu; Choi, Hong Seo; Ryu, Chun Jeih

    2016-01-01

    Background The normal cells derived from human embryonic stem cells (hESCs) are regarded as substitutes for damaged or dysfunctional adult cells. However, tumorigenicity of hESCs remains a major challenge in clinical application of hESC-derived cell transplantation. Previously, we generated monoclonal antibody (MAb) 57-C11 specific to the surface molecule on undifferentiated hESCs. The aim of this study is to prove whether 57-C11-positive hESCs are pluripotent and tumorigenic in immunodeficient mice. Methods Undifferentiated hESCs were mixed with retinoic acid (RA)-differentiated hESCs at different ratios prior to 57-C11-mediated separation. To isolate 57-C11-positive hESCs from the mixture, biotinylated 57-C11 and streptavidin-coated magnetic beads were added to the mixture. Unbound 57-C11-negative hESCs were first isolated after applying magnet to the cell mixture, and 57-C11-bound hESCs were then released from the magnetic beads. In order to measure the efficiency of separation, 57-C11-positive or -negative hESCs were counted after isolation. To evaluate the efficiency of teratoma formation in vivo, 57-C11-positive or negative cells were further injected into left and right, respectively, testes of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Results Approximately 77~100% of undifferentiated hESCs were isolated after applying 57-C11-coated magnetic beads to the mixed cell populations. Importantly, teratomas were not observed in NOD/SCID mice after the injection of isolated 57-C11-negative hESCs, whereas teratomas were observed with 57-C11-positive hESCs. Conclusion 57-C11-positive hESCs are pluripotent and tumorigenic. The combination of 57-C11 and magnetic beads will be useful to eliminate remaining undifferentiated hESCs for the safe cell transplantation. PMID:27871153

  20. Human pancreatic cancer fusion 2 (HPC2) 1-B3: a novel monoclonal antibody to screen for pancreatic ductal dysplasia.

    PubMed

    Morgan, Terry K; Hardiman, Karin; Corless, Christopher L; White, Sandra L; Bonnah, Robert; Van de Vrugt, Henry; Sheppard, Brett C; Grompe, Markus; Cosar, Ediz F; Streeter, Philip R

    2013-01-01

    BACKGROUND.: Pancreatic ductal adenocarcinoma is rarely detected early enough for patients to be cured. The objective of the authors was to develop a monoclonal antibody to distinguish adenocarcinoma and precancerous intraductal papillary mucinous neoplasia (IPMN) from benign epithelium. METHODS.: Mice were immunized with human pancreatic adenocarcinoma cells and monoclonal antibodies were screened against a panel of archived pancreatic tissue sections, including pancreatitis (23 cases), grade 1 IPMN (16 cases), grade 2 IPMN (9 cases), grade 3 IPMN (13 cases), and various grades of adenocarcinoma (17 cases). One monoclonal antibody, human pancreatic cancer fusion 2 (HPC2) 1-B3, which specifically immunostained adenocarcinoma and all grades of IPMN, was isolated. Subsequently, HPC2 1-B3 was evaluated in a retrospective series of 31 fine-needle aspiration (FNA) biopsies from clinically suspicious pancreatic lesions that had long-term clinical follow-up. RESULTS.: HPC2 1-B3 was negative in all 31 cases of chronic pancreatitis that were tested. In contrast, HPC2 1-B3 immunostained the cytoplasm and luminal surface of all 16 well- to moderately differentiated pancreatic ductal adenocarcinomas. It demonstrated only weak focal staining of poorly differentiated carcinomas. All high-grade IPMNs were found to be positive for HPC2 1-B3. The majority of low-grade to intermediate-grade IPMNs were positive (66% of cases). Immunostaining a separate series of pancreatic FNA cell blocks for HPC2 1-B3 demonstrated that the relative risk for detecting at least low-grade dysplasia (2.0 [95% confidence interval, 1.23-3.26]) was statistically significant (P = .002 by the Fisher exact test). CONCLUSIONS.: To reduce the mortality of pancreatic cancer, more effective early screening methods are necessary. The data from the current study indicate that a novel monoclonal antibody, HPC2 1-B3, may facilitate the diagnosis of early pancreatic dysplasia.

  1. Monoclonal antibodies reveal multiple forms of expression of human microsomal epoxide hydrolase

    SciTech Connect

    Duan, Hongying; Takagi, Akira; Kayano, Hidekazu; Koyama, Isamu; Morisseau, Christophe; Hammock, Bruce D.; Akatsuka, Toshitaka

    2012-04-01

    In a previous study, we developed five kinds of monoclonal antibodies against different portions of human mEH: three, anti-N-terminal; one, anti-C-terminal; one, anti-conformational epitope. Using them, we stained the intact and the permeabilized human cells of various kinds and performed flow cytometric analysis. Primary hepatocytes and peripheral blood mononuclear cells (PBMC) showed remarkable differences. On the surface, hepatocytes exhibited 4 out of 5 epitopes whereas PBMC did not show any of the epitopes. mEH was detected inside both cell types, but the most prominent expression was observed for the conformational epitope in the hepatocytes and the two N-terminal epitopes in PBMC. These differences were also observed between hepatocyte-derived cell lines and mononuclear cell-derived cell lines. In addition, among each group, there were several differences which may be related to the cultivation, the degree of differentiation, or the original cell subsets. We also noted that two glioblastoma cell lines reveal marked expression of the conformational epitope on the surface which seemed to correlate with the brain tumor-associated antigen reported elsewhere. Several cell lines also underwent selective permeabilization before flow cytometric analysis, and we noticed that the topological orientation of mEH on the ER membrane in those cells was in accordance with the previous report. However, the orientation on the cell surface was inconsistent with the report and had a great variation between the cells. These findings show the multiple mode of expression of mEH which may be possibly related to the multiple roles that mEH plays in different cells. -- Highlights: ► We examine expression of five mEH epitopes in human cells. ► Remarkable differences exist between hepatocytes and PBMC. ► mEH expression in cell lines differs depending on several factors. ► Some glioblastoma cell lines reveal marked surface expression of mEH. ► Topology of mEH on the cell

  2. Porphyrin conjugated with serum albumins and monoclonal antibodies boosts efficiency in targeted destruction of human bladder cancer cells.

    PubMed

    Pereira, Patrícia M R; Carvalho, José J; Silva, Sandrina; Cavaleiro, José A S; Schneider, Rudolf J; Fernandes, Rosa; Tomé, João P C

    2014-03-21

    The synthesis of a novel PS conjugated with bovine and human serum albumin (BSA and HSA) and a monoclonal antibody anti-CD104 is reported, as well as their biological potential against the human bladder cancer cell line UM-UC-3. No photodynamic effect was detected when the non-conjugated porphyrin was used. Yet, when it was coupled covalently with the mAb anti-CD104, BSA and HSA, the resulting photosensitizer conjugates demonstrated high efficacy in destroying the cancer cells, the mAb anti-CD104 efficacy overruling the albumins.

  3. Antigenic characterization of human hepatocellular carcinoma. Development of in vitro and in vivo immunoassays that use monoclonal antibodies.

    PubMed Central

    Carlson, R I; Ben-Porath, E; Shouval, D; Strauss, W; Isselbacher, K J; Wands, J R

    1985-01-01

    Several libraries of monoclonal antibodies have been produced by immunization of Balb/c mice with single cell suspensions of nontrypsin-treated human hepatocellular carcinoma cell (HCC) lines in order to study the antigenic properties of transformed hepatocytes. The antibodies were characterized with regards to specificity for hepatoma-associated antigens and their capability for use as reagents in radioimmunoassays (RIAs) and tumor localization in vivo. Three such antibodies namely, P215457, PM4E9917, P232524 of the IgG2a, IgG2a, and IgG1 isotypes, respectively, not only recognized separate and distinct antigenic determinants on four human hepatoma cell lines but also reacted with epitopes present on chemically induced rat hepatoma cell lines. In contrast, only 1 of 38 other human malignant and transformed cell lines demonstrated reactivity with the three antibodies; normal human tissues were also found to be unreactive. Monoclonal antibody P215457 densely stained the plasma membrane by indirect immunofluorescence, showed rapid binding activity to HCC cells in suspension, and precipitated a 50,000-mol wt cell surface protein; antibody PM4E9917 also stained the plasma membrane and precipitated a 65,000-mol wt protein, whereas P232534 recognized cytoplasmic antigenic determinants. With these antibodies "simultaneous sandwich" RIAs were established that detect soluble hepatoma-associated antigens in culture supernatants. Finally, the Fab fragment of P215457 was found to be useful in tumor localization in vivo. This antibody fragment when labeled with 131I was shown to localize by radionuclide-imaging studies in human hepatoma grown in nude mice. Thus, these investigations demonstrate that monoclonal antibodies may be produced against epitopes that reside almost exclusively on transformed hepatocytes and such antibodies may be successfully employed in the development of in vitro and in vivo immunoassays. Images PMID:2991342

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

    NASA Astrophysics Data System (ADS)

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

    1992-09-01

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

  5. Monoclonal antibody "gold rush".

    PubMed

    Maggon, Krishan

    2007-01-01

    The market, sales and regulatory approval of new human medicines, during the past few years, indicates increasing number and share of new biologics and emergence of new multibillion dollar molecules. The global sale of monoclonal antibodies in 2006 were $20.6 billion. Remicade had annual sales gain of $1 billion during the past 3 years and five brands had similar increase in 2006. Rituxan with 2006 sales of $4.7 billion was the best selling monoclonal antibody and biological product and the 6th among the top selling medicinal brand. It may be the first biologic and monoclonal antibody to reach $10 billion annual sales in the near future. The strong demand from cancer and arthritis patients has surpassed almost all commercial market research reports and sales forecast. Seven monoclonal antibody brands in 2006 had sales exceeding $1 billion. Humanized or fully human monoclonal antibodies with low immunogenicity, enhanced antigen binding and reduced cellular toxicity provide better clinical efficacy. The higher technical and clinical success rate, overcoming of technical hurdles in large scale manufacturing, low cost of market entry and IND filing, use of fully human and humanized monoclonal antibodies has attracted funds and resources towards R&D. Review of industry research pipeline and sales data during the past 3 years indicate a real paradigm shift in industrial R&D from pharmaceutical to biologics and monoclonal antibodies. The antibody bandwagon has been joined by 200 companies with hundreds of new projects and targets and has attracted billions of dollars in R&D investment, acquisitions and licensing deals leading to the current Monoclonal Antibody Gold Rush.

  6. STRUCTURAL ELUCIDATION OF CRITICAL RESIDUES INVOLVED IN BINDING OF HUMAN MONOCLONAL ANTIBODIES TO HEPATITIS C VIRUS E2 ENVELOPE GLYCOPROTEIN

    PubMed Central

    Iacob, Roxana E.; Keck, Zhenyong; Olson, Oakley; Foung, Steven K.H.; Tomer, Kenneth B.

    2008-01-01

    Human monoclonal antibodies derived from B cells of HCV infected individuals provide information on the immune response to native HCV envelope proteins as they are recognized during infection. Monoclonal antibodies have been useful in the determination of the function and structure of specific immunogenic domains of proteins and should also be useful for the structure/function characterization of HCV E1 and E2 envelope glycoproteins. The HCV E2 envelope glycoprotein has at least three immunodistinctive conformation domains, designated A, B, and C. Conformational epitopes within domain B and C are neutralizing antibody targets on HCV pseudoparticles as well as from infectious cell culture virus. In this study, a combination of differential surface modification and mass spectrometric limited proteolysis followed by alanine mutagenesis was used to provide insight into potential conformational changes within the E2 protein upon antibody binding. The arginine guanidine groups in the E2 protein were modified with CHD in both the affinity bound and free states followed by mass spectrometric analysis, and the regions showing protection upon antibody binding were identified. This protection can arise by direct contact between the residues and the monoclonal antibody, or by antibody-induced conformational changes. Based on the mass spectrometric data, site-directed mutagenesis experiments were performed which clearly identified additional amino acids residues on E2 distant from the site of antibody interaction, whose change to alanine inhibited antibody recognition by inducing conformational changes within the E2 protein. PMID:18230369

  7. Seroepidemiology of Human Papillomavirus 16 (HPV16) L2 and Generation of L2-Specific Human Chimeric Monoclonal Antibodies

    PubMed Central

    Wang, Joshua W.; Jagu, Subhashini; Wu, Wai-Hong; Viscidi, Raphael P.; Macgregor-Das, Anne; Fogel, Jessica M.; Kwak, Kihyuck; Daayana, Sai; Kitchener, Henry; Stern, Peter L.; Gravitt, Patti E.; Trimble, Cornelia L.

    2015-01-01

    Presently, the seroprevalence of human papillomavirus (HPV) minor capsid antigen L2-reactive antibody is not well understood, and no serologic standard exists for L2-specific neutralizing antibodies. Therefore, we screened a total of 1,078 serum samples for HPV16 L2 reactivity, and these were obtained from four prior clinical studies: a population-based (n = 880) surveillance study with a high-risk HPV DNA prevalence of 10.8%, a cohort study of women (n = 160) with high-grade cervical intraepithelial neoplasia (CIN), and two phase II trials in women with high-grade vulvar intraepithelial neoplasia (VIN) receiving imiquimod therapy combined with either photodynamic therapy (PDT) (n = 19) or vaccination with a fusion protein comprising HPV16 L2, E7, and E6 (TA-CIN) (n = 19). Sera were screened sequentially by HPV16 L2 enzyme-linked immunosorbent assay (ELISA) and then Western blot. Seven of the 1,078 serum samples tested had L2-specific antibodies, but none were detectably neutralizing for HPV16. To develop a standard, we substituted human IgG1 sequences into conserved regions of two rodent monoclonal antibodies (MAbs) specific for neutralizing epitopes at HPV16 L2 residues 17 to 36 and 58 to 64, creating JWW-1 and JWW-2, respectively. These chimeric MAbs retained neutralizing activity and together reacted with 33/34 clinically relevant HPV types tested. In conclusion, our inability to identify an HPV16 L2-specific neutralizing antibody response even in the sera of patients with active genital HPV disease suggests the subdominance of L2 protective epitopes and the value of the chimeric MAbs JWW-1 and JWW-2 as standards for immunoassays to measure L2-specific human antibodies. PMID:25972404

  8. Evaluation of monoclonal antibodies to human plasma low density lipoproteins. A requirement for lipids to maintain antigenic structure.

    PubMed

    Patton, J G; Alley, M C; Mao, S J

    1982-12-17

    Human plasma low density lipoproteins (LDL) are composed of approximately 25% apoproteins and 75% lipids (w/w). Immunochemical properties of LDL were studied using monoclonal antibodies. BALB/c mice were immunized with LDL and the spleen cells from these mice were then fused with a non-immunoglobulin secreting myeloma cell line (F0). The clones producing desirable antibodies were selected to study the antigenic properties of LDL by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay. First, it was found that the maximal binding of 125I-labeled LDL to polyvinyl chloride microtiter dishes was not temperature dependent. The binding affinity was high with a Ka value of approximately 1.9 X 10(10) M-1 while the monoclonal antibodies possessed an affinity to LDL of 5 X 10(8) M-1 which was 2 orders less than the affinity of LDL to the dishes. The former binding, once established, was irreversible as judged by a subsequent incubation with an excess of unlabeled LDL. The latter binding could be displaced by unlabeled LDL. Therefore, the ELISA technique offered a satisfactory approach to study the interaction between LDL and monoclonal antibodies. Removal of lipids from bound LDL by organic extraction resulted in a 50% loss of immunoreactivity, suggesting that the lipids of LDL are important in maintaining the antigenic structure of LDL. Since the apoprotein of LDL also constitutes approximately 40% of the mass (w/w) of very low density lipoproteins (VLDL), the immunoreactivity of VLDL assessed by LDL-monoclonal antibodies was also carried out. Removal of triglycerides from VLDL by lipoprotein lipase resulted in a substantial loss of immunoreactivity as determined by radioimmunoassay. These findings are consistent with the concept that lipids play a role in maintaining the integrity of the antigenic structure of LDL.

  9. Lymphocyte mitogenesis induced by monoclonal antibodies to the T3 complex. Differential modulation by human IgG.

    PubMed

    Tsoukas, C D; Lambris, J; Lotz, M; Valentine, M A; Vaughan, J H; Carson, D A

    1984-11-01

    Murine monoclonal antibodies OKT3 (IgG2), 64.1 (IgG2), and Leu 4 (IgG1) react with a common membrane antigen on human T cells and induce potent mitogenesis at concentrations of 1 ng/ml, 10 ng/ml, and 100 ng/ml, respectively. Human serum inhibits the mitogenic effect of antibodies OKT3 and 64.1, but not that of Leu 4. The inhibitor in serum has been identified as immunoglobulin G (IgG) as evidenced by the ability of anti-human IgG-Sepharose affinity columns to retain the inhibitory activity. Various immunoglobulin classes and subclasses obtained from human myelomas differ in their ability to inhibit the OKT3-induced activation. The best inhibition is obtained with the IgG subclasses IgG1 and IgG3, followed by IgG2; IgG4, IgM, and IgA have little if any effect. None of the IgG subclasses inhibit the Leu 4-induced mitogenesis. Indomethacin as well as supernatants containing interleukin 2 (IL-2) can reverse the inhibitory effects of IgG. Prostaglandins (PGE1 and PGE2) inhibit both the OKT3- and Leu 4-induced mitogenesis, thus lacking the selectivity seen with IgG. Since stimulation by the monoclonal antibodies requires the participation of monocytes, an interpretation consistent with the present data is that IgG stimulates monocytes via its Fc portion to release prostaglandins and/or other suppressor factors via an indomethacin-sensitive pathway. The inability of IgG to inhibit Leu 4-induced mitogenesis may therefore relate to an inability of the monocyte subpopulation, which mediates the Leu 4 response, to secrete suppressor factors. These data suggest a potential value of the mitogenic monoclonal antibodies as probes in studying monocyte heterogeneity and T-cell-monocyte interactions.

  10. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning.

    PubMed

    Tiller, Thomas; Meffre, Eric; Yurasov, Sergey; Tsuiji, Makoto; Nussenzweig, Michel C; Wardemann, Hedda

    2008-01-01

    We have developed an efficient strategy that combines immunoglobulin (Ig) gene repertoire analysis and Ig reactivity profiling at the single cell level. Based on surface marker expression individual cells at different stages of human B cell development are isolated by fluorescence-activated cell sorting. For each cell Ig heavy and corresponding Ig light chain gene transcripts are amplified by nested RT-PCR and cloned into eukaryotic expression vectors to produce monoclonal human antibodies of the same specificity in vitro. All reactions are performed in 96-well plates and allow cloning of large numbers of Ig genes. The recombinant antibodies are tested for reactivity with diverse self- and non-self antigens and the reactivity profile can be directly linked to the complete Ig heavy and Ig light chain gene sequence information that is obtained as part of the cloning strategy. In summary, our method to clone and express human monoclonal antibodies is unbiased, highly efficient, requires only small cell numbers and the recombinant antibodies allow direct conclusions on the frequency of specific human B cells in a diverse repertoire.

  11. A neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection.

    PubMed

    Bossart, Katharine N; Zhu, Zhongyu; Middleton, Deborah; Klippel, Jessica; Crameri, Gary; Bingham, John; McEachern, Jennifer A; Green, Diane; Hancock, Timothy J; Chan, Yee-Peng; Hickey, Andrew C; Dimitrov, Dimiter S; Wang, Lin-Fa; Broder, Christopher C

    2009-10-01

    Nipah virus is a broadly tropic and highly pathogenic zoonotic paramyxovirus in the genus Henipavirus whose natural reservoirs are several species of Pteropus fruit bats. Nipah virus has repeatedly caused outbreaks over the past decade associated with a severe and often fatal disease in humans and animals. Here, a new ferret model of Nipah virus pathogenesis is described where both respiratory and neurological disease are present in infected animals. Severe disease occurs with viral doses as low as 500 TCID(50) within 6 to 10 days following infection. The underlying pathology seen in the ferret closely resembles that seen in Nipah virus infected humans, characterized as a widespread multisystemic vasculitis, with virus replicating in highly vascular tissues including lung, spleen and brain, with recoverable virus from a variety of tissues. Using this ferret model a cross-reactive neutralizing human monoclonal antibody, m102.4, targeting the henipavirus G glycoprotein was evaluated in vivo as a potential therapeutic agent. All ferrets that received m102.4 ten hours following a high dose oral-nasal Nipah virus challenge were protected from disease while all controls died. This study is the first successful post-exposure passive antibody therapy for Nipah virus using a human monoclonal antibody.

  12. Generation, affinity maturation, and characterization of a human anti-human NKG2D monoclonal antibody with dual antagonistic and agonistic activity.

    PubMed

    Kwong, Ka Yin; Baskar, Sivasubramanian; Zhang, Hua; Mackall, Crystal L; Rader, Christoph

    2008-12-31

    In humans, NKG2D is an activating receptor on natural killer (NK) cells and a costimulatory receptor on certain T cells and plays a central role in mediating immune responses in autoimmune diseases, infectious diseases, and cancer. Monoclonal antibodies that antagonize or agonize immune responses mediated by human NKG2D are considered to be of broad and potent therapeutic utility. Nonetheless, monoclonal antibodies to NKG2D that are suitable for clinical investigations have not been published yet. Here, we describe the generation, affinity maturation, and characterization of a fully human monoclonal antibody to human NKG2D. Using phage display technology based on a newly generated naïve human Fab library in phage display vector pC3C followed by a tandem chain shuffling process designed for minimal deviation from natural human antibody sequences, we selected a human Fab, designated KYK-2.0, with high specificity and affinity to human NKG2D. KYK-2.0 Fab blocked the binding of the natural human NKG2D ligands MICA, MICB, and ULBP2 as potently as a commercially available mouse anti-human NKG2D monoclonal antibody in immunoglobulin G (IgG) format. Conversion of KYK-2.0 Fab to IgG1 resulted in subnanomolar avidity for human NKG2D. KYK-2.0 IgG1 was found to selectively recognize defined subpopulations of human lymphocytes known to express NKG2D, that is, the majority of human CD8+, CD16+, and CD56+ cells as well as a small fraction of human CD4+ cells. In solution, KYK-2.0 IgG1 interfered with the cytolytic activity of ex vivo expanded human NK cells. By contrast, immobilized KYK-2.0 IgG1 was found to strongly induce human NK cell activation. The dual antagonistic and agonistic activity promises a wide range of therapeutic applications for KYK-2.0 IgG1 and its derivatives.

  13. Monoclonal antibodies to human growth hormone induce an allosteric conformational change in the antigen.

    PubMed Central

    Mazza, M M; Retegui, L A

    1989-01-01

    We re-investigated the properties of a monoclonal antibody (mAb), 4D11, to human growth hormone (hGH) that showed a very weak affinity, recognizing hGH only when the hormone was solubilized on a solid surface. MAb4D11 did not significantly bind 125I-hGH. It was found that three mAb directed to different hGH epitopes (mAb 3C11, 10C1 and NA71) were able to induce the binding of the soluble antigen to mAb 4D11. The co-operative effect could be demonstrated by the formation of binary complexes (Ag:Ab, 1:2) detected by high-performance liquid chromatography (HPLC) and by the increase of radioactivity found when the synergistic mAb were added to 125I-hGH incubated with mAb 4D11 immobilized on polyvinyl microplates. Other possible explanations, such as the formation of cyclic complexes or the generation of a new epitope in the Fc fragment of the first antibody (Ab), were dismissed because the Fab fragment of one of the enhancing mAb (3C11) gave the same effect as the intact Ab. The data suggest that the hGH molecule undergoes a localized conformational change after binding to mAb 3C11, NA71 or 10C1 and that mAb 4D11 binds with high affinity to the modified region of the hormone. The formation or not of ternary complexes (Ag:Ab, 1:3) was used to localize the 4D11 epitope on the surface of the Ag. It is suggested that mAb 4D11 recognizes a conformational change produced in the region defined by the AE5/AC8 epitopes, which is close to the hGH antigenic domain only expressed when the protein is immobilized on plastic surfaces. PMID:2473953

  14. Adsorption behavior of a human monoclonal antibody at hydrophilic and hydrophobic surfaces

    PubMed Central

    Couston, Ruairidh G.; Skoda, Maximilian W.; Uddin, Shahid; van der Walle, Christopher F.

    2013-01-01

    One aspiration for the formulation of human monoclonal antibodies (mAb) is to reach high solution concentrations without compromising stability. Protein surface activity leading to instability is well known, but our understanding of mAb adsorption to the solid-liquid interface in relevant pH and surfactant conditions is incomplete. To investigate these conditions, we used total internal reflection fluorescence (TIRF) and neutron reflectometry (NR). The mAb tested (“mAb-1”) showed highest surface loading to silica at pH 7.4 (~12 mg/m2), with lower surface loading at pH 5.5 (~5.5 mg/m2, further from its pI of 8.99) and to hydrophobized silica (~2 mg/m2). The extent of desorption of mAb-1 from silica or hydrophobized silica was related to the relative affinity of polysorbate 20 or 80 for the same surface. mAb-1 adsorbed to silica on co-injection with polysorbate (above its critical micelle concentration) and also to silica pre-coated with polysorbate. A bilayer model was developed from NR data for mAb-1 at concentrations of 50–5000 mg/L, pH 5.5, and 50–2000 mg/L, pH 7.4. The inner mAb-1 layer was adsorbed to the SiO2 surface at near saturation with an end-on” orientation, while the outer mAb-1 layer was sparse and molecules had a “side-on” orientation. A non-uniform triple layer was observed at 5000 mg/L, pH 7.4, suggesting mAb-1 adsorbed to the SiO2 surface as oligomers at this concentration and pH. mAb-1 adsorbed as a sparse monolayer to hydrophobized silica, with a layer thickness increasing with bulk concentration - suggesting a near end-on orientation without observable relaxation-unfolding. PMID:23196810

  15. Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS.

    PubMed

    He, Lidong; Anderson, Lissa C; Barnidge, David R; Murray, David L; Hendrickson, Christopher L; Marshall, Alan G

    2017-05-01

    With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2-0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error ~4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background. Graphical Abstract ᅟ.

  16. Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS

    NASA Astrophysics Data System (ADS)

    He, Lidong; Anderson, Lissa C.; Barnidge, David R.; Murray, David L.; Hendrickson, Christopher L.; Marshall, Alan G.

    2017-05-01

    With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2-0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error 4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background.

  17. A novel multipurpose monoclonal antibody for evaluating human c-MET expression in preclinical and clinical settings

    PubMed Central

    Knudsen, Beatrice S.; Zhao, Ping; Resau, James; Cottingham, Sandra; Gherardi, Ermanno; Xu, Eric; Berghuis, Bree; Daugherty, Jennifer; Grabinski, Tessa; Toro, Jose; Giambernardi, Troy; Skinner, R. Scot; Gross, Milton; Hudson, Eric; Kort, Eric; Lengyel, Ernst; Ventura, Aviva; Xie, Qian; Hay, Rick; Woude, George Vande; Cao, Brian

    2010-01-01

    The inappropriate expression of the c-MET cell surface receptor in many human solid tumors necessitates the development of companion diagnostics to identify those patients who could benefit from c-MET targeted therapies. Tumor tissues are formalin-fixed and paraffin embedded (FFPE) for histopathological evaluation, making the development of an antibody against c-MET that accurately and reproducibly detects the protein in FFPE samples an urgent need. We have developed a monoclonal antibody, designated MET4, from a panel of MET-avid monoclonal antibodies, based on its specific staining pattern in FFPE preparations of normal human prostate tissues. The accuracy of MET4 immunohistochemistry (MET4-IHC) was assessed by comparing MET4-IHC in FFPE cell pellets with immunoblotting analysis. The technical reproducibility of MET4-IHC possessed a percentage coefficient of variability (%CV) of 6.25% in intra-assay and inter-assay testing. Comparison with other commercial c-MET antibody detection reagents demonstrated equal specificity and increased sensitivity for c-MET detection in prostate tissues. In two cohorts of ovarian cancers and gliomas, MET4 reacted with ovarian cancers of all histological subtypes (strong staining in 25%) and with 63% of gliomas. In addition, MET4 bound c-Met on the surfaces of cultured human cancer cells and tumor xenografts. In summary, the MET4 monoclonal antibody accurately and reproducibly measures c-MET expression by IHC in FFPE tissues and can be used for molecular imaging in-vivo. These properties encourage further development of MET4 as a multipurpose molecular diagnostics reagent to help to guide appropriate selection of patients being considered for treatment with c-MET-antagonistic drugs. PMID:18815565

  18. Humanization of a mouse monoclonal antibody by CDR-grafting: the importance of framework residues on loop conformation.

    PubMed

    Kettleborough, C A; Saldanha, J; Heath, V J; Morrison, C J; Bendig, M M

    1991-10-01

    A mouse monoclonal antibody (mAb 425) with therapeutic potential was 'humanized' in two ways. Firstly the mouse variable regions from mAb 425 were spliced onto human constant regions to create a chimeric 425 antibody. Secondly, the mouse complementarity-determining regions (CDRs) from mAb 425 were grafted into human variable regions, which were then joined to human constant regions, to create a reshaped human 425 antibody. Using a molecular model of the mouse mAb 425 variable regions, framework residues (FRs) that might be critical for antigen-binding were identified. To test the importance of these residues, nine versions of the reshaped human 425 heavy chain variable (VH) regions and two versions of the reshaped human 425 light chain variable (VL) regions were designed and constructed. The recombinant DNAs coding for the chimeric and reshaped human light and heavy chains were co-expressed transiently in COS cells. In antigen-binding assays and competition-binding assays, the reshaped human antibodies were compared with mouse 425 antibody and to chimeric 425 antibody. The different versions of 425-reshaped human antibody showed a wide range of avidities for antigen, indicating that substitutions at certain positions in the human FRs significantly influenced binding to antigen. Why certain individual FR residues influence antigen-binding is discussed. One version of reshaped human 425 antibody bound to antigen with an avidity approaching that of the mouse 425 antibody.

  19. Anti-human embryonic stem cell monoclonal antibody Hesca-2 binds to a glycan epitope commonly found on carcinomas.

    PubMed

    Shoreibah, Mohamed G; Jackson, Crystal L; Price, Paul W; Meagher, Richard; Godwin, Andrew K; Cai, Qi; Gildersleeve, Jeffrey C

    2011-03-01

    Hesca-2, a monoclonal antibody (mAb) IgM raised to the human embryonic stem cell (hESC) line BG-01v, binds with high affinity (nM) to the disaccharide epitope (Galβ1-3GlcNAc) on a glycan microarray. This epitope was expressed on pluripotent progenitor hESCs in culture, but not in various differentiated cells derived from hESC based on immunofluorescence microscopy. Hesca-2 stains a limited subset of cells in adult human tissues (eg, esophagus and breast). This mAb also crossreacts in immunofluorescence microscopy studies with several human ovarian cancer cell lines and is cytotoxic to them based on the release of cytosolic enzyme lactate dehydrogenase into the media. Hesca-2 immunohistochemically stained tissue from a number of human tumors, including ovary, breast, colon, and esophageal cancer. These data suggest that Hesca-2 recognizes a surface marker found both in stem cells and certain cancer cells.

  20. Monoclonal Antibodies That Recognize Various Folding States of Pure Human Butyrylcholinesterase Can Immunopurify Butyrylcholinesterase from Human Plasma Stored at Elevated Temperatures

    PubMed Central

    2016-01-01

    Human plasma to be analyzed for exposure to cholinesterase inhibitors is stored at 4 °C or lower to prevent denaturation of human butyrylcholinesterase (HuBChE), the biomarker of exposure. Currently published protocols immunopurify HuBChE using antibodies that bind native HuBChE before analysis by mass spectrometry. It is anticipated that the plasma collected from human casualties may be stored nonideally at elevated temperatures of up to 45 °C for days or maybe weeks. At 45 °C, the plasma loses 50% of its HuBChE activity in 8 days and 95% in 40 days. Our goal was to identify a set of monoclonal antibodies that could be used to immunopurify HuBChE from plasma stored at 45 °C. The folding states of pure human HuBChE stored at 4 and 45 °C and boiled at 100 °C were visualized on nondenaturing gels stained with Coomassie blue. Fully active pure HuBChE tetramers had a single band, but pure HuBChE stored at 45 °C had four bands, representing native, partly unfolded, aggregated, and completely denatured, boiled tetramers. The previously described monoclonal B2 18-5 captured native, partly unfolded, and aggregated HuBChE tetramers, whereas a new monoclonal, C191 developed in our laboratory, was found to selectively capture completely denatured, boiled HuBChE. The highest quantity of HuBChE protein was extracted from 45 °C heat-denatured human plasma when HuBChE was immunopurified with a combination of monoclonals B2 18-5 and C191. Using a mixture of these two antibodies in future emergency response assays may increase the capability to confirm exposure to cholinesterase inhibitors. PMID:28058292

  1. Limited proteolysis of human leukocyte interferon-. cap alpha. 2 and localization of the monoclonal antibody-binding antigenic determinant

    SciTech Connect

    Kostrov, S.V.; Chernovskaya, T.V.; Khodova, O.M.; Borukhov, S.I.; Ryzhavskaya, A.S.; Izotova, L.S.; Strongin, A.Ya.

    1986-05-20

    Large peptide fragments of human leukocyte interferon-..cap alpha..2 (INF-..cap alpha..2) were produced by limited proteolysis with trypsin, pepsin, thermolysin, and Bacillus amyloliquefaciens serine proteinase, and the ability of the fragments to react with murine monoclonal antibodies NK2, directed toward INF-..cap alpha..2, was studied by the immunoblotting technique. The region of the sequence 110-149 is the most sensitive to proteinase attack and evidently is exposed on the surface of the INF-..cap alpha..2 molecule. The INF-..cap alpha..2 fragments 1-139, 1-147, and 1-149 react with antibodies, whereas the fragments 1-109 and 1-112 do not bind NK2 antibodies. A comparison of the primary structure of the families of human leukocyte and murine leukocyte INF in the region of the sequence 110-139 and an analysis of the ability of human INF differing in amino acid sequence to interact with NK2 antibodies suggested that the antigenic determinant that binds monoclonal antibodies NK2 is the sequence Glu/sub 114/-Asp/sub 115/-Ser/sub 116/-He/sub 117/ of the INF-..cap alpha..2 molecule.

  2. Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours

    PubMed Central

    Ahlskog, J K J; Schliemann, C; Mårlind, J; Qureshi, U; Ammar, A; Pedley, R B; Neri, D

    2009-01-01

    Background: Hypoxia, which is commonly observed in areas of primary tumours and of metastases, influences response to treatment. However, its characterisation has so far mainly been restricted to the ex vivo analysis of tumour sections using monoclonal antibodies specific to carbonic anhydrase IX (CA IX) or by pimonidazole staining, after the intravenous administration of this 2-nitroimidazole compound in experimental animal models. Methods: In this study, we describe the generation of high-affinity human monoclonal antibodies (A3 and CC7) specific to human CA IX, using phage technology. Results: These antibodies were able to stain CA IX ex vivo and to target the cognate antigen in vivo. In one of the two animal models of colorectal cancer studied (LS174T), CA IX imaging closely matched pimonidazole staining, with a preferential staining of tumour areas characterised by little vascularity and low perfusion. In contrast, in a second animal model (SW1222), distinct staining patterns were observed for pimonidazole and CA IX targeting. We observed a complementary pattern of tumour regions targeted in vivo by the clinical-stage vascular-targeting antibody L19 and the anti-CA IX antibody A3, indicating that a homogenous pattern of in vivo tumour targeting could be achieved by a combination of the two antibodies. Conclusion: The new human anti-CA IX antibodies are expected to be non-immunogenic in patients with cancer and may serve as broadly applicable reagents for the non-invasive imaging of hypoxia and for pharmacodelivery applications. PMID:19623173

  3. Purification of human adult and foetal intestinal alkaline phosphatases by monoclonal antibody immunoaffinity chromatography.

    PubMed Central

    Vockley, J; Harris, H

    1984-01-01

    We have used the technique of monoclonal antibody immunoaffinity chromatography to purify adult and foetal intestinal alkaline phosphatases. Pure adult intestinal enzyme was obtained from a crude tissue extract with a single immunoaffinity chromatographic step in yields exceeding 95%. An additional ion-exchange chromatographic step was necessary for purification of the foetal enzyme, but yields still exceeded 70%. Experiments to optimize the efficiency of the monoclonal antibody immunoaffinity chromatography procedure suggest that the relative strength of binding of an antibody to its antigen is the most important factor to consider when constructing such columns. A column made from an antibody of too low an avidity will not retain the enzyme, while one of too high an avidity will make elution of enzyme in the active state difficult. A scheme is suggested for the application of this technique to a general approach to enzyme purification. Images Fig. 2. PMID:6365087

  4. Anti-(+)-methamphetamine monoclonal antibody antagonists designed to prevent the progression of human diseases of addiction.

    PubMed

    Gentry, W B; Rüedi-Bettschen, D; Owens, S M

    2010-09-01

    Anti-(+)-methamphetamine monoclonal antibodies (mAbs) have the potential to reduce the devastating behavioral and societal effects of the worldwide epidemic of (+)-methamphetamine (METH) addiction and transform the treatment paradigm for diseases of addiction. These novel, protein-based medications could play a vital role in helping patients to achieve sustainable abstinence from METH abuse by serving as an in vivo, around-the-clock sentry against a patient's vulnerability to relapse.

  5. Molecular cloning of the first human monoclonal antibodies neutralizing with high potency swine-origin influenza A pandemic virus (S-OIV).

    PubMed

    Burioni, Roberto; Canducci, Filippo; Mancini, Nicasio; Clementi, Nicola; Sassi, Monica; De Marco, Donata; Saita, Diego; Diotti, Roberta Antonia; Sautto, Giuseppe; Sampaolo, Michela; Clementi, Massimo

    2009-10-01

    The pandemic caused by the new H1N1 swine-origin influenza virus (S-OIV) strain is a worldwide health emergency and alternative therapeutic and prophylactic options are greatly needed. Two human monoclonal antibody Fab fragments (HMab) neutralizing the novel H1N1 influenza strain at very low concentrations were cloned from a patient who had a broad-range anti-H1N1 serum neutralizing activity. The two HMabs neutralized S-OIV with an IC50 of 2.8 and 4 microg/mL. The genes coding for the neutralizing HMabs could be used for generating full human monoclonal IgGs that can be safely administered with the potentially of representing a novel drug to be used in the prophylaxis and the treatment of this human infection. This is the first report of molecular cloning of human monoclonal antibodies against the new pandemic swine-origin influenza virus.

  6. Broad-range neutralizing anti-influenza A human monoclonal antibodies: new perspectives in therapy and prophylaxis.

    PubMed

    Clementi, Nicola; Criscuolo, Elena; Castelli, Matteo; Clementi, Massimo

    2012-10-01

    Broadly neutralizing monoclonal antibodies (mAbs) directed against different subtypes of influenza A viruses are novel tools for the potential development of effective anti-influenza prophylactic and therapeutic strategies. In both cases, the main candidates for passive transfer and new vaccine development are represented by protective mAbs directed against influenza hemagglutinin (HA). A large number of mAbs directed against influenza HA has been developed to date. However, even if they can be useful and contribute to develop new vaccinal strategies, only few of them can be a good candidate for human administration. In this review, we will describe the most relevant human mAb directed against influenza HA able to recognize highly divergent influenza isolates and possibly useful for human therapy and prophylaxis.

  7. Recombinant expression of a novel human transcriptional repressor HMBOX1 and preparation of anti-HMBOX1 monoclonal antibody.

    PubMed

    Dai, Jun; Wu, Longyan; Zhang, Cai; Zheng, Xiaodong; Tian, Zhigang; Zhang, Jian

    2009-08-01

    HMBOX1 was a novel transcription factor possibly involving in function of pancreas and cytotoxicity of NK cells. For function determination, recombinant human HMBOX1 protein was obtained and purified, and the monoclonal antibodies against HMBOX1 were prepared. The full-length cDNA fragment encoding HMBOX1 was amplified from NK-92 cells and inserted into prokaryotic expression vector pET22b. The pET22b-HMBOX1-6his vector was then transformed into E. coli Rosetta (DE3) and induced by 1 mM IPTG for 4 h at 37 degrees Celsius. The fusion HMBOX1 protein was mainly expressed in inclusion bodies, which was purified and refolded using Ni2+-affinity chromatography. With the purified fusion HMBOX1 protein as antigen, monoclonal antibodies against HMBOX1 were generated, providing a potentially useful tool for further study in HMBOX1 functions. Using these anti-HMBOX1 mAbs, we identified that HMBOX1 is located in both cytoplasm and nucleus and could be detected in 10 human normal tissues, including cerebrum, pancreas, kidney and liver tissues. Moreover, the expression in hepatic carcinoma was significantly lower than that in adjacent tissues.

  8. A monoclonal antibody recognizes undifferentiation-specific carbohydrate moieties expressed on cell surface of the human dental pulp cells.

    PubMed

    Kang, Kyung-Jung; Ko, Seon-Yle; Ryu, Chun-Jeih; Jang, Young-Joo

    2017-05-01

    Human dental pulp cells are obtained from dental pulp tissue, and have the ability to form dentin and a pulp-like complex. Although adult stem cells have been identified from the primary culture by using specific cell surface markers, the identity of surface markers for the purification of stem cells within the dental pulp population are still unclear. Previously, we had constructed monoclonal antibodies against the undifferentiated cell-specific surface markers of human dental pulp cells (hDPCs) by performing decoy immunization. Among them, a monoclonal antibody against the cell surface antigen of the undifferentiated hDPCs (named UPSA-1) was purified and its heavy and light chain consensus regions were analyzed. The cell surface binding affinity of UPSA-1 mAb on the undifferentiated hDPCs was stronger than that on the differentiated cells. When tunicamycin was applied to hDPSCs during culture, the cell surface binding affinity of the antibody was dramatically decreased, and dentinogenic differentiation was reduced. The purified UPSA-1 antigen band resulting from immunoprecipitation disappeared or shifted down on the SDS-PAGE by deglycosylation. These data suggested that glycosylation on the cell surface might be a marker of an undifferentiated state, and that UPSA-1 mAb might be useful for identifying the carbohydrate moiety on the cell surface of undifferentiated pulp cells. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  9. A Monoclonal Antibody to the Amebic Lipophosphoglycan-Proteophosphoglycan Antigens Can Prevent Disease in Human Intestinal Xenografts Infected with Entamoeba histolytica

    PubMed Central

    Zhang, Zhi; Duchêne, Michael; Stanley, Samuel L.

    2002-01-01

    Entamoeba histolytica trophozoites are covered by lipophosphoglycan-peptidoglycan molecules which may be key virulence factors. We found that pretreatment of severe combined immunodeficient mice bearing human intestinal xenografts with a monoclonal antibody to the amebic lipophosphoglycan-peptidoglycan molecules can prevent or significantly reduce the human intestinal inflammation and tissue damage that are normally seen with E. histolytica colonic infection. PMID:12228321

  10. Microdistribution of specific rat monoclonal antibodies to mouse tissues and human tumor xenografts.

    PubMed

    Kennel, S J; Falcioni, R; Wesley, J W

    1991-03-01

    Detailed evaluations of the microdistribution of 125I-labeled monoclonal antibodies (MoAbs) to normal tissue antigens were conducted in BALB/c mice. MoAb 273-34A, which binds to a target molecule on the lumenal surface of lung endothelial cells, localizes quickly and efficiently throughout the lung vasculature. MoAb 133-13A, which binds to an antigen on macrophage-like cells expressed in nearly equal amounts in lung, liver, and spleen, localizes most efficiently to spleen and less well to liver and lung. The microdistribution of MoAb 133-13A in liver and spleen is consistent with the antigen distribution in these organs, but in the lung a more diffuse microdistribution is observed, indicating poor access of MoAb to the antigen-positive alveolar macrophages. These findings are consistent with the hypothesis that tight endothelium (lung) represents a significant barrier to extravasation of MoAb into tissue while fenestrated (spleen) and sinusoidal (liver) endothelium are more easily penetrated. In human tumor bearing nu/nu mice, the microdistribution of MoAb to the beta 4 and alpha 6 subunits of integrin was studied. These MoAbs do not cross-react with murine integrins and thus are tumor-specific in the nu/nu mouse model. Localization of 125I-labeled MoAb 450-11A, which reacts with an intercellular domain of beta 4 integrin, is very weak and diffuse. All MoAbs to extracellular domains (mouse 450-9D, 450-30A1, and rat 439-9B) localize well to the tumor. Microdistribution of these MoAbs in the 3 different tumors is nonuniform with heavy distribution near the blood vessels, whereas antigen distribution as determined by immunoperoxidase shows a much more uniform pattern throughout the tumors. In experiments with 125I-labeled MoAb 439-9B F(ab')2, the nonuniform pattern of distribution was not changed. Gross and microdistribution of different doses of 125I-labeled MoAb 439-9B were studied. The percent of injected dose per g of MoAb in the tumor at 48 h did not vary

  11. Efficient generation in vitro, from human peripheral blood cells, of monoclonal Epstein-Barr virus transformants producing specific antibody to a variety of antigens without prior deliberate immunization.

    PubMed Central

    Winger, L; Winger, C; Shastry, P; Russell, A; Longenecker, M

    1983-01-01

    This paper describes a simple protocol for the efficient generation of large numbers of human monoclonal antibody-producing cells. This system is based on initial limiting-dilution culture after Epstein-Barr virus exposure of highly enriched precursors selected from peripheral blood mononuclear cells. Precursors can be enriched by using rosetting or panning approaches. Antibodies to erythrocytes, a mouse mammary carcinoma, DNA, and sperm antigens, produced without any deliberate immunization, are described. Large-scale human monoclonal antibody production may be facilitated by a combination of this protocol with a human cellular fusion system. For efficient precursor analysis and short-term (2 months or more) monoclonal antibody production, however, the system described here may be sufficient. Images PMID:6308627

  12. Reevaluation of sst₁ somatostatin receptor expression in human normal and neoplastic tissues using the novel rabbit monoclonal antibody UMB-7.

    PubMed

    Lupp, Amelie; Nagel, Falko; Schulz, Stefan

    2013-05-10

    The somatostatin receptor 1 (sst1) is widely distributed throughout the body and is also present in neoplastic tissues. However, little is known about its precise tissue distribution, regulation and function, which may in part be due to the lack of specific monoclonal anti-sst1 antibodies. We have characterized the novel rabbit monoclonal anti-human sst1 antibody UMB-7 using sst1-expressing cells and human pituitary samples. The antibody was then used for immunohistochemical staining of a large panel of formalin-fixed, paraffin-embedded human tissues. Western blot analyses of BON-1 cells and human pituitary revealed a broad band migrating at a molecular weight of 45,000-60,000. After enzymatic deglycosylation the size of this band decreased to a molecular weight of 45,000. UMB-7 yielded an efficient immunostaining of distinct cell populations in the human tissue samples with a predominance of plasma membrane staining, which was completely abolished by preadsorption of UMB-7 with its immunizing peptide. The sst1 receptor was detected in anterior pituitary, pancreatic islets, distal tubules, enteric ganglion cells and nerve fibers, chief cells of the gastric mucosa, macrophages and mast cells. In addition, sst1 was observed in pituitary adenomas, gastrointestinal neuroendocrine tumors and pheochromocytoma as well as in pancreatic adenocarcinomas, gastric carcinomas, urinary bladder carcinomas and sarcomas. UMB-7 may prove of great value in the identification of sst1-expressing tumors during routine histopathological examinations. This may open up new routes for diagnostic and therapeutic intervention. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Antigenic structure of human hepatitis A virus defined by analysis of escape mutants selected against murine monoclonal antibodies.

    PubMed Central

    Ping, L H; Lemon, S M

    1992-01-01

    We examined the antigenic structure of human hepatitis A virus (HAV) by characterizing a series of 21 murine monoclonal-antibody-resistant neutralization escape mutants derived from the HM175 virus strain. The escape phenotype of each mutant was associated with reduced antibody binding in radioimmunofocus assays. Neutralization escape mutations were identified at the Asp-70 and Gln-74 residues of the capsid protein VP3, as well as at Ser-102, Val-171, Ala-176, and Lys-221 of VP1. With the exception of the Lys-221 mutants, substantial cross-resistance was evident among escape mutants tested against a panel of 22 neutralizing monoclonal antibodies, suggesting that the involved residues contribute to epitopes composing a single antigenic site. As mutations at one or more of these residues conferred resistance to 20 of 22 murine antibodies, this site appears to be immunodominant in the mouse. However, multiple mutants selected independently against any one monoclonal antibody had mutations at only one or, at the most, two amino acid residues within the capsid proteins, confirming that there are multiple epitopes within this antigenic site and suggesting that single-amino-acid residues contributing to these epitopes may play key roles in the binding of individual antibodies. A second, potentially independent antigenic site was identified by three escape mutants with different substitutions at Lys-221 of VP1. These mutants were resistant only to antibody H7C27, while H7C27 effectively neutralized all other escape mutants. These data support the existence of an immunodominant neutralization site in the antigenic structure of hepatitis A virus which involves residues of VP3 and VP1 and a second, potentially independent site involving residue 221 of VP1. PMID:1312628

  14. Stable, continuous large-scale production of human monoclonal HIV-1 antibody using a computer-controlled pilot plant.

    PubMed

    Unterluggauer, F; Doblhoff-Dier, O; Tauer, C; Jungbauer, A; Gaida, T; Reiter, M; Schmatz, C; Zach, N; Katinger, H

    1994-01-01

    A completely automated pilot plant used for fermentation has been employed with direct digital control (DDC) technology for monitoring and regulating growth of human cells. A human hybridoma cell line (3D6) producing anti-human immunodeficiency virus (HIV)-1 antibodies was used as a model for large-scale production (300-liter airlift fermentor) in continuous culture. Parameters controlled were pH, dissolved oxygen, temperature and the flow rate of four gases used in the process. A control strategy was implemented to achieve constant fluid velocity and mixing by maintaining the rate of gas flow at a constant level. Another advantage of this approach was that the total gas flow required for optimal fluid circulation was reduced from 1 volume gas/volume fermenter/hour (vvh) to 0.3 vvh. Use of a low flow rate eliminated the serious problems of foaming, which contributed significantly to cell destruction, shorter filter-life and other considerations. Dilution rate was optimized at laboratory scale for maximum productivity, which results in relatively low viability. At a dilution rate of 0.0076 h-1, a total cell density of 6-7 x 10(5) cells/ml with a viability of approximately 75% was maintained during long-term continuous cultivation. These growth conditions resulted in a product titer stabilized in the range of 35 micrograms IgG/ml. Batchwise purification was achieved with a recovery of more than 50% and a final purification of active monoclonal antibody representing about 99% product. Results from isoelectric focusing and Western blotting demonstrated batch-to-batch consistency of the purified human monoclonal antibody to HIV-1 during the continuous growth process.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Aberrant expression of the embryonic transcription factor brachyury in human tumors detected with a novel rabbit monoclonal antibody.

    PubMed

    Hamilton, Duane H; Fernando, Romaine I; Schlom, Jeffrey; Palena, Claudia

    2015-03-10

    The embryonic transcription factor brachyury is overexpressed in a variety of human tumors, including lung, breast, colon and prostate carcinomas, chordomas and hemangioblastomas. In human carcinoma cells, overexpression of brachyury associates with the occurrence of the phenomenon of epithelial-mesenchymal transition (EMT), acquisition of metastatic propensity and resistance to a variety of anti-cancer therapeutics. Brachyury is preferentially expressed in human tumors vs. normal adult tissues, and high levels of this molecule associate with poor prognosis in patients with lung, colon and prostate carcinomas, and in breast cancer patients treated with adjuvant tamoxifen. Brachyury is immunogenic in humans and vaccines against this novel oncotarget are currently undergoing clinical investigation. While our group and others have employed various anti-brachyury antibodies to interrogate the above findings, we report here on the development and thorough characterization of a novel rabbit monoclonal antibody (MAb 54-1) that reacts with distinct high affinity and specificity with human brachyury. MAb 54-1 was successfully used in ELISA, western blot, immunofluorescence and immunohistochemistry assays to evaluate expression of brachyury in various human tumor cell lines and tissues. We propose the use of this antibody to assist in research studies of EMT and in prognostic studies for a range of human tumors.

  16. Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo

    PubMed Central

    Dobson, Claire L.; Devine, Paul W. A.; Phillips, Jonathan J.; Higazi, Daniel R.; Lloyd, Christopher; Popovic, Bojana; Arnold, Joanne; Buchanan, Andrew; Lewis, Arthur; Goodman, Joanne; van der Walle, Christopher F.; Thornton, Peter; Vinall, Lisa; Lowne, David; Aagaard, Anna; Olsson, Lise-Lotte; Ridderstad Wollberg, Anna; Welsh, Fraser; Karamanos, Theodoros K.; Pashley, Clare L.; Iadanza, Matthew G.; Ranson, Neil A.; Ashcroft, Alison E.; Kippen, Alistair D.; Vaughan, Tristan J.; Radford, Sheena E.; Lowe, David C.

    2016-01-01

    Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins. PMID:27995962

  17. Local distribution and concentration of intravenously injected sup 131 I-9. 2. 27 monoclonal antibody in human malignant melanoma

    SciTech Connect

    Del Vecchio, S.; Reynolds, J.C.; Carrasquillo, J.A.; Blasberg, R.G.; Neumann, R.D.; Lotze, M.T.; Bryant, G.J.; Farkas, R.J.; Larson, S.M. )

    1989-05-15

    Regional measurements of {sup 131}I-9.2.27 distribution in human melanoma tumors were obtained using quantitative autoradiography. Tumors were removed from patients 72-96 h after they had received an i.v. injection of 9.15 mCi (100 mg) of {sup 131}I-9.2.27. The autoradiographic images showed that the radioactivity reaching the tumor was heterogeneously distributed. Areas of relative high and low uptake were selected in each tumor. Regions of high activity contained from 51 to 1371 nCi/g, while areas with low uptake had radioactivity ranging from 12 to 487 nCi/g. The reliability of the autoradiographic measurements was demonstrated by the strong positive correlation with direct tissue sample counting (r = 0.994 P less than 0.001). Since comparative immunocytochemistry showed a homogeneous and diffuse staining of target antigen on viable tumor cells, variability of monoclonal antibody uptake within individual tumors was not primarily due to heterogeneity of antigen expression in these cases. However, antigen levels accounted for some of the variation from tumor to tumor. When immunoperoxidase staining was repeated on adjacent sections without the addition of 9.2.27, it confirmed the nonuniform distribution of monoclonal antibody found at autoradiography. Thus, quantitative autoradiography gives information about the distribution and the local concentration of radioactive antibody in tumors allowing calculation of the radiation dose delivered to small regions within tumors.

  18. Effects of nutrient levels and average culture pH on the glycosylation pattern of camelid-humanized monoclonal antibody.

    PubMed

    Aghamohseni, Hengameh; Ohadi, Kaveh; Spearman, Maureen; Krahn, Natalie; Moo-Young, Murray; Scharer, Jeno M; Butler, Mike; Budman, Hector M

    2014-09-30

    The impact of operating conditions on the glycosylation pattern of humanized camelid monoclonal antibody, EG2-hFc produced by Chinese hamster ovary (CHO) cells has been evaluated by a combination of experiments and modeling. Cells were cultivated under different levels of glucose and glutamine concentrations with the goal of investigating the effect of nutrient depletion levels and ammonia build up on the cell growth and the glycoprofiles of the monoclonal antibody (Mab). The effect of average pH reduction on glycosylation level during the entire culture time or during a specific time span was also investigated. The relative abundance of glycan structures was quantified by hydrophilic interaction liquid chromatography (HILIC) and the galactosylation index (GI) and the sialylation index (SI) were determined. Lower initial concentrations of glutamine resulted in lower glucose consumption and lower cell yield but increased GI and SI levels when compared to cultures started with higher initial glutamine levels. Similarly, reducing the average pH of culture resulted in lower growth but higher SI and GI levels. These findings indicate that there is a tradeoff between cell growth, resulting Mab productivity and the achievement of desirable higher glycosylation levels. A dynamic model, based on a metabolic flux analysis (MFA), is proposed to describe the metabolism of nutrients, cell growth and Mab productivity. Finally, existing software (GLYCOVIS) that describes the glycosylation pathways was used to illustrate the impact of extracellular species on the glycoprofiles. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo.

    PubMed

    Dobson, Claire L; Devine, Paul W A; Phillips, Jonathan J; Higazi, Daniel R; Lloyd, Christopher; Popovic, Bojana; Arnold, Joanne; Buchanan, Andrew; Lewis, Arthur; Goodman, Joanne; van der Walle, Christopher F; Thornton, Peter; Vinall, Lisa; Lowne, David; Aagaard, Anna; Olsson, Lise-Lotte; Ridderstad Wollberg, Anna; Welsh, Fraser; Karamanos, Theodoros K; Pashley, Clare L; Iadanza, Matthew G; Ranson, Neil A; Ashcroft, Alison E; Kippen, Alistair D; Vaughan, Tristan J; Radford, Sheena E; Lowe, David C

    2016-12-20

    Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins.

  20. Characterization of two anti-dengue human monoclonal antibodies prepared from PBMCs of patients with dengue illness in Thailand.

    PubMed

    Li, Z-Y; Yamashita, A; Kawashita, N; Sasaki, T; Pan, Y; Ono, K-I; Ikuta, K; Li, Y-G

    2016-06-01

    The global spread of the four dengue virus (DENV) serotypes (dengue-1 to -4) has made this virus a major and growing public health concern. Generally, pre-existing neutralizing antibodies derived from primary infection play a significant role in protecting against subsequent infection with the same serotype. By contrast, these pre-existing antibodies are believed to mediate a non-protective response to subsequent heterotypic DENV infections, leading to the onset of dengue illness. In this study, two monoclonal antibodies prepared by using peripheral blood mononuclear cells (PBMCs) from patients with dengue fever were characterized. Epitope mapping revealed that amino acid residues 254-278 in domain II of the viral envelope protein E were the target region of these antibodies. A database search revealed that certain sequences in this epitope region showed high conservation among the four serotypes of DENV. These two human monoclonal antibodies could neutralize DENV-2,-4 more effectively than DENV-1,-3. The amino acid sequences could not explain this difference in neutralizing activity. However, the 3D structure results showed that amino acid 274 could be the critical residue for the difference in neutralization. These results may provide basic information for the development of a dengue vaccine.

  1. A Unique Report: Development of Super Anti-Human IgG Monoclone with Optical Density Over Than 3

    PubMed Central

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

    2013-01-01

    Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity with IgM & IgA by ELISA and confirmed by immunoblotting. The subclasses of the selected mAbs were determined. The best clone was injected intraperitoneally to some pristane-injected mice. Anti-IgG mAb was purified from the animals' ascitic fluid by Ion exchange chromatography and then, mAb was conjugated with HRP. Results: In the present study, over than 50 clones were obtained that 1 clone had optical density over than 3. We named this clone as supermonoclone which was selected for limiting dilution. The result of the immunoblotting, showed sharp band in IgG position and did not show any band in IgM&IgA position. Conclusion: Based on the findings of this study, the conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of other infectious and non- infectious diseases. PMID:24312857

  2. A unique report: development of super anti-human IgG monoclone with optical density over than 3.

    PubMed

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

    2013-01-01

    Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity with IgM & IgA by ELISA and confirmed by immunoblotting. The subclasses of the selected mAbs were determined. The best clone was injected intraperitoneally to some pristane-injected mice. Anti-IgG mAb was purified from the animals' ascitic fluid by Ion exchange chromatography and then, mAb was conjugated with HRP. In the present study, over than 50 clones were obtained that 1 clone had optical density over than 3. We named this clone as supermonoclone which was selected for limiting dilution. The result of the immunoblotting, showed sharp band in IgG position and did not show any band in IgM&IgA position. Based on the findings of this study, the conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of other infectious and non- infectious diseases.

  3. Production and characterization of monoclonal antibodies against α-globin chain-containing human hemoglobins for detecting α-thalassemia disease.

    PubMed

    Pakdeepak, Kanet; Pata, Supansa; Chiampanichayakul, Sawitree; Kasinrerk, Watchara; Tatu, Thanusak

    2016-01-01

    Monoclonal antibodies against α-globin containing human Hbs, named AMS-Alpha1 and AMS-Alpha 2, were produced by the hybridoma technique using spleen cells enriched by the newly developed B lymphocyte enrichment protocol. These two monoclonal antibodies were of IgM class, reacting to only intact form of human Hbs A, A2, E, and F, which contain α-globin chain. By the indirect ELISA, the AMS-Alpha1 and AMS-Alpha 2 quantified less amount of α-globin chain containing hemoglobins in HbH disease than the SEA-α thalassemia 1 carriers and normal individuals. It was thus anticipated that these monoclonal antibodies can be used for detecting Hb Bart's hydrops fetalis in which no α-globin chain is produced.

  4. Identification, immunoaffinity purification and initial characterization of a novel 71 kD human decidua associated protein by use of specific monoclonal antibodies.

    PubMed

    Halperin, R; Fleminger, G; Ovadia, Y; Kraicer, P F; Hadas, E

    1990-01-01

    This study is part of an ongoing attempt to identify and characterize proteins associated with the human decidual tissue. A novel decidual-associated glycoprotein with an apparent molecular weight of 71 kD named hDP71 (human decidual-protein 71), has been identified and purified by immunoaffinity technique using monoclonal antibodies. The monoclonal antibodies recognizing the hDP71 were raised against a partly purified preparation of decidual associated proteins, which was obtained by immunoabsorption of serum proteins from crude decidual extract. Although the hDP71 was copurified with another decidual-associated glycoprotein, the previously described hDP200 (Halperin et al., 1989), evidence is presented showing that the monoclonal antibodies described above are specific for hDP71.

  5. Epitope analysis of Japanese cedar pollen allergen Cry j2 with a human IgM class monoclonal antibody 404-117.

    PubMed

    Chiba, Seiya; Yokoyama, Haruka; Kumazawa, Ayane; Shimmoto, Michie; Naganawa, Yasunori; Shinmoto, Hiroshi

    2017-01-01

    Japanese cedar pollen allergen Cry j2 is a causal allergen of seasonal pollinosis in Japan. To analyze B cell epitopes of Cry j2, we established two human-mouse hybridomas secreting IgM class human monoclonal antibodies to Cry j2. A pin-peptide enzyme-linked immunosorbent assay with synthesized icosa peptides showed that 404-117 monoclonal antibody bound to peptides #11-13 with cry j2 amino acid sequence of 101F-L140. Detailed analysis with octa peptides and alanine substituted peptides indicated that an amino acid sequence of 118FKVD121 was an essential for antibody binding. When K119 (Asn) was substituted with alanine, 404-117 monoclonal antibody did not bind to the alanine substituted peptide. We concluded that the 118FKVD121 sequence might have a very important role in early recognition by Cry j2-specific B cells, which could act as antigen presenting cells.

  6. Immunohistological analysis of human bone marrow trephine biopsies using monoclonal antibodies.

    PubMed

    Falini, B; Martelli, M F; Tarallo, F; Moir, D J; Cordell, J L; Gatter, K C; Loreti, G; Stein, H; Mason, D Y

    1984-03-01

    This paper describes the use of a recently developed immuno-alkaline phosphatase method (the 'APAAP' technique) for labelling frozen sections of undecalcified bone marrow biopsies with monoclonal antibodies, including reagents reactive with T cells and their subsets, B cells, glycophorin, HLA-DR antigen, common ALL antigen, epithelial cells and megakaryocytes. Use of an immuno-alkaline phosphatase technique avoids problems due to endogenous enzyme activity encountered when staining bone marrow by immunoperoxidase procedures. Immunohistological labelling of frozen trephine biopsies is of particular value when it is impossible to aspirate marrow particles and for identifying cells which do not readily enter suspension (e.g. dendritic reticulum cells or stromal cells). Details are given of cases in which immunohistological analysis was used for the phenotyping of acute leukaemias, for the differential diagnosis of intramedullary T and B cell proliferations, and for identifying bone marrow metastases.

  7. Characterization of site-specific glycation during process development of a human therapeutic monoclonal antibody.

    PubMed

    Miller, Amanda K; Hambly, David M; Kerwin, Bruce A; Treuheit, Michael J; Gadgil, Himanshu S

    2011-07-01

    A therapeutic recombinant monoclonal antibody (mAb1) was found to be highly susceptible to glycation during production. Up to 42% glycation was observed in mAb1, which was significantly greater than the glycation observed in 17 other monoclonal antibodies (mAbs). The majority of the glycation was localized to lysine 98 of a unique sequence in the heavy chain complementarity determining region 3. Upon incubation with 5% glucose at 37 °C for 5 days, the level of glycation rose to 80% of the total protein where the majority of the additional glycation was on the lysine 98 residue. These data suggested that the lysine 98 residue was highly susceptible to glycation. However, three other mAbs with a lysine residue in the same position did not show high rates of glycation in the forced glycation assay, suggesting that primary and perhaps secondary structural constraints could contribute to the rate of glycation at that lysine. Interestingly, a portion of the glycation in mAb1 was found to be reversible and upon incubation in phosphate buffer (pH 7) at 37 °C for 5 days, the glycation dropped from starting levels of 42% to 20%. Variation was observed in the total glycation levels between different lots of mAb1. The variability in glycation introduced charge heterogeneity in the form of an acidic peak on cation exchange chromatography and lead to product inconsistency. Mutation of lysine 98 to arginine reduced the starting level of glycation without any impact on potency. Copyright © 2011 Wiley-Liss, Inc. and the American Pharmacists Association

  8. Efficacy and safety evaluation of claudin-4-targeted antitumor therapy using a human and mouse cross-reactive monoclonal antibody.

    PubMed

    Hashimoto, Yosuke; Kawahigashi, Yumi; Hata, Tomoyuki; Li, Xiangru; Watari, Akihiro; Tada, Minoru; Ishii-Watabe, Akiko; Okada, Yoshiaki; Doi, Takefumi; Fukasawa, Masayoshi; Kuniyasu, Hiroki; Yagi, Kiyohito; Kondoh, Masuo

    2016-10-01

    Claudin-4 (CLDN-4), a tight-junction protein, is overexpressed in various malignant tumors, including gastric, colorectal, pancreatic, and breast cancers. However, CLDN-4 is also expressed in normal tissues, including the liver, pancreas, kidney, and small intestine. Whether CLDN-4 is an effective and safe target for cancer therapy has been unclear owing to the lack of a binder with both CLDN-4 specificity and cross-reactivity to human and murine cells. In this study, we successfully generated a rat anti-CLDN-4 monoclonal antibody (5D12) that was specific to, and cross-reactive with, human and mouse CLDN-4. 5D12 recognized the second extracellular domain of human CLDN-4 in a conformation-dependent manner. A human-rat chimeric IgG1 of 5D12 (xi-5D12) activated the Fcγ IIIa receptor, indicating the activation of antibody-dependent cellular cytotoxicity in CLDN-4-expressing cells. Moreover, xi-5D12 significantly suppressed tumor growth in mice bearing human colorectal and gastric tumors without apparent adverse effects, such as weight loss or liver and kidney damage. These results suggest that CLDN-4 is a potent target for cancer therapy and that an anti-CLDN-4 antibody is a promising candidate anticancer agent.

  9. Characterization in vitro and in vivo of the pig analogue of human CD59 using new monoclonal antibodies.

    PubMed Central

    Hanna, S M; Williams, G T; Van Den Berg, C W; Morgan, B P

    1998-01-01

    CD59 is the sole characterized regulator of the complement membrane attack complex in humans. It is very widely and abundantly distributed, being present on all circulating cells, endothelia and epithelia, and in most tissues. CD59 analogues in rodents are distributed similarly. Interest in complement regulation in the pig has developed out of the current enthusiasm to exploit this species as a donor in xenotransplantation of organs to humans. We have recently isolated and cloned the pig analogue of human CD59. We here report the development and characterization of monoclonal antibodies against pig CD59. We have used these antibodies to develop efficient methods for the purification of pig CD59 to homogeneity from erythrocyte membranes and have obtained new information on the structure and function of the purified protein. The antibodies were found to function well in immunohistochemistry and have been used to perform a comprehensive survey of the expression and distribution of pig CD59 on cells and in organs of normal pigs. Pig CD59, like human CD59, is broadly expressed but there are some striking differences in tissue distribution, notably the apparent lack of pig CD59 on circulating platelets and on a subset of leucocytes in blood and lymphoid organs. The reported findings have important implications for the current approaches to avoiding complement-mediated hyperacute rejection in pig-to-human xenografts. Images Figure 1 Figure 4 Figure 5 PMID:9824510

  10. Preclinical and clinical evaluation of elotuzumab, a SLAMF7-targeted humanized monoclonal antibody in development for multiple myeloma.

    PubMed

    Palumbo, Antonio; Sonneveld, Pieter

    2015-08-01

    Although multiple myeloma has historically been treated with chemotherapy, prolonged survival has only been possible since the introduction of thalidomide, lenalidomide and bortezomib. However, multiple myeloma remains largely incurable, and new treatments are needed to improve long-term outcome. Elotuzumab is a humanized IgG1 monoclonal antibody that targets Signaling Lymphocyte Activation Molecule Family member 7 (SLAMF7) to activate NK cells, enabling selective killing of myeloma cells with minimal effects on normal tissue. The combination of elotuzumab with antimyeloma therapies that stimulate host immunity may be an attractive treatment option for patients with newly diagnosed or relapsed/refractory multiple myeloma. Here, we review the role of SLAMF7 in the pathogenesis of multiple myeloma and the preclinical and clinical development of elotuzumab.

  11. Control of pro-inflammatory cytokine release from human monocytes with the use of an interleukin-10 monoclonal antibody.

    PubMed

    Patel, Hardik; Davidson, Dennis

    2014-01-01

    The monocytes (MONOs) can be considered as "double-edge swords"; they have both important pro-inflammatory and anti-inflammatory functions manifested in part by cytokine production and release. Although MONOs are circulating cells, they are the major precursors of a variety of tissue-specific immune cells such as the alveolar macrophage, dendritic cells, microglial cells, and Kupffer cells. Unlike the polymorphonuclear leukocyte, which produces no or very little interleukin-10 (IL-10), the monocyte can produce this potent anti-inflammatory cytokine to control inflammation. IL-10, on an equimolar basis, is a more potent inhibitor of pro-inflammatory cytokines produced by monocytes than many anti-inflammatory glucocorticoids which are used clinically. This chapter describes how to isolate monocytes from human blood and the use of IL-10 monoclonal antibody to determine the effect and timing of endogenous IL-10 release on the production and release of pro-inflammatory cytokines.

  12. Anti-phospholipid human monoclonal antibodies inhibit CCR5-tropic HIV-1 and induce β-chemokines

    PubMed Central

    Liao, Hua-Xin; Alam, S. Munir; Scearce, Richard M.; Plonk, M. Kelly; Kozink, Daniel M.; Drinker, Mark S.; Zhang, Ruijun; Xia, Shi-Mao; Sutherland, Laura L.; Tomaras, Georgia D.; Giles, Ian P.; Kappes, John C.; Ochsenbauer-Jambor, Christina; Edmonds, Tara G.; Soares, Melina; Barbero, Gustavo; Forthal, Donald N.; Landucci, Gary; Chang, Connie; King, Steven W.; Kavlie, Anita; Denny, Thomas N.; Hwang, Kwan-Ki; Chen, Pojen P.; Thorpe, Philip E.; Montefiori, David C.

    2010-01-01

    Traditional antibody-mediated neutralization of HIV-1 infection is thought to result from the binding of antibodies to virions, thus preventing virus entry. However, antibodies that broadly neutralize HIV-1 are rare and are not induced by current vaccines. We report that four human anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1) inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to ∼10 µg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1 infection in vitro by mechanisms involving binding to monocytes and triggering the release of MIP-1α and MIP-1β. The release of these β-chemokines explains both the specificity for R5 HIV-1 and the activity of these mAbs in PBMC cultures containing both primary lymphocytes and monocytes. PMID:20368576

  13. Monoclonal antibodies raised against 167-180 aa sequence of human carbonic anhydrase XII inhibit its enzymatic activity.

    PubMed

    Dekaminaviciute, Dovile; Kairys, Visvaldas; Zilnyte, Milda; Petrikaite, Vilma; Jogaite, Vaida; Matuliene, Jurgita; Gudleviciene, Zivile; Vullo, Daniela; Supuran, Claudiu T; Zvirbliene, Aurelija

    2014-12-01

    Abstract Human carbonic anhydrase XII (CA XII) is a single-pass transmembrane protein with an extracellular catalytic domain. This enzyme is being recognized as a potential biomarker for different tumours. The current study was aimed to generate monoclonal antibodies (MAbs) neutralizing the enzymatic activity of CA XII. Bioinformatics analysis of CA XII structure revealed surface-exposed sequences located in a proximity of its catalytic centre. Two MAbs against the selected antigenic peptide spanning 167-180 aa sequence of CA XII were generated. The MAbs were reactive with recombinant catalytic domain of CA XII expressed either in E. coli or mammalian cells. Inhibitory activity of the MAbs was demonstrated by a stopped flow CO2 hydration assay. The study provides new data on the surface-exposed linear CA XII epitope that may serve as a target for inhibitory antibodies with a potential immunotherapeutic application.

  14. Quantitative autoradiographic evaluation of the influence of protein dose on monoclonal antibody distribution in human ovarian adenocarcinoma xenografts.

    PubMed

    Yang, F E; Brown, R S; Koral, K F; Clavo, A C; Jackson, G A; Wahl, R L

    1992-01-01

    We studied the effect of monoclonal antibody protein dose on the uniformity of radioiodinated antibody distribution within tumor masses using quantitative autoradiography. Groups (n = 11-13/group) of athymic nude mice with subcutaneous HTB77 human ovarian carcinoma xenografts were injected intraperitoneally with an 125I-labeled anticarcinoma-associated antigen murine monoclonal antibody, 5G6.4 using a high or a low protein dose (500 micrograms or 5 micrograms). At 6 days post-injection the macroscopic and microscopic intratumoral biodistribution of radiolabeled antibody was determined. The degree of heterogeneity of the labeled antibody distribution within each tumor was quantified and expressed as the coefficient of variation (CV) of the activity levels in serial histological sections. Tumors from mice given the 500-micrograms protein doses had substantially lower CV values, 0.327 +/- 0.027, than did tumors from animals given 5-micrograms protein doses, 0.458 +/- 0.041, (P = 0.0078), indicating that the higher protein dose resulted in more homogeneous distribution of radioactivity in tumors than did the lower dose. While the percentage of the injected dose reaching the tumor was comparable between groups, injecting the higher dose of protein resulted in significantly lower tumor to non-tumor uptake ratios than those obtained for the lower protein dose. These data indicate, in this system, that to achieve more uniform intratumoral antibody (and radiation for radioimmunotherapy) delivery, a relatively high protein dose must be administered. However, to obtain this increased uniformity, a substantial drop in tumor/background uptake ratios was seen. Quantitative autoradiographic evaluation of human tumor xenografts is a useful method to assess the intratumoral distribution of antibodies.

  15. Characterization of human-type monoclonal antibodies against reduced form of hemin binding protein 35 from Porphyromonas gingivalis.

    PubMed

    Shibata, Y; Okano, S; Shiroza, T; Tahara, T; Nakazawa, K; Kataoka, S; Ishida, I; Kobayashi, T; Yoshie, H; Abiko, Y

    2011-12-01

    The gram-negative anaerobe Porphyromonas gingivalis has been implicated as an important pathogen in the development of adult periodontitis, and its colonization of subgingival sites is critical in the pathogenic process. We previously identified a 35 kDa surface protein (hemin binding protein 35; HBP35) from P. gingivalis that exhibited coaggregation activity, while additional analysis suggested that this protein possessed an ability to bind heme molecules. For development of passive immunotherapy for periodontal diseases, human-type monoclonal antibodies have been prepared using HBP35 as an antigen in TransChromo mice. In the present study, we focused on a single antibody, TCmAb-h13, which is known to inhibit heme binding to recombinant HBP35. The aim of our investigation was to clarify the redox-related function of HBP35 and consider the benefits of human-type monoclonal antibodies. To examine the antigen recognition capability of TCmAbs with immunoblotting and Biacore techniques, we used the native form as well as several Cys-to-Ser variants of recombinant HBP35. We found that the redox state of recombinant HBP35 was dependent on two Cys residues, (48) C and (51) C, in the thioredoxin active center (WCGxCx). Furthermore, TCmAb-h13 recognized the reduced forms of recombinant HBP35, indicating its inhibitory effect on P. gingivalis growth. Hemin binding protein 35 appears to be an important molecule involved in recognition of the redox state of environmental conditions. In addition, TCmAb-h13 had an inhibitory effect on heme binding to recombinant HBP35, thereby interfering with P. gingivalis growth. © 2011 John Wiley & Sons A/S.

  16. Safety, pharmacokinetics and neutralization of the broadly neutralizing HIV-1 human monoclonal antibody VRC01 in healthy adults.

    PubMed

    Ledgerwood, J E; Coates, E E; Yamshchikov, G; Saunders, J G; Holman, L; Enama, M E; DeZure, A; Lynch, R M; Gordon, I; Plummer, S; Hendel, C S; Pegu, A; Conan-Cibotti, M; Sitar, S; Bailer, R T; Narpala, S; McDermott, A; Louder, M; O'Dell, S; Mohan, S; Pandey, J P; Schwartz, R M; Hu, Z; Koup, R A; Capparelli, E; Mascola, J R; Graham, B S

    2015-12-01

    VRC-HIVMAB060-00-AB (VRC01) is a broadly neutralizing HIV-1 monoclonal antibody (mAb) isolated from the B cells of an HIV-infected patient. It is directed against the HIV-1 CD4 binding site and is capable of potently neutralizing the majority of diverse HIV-1 strains. This Phase I dose-escalation study in healthy adults was conducted at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Primary objectives were the safety, tolerability and pharmacokinetics (PK) of VRC01 intravenous (i.v.) infusion at 5, 20 or 40 mg/kg, given either once (20 mg/kg) or twice 28 days apart (all doses), and of subcutaneous (s.c.) delivery at 5 mg/kg compared to s.c. placebo given twice, 28 days apart. Cumulatively, 28 subjects received 43 VRC01 and nine received placebo administrations. There were no serious adverse events or dose-limiting toxicities. Mean 28-day serum trough concentrations after the first infusion were 35 and 57 μg/ml for groups infused with 20 mg/kg (n = 8) and 40 mg/kg (n = 5) doses, respectively. Mean 28-day trough concentrations after the second infusion were 56 and 89 μg/ml for the same two doses. Over the 5-40 mg/kg i.v. dose range (n = 18), the clearance was 0.016 l/h and terminal half-life was 15 days. After infusion VRC01 retained expected neutralizing activity in serum, and anti-VRC01 antibody responses were not detected. The human monoclonal antibody (mAb) VRC01 was well tolerated when delivered i.v. or s.c. The mAb demonstrated expected half-life and pharmacokinetics for a human immunoglobulin G. The safety and PK results support and inform VRC01 dosing schedules for planning HIV-1 prevention efficacy studies.

  17. Structural basis of clade-specific HIV-1 neutralization by humanized anti-V3 monoclonal antibody KD-247

    PubMed Central

    Kirby, Karen A.; Ong, Yee Tsuey; Hachiya, Atsuko; Laughlin, Thomas G.; Chiang, Leslie A.; Pan, Yun; Moran, Jennifer L.; Marchand, Bruno; Singh, Kamalendra; Gallazzi, Fabio; Quinn, Thomas P.; Yoshimura, Kazuhisa; Murakami, Toshio; Matsushita, Shuzo; Sarafianos, Stefan G.

    2015-01-01

    Humanized monoclonal antibody KD-247 targets the Gly312-Pro313-Gly314-Arg315 arch of the third hypervariable (V3) loop of the HIV-1 surface glycoprotein. It potently neutralizes many HIV-1 clade B isolates, but not of other clades. To understand the molecular basis of this specificity, we solved a high-resolution (1.55 Å) crystal structure of the KD-247 antigen binding fragment and examined the potential interactions with various V3 loop targets. Unlike most antibodies, KD-247 appears to interact with its target primarily through light chain residues. Several of these interactions involve Arg315 of the V3 loop. To evaluate the role of light chain residues in the recognition of the V3 loop, we generated 20 variants of KD-247 single-chain variable fragments with mutations in the antigen-binding site. Purified proteins were assessed for V3 loop binding using AlphaScreen technology and for HIV-1 neutralization. Our data revealed that recognition of the clade-specificity defining residue Arg315 of the V3 loop is based on a network of interactions that involve TyrL32, TyrL92, and AsnL27d that directly interact with Arg315, thus elucidating the molecular interactions of KD-247 with its V3 loop target.—Kirby, K. A., Ong, Y. T., Hachiya, A., Laughlin, T. G., Chiang, L. A., Pan, Y., Moran, J. L., Marchand, B., Singh, K., Gallazzi, F., Quinn, T. P., Yoshimura, K., Murakami, T., Matsushita, S., Sarafianos, S. G. Structural basis of clade-specific HIV-1 neutralization by humanized anti-V3 monoclonal antibody KD-247. PMID:25351987

  18. Monoclonal antibody-induced ErbB3 receptor internalization and degradation inhibits growth and migration of human melanoma cells.

    PubMed

    Belleudi, Francesca; Marra, Emanuele; Mazzetta, Francesca; Fattore, Luigi; Giovagnoli, Maria Rosaria; Mancini, Rita; Aurisicchio, Luigi; Torrisi, Maria Rosaria; Ciliberto, Gennaro

    2012-04-01

    Members of the ErbB receptor family are targets of a growing numbers of small molecules and monoclonal antibodies inhibitors currently under development for the treatment of cancer. Although historical efforts have been directed against ErbB1 (EGFR) and ErbB2 (HER2/neu), emerging evidences have pointed to ErbB3 as a key node in the activation of proliferation/survival pathways from the ErbB receptor family and have fueled enthusiasm toward the clinical development of anti-ErbB3 agents. In this study, we have evaluated the potential therapeutic efficacy of a set of three recently generated anti-human ErbB3 monoclonals, A2, A3 and A4, in human primary melanoma cells. We show that in melanoma cells expressing ErbB1, ErbB3 and ErbB4 but not ErbB2 receptor ligands activate the PI3K/AKT pathway, and this leads to increased cell proliferation and migration. While antibodies A3 and A4 are able to potently inhibit ligand-induced signaling, proliferation and migration, antibody A2 is unable to exert this effect. In attempt to understand the mechanism of action and the basis of this different behavior, we demonstrate, through a series of combined approaches, that antibody efficacy strongly correlates with antibody-induced receptor internalization, degradation and inhibition of receptor recycling to the cell surface. Finally, fine epitope mapping studies through a peptide array show that inhibiting vs. non-inhibiting antibodies have a dramatically different mode of binding to the to the receptor extracellular domain. Our study confirms the key role of ErbB3 and points to exploitation of novel combination therapies for treatment of malignant melanoma.

  19. A human monoclonal IgG1 potently neutralizing the pro-inflammatory cytokine GM-CSF.

    PubMed

    Krinner, Eva-Maria; Raum, Tobias; Petsch, Silke; Bruckmaier, Sandra; Schuster, Ioana; Petersen, Laetitia; Cierpka, Ronny; Abebe, Derege; Mølhøj, Michael; Wolf, Andreas; Sørensen, Poul; Locher, Mathias; Baeuerle, Patrick A; Hepp, Julia

    2007-02-01

    The pro-inflammatory cytokine GM-CSF is aberrantly produced in many autoimmune and chronic inflammatory human diseases. GM-CSF neutralization by antibodies has been shown to have a profound therapeutic effect in animal models of rheumatoid arthritis, inflammatory lung diseases, psoriasis and multiple sclerosis. Moreover, the absence of GM-CSF in null mutant mice ameliorates or prevents certain of these diseases. Here we describe the biophysical and biological properties of a human anti-GM-CSF IgG1 antibody designated MT203, which was derived by phage display guided selection. MT203 bound with picomolar affinity to an epitope on human and macaque GM-CSF involved in high-affinity receptor interaction. As a consequence, the antibody potently prevented both GM-CSF-induced proliferation of TF-1 cells with a sub-nanomolar IC50 value and the production of the chemokine IL-8 by U937 cells. MT203 neutralized equally well recombinant (r) human (h) GM-CSF from Escherichia coli and yeast, and also normally glycosylated GM-CSF secreted by human lung epithelial cells in response to IL-1beta stimulation. Furthermore, MT203 significantly reduced both survival and activation of peripheral human eosinophils as may be required for effective treatment of inflammatory lung diseases. The antibody did not show a detectable loss of neutralizing activity after 5 days in human serum at 37 degrees C. Based on its favorable properties, MT203 has been selected for development as a novel anti-inflammatory human monoclonal antibody with therapeutic potential in a multitude of human autoimmune and inflammatory diseases.

  20. Monoclonal antibodies against bacteria.

    PubMed

    Macario, A J; Conway de Macario, E

    1988-01-01

    Highlights are presented of most recent work in which monoclonal antibodies have been instrumental in the study of bacteria and their products. Topics summarized pertain to human and veterinary medicines, dentistry, phytopathology, ichthyology, and bacterial ecophysiology, differentiation, evolution and methanogenic biotechnology.

  1. Properties and structure-function relationships of veltuzumab (hA20), a humanized anti-CD20 monoclonal antibody

    PubMed Central

    Rossi, Edmund A.; Stein, Rhona; Cardillo, Thomas M.; Czuczman, Myron S.; Hernandez-Ilizaliturri, Francisco J.; Hansen, Hans J.; Chang, Chien-Hsing

    2009-01-01

    Veltuzumab is a humanized anti-CD20 monoclonal antibody with complementarity-determining regions (CDRs) identical to rituximab, except for one residue at the 101st position (Kabat numbering) in CDR3 of the variable heavy chain (VH), having aspartic acid (Asp) instead of asparagine (Asn), with framework regions of epratuzumab, a humanized anti-CD22 antibody. When compared with rituximab, veltuzumab has significantly reduced off-rates in 3 human lymphoma cell lines tested, aswell as increased complement-dependent cytotoxicity in 1 of 3 cell lines, but no other in vitro differences. Mutation studies confirmed that the differentiation of the off-rate between veltuzumab and rituximab is related to the single amino acid change in CDR3-VH. Studies of intraperitoneal and subcutaneous doses in mouse models of human lymphoma and in normal cynomolgus monkeys disclosed that low doses of veltuzumab control tumor growth or deplete circulating or sessile B cells. Low- and high-dose veltuzumab were significantly more effective in vivo than rituximab in 3 lymphoma models. These findings are consistent with activity in patients with non-Hodgkin lymphoma given low intravenous or subcutaneous doses of veltuzumab. Thus, changing Asn101 to Asp101 in CDR3-VH of rituximab is responsible for veltuzumab's lower off-rate and apparent improved potency in preclinical models that could translate into advantages in patients. PMID:18941114

  2. Therapeutic administration of a recombinant human monoclonal antibody reduces the severity of chikungunya virus disease in rhesus macaques

    PubMed Central

    Kreklywich, Craig N.; Sukulpovi-Petty, Soila; Legasse, Alfred; Smith, Patricia P.; Denton, Michael; Corvey, Carsten; Krishnan, Shiv; Colgin, Lois M. A.; Ducore, Rebecca M.; Lewis, Anne D.; Axthelm, Michael K.; Mandron, Marie; Cortez, Pierre; Rothblatt, Jonathan; Rao, Ercole; Focken, Ingo; Carter, Kara; Sapparapau, Gopal; Crowe, James E.; Diamond, Michael S.

    2017-01-01

    Chikungunya virus (CHIKV) is a mosquito-borne virus that causes a febrile syndrome in humans associated with acute and chronic debilitating joint and muscle pain. Currently no licensed vaccines or therapeutics are available to prevent or treat CHIKV infections. We recently isolated a panel of potently neutralizing human monoclonal antibodies (mAbs), one (4N12) of which exhibited prophylactic and post-exposure therapeutic activity against CHIKV in immunocompromised mice. Here, we describe the development of an engineered CHIKV mAb, designated SVIR001, that has similar antigen binding and neutralization profiles to its parent, 4N12. Because therapeutic administration of SVIR001 in immunocompetent mice significantly reduced viral load in joint tissues, we evaluated its efficacy in a rhesus macaque model of CHIKV infection. Rhesus macaques that were treated after infection with SVIR001 showed rapid elimination of viremia and less severe joint infiltration and disease compared to animals treated with SVIR002, an isotype control mAb. SVIR001 reduced viral burden at the site of infection and at distant sites and also diminished the numbers of activated innate immune cells and levels of pro-inflammatory cytokines and chemokines. SVIR001 therapy; however, did not substantively reduce the induction of CHIKV-specific B or T cell responses. Collectively, these results show promising therapeutic activity of a human anti-CHIKV mAb in rhesus macaques and provide proof-of-principle for its possible use in humans to treat active CHIKV infections. PMID:28628616

  3. Crystal Structure of the Hendra Virus Attachment G Glycoprotein Bound to a Potent Cross-Reactive Neutralizing Human Monoclonal Antibody

    PubMed Central

    Xu, Kai; Rockx, Barry; Xie, Yihu; DeBuysscher, Blair L.; Fusco, Deborah L.; Zhu, Zhongyu; Chan, Yee-Peng; Xu, Yan; Luu, Truong; Cer, Regina Z.; Feldmann, Heinz; Mokashi, Vishwesh; Dimitrov, Dimiter S.; Bishop-Lilly, Kimberly A.; Broder, Christopher C.; Nikolov, Dimitar B.

    2013-01-01

    The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines. PMID:24130486

  4. The transcriptional profiling of human in vivo-generated plasma cells identifies selective imbalances in monoclonal gammopathies.

    PubMed

    Valor, Luis M; Rodríguez-Bayona, Beatriz; Ramos-Amaya, Ana B; Brieva, José A; Campos-Caro, Antonio

    2017-01-01

    Plasma cells (PC) represent the heterogeneous final stage of the B cells (BC) differentiation process. To characterize the transition of BC into PC, transcriptomes from human naïve BC were compared to those of three functionally-different subsets of human in vivo-generated PC: i) tonsil PC, mainly consisting of early PC; ii) PC released to the blood after a potent booster-immunization (mostly cycling plasmablasts); and, iii) bone marrow CD138+ PC that represent highly mature PC and include the long-lived PC compartment. This transcriptional transition involves subsets of genes related to key processes for PC maturation: the already known protein processing, apoptosis and homeostasis, and of new discovery including histones, macromolecule assembly, zinc-finger transcription factors and neuromodulation. This human PC signature is partially reproduced in vitro and is conserved in mouse. Moreover, the present study identifies genes that define PC subtypes (e.g., proliferation-associated genes for circulating PC and transcriptional-related genes for tonsil and bone marrow PC) and proposes some putative transcriptional regulators of the human PC signatures (e.g., OCT/POU, XBP1/CREB, E2F, among others). Finally, we also identified a restricted imbalance of the present PC transcriptional program in monoclonal gammopathies that correlated with PC malignancy.

  5. Chimeric human/murine monoclonal IgM antibodies to HIV-1 Nef antigen expressed on chronically infected cells.

    PubMed

    Kawai, Masahiro; He, Lianying; Kawamura, Takeshi; Omoto, Shinya; Fujii, Yoichi R; Okada, Noriko

    2003-01-01

    Human IgM antibody (Ab) to gangliosides induced cytolysis of HIV-1-infected cells by homologous human complement. We expected that any human IgM Ab reactive with HIV-1 infected cells could cause complement-mediated cytolysis. The trans-chromosome mouse (TC mouse) contains human chromosomes harboring genes responsible for immunoglobulin production. Spleen cells from TC mice immunized with recombinant Nef were fused with mouse myeloma cells to generate hybridomas, and we selected those that produced human mu-chain-positive Abs reactive with Nef fixed on an ELISA plate. However, the L-chain of the monoclonal Abs (mAbs) were murine lambda in type and were chimeric, and we could not succeed in obtaining mAb with human mu- and human kappa-chains. The chimeric mAbs reacted with the HIV-1 infected cells as seen with flow cytometric analysis, and the surface expression of Nef was also detectable on chronically infected OM10.1 cells which had no detectable gp120. However, although the reaction of the chimeric IgM mAb with HIV-1-infected MOLT4 cells induced C3 deposition on cell surfaces on incubation with fresh human serum, the cells remained unlysed, as determined by 51Cr release assay. The amount of Nef antigen on the cells might not have been high enough to overcome the function of HRF20 (CD59) that restricts formation of membrane attack complexes of homologous complement. However, combination of anti-Nef IgM mAb with other IgM mAbs reactive with the surface of HIV-1-infected cells may induce a synergistic effect in complement mediated cytolysis.

  6. Enhancing effect of human thyroid stimulating hormone (hTSH) monoclonal antibody in the hTSH immunoradiometric assay (IRMA) system.

    PubMed

    Mirapurkar, Shubhangi; Samuel, G; Borkute, S D; Sivaprasad, N

    2012-01-01

    Different monoclonal antibodies, both commercial and indigenously produced, were evaluated in various combinations to optimize an immunoradiometric assay (IRMA) system for human thyroid stimulating hormone (hTSH). During these studies, it was observed that mixing one of the indigenously produced hTSH monoclonal antibody (2B11) in the hTSH IRMA system using Immunotech (Beckman Coulter, Czech Republic) kit reagents, led to an overall increase in the assay binding and sensitivity (from 0.025 mIU/L to 0.015mIU/L) of this IRMA system. This is not a general property of all monoclonal antibodies against hTSH. The mechanism for this enhancement can be attributed to the formation of a multicomponent complex.

  7. [Evolution of monoclonal antibodies in cancer treatment].

    PubMed

    Kubczak, Małgorzata; Rogalińska, Małgorzata

    2016-01-01

    Since late 90s of last century the new age of directed therapy began using mainly biological constructs produced in rodents called monoclonal antibodies. The side effects of monoclonal antibodies were a challenge for pharmaceutical companies to improve the biological properties of these biological drugs. The humanization of monoclonal constructs was an idea to improve monoclonal antibodies next generation activity cancer cell reduction in humans. Moreover for some other patients sensitive for monoclonal antibodies therapy could also potentially induce immunological differences that might imply on human health. The new idea related to monoclonal antibodies was to design a small molecule constructs of nanoantibodies with ability to enter into cells. Such small molecules could find their targets inside human cells, even in nuclei leading to differences in cancer cells expression. The existing knowledge on monoclonal antibodies as well as directed activity of nanoantibodies could improve anticancer treatment efficancy of diseases.

  8. Anti-tax interacting protein-1 (TIP-1) monoclonal antibody targets human cancers.

    PubMed

    Yan, Heping; Kapoor, Vaishali; Nguyen, Kim; Akers, Walter J; Li, Hua; Scott, Jalen; Laforest, Richard; Rogers, Buck; Thotala, Dinesh; Hallahan, Dennis

    2016-07-12

    Radiation-inducible neo-antigens are proteins expressed on cancer cell surface after exposure to ionizing radiation (IR). These neo-antigens provide opportunities to specifically target cancers while sparing normal tissues. Tax interacting protein-1 (TIP-1) is induced by irradiation and is translocated to the surface of cancer cells. We have developed a monoclonal antibody, 2C6F3, against TIP-1.Epitope mapping revealed that 2C6F3 binds to the QPVTAVVQRV epitope of the TIP-1 protein. 2C6F3 binds to the surface of lung cancer (A549, LLC) and glioma (D54, GL261) cell lines. 2C6F3 binds specifically to TIP-1 and ELISA analysis showed that unconjugated 2C6F3 efficiently blocked binding of radiolabeled 2C6F3 to purified TIP-1 protein. To study in vivo tumor binding, we injected near infrared (NIR) fluorochrome-conjugated 2C6F3 via tail vein in mice bearing subcutaneous LLC and GL261 heterotopic tumors. The NIR images indicated that 2C6F3 bound specifically to irradiated LLC and GL261 tumors, with little or no binding in un-irradiated tumors.We also determined the specificity of 2C6F3 to bind tumors in vivo using SPECT/CT imaging. 2C6F3 was conjugated with diethylene triamine penta acetic acid (DTPA) chelator and radiolabeled with 111Indium (111In). SPECT/CT imaging revealed that 111In-2C6F3 bound more to the irradiated LLC tumors compared to un-irradiated tumors. Furthermore, injection of DTPA-2C6F3 labeled with the therapeutic radioisotope, 90Y, (90Y-DTPA-2C6F3) significantly delayed LLC tumor growth. 2C6F3 mediated antibody dependent cell-mediated cytotoxicity (ADCC) and antibody dependent cell-mediated phagocytosis (ADCP) in vitro.In conclusion, the monoclonal antibody 2C6F3 binds specifically to TIP-1 on cancer and radio-immunoconjugated 2C6F3 improves tumor control.

  9. Anti-tax interacting protein-1 (TIP-1) monoclonal antibody targets human cancers

    PubMed Central

    Yan, Heping; Kapoor, Vaishali; Nguyen, Kim; Akers, Walter J.; Li, Hua; Scott, Jalen; Laforest, Richard; Rogers, Buck; Thotala, Dinesh; Hallahan, Dennis

    2016-01-01

    Radiation-inducible neo-antigens are proteins expressed on cancer cell surface after exposure to ionizing radiation (IR). These neo-antigens provide opportunities to specifically target cancers while sparing normal tissues. Tax interacting protein-1 (TIP-1) is induced by irradiation and is translocated to the surface of cancer cells. We have developed a monoclonal antibody, 2C6F3, against TIP-1. Epitope mapping revealed that 2C6F3 binds to the QPVTAVVQRV epitope of the TIP-1 protein. 2C6F3 binds to the surface of lung cancer (A549, LLC) and glioma (D54, GL261) cell lines. 2C6F3 binds specifically to TIP-1 and ELISA analysis showed that unconjugated 2C6F3 efficiently blocked binding of radiolabeled 2C6F3 to purified TIP-1 protein. To study in vivo tumor binding, we injected near infrared (NIR) fluorochrome-conjugated 2C6F3 via tail vein in mice bearing subcutaneous LLC and GL261 heterotopic tumors. The NIR images indicated that 2C6F3 bound specifically to irradiated LLC and GL261 tumors, with little or no binding in un-irradiated tumors. We also determined the specificity of 2C6F3 to bind tumors in vivo using SPECT/CT imaging. 2C6F3 was conjugated with diethylene triamine penta acetic acid (DTPA) chelator and radiolabeled with 111Indium (111In). SPECT/CT imaging revealed that 111In-2C6F3 bound more to the irradiated LLC tumors compared to un-irradiated tumors. Furthermore, injection of DTPA-2C6F3 labeled with the therapeutic radioisotope, 90Y, (90Y-DTPA-2C6F3) significantly delayed LLC tumor growth. 2C6F3 mediated antibody dependent cell-mediated cytotoxicity (ADCC) and antibody dependent cell-mediated phagocytosis (ADCP) in vitro. In conclusion, the monoclonal antibody 2C6F3 binds specifically to TIP-1 on cancer and radio-immunoconjugated 2C6F3 improves tumor control. PMID:27270318

  10. In Vitro and In Vivo Pharmacology and Pharmacokinetics of a Human Engineered™ Monoclonal Antibody to Epithelial Cell Adhesion Molecule

    PubMed Central

    Ammons, W Steve; Bauer, Robert J; Horwitz, Arnold H; Chen, Zhi J; Bautista, Eddie; Ruan, Harry H; Abramova, Marina; Scott, Kristen R; Dedrick, Russell L

    2003-01-01

    Abstract ING-1(heMAb), a Human Engineered™ monoclonal antibody to epithelial cell adhesion molecule (Ep-CAM), was evaluated for its in vitro and in vivo activity. The dissociation constant of ING-1(heMAb) for binding to Ep-CAM on HT-29 human colon tumor cells was 2 to 5 nM, similar to chimeric ING-1. In antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays, ING-1(heMAb) caused a concentration -dependent lysis of BT-20 breast, MCF-7 breast, HT-29 colon, and CACO-2 colon tumor cells, with maximum cytolysis at approximately 1 µg/ml. After an intravenous injection in rats, plasma ING-1(heMAb) levels declined with an alpha half-life of 8 to 11 hours, and a beta half-life of 20 days, typical of an IgG in a species without the target for ING-1. In nude mice with human HT-29 colon tumors, plasma ING-1(heMAb) levels declined more rapidly than in non-tumor-bearing mice, suggesting an enhanced clearance via the tumor-associated human Ep-CAM. In nude mice, intravenous treatments with ING-1(heMAb) twice a week for 3 weeks significantly suppressed the growth of human HT-29 colon and PC-3 prostate tumors in a dose-dependent manner, with 1.0 mg/kg providing the greatest benefit. These results indicate that Human Engineered™ ING-1(heMAb) is a high-affinity antibody with potent in vitro activity that targets and suppresses the growth of human tumors in vivo. PMID:12659687

  11. The monoclonal antibody GZS-1 detects a maturation-associated antigen of human spermatozoa that is also present on the surface of human mononuclear blood cells.

    PubMed

    Hutter, H; Hammer, A; Blaschitz, A; Hartmann, M; Mahnert, W; Sedlmayr, P; Primus, G; Rosenkranz, C; Gebru, G; Henkel, R; Dohr, G

    1996-05-01

    A monoclonal antibody (GZS-1) has been generated by fusion of mouse myeloma cells with spleen cells from BALB/c mice immunised with human sperm cells. The antibody was determined to be an IgG1. The corresponding antigen is present on the whole surface of ejaculated human spermatozoa. It is not detectable on spermatozoa of other mammalian species (rabbit, cat, dog, sheep, boar, bull, horse). In human male genital organs, immunostaining with GZS-1 is observed on sperm cells in the epididymis and the ductus deferens together with the lining epithelium of those organs. No reactivity of sperm cells or germ cell precursors in the testis has been detected. Functional tests using the antibody show a strong inhibitory effect of human sperm in the hamster egg penetration assay. Furthermore, the GZS-1 antigen is detectable on the surface of human lymphocytes and monocytes by immunogold electron microscopy and FACS analysis. By Western blotting of human sperm and seminal plasma performed under reducing conditions immunostaining was detected at 21-25, 31, 51-54, and 62 kDa. The reaction with human lymphocytes shows one major band at 62 kDa and additional bands at 31 and 54 kDa. The results suggest that the monoclonal antibody GZS-1 may recognise an antigen which is secreted from the epithelial cells of the epididymis and binds to ejaculated spermatozoa as a sperm coating antigen. This component may be involved in the capacitation of the sperm and the acrosome reaction. Molecules that are expressed both on sperm and on immunocompetent cells may be relevant for the regulation of immunological processes or for the development of the related immunological tolerance of sperm in the female reproductive tract.

  12. First-in-man Study With Inclacumab, a Human Monoclonal Antibody Against P-selectin

    PubMed Central

    Abt, Markus; Ciorciaro, Cornelia; Kling, Dorothee; Jamois, Candice; Schick, Eginhard; Solier, Corinne; Benghozi, Renée; Gaudreault, Jacques

    2015-01-01

    Abstract: Inclacumab, a novel monoclonal antibody against P-selectin in development for the treatment and prevention of atherosclerotic cardiovascular diseases, was administered in an ascending single-dose study as intravenous infusion to evaluate safety, pharmacokinetics, and pharmacodynamics. Fifty-six healthy subjects were enrolled in this randomized, double-blind placebo-controlled study. Each dose level (0.03–20 mg/kg) was investigated in separate groups of 8 subjects (6 on inclacumab, 2 on placebo). Platelet–leukocyte aggregates, free/total soluble P-selectin concentration ratio, drug concentrations, bleeding time, platelet aggregation, antibody formation, and routine laboratory parameters were measured frequently until 32 weeks. Pharmacokinetic profiles were indicative of target-mediated drug disposition. Platelet–leukocyte aggregate inhibition and soluble P-selectin occupancy showed dose dependency and were strongly correlated to inclacumab plasma concentrations, with IC50 of 740 and 4600 ng/mL, respectively. Inclacumab was well tolerated by the majority of subjects and did neither affect bleeding time nor platelet aggregation. These findings allowed the investigation of the potential beneficial therapeutic use of inclacumab in patient study. PMID:25714598

  13. First-in-Man Study With Inclacumab, a Human Monoclonal Antibody Against P-selectin.

    PubMed

    Schmitt, Christophe; Abt, Markus; Ciorciaro, Cornelia; Kling, Dorothee; Jamois, Candice; Schick, Eginhard; Solier, Corinne; Benghozi, Renée; Gaudreault, Jacques

    2015-06-01

    Inclacumab, a novel monoclonal antibody against P-selectin in development for the treatment and prevention of atherosclerotic cardiovascular diseases, was administered in an ascending single-dose study as intravenous infusion to evaluate safety, pharmacokinetics, and pharmacodynamics. Fifty-six healthy subjects were enrolled in this randomized, double-blind placebo-controlled study. Each dose level (0.03-20 mg/kg) was investigated in separate groups of 8 subjects (6 on inclacumab, 2 on placebo). Platelet-leukocyte aggregates, free/total soluble P-selectin concentration ratio, drug concentrations, bleeding time, platelet aggregation, antibody formation, and routine laboratory parameters were measured frequently until 32 weeks. Pharmacokinetic profiles were indicative of target-mediated drug disposition. Platelet-leukocyte aggregate inhibition and soluble P-selectin occupancy showed dose dependency and were strongly correlated to inclacumab plasma concentrations, with IC50 of 740 and 4600 ng/mL, respectively. Inclacumab was well tolerated by the majority of subjects and did neither affect bleeding time nor platelet aggregation. These findings allowed the investigation of the potential beneficial therapeutic use of inclacumab in patient study.

  14. Anti-human AFP variant monoclonal antibody in radioimmunodetection of primary hepatocellular carcinoma

    PubMed Central

    Liu, Yang; Wu, Meng-Chao; Chen, Han; Zhang, Bai-He; Qian, Guang-Xiang; Pan, Wen-Zhou; Qiang, Mei-Yu

    1997-01-01

    AIM: To investigate the affinity of AFP-R-LCA monoclonal antibody (AFP-R-LCA McAb) for AFP-positive primary hepatocellular carcinoma (HCC) cells. METHODS: AFP-R-LCA McAb was labeled by 131I. Eleven cases of HCC with AFP positivity, 6 with AFP negativity, and 4 with hepatitis B-related cirrhosis were investigated by radioimmunodetection. RESULTS: The 131I-AFP-R-LCA McAb immunoreacted with 9 of the HCC AFP-positive cases (9/11), but with none of the 6 AFP negative HCC cases or of the 4 cirrhosis patients. 131I-AFP-R-LCA McAb at a small dose (7.4 × 107 Bq/300 μg) was associated with no side effects as determined by the liver function test, prothrombin time (Pt) test and thyroid gland function test (P > 0.05). Two cases of AFP-positive HCC were not imaged because of large tumor size (diameter > 10 cm) and higher AFP concentration in serum (20000 μg/L). CONCLUSION: AFP-R-LCA McAb has a strong and special affinity to AFP-positive HCC cells and may be useful as a carrier for radioimmunodetection and radioimmunotherapy. PMID:27053873

  15. Patterns of evolution of myocyte damage after human heart transplantation detected by indium-111 monoclonal antimyosin

    SciTech Connect

    Ballester-Rodes, M.; Carrio-Gasset, I.; Abadal-Berini, L.; Obrador-Mayol, D.; Berna-Roqueta, L.; Caralps-Riera, J.M.

    1988-09-15

    The indium-111 labeled Fab fragment of antimyosin monoclonal antibody was used to study cardiac rejection and the time course of myocyte damage after transplantation. Fifty-three studies were performed in 21 patients, 17 men and 4 women, aged 19 to 54 years (mean 37 +/- 8), from 7 to 40 months after transplantation. Repeat studies were available in 8, and 10 were studied after the first year of transplantation. A heart-to-lung ratio was used for quantitation of uptake (normal 1.46 +/- 0.04). Differences between absent (1.69 +/- 0.29) and moderate (1.90 +/- 0.36) rejection were significant (p less than 0.03). Antimyosin ratio at 1 to 3 months (1.89 +/- 0.35) differed from that at greater than 12 months (1.65 +/- 0.2) (p less than 0.01). Repeat studies revealed a decrease in antimyosin ratio in 5 patients with uneventful clinical course; 2 had persistent activity after transplantation and suffered heart failure from rejection. After 1 year of transplantation uptake was within normal limits in 7 of 10 patients, and high uptake was associated with vascular rejection in 1. Because they can define evolving patterns of myocardial lesion activity, antimyosin studies could be useful both in patient management and in concentrating resources for those patients who most require them. The heart-to-lung ratio is suggested to monitor sequentially the degree of myocyte damage after transplantation.

  16. Characterization of binding of four monoclonal antibodies to the human ovarian adenocarcinoma cell line HEY.

    PubMed

    Sheldon, K; Marks, A; Baumal, R

    1987-05-01

    Four mouse monoclonal antibodies (mAb) (10B, IgG1; 8C, IgG2a; M2A, IgG2a; M2D, IgG2b) were characterized with respect to their binding to the ovarian adenocarcinoma cell line HEY, using displacement assays and Scatchard plot analyses. The four mAb reacted with different antigens on the surface of HEY cells, with affinity constants ranging from 1 X 10(9) to 3 X 10(9) M-1. The number of binding sites per cell for each antibody was approximately 2 X 10(4). mAb 8C and M2D remained associated with the cell surface following binding to their respective antigens, while mAb 10B was rapidly internalized, with 50% of the bound mAb being lost from the cell surface during 4 h of incubation at 37 degrees C. These different binding characteristics of the mAb may influence their ability to target radioactivity and cytotoxic drugs to HEY cells.

  17. Monoclonal antibody Po66 uptake by human lung tumours implanted in nude mice: effect of co-administration with doxorubicin.

    PubMed Central

    Desrues, B.; Léna, H.; Brichory, F.; Ramée, M. P.; Toujas, L.; Delaval, P.; Dazord, L.

    1995-01-01

    The efficacy of radioimmunotherapy of tumours with radiolabelled monoclonal antibodies (MAbs) depends on the amount of antibody taken up by the tumour and on its intratumoral distribution. In the case of MAbs directed against intracellular antigens, increasing the permeability of the cytoplasmic membrane may augment the bioavailability of the antigen for the antibody. This raises the question whether the induction of tumour necrosis by chemotherapy can enhance the tumour uptake of radiolabelled monoclonal antibodies. In this work, the effect of doxorubicin on the biodistribution of Po66, an MAb directed against an intracellular antigen, was studied in nude mice grafted with the human non-small-cell lung carcinoma cell line SK-MES-1. After injection on day 0 of 125I-labelled Po66, tumour radioactivity increased up to days 3-5, and then remained unchanged to day 14. The combined administration of 125I-labelled Po66 with 8 mg kg-1 doxorubicin, in two doses separated by 7 days, doubled the radioactivity retained by the tumour. Histological and historadiographic analysis showed, however, that the drug induced cellular damage. In the absence of doxorubicin, the accumulation of Po66 was restricted to some necrotic areas, whereas with doxorubicin the necrosis was more extensive and the antibody more evenly distributed. These results suggest that chemotherapy and immunoradiotherapy combined would enhance tumour uptake of radioisotope and promote more homogenous distribution of the radiolabelled MAb. This would promote eradication of the remaining drug-resistant cells in tumours. Images Figure 2 Figure 5 PMID:7577450

  18. Lymphoscintigraphy of human colorectal carcinoma metastases in athymic mice by use of radioiodinated B72. 3 monoclonal antibody

    SciTech Connect

    Shah, S.A.; Gallagher, B.M.; Sands, H.

    1987-06-01

    The potential of radioiodinated monoclonal antibody B72.3 for lymphoscintigraphy was evaluated, using suitable animal models of a human colorectal carcinoma. LS174T xenografts were grown at various sites in beta-estradiol-pretreated athymic mice, and the development of metastases in different organs was assessed histologically. After iv inoculation of the mice, 66% of the animals developed ''metastases'' to the axillary lymph nodes. Of these mice, 100% also developed multiple tumors on their backs and 79% had lung micrometastases. Livers, kidneys, and spleens showed no evidence of tumor growth. In 33% of the mice in which primary LS174T tumors had been removed from the hindfoot pad, metastases to the popliteal lymph nodes were observed 3 1/2 weeks after tumor implantation. BALB/c (nu/nu) female mice bearing axillary and popliteal lymph node metastases were used to test the potential of radiolabeled B72.3 antibody (an IgG1) as a lymphoscintigraphic agent. A monoclonal antibody against horseradish peroxidase (also an IgG1), which did not bind LS174T tumor cells in vitro, served as a control. Both normal and tumor-bearing axillary and popliteal lymph nodes imaged up to 6 hours after the sc injection of 20-40 mu Ci of /sup 125/I-labeled B72.3 into either the forefoot or hindfoot pads. The localization index (L.I.) (specific/nonspecific antibody in tumor divided by specific/nonspecific antibody in blood) for LS174T tumors in lymph nodes was approximately 1 during the first 6 hours after antibody injection, thus indicating no specific antibody accumulation.

  19. High-yield production of a human monoclonal IgG by rhizosecretion in hydroponic tobacco cultures.

    PubMed

    Madeira, Luisa M; Szeto, Tim H; Henquet, Maurice; Raven, Nicole; Runions, John; Huddleston, Jon; Garrard, Ian; Drake, Pascal M W; Ma, Julian K-C

    2016-02-01

    Rhizosecretion of recombinant pharmaceuticals from in vitro hydroponic transgenic plant cultures is a simple, low cost, reproducible and controllable production method. Here, we demonstrate the application and adaptation of this manufacturing platform to a human antivitronectin IgG1 monoclonal antibody (mAb) called M12. The rationale for specific growth medium additives was established by phenotypic analysis of root structure and by LC-ESI-MS/MS profiling of the total protein content profile of the hydroponic medium. Through a combination of optimization approaches, mAb yields in hydroponic medium reached 46 μg/mL in 1 week, the highest figure reported for a recombinant mAb in a plant secretion-based system to date. The rhizosecretome was determined to contain 104 proteins, with the mAb heavy and light chains the most abundant. This enabled evaluation of a simple, scalable extraction and purification protocol and demonstration that only minimal processing was necessary prior to protein A affinity chromatography. MALDI-TOF MS revealed that purified mAb contained predominantly complex-type plant N-glycans, in three major glycoforms. The binding of M12 purified from hydroponic medium to vitronectin was comparable to its Chinese hamster ovary (CHO)-derived counterpart. This study demonstrates that in vitro hydroponic cultivation coupled with recombinant protein rhizosecretion can be a practical, low-cost production platform for monoclonal antibodies. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  20. Broadly neutralizing human monoclonal JC polyomavirus VP1–specific antibodies as candidate therapeutics for progressive multifocal leukoencephalopathy

    PubMed Central

    Jelcic, Ivan; Combaluzier, Benoit; Jelcic, Ilijas; Faigle, Wolfgang; Senn, Luzia; Reinhart, Brenda J.; Ströh, Luisa; Nitsch, Roger M.; Stehle, Thilo; Sospedra, Mireia; Grimm, Jan; Martin, Roland

    2016-01-01

    In immunocompromised individuals, JC polyomavirus (JCPyV) may mutate and gain access to the central nervous system resulting in progressive multifocal leukoencephalopathy (PML), an often fatal opportunistic infection for which no treatments are currently available. Despite recent progress, the contribution of JCPyV-specific humoral immunity to controlling asymptomatic infection throughout life and to eliminating JCPyV from the brain is poorly understood. We examined antibody responses against JCPyV major capsid protein VP1 (viral protein 1) variants in the serum and cerebrospinal fluid (CSF) of healthy donors (HDs), JCPyV-positive multiple sclerosis patients treated with the anti-VLA-4 monoclonal antibody natalizumab (NAT), and patients with NAT-associated PML. Before and during PML, CSF antibody responses against JCPyV VP1 variants show “recognition holes”; however, upon immune reconstitution, CSF antibody titers rise, then recognize PML-associated JCPyV VP1 variants, and may be involved in elimination of the virus. We therefore reasoned that the memory B cell repertoire of individuals who recovered from PML could be a source for the molecular cloning of broadly neutralizing antibodies for passive immunization. We generated a series of memory B cell-derived JCPyV VP1-specific human monoclonal antibodies from HDs and a patient with NAT-associated PML-immune reconstitution inflammatory syndrome (IRIS). These antibodies exhibited diverse binding affinity, cross-reactivity with the closely related BK polyomavirus, recognition of PML-causing VP1 variants, and JCPyV neutralization. Almost all antibodies with exquisite specificity for JCPyV, neutralizing activity, recognition of all tested JCPyV PML variants, and high affinity were derived from one patient who had recovered from PML. These antibodies are promising drug candidates for the development of a treatment of PML. PMID:26400911

  1. Human anti-murine antibody responses in ovarian cancer patients undergoing radioimmunotherapy with the murine monoclonal antibody OC-125

    SciTech Connect

    Muto, M.G.; Finkler, N.J.; Kassis, A.I.; Lepisto, E.M.; Knapp, R.C. )

    1990-08-01

    Human anti-murine antibody (HAMA) responses were monitored in 23 patients with recurrent or persistent epithelial ovarian carcinoma undergoing single-dose intraperitoneal radioimmunotherapy (RIT) with the murine monoclonal antibody OC-125. Sera of patients receiving escalating doses of OC-125 F(ab')2 (10-70 mg) radiolabeled with 18 to 141 mCi of iodine-131 were assayed for HAMA by a protein A-based radioimmunoassay. Overall, 70% of patients (16/23) developed HAMA within 10 to 46 days (median = 29) postinfusion, with peak values (23 +/- 6 to 325 +/- 10 micrograms/ml) at 32 to 102 days (median = 38). HAMA was undetectable prior to infusion in all cases and persisted up to 76 weeks. Of patients receiving a dose of 123 mCi or less, 80% (16/20) developed HAMA, whereas in the 140-mCi group, none of the three patients had detectable levels. Two patients in the 140-mCi group demonstrated dose-limiting bone marrow toxicity (severe thrombocytopenia and neutropenia). It is concluded that a single intraperitoneal dose of monoclonal antibody leads to a high incidence of HAMA production. The results also suggest that the likelihood of HAMA formation in patients who either had undergone recent chemotherapy or had received the highest dose of the radioimmunoconjugate is reduced. These observations may be of significance in designing multiple-dose therapy trials as HAMA has been demonstrated to decrease antibody-to-tumor binding and may potentially increase renal, hepatic, and hematologic toxicity associated with radioimmunotherapy.

  2. [Towards an industrial control of the cloning of lymphocytes B human for the manufacturing of monoclonal antibodies stemming from the human repertoire].

    PubMed

    Guillot-Chene, P; Lebecque, S; Rigal, D

    2009-05-01

    Monoclonal antibodies (mAbs) are efficient drugs for treating infectious, inflammatory and cancer diseases. Antibodies secreted by human lymphocytes that have been isolated from either peripheral blood or tissues present the definite interest of being part of the physiological or disease-related response to antigens present in the human body. However, attempts to generate hybridomas with human B cells have been largely unsuccessful, and cloning of human B cells has been achieved only via their inefficient immortalization with Epstein Barr Virus (EBV). However, recent progress in our understanding of the molecular mechanisms of polyclonal B cell activation has dramatically increased the capacity to clone human B cells. In particular, activation of human naïve and memory B cells through CD40 or memory B cells only through TLR9 was shown to greatly facilitate their immortalization by EBV. Industrial development based on these observations will soon provide large collections of high affinity human mAbs of every isotype directly selected by the human immune system directed to recognize epitopes relevant for individual patients. Moreover, after CD40 activation, these mAbs will cover the full human repertoire, including the natural auto-immune repertoire. Full characterization of the biological activity of these mAbs will in turn bring useful information for selecting vaccine epitopes. This breakthrough in human B cell cloning opens the way into new areas for therapeutic use of mAbs.

  3. Cross-protection of newly emerging HPAI H5 viruses by neutralizing human monoclonal antibodies: A viable alternative to oseltamivir

    PubMed Central

    Ren, Huanhuan; Wang, Guiqin; Wang, Shuangshuang; Chen, Honglin; Chen, Zhiwei; Hu, Hongxing; Cheng, Genhong; Zhou, Paul

    2016-01-01

    ABSTRACT Newly emerging highly pathogenic avian influenza (HPAI) H5N2, H5N3, H5N5, H5N6, H5N8 and H5N9 viruses have been spreading in poultry and wild birds. The H5N6 viruses have also caused 10 human infections with 4 fatal cases in China. Here, we assessed the cross-neutralization and cross-protection of human and mouse monoclonal antibodies against 2 viruses: a HPAI H5N8 virus, A/chicken/Netherlands/14015526/2014 (NE14) and a HPAI H5N6 virus, A/Sichuan/26221/2014 (SC14). The former was isolated from an infected chicken in Netherlands in 2014 and the latter was isolated from an infected human patient in Sichuan, China. We show that antibodies FLA5.10, FLD21.140, 100F4 and 65C6, but not AVFluIgG01, AVFluIgG03, S139/1 and the VRC01 control, potently cross-neutralize the H5N8 NE14 and H5N6 SC14 viruses. Furthermore, we show that a single injection of >1 mg/kg of antibody 100F4 at 4 hours before, or 20 mg/kg antibody 100F4 at 72 hours after, a lethal dose of H5N8 NE14 enables mice to withstand the infection. Finally, we show that a single injection of 0.5 or 1 mg/kg antibody 100F4 prophylactically or 10 mg/kg 100F4 therapeutically outperforms a 5-day course of 10 mg/kg/day oseltamivir treatment against lethal H5N8 NE14 or H5N6 SC14 infection in mice. Our results suggest that further preclinical evaluation of human monoclonal antibodies against newly emerging H5 viruses is warranted. PMID:27167234

  4. Tumor localization by combinations of monoclonal antibodies in a new human colon carcinoma cell line (LIM1899)

    SciTech Connect

    Andrew, S.M.; Teh, J.G.; Johnstone, R.W.; Russell, S.M.; Whitehead, R.H.; McKenzie, I.F.; Pietersz, G.A. )

    1990-09-01

    One of the problems of in vivo diagnosis and therapy of tumors with monoclonal antibodies is their heterogeneity with respect to antigen expression, with some cells expressing no antigen and others being weakly or strongly positive. Selected mixtures of antibodies to different antigens are therefore likely to react with more cells than single antibodies and be more effective for imaging and therapy. With this in mind, we have examined a new human colon cancer cell line (LIM1899) which has a heterogeneous expression of several cell surface molecules: by flow cytometry 38% were carcinoembryonic antigen positive; 64%, human milk fat globule positive, and 73%, CD46 positive; 87% of tumor cells bound a mixture of all three antibodies in vitro. Some blocking of the binding of anti-human milk fat globule antibody by the anti-CD46 antibody was noted. LIM1899 was established as a xenograft in nude mice and in vivo biodistribution studies performed using antibodies alone or in combination. Mixtures of antibodies clearly showed a higher percentage of injected dose of antibody in the tumor than did single antibodies: one antibody gave 10%; two together, 17 to 21%; and all three together gave 29% of the injected dose in the tumor. Tumor:blood ratios were also superior for combinations of antibodies, provided that low doses of the antibodies were used; at higher doses the effect was lost. The study demonstrates that combinations of antibodies are better than single antibodies for localization, provided that the dose used is carefully selected.

  5. New method to quantitate platelets adhered on biomaterials using monoclonal antibodies to human platelet membrane glycoprotein SZ-21

    SciTech Connect

    Xi, T.F.; Zhang, J.C.; Tian, W.H.; Wang, C.R.; Lei, X.H.; Wai, H.Y.; Ruan, C.G. )

    1990-01-01

    This study developed a new technique to quantitate platelets adhered on biomaterials surfaces in vitro, based on a surface phased radioimmunoassay using a monoclonal antibody SZ-21, directed specifically against the membrane glycoprotein complex IIIa of human platelets. In vitro perfusion is performed in system which consists of testing tubes and infusion pump. After 5 minutes perfusion with fresh ACD anticoagulated human whole blood at 2,000s-1 platelets deposition on surface precoated with proteins determined using anti-human platelet antibody (125 I-SZ-21) are 4,173 +/- 932 (Albumin), 59,032 +/- 25,554 (Fibrinogen), and 71,253 +/- 11,484 (Collagen). Meanwhile, platelets adhered on surfaces of four polymers were determined (platelet/mm2): 19,493 +/- 2,050 (Silicone), 48,193 +/- 4,055 (Polytetrafluoroethylene), 50,375 +/- 8,675 (Polyvinyl chloride) and 101,906 +/- 5,916 (Polyethylene). These results were confirmed by SEM. This method is not only applied for evaluating rapidly and reliably blood compatibility of biomaterials in vitro, but will be used at basic study for interaction of blood materials.

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

    SciTech Connect

    Long, Feng; Fong, Rachel H.; Austin, Stephen K.; Chen, Zhenguo; Klose, Thomas; Fokine, Andrei; Liu, Yue; Porta, Jason; Sapparapu, Gopal; Akahata, Wataru; Doranz, Benjamin J.; Crowe, James E.; Diamond, Michael S.; Rossmann, Michael G.

    2015-10-26

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

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

    DOE PAGES

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

    2015-10-26

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

  8. Tumor localization by combinations of monoclonal antibodies in a new human colon carcinoma cell line (LIM1899).

    PubMed

    Andrew, S M; Teh, J G; Johnstone, R W; Russell, S M; Whitehead, R H; McKenzie, I F; Pietersz, G A

    1990-09-01

    One of the problems of in vivo diagnosis and therapy of tumors with monoclonal antibodies is their heterogeneity with respect to antigen expression, with some cells expressing no antigen and others being weakly or strongly positive. Selected mixtures of antibodies to different antigens are therefore likely to react with more cells than single antibodies and be more effective for imaging and therapy. With this in mind, we have examined a new human colon cancer cell line (LIM1899) which has a heterogeneous expression of several cell surface molecules: by flow cytometry 38% were carcinoembryonic antigen positive; 64%, human milk fat globule positive, and 73%, CD46 positive; 87% of tumor cells bound a mixture of all three antibodies in vitro. Some blocking of the binding of anti-human milk fat globule antibody by the anti-CD46 antibody was noted. LIM1899 was established as a xenograft in nude mice and in vivo biodistribution studies performed using antibodies alone or in combination. Mixtures of antibodies clearly showed a higher percentage of injected dose of antibody in the tumor than did single antibodies: one antibody gave 10%; two together, 17 to 21%; and all three together gave 29% of the injected dose in the tumor. Tumor:blood ratios were also superior for combinations of antibodies, provided that low doses of the antibodies were used; at higher doses the effect was lost. The study demonstrates that combinations of antibodies are better than single antibodies for localization, provided that the dose used is carefully selected.

  9. Affinity maturation of a broadly neutralizing human monoclonal antibody that prevents acute hepatitis C virus infection in mice.

    PubMed

    Keck, Zhen-Yong; Wang, Yong; Lau, Patrick; Lund, Garry; Rangarajan, Sneha; Fauvelle, Catherine; Liao, Grant C; Holtsberg, Frederick W; Warfield, Kelly L; Aman, M Javad; Pierce, Brian G; Fuerst, Thomas R; Bailey, Justin R; Baumert, Thomas F; Mariuzza, Roy A; Kneteman, Norman M; Foung, Steven K H

    2016-12-01

    Direct-acting antivirals (DAAs) have led to a high cure rate in treated patients with chronic hepatitis C virus (HCV) infection, but this still leaves a large number of treatment failures secondary to the emergence of resistance-associated variants (RAVs). To increase the barrier to resistance, a complementary strategy is to use neutralizing human monoclonal antibodies (HMAbs) to prevent acute infection. However, earlier efforts with the selected antibodies led to RAVs in animal and clinical studies. Therefore, we identified an HMAb that is less likely to elicit RAVs for affinity maturation to increase potency and, more important, breadth of protection. Selected matured antibodies show improved affinity and neutralization against a panel of diverse HCV isolates. Structural and modeling studies reveal that the affinity-matured HMAb mediates virus neutralization, in part, by inducing conformational change to the targeted epitope, and that the maturated light chain is responsible for the improved affinity and breadth of protection. A matured HMAb protected humanized mice when challenged with an infectious HCV human serum inoculum for a prolonged period. However, a single mouse experienced breakthrough infection after 63 days when the serum HMAb concentration dropped by several logs; sequence analysis revealed no viral escape mutation. The findings suggest that a single broadly neutralizing antibody can prevent acute HCV infection without inducing RAVs and may complement DAAs to reduce the emergence of RAVs. (Hepatology 2016;64:1922-1933). © 2016 by the American Association for the Study of Liver Diseases.

  10. Human macrophages produce dimeric forms of IL-18 which can be detected with monoclonal antibodies specific for inactive IL-18.

    PubMed

    Kikkawa, S; Matsumoto, M; Shida, K; Fukumori, Y; Toyoshima, K; Seya, T

    2001-02-23

    We established two monoclonal antibodies (mAbs) which specifically recognize human 'functionally inactive' recombinant IL-18, and IL-18 protein polymorphism was examined using human monocytes and macrophages (M phi). In 6 day GM-CSF-treated M phi, an 'inactive' IL-18-recognizing mAb 21 detected the IL-18 proform (24 kDa) and a 48-kDa protein, which were gradually increased concomitant with maturation stage. Majority of the 24- and 48-kDa forms were barely detectable with other mAbs recognizing 'active' IL-18. No reagents including Toll stimulators up-regulated these IL-18 populations in M phi. The 21-recognizable IL-18 species were separated using an anion-exchanger column and their IFN gamma-inducing activity was assessed with human lymphocytes plus IL-12. Virtually no as yet known activity was detected with these IL-18 species. After processed with M phi proteases, an 18-kDa form was generated to express the IFN gamma-inducing activity, although the activity was far weaker than that of control 'active' IL-18. These observations suggested that large amounts of various IL-18 species are produced with monocyte-M phi differentiation and most of these IL-18 species are functionally 'inactive' in terms of the reported IL-18 function even after proteolytic 18-kDa conversion.

  11. Construction of scFv derived from a tumor-associated monoclonal antibody having tumoricidal activity on human hepatocellular carcinoma.

    PubMed

    Tungpradabkul, Sumalee; Sandee, Duanpen; Puthong, Songchan; Laohathai, Kingkarn

    2005-04-01

    A mouse monoclonal antibody (Mab-HepTAA43), classified as an anti-tumor-associated antigen, was raised by immunizing BALB/c mice with the Thai human hepatocellular carcinoma S102 (HCC-S102) cell line cells using hybridoma techniques. The Mab-HepTAA43 reacted with and markedly inhibited the growth of human hepatocellular carcinoma cell lines as well as a tumor mass in an animal model. Human hepatoma transplanted into nude mice did not show metastasis after 20 injections amounting to a total of about 4 mg of the Mab over 1-month period. A single-chain variable fragment (scFv) molecule derived from the Mab was constructed by phage display method. DNA sequence analysis of the active variable regions of both heavy- and light-chains of the cDNA clone was subsequently performed. The scFv43 molecule contains a V(L) kappa type and a unique V(H) sequence having 88% amino acid homology to that of Mab-MAK B raised against tumor-associated antigen. Immunohistochemical staining on frozen sections of paired hepatoma (NCI-I) and normal liver tissue from the same individual showed that both scFv43 and Mab-HepTAA43 antibodies reacted with hepatoma but not with normal liver tissue. The results suggest that scFv43 may be useful in the immunotherapy of hepatocellular carcinoma.

  12. A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words.

    PubMed

    Mirick, G R; Bradt, B M; Denardo, S J; Denardo, G L

    2004-12-01

    The United States Food and Drug Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ((90)yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ((131)I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) anti-CD20 MAbs for use in radioimmunotherapy (RIT) of non-Hodgkin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, assays were developed to determine HAGA (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to ''humanize'' MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades.

  13. Cloning, characterization, and modeling of a monoclonal anti-human transferrin antibody that competes with the transferrin receptor.

    PubMed Central

    Orlandini, M.; Santucci, A.; Tramontano, A.; Neri, P.; Oliviero, S.

    1994-01-01

    In this report we describe the isolation and characterization of a monoclonal antibody against human serum transferrin (Tf) and the cloning and sequencing of its cDNA. The antibody competes with the transferrin receptor (TR) for binding to human Tf and is therefore expected to bind at or very close to a region of interaction between Tf and its receptor. From the deduced amino acid sequence, we constructed a 3-dimensional model of the variable domains of the antibody based on the canonical structure model for the hypervariable loops. The proposed structure of the antibody is a first step toward a more detailed characterization of the antibody-Tf complex and possibly toward a better understanding of the Tf interaction with its receptor. The model might prove useful in guiding site-directed mutagenesis studies, simplifying the experimental elucidation of the antibody structure, and in the use of automatic procedures to dock the interacting molecules as soon as structural information about the structure of the human Tf molecule will be available. PMID:7530542

  14. Comprehensive Identification of Tumor-associated Antigens via Isolation of Human Monoclonal Antibodies that may be Therapeutic

    PubMed Central

    2009-01-01

    Although the success of trastuzumab and rituximab for treatment of breast cancer and non-Hodgkins lymphoma, respectively, suggests that monoclonal antibodies (mAbs) will become important therapeutic agents against a wider range of cancers, useful therapeutic Abs are not yet available for the majority of the human cancers because of our lack of knowledge of which antigens (Ags) are likely to become useful targets. We established a procedure for comprehensive identification of such Ags through the extensive isolation of human mAbs that may be therapeutic. Using the phage-display Ab library we isolated a large number of human mAbs that bind to the surface of tumor cells. They were individually screened by immunostaining, and clones that preferentially and strongly stained the malignant cells were chosen. The Ags recognized by those clones were isolated by immunoprecipitation and identified by mass spectrometry (MS). We isolated 2,114 mAbs with unique sequences and identified 25 distinct Ags highly expressed on several carcinomas. Of those 2,114 mAbs 434 bound to specifically to one of the 25 Ags. I am going to discuss how we could select proper target Ags for therapeutic Abs and candidate clones as therapeutic agents. PMID:20107531

  15. Comprehensive Identification of Tumor-associated Antigens via Isolation of Human Monoclonal Antibodies that may be Therapeutic.

    PubMed

    Kurosawa, Yoshikazu

    2009-02-01

    Although the success of trastuzumab and rituximab for treatment of breast cancer and non-Hodgkins lymphoma, respectively, suggests that monoclonal antibodies (mAbs) will become important therapeutic agents against a wider range of cancers, useful therapeutic Abs are not yet available for the majority of the human cancers because of our lack of knowledge of which antigens (Ags) are likely to become useful targets. We established a procedure for comprehensive identification of such Ags through the extensive isolation of human mAbs that may be therapeutic. Using the phage-display Ab library we isolated a large number of human mAbs that bind to the surface of tumor cells. They were individually screened by immunostaining, and clones that preferentially and strongly stained the malignant cells were chosen. The Ags recognized by those clones were isolated by immunoprecipitation and identified by mass spectrometry (MS). We isolated 2,114 mAbs with unique sequences and identified 25 distinct Ags highly expressed on several carcinomas. Of those 2,114 mAbs 434 bound to specifically to one of the 25 Ags. I am going to discuss how we could select proper target Ags for therapeutic Abs and candidate clones as therapeutic agents.

  16. Distinct expression profiles of Notch-1 protein in human solid tumors: Implications for development of targeted therapeutic monoclonal antibodies

    PubMed Central

    Li, Yuan; Burns, Janine A; Cheney, Carol A; Zhang, Ningyan; Vitelli, Salvatore; Wang, Fubao; Bett, Andrew; Chastain, Michael; Audoly, Laurent P; Zhang, Zhi-Qiang

    2010-01-01

    Biological therapies, such as monoclonal antibodies (mAbs) that target tumor-associated antigens have been considered an effective therapeutic approach in oncology. In considering Notch-1 receptor as a potential target, we performed immunohistochemistry on tissue microarrays to determine 1) whether the receptor is overexpressed in tumor cells as compared to their corresponding normal tissues and 2) the clinical significance of its expression levels in human breast, colorectal, lung and prostate cancers. We found that the expression of Notch-1 protein was overexpressed in primary colorectal adenocarcinoma and nonsmall cell lung carcinoma (NSCLC), but not in primary ductal breast carcinoma or prostate adenocarcinoma. Further analysis revealed that higher levels of Notch-1 protein expression were significantly associated with poorer differentiation of breast and prostate tumors. Strikingly, for NSCLC, the expression levels of Notch-1 protein were found to be inversely correlated with tumor differentiation and progression. For colorectal tumors, however, no correlation of Notch-1 protein expression was found with any tumor clinicopathological parameters, in spite of its overexpression in tumor cells. Our data demonstrated the complexity of Notch-1 protein expression in human solid tumors and further supported the notion that the roles of Notch-1 expression in tumorigenesis are highly context-dependent. The findings could provide the basis for development of distinct therapeutic strategies of Notch-1 mAbs for its applications in the treatment of suitable types of human cancers. PMID:20631820

  17. Production and characterization of high-affinity human monoclonal antibodies to human immunodeficiency virus type 1 envelope glycoproteins in a mouse model expressing human immunoglobulins.

    PubMed

    Sheppard, Neil C; Davies, Sarah L; Jeffs, Simon A; Vieira, Sueli M; Sattentau, Quentin J

    2007-02-01

    Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-1(97CN54)), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-1(97CN54) Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies.

  18. Cell lines for the production of monoclonal antibodies to human glycophorin A

    SciTech Connect

    Bigbee, W.L.; Fong, S.S.N.; Jensen, R.H.; Vanderlaan, M.; Langlois, R.G.

    1988-08-30

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that differentiate between the M and N forms of human glycophorin A. These antibodies have potential application as human blood group reagents, as markers for terminally differentiated erythroid cells and as immunofluorescent labels of somatically variant human erythrocytes.

  19. Cell lines for the production of monoclonal antibodies to human glycophorin A

    DOEpatents

    Bigbee, William L.; Fong, Stella S. N.; Jensen, Ronald H.; Vanderlaan, Martin; Langlois, Richard G.

    1988-01-01

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that differentiate between the M and N forms of human glycophorin A. These antibodies have potential application as human blood group reagents, as markers for terminally differentiated erythroid cells and as immunofluorescent labels of somatically variant human erythrocytes.

  20. Method and cell lines for the production of monoclonal antibodies to human glycophorin A

    DOEpatents

    Bigbee, W.L.; Fong, S.S.N.; Jensen, R.H.; Vanderlaan, M.

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that differentiate between the M and N forms of human glycophorin A. These antibodies have potential application as human blood group reagents, as markers for terminally differentiated erythroid cells and as immunofluorescent labels of somatically variant human erythrocytes.

  1. Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum

    PubMed Central

    Vanier, Gaëtan; Hempel, Franziska; Chan, Philippe; Rodamer, Michael; Vaudry, David; Maier, Uwe G.; Lerouge, Patrice; Bardor, Muriel

    2015-01-01

    Monoclonal antibodies (mAbs) represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fueled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharmaceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C- and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro- and microheterogeneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing recombinant proteins bearing high mannose-type N-glycans. PMID:26437211

  2. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody.

    PubMed

    Ye, Xiaohua; Fan, Chen; Ku, Zhiqiang; Zuo, Teng; Kong, Liangliang; Zhang, Chao; Shi, Jinping; Liu, Qingwei; Chen, Tan; Zhang, Yingyi; Jiang, Wen; Zhang, Linqi; Huang, Zhong; Cong, Yao

    2016-03-01

    Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.

  3. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody

    PubMed Central

    Ku, Zhiqiang; Zuo, Teng; Kong, Liangliang; Zhang, Chao; Shi, Jinping; Liu, Qingwei; Chen, Tan; Zhang, Yingyi; Jiang, Wen; Zhang, Linqi; Huang, Zhong; Cong, Yao

    2016-01-01

    Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection. PMID:26938634

  4. Use of monoclonal antibodies to investigate the immunochemical heterogeneity of human plasma high density lipoproteins

    SciTech Connect

    Krul, E.S.; Keller, J.; Melton, M.; Schonfeld, G.

    1986-03-01

    Immunoaffinity chromatography of high density lipoproteins (HDL) on columns prepared with seven different anti-apoAI monoclonal antibodies (MAbs) was used to investigate the nature of HDL particle heterogeneity. Using subsaturating amounts of /sup 125/I-HDL, the MAbs coupled to the affinity columns could be divided into 2 groups: Group A, those MAbs that retained approx.75% of the total radioactive counts applied and Group B, those MAbs that retained approx.40% of the counts. The percent of total counts retained correlated (r/sup 2/ = 0.93) with the AI/AII molar ratios of the retained HDL fractions. The HDL particles retained by Group A had high AI/AII ratios, whereas the particles retained by Group B had AI/AII ratios similar to the starting HDL population. These results are consistent with the concept that HDL consists of two types of particles, namely Lp(AI without AII) and Lp(AI with AII). Group A MAbs preferentially bind Lp(AI without AII) but may bind some Lp(AI with AII). These MAbs are directed towards the COOH terminal half of apoAI. Group B MAbs recognize an epitope on apoAI that is expressed on HDL particles also containing apoAII. The specificities of some of these MAbs are towards the NH/sub 2/ terminal half of apoAI is similar on HDL particles containing or not containing apoAII. Heterogeneity of apoAI epitope expression on HDL would appear to be due to interactions with the apoAII molecule that probably occur towards the NH/sub 2/ end of apoAI.

  5. Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum.

    PubMed

    Vanier, Gaëtan; Hempel, Franziska; Chan, Philippe; Rodamer, Michael; Vaudry, David; Maier, Uwe G; Lerouge, Patrice; Bardor, Muriel

    2015-01-01

    Monoclonal antibodies (mAbs) represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fueled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharmaceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C- and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro- and microheterogeneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing recombinant proteins bearing high mannose-type N-glycans.

  6. Molecular Mechanisms and Translational Therapies for Human Epidermal Receptor 2 Positive Breast Cancer

    PubMed Central

    Lv, Quanxia; Meng, Ziyuan; Yu, Yuanyuan; Jiang, Feng; Guan, Daogang; Liang, Chao; Zhou, Junwei; Lu, Aiping; Zhang, Ge

    2016-01-01

    Breast cancer is the second leading cause of cancer death among women. Human epidermal receptor 2 (HER2) positive breast cancer (HER2+ BC) is the most aggressive subtype of breast cancer, with poor prognosis and a high rate of recurrence. About one third of breast cancer is HER2+ BC with significantly high expression level of HER2 protein compared to other subtypes. Therefore, HER2 is an important biomarker and an ideal target for developing therapeutic strategies for the treatment HER2+ BC. In this review, HER2 structure and physiological and pathological roles in HER2+ BC are discussed. Two diagnostic tests, immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH), for evaluating HER2 expression levels are briefly introduced. The current mainstay targeted therapies for HER2+ BC include monoclonal antibodies, small molecule tyrosine kinase inhibitors, antibody–drug conjugates (ADC) and other emerging anti-HER2 agents. In clinical practice, combination therapies are commonly adopted in order to achieve synergistic drug response. This review will help to better understand the molecular mechanism of HER2+ BC and further facilitate the development of more effective therapeutic strategies against HER2+ BC. PMID:27983617

  7. Brain metastasis in human epidermal growth factor receptor 2-positive breast cancer: from biology to treatment

    PubMed Central

    Koo, Taeryool

    2016-01-01

    Overexpression of human epidermal growth factor receptor 2 (HER2) is found in about 20% of breast cancer patients. With treatment using trastuzumab, an anti-HER2 monoclonal antibody, systemic control is improved. Nonetheless, the incidence of brain metastasis does not be improved, rather seems to be increased in HER2-positive breast cancer. The mainstay treatment for brain metastases is radiotherapy. According to the number of metastatic lesions and performance status of patients, radiosurgery or whole brain radiotherapy can be performed. The concurrent use of a radiosensitizer further improves intracranial control. Due to its large molecular weight, trastuzumab has a limited ability to cross the blood-brain barrier. However, small tyrosine kinase inhibitors such as lapatinib, has been noted to be a promising agent that can be used as a radiosensitizer to affect HER2-positive breast cancer. This review will outline general management of brain metastases and will focus on preclinical findings regarding the radiosensitizing effect of small molecule HER2 targeting agents. PMID:27104161

  8. Generation of a novel high-affinity monoclonal antibody with conformational recognition epitope on human IgM.

    PubMed

    Sarikhani, Sina; Mirshahi, Manouchehr; Gharaati, Mohammad Reza; Mirshahi, Tooran

    2010-11-01

    As IgM is the first isotype of antibody which appears in blood after initial exposure to a foreign antigen in the pattern of primary response, detection, and quantification of this molecule in blood seems invaluable. To approach these goals, generation, and characterization of a highly specific mAb (monoclonal antibody) against human IgM were investigated. Human IgM immunoglobulins were used to immunize Balb/c mice. Spleen cells taken from the immunized animals were fused with SP2/O myeloma cells using PEG (polyethylene glycol, MW 1450) as fusogen. The hybridomas were cultured in HAT containing medium and supernatants from the growing hybrids were screened by enzyme-linked immunosorbent assay (ELISA) using plates coated with pure human IgM and the positive wells were then cloned at limiting dilutions. The best clone designated as MAN-1, was injected intraperitoneally to some Pristane-injected mice. Anti-IgM mAb was purified from the animals' ascitic fluid by protein-G sepharose followed by DEAE-cellulose ion exchange chromatography. MAN-1 interacted with human IgM with a very high specificity and affinity. The purity of the sample was tested by SDS-PAGE and the affinity constant was measured (K(a) = 3.5 x 10(9)M(-1). Immunoblotting and competitive ELISA were done and the results showed that the harvested antibody recognizes a conformational epitope on the mu chain of human IgM and there was no cross-reactivity with other subclasses of immunoglobulins. Furthermore, isotyping test was done and the results showed the subclass of the obtained mAb which was IgG(1)kappa.

  9. Structures of complexes formed by H5 influenza hemagglutinin with a potent broadly neutralizing human monoclonal antibody.

    PubMed

    Xiong, Xiaoli; Corti, Davide; Liu, Junfeng; Pinna, Debora; Foglierini, Mathilde; Calder, Lesley J; Martin, Stephen R; Lin, Yi Pu; Walker, Philip A; Collins, Patrick J; Monne, Isabella; Suguitan, Amorsolo L; Santos, Celia; Temperton, Nigel J; Subbarao, Kanta; Lanzavecchia, Antonio; Gamblin, Steven J; Skehel, John J

    2015-07-28

    H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer's formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion.

  10. Rapid detection of human group C rotaviruses by reverse passive hemagglutination and latex agglutination tests using monoclonal antibodies.

    PubMed Central

    Kuzuya, M; Fujii, R; Hamano, M; Nagabayashi, T; Tsunemitsu, H; Yamada, M; Nii, S; Mori, T

    1993-01-01

    Reverse passive hemagglutination (RPHA) tests and a latex agglutination test with monoclonal antibodies (MAbs) were developed for the rapid detection of noncultivatable human group C rotaviruses. For RPHA tests, two MAbs, MAb 5A12 recognizing the outer capsid and MAb 13A3 recognizing the inner capsid, were separately used for the coating of sheep erythrocytes (SRBCs). Forty-six fecal samples were examined to confirm the practicality of the tests. As a result, there was concordance between the RPHA test with SRBCs coated with MAb 5A12 and polyacrylamide gel electrophoresis of viral RNA (RNA-PAGE) in 44 (95.6%) of 46 samples, while the diagnoses by the RPHA test with SRBCs coated with MAb 13A3 were in complete agreement with those by RNA-PAGE. Furthermore, a latex agglutination test with MAb 13A3 was also developed, and this test was fast enough and sensitive enough to successfully detect the viruses from most fecal samples within 2 min. The present procedures would be useful for the diagnosis of human group C rotavirus infections in clinical laboratories which are not well equipped. Images PMID:8388891

  11. Antigenic implications of human immunodeficiency virus type 1 envelope quaternary structure: oligomer-specific and -sensitive monoclonal antibodies.

    PubMed Central

    Broder, C C; Earl, P L; Long, D; Abedon, S T; Moss, B; Doms, R W

    1994-01-01

    A majority of monoclonal antibodies (mAbs) raised against soluble oligomeric human immunodeficiency virus type 1 isolate IIIB (HIV-1IIIB) envelope (env) glycoprotein reacted with conformational epitopes within the gp120 or gp41 subunits. Of 35 mAbs directed against gp41, 21 preferentially reacted with oligomeric env. A subset of these mAbs reacted only with env oligomers (oligomer-specific mAbs). In contrast, only 1 of 27 mAbs directed against the gp120 subunit reacted more strongly with env oligomers than with monomers, and none were oligomer-specific. However, 50% of anti-gp120 mAbs preferentially recognized monomeric env, suggesting that some epitopes in gp120 are partially masked or altered by intersubunit contacts in the native env oligomer. Two mAbs to oligomer-dependent epitopes in gp41 neutralized HIV-1IIIB and HIV-1SF2, and binding of these mAbs to env was blocked by preincubation with HIV-1-positive human serum. Thus, immunization with soluble, oligomeric env elicits antibodies to conserved, conformational epitopes including a newly defined class of neutralizing antibodies that bind to oligomer-specific epitopes in gp41, and may also minimize the production of antibodies that preferentially react with monomeric env protein. Images PMID:7972127

  12. Human Monoclonal Antibodies Generated Following Vaccination with AVA Provide Neutralization by Blocking Furin Cleavage but not by Preventing Oligomerization

    PubMed Central

    Smith, Kenneth; Crowe, Sherry R.; Garman, Lori; Guthridge, Carla; Muther, Jennifer J.; McKee, Emily; Farris, A. Darise; Guthridge, Joel M.; Wilson, Patrick C.; James, Judith A.

    2012-01-01

    In order to identify the combination of antibody-mediated mechanisms of neutralization that result from vaccination with anthrax vaccine adsorbed (AVA), we isolated antibody secreting cells from a single donor seven days after booster vaccination with AVA and generated nine fully human monoclonal antibodies (hmAb) with high specificity for protective antigen (PA). Two of the antibodies were able to neutralize lethal toxin in vitro at low concentrations (IC50: p6C01, 0.12 µg/ml and p6F01, 0.45 µg/ml). Passive transfer of either of these hmAbs to A/J mice prior to challenge with lethal toxin conferred 80–90% protection. We demonstrate that hmAb p6C01 is neutralizing by preventing furin cleavage of PA in a dose-dependent manner, but the mechanism of p6F01 is unclear. Three additional antibodies were found to bind to domain 3 of PA and prevent oligomerization, although they did not confer significant protection in vivo and showed a significant prozone-like effect in vitro. These fully human antibodies provide insight into the neutralizing response to AVA for future subunit vaccine and passive immunotherapeutic cocktail design. PMID:22425791

  13. Rozrolimupab, a mixture of 25 recombinant human monoclonal RhD antibodies, in the treatment of primary immune thrombocytopenia.

    PubMed

    Robak, Tadeusz; Windyga, Jerzy; Trelinski, Jacek; von Depka Prondzinski, Mario; Giagounidis, Aristoteles; Doyen, Chantal; Janssens, Ann; Alvarez-Román, María Teresa; Jarque, Isidro; Loscertales, Javier; Rus, Gloria Pérez; Hellmann, Andrzej; Jêdrzejczak, Wieslaw Wiktor; Kuliczkowski, Kazimierz; Golubovic, Lana M; Celeketic, Dusica; Cucuianu, Andrei; Gheorghita, Emanuil; Lazaroiu, Mihaela; Shpilberg, Ofer; Attias, Dina; Karyagina, Elena; Svetlana, Kalinina; Vilchevska, Kateryna; Cooper, Nichola; Talks, Kate; Prabhu, Mukhyaprana; Sripada, Prasad; Bharadwaj, T P R; Næsted, Henrik; Skartved, Niels J Ø; Frandsen, Torben P; Flensburg, Mimi F; Andersen, Peter S; Petersen, Jørgen

    2012-11-01

    Rozrolimupab, a recombinant mixture of 25 fully human RhD-specific monoclonal antibodies, represents a new class of recombinant human antibody mixtures. In a phase 1 or 2 dose escalation study, RhD(+) patients (61 subjects) with primary immune thrombocytopenia received a single intravenous dose of rozrolimupab ranging from 75 to 300 μg/kg. The primary outcome was the occurrence of adverse events. The principal secondary outcome was the effect on platelet levels 7 days after the treatment. The most common adverse events were headache and pyrexia, mostly mild, and reported in 20% and 13% of the patients, respectively, without dose relationship. Rozrolimupab caused an expected transient reduction of hemoglobin concentration in the majority of the patients. At the dose of 300 μg/kg platelet responses, defined as platelet count ≥ 30 × 10(9)/L and an increase in platelet count by > 20 × 10(9)/L from baseline were observed after 72 hours and persisted for at least 7 days in 8 of 13 patients (62%). Platelet responses were observed within 24 hours in 23% of patients and lasted for a median of 14 days. Rozrolimupab was well tolerated and elicited rapid platelet responses in patients with immune thrombocytopenia and may be a useful alternative to plasma-derived products. This trial is registered at www.clinicaltrials.gov as #NCT00718692.

  14. Epitope mapping of anti-human transferrin monoclonal antibodies: potential uses for transferrin-transferrin receptor interaction studies.

    PubMed

    Perera, Yasser; García, Darién; Guirola, Osmany; Huerta, Vivian; García, Yanet; Muñoz, Yasmiana

    2008-01-01

    Human transferrin (hTf) is an 80 kDa glycoprotein involved in iron transport from the absorption sites to the sites of storage and utilization. Additionally, transferrin also plays a relevant role as a bacteriostatic agent preventing uncontrolled bacterial growth in the host. In this work we describe a well-characterized Mabs panel in terms of precise epitope localization and estimate affinity for the two major hTf isoforms. We found at least four antigenic regions in the hTf molecule, narrowed down the interacting antigen residues within three of such regions, and located them on a molecular model of hTf. Two of the antigenic regions partially overlap with previously described transferrin-binding sites for both human receptor (antigenic region I: containing amino acid residues from Asp-69 to Asn-76 at the N-lobe) and bacterial receptors from two pathogenic species (antigenic region III: amino acid residues from Leu-665 to Ser-672 at the C-lobe). Hence, such monoclonal antibodies (Mabs) could be used as an additional tool for conformational studies and/or the characterization of the interaction between hTf and both types of receptor molecules.

  15. Structural basis for the neutralization of MERS-CoV by a human monoclonal antibody MERS-27

    PubMed Central

    Yu, Xiaojuan; Zhang, Senyan; Jiang, Liwei; Cui, Ye; Li, Dongxia; Wang, Dongli; Wang, Nianshuang; Fu, Lili; Shi, Xuanlin; Li, Ziqiang; Zhang, Linqi; Wang, Xinquan

    2015-01-01

    The recently reported Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness in humans with an approximately 30% mortality rate. The envelope spike glycoprotein on the surface of MERS-CoV mediates receptor binding, membrane fusion, and viral entry. We previously reported two human monoclonal antibodies that target the receptor binding domain (RBD) of the spike and exhibit strong neutralization activity against live and pesudotyped MERS-CoV infection. Here we determined the crystal structure of MERS-CoV RBD bound to the Fab fragment of MERS-27 antibody at 3.20 Å resolution. The MERS-27 epitope in the RBD overlaps with the binding site of the MERS-CoV receptor DPP4. Further biochemical, viral entry, and neutralization analyses identified two critical residues in the RBD for both MERS-27 recognition and DPP4 binding. One of the residues, Trp535, was found to function as an anchor residue at the binding interface with MERS-27. Upon receptor binding, Trp535 interacts with the N-linked carbohydrate moiety of DPP4. Thus, MERS-27 inhibits MERS-CoV infection by directly blocking both protein-protein and protein-carbohydrate interactions between MERS-CoV RBD and DPP4. These results shed light on the molecular basis of MERS-27 neutralization and will assist in the optimization of MERS-27 as a tool to combat MERS-CoV infection. PMID:26281793

  16. Appearance of cross linked proteins in human atheroma and rat pre-fibrotic liver detected by a new monoclonal antibody.

    PubMed

    Itabe, H; Jimi, S; Kamimura, S; Suzuki, K; Uesugi, N; Imanaka, T; Shijo, H; Takano, T

    1998-02-27

    A new monoclonal antibody against malondialdehyde (MDA)-treated low density lipoprotein (LDL) was raised using homogenate of human atheroma as immunogen. This antibody, DLH2, was obtained by selecting the clones which did not react to native LDL but did react to copper-induced oxidized LDL (OxLDL). DLH2 showed a greater reactivity to MDA-LDL than to OxLDL. When LDL was treated with various aldehyde containing reagents, treatment of LDL with glutaraldehyde or MDA greatly increased the reactivity to the antibody, while LDL treated with 2,4-hexadienal or 4-hydroxynonenal was not reactive. Among many proteins tested, high density lipoprotein, bovine serum albumin and hemoglobin showed significant reactivity to DLH2 after they were treated with MDA or glutaraldehyde. When low density and high density lipoproteins treated with MDA were subjected to immunoblot analysis, newly formed products larger than the original apolipoproteins were detected with the antibody, suggesting that this antibody recognizes aggregated proteins with divalent short chain cross linkers. The antigenic materials were shown by immunohistochemical analysis to be present in foamy macrophages in human atheromatous lesions. DLH2 antigen did not colocalize either with apolipoprotein B. Furthermore, we found a massive accumulation of the antigenic material in Kupffer cells in the liver of rats treated with alcohol and carbonyl iron, a model of hepatic fibrosis due to oxidative stress. These results suggest the presence of cross linked proteins in damaged tissues.

  17. Human tenascin: primary structure, pre-mRNA splicing patterns and localization of the epitopes recognized by two monoclonal antibodies.

    PubMed Central

    Siri, A; Carnemolla, B; Saginati, M; Leprini, A; Casari, G; Baralle, F; Zardi, L

    1991-01-01

    By sequencing cDNA clones which cover the complete coding region of human tenascin (TN), we have established its primary structure. This confirms that, as in the case of chicken, TN is mainly made up of three groups of sequences with a high homology to Epidermal Growth Factor (EGF) fibronectin (FN) type III repeat and fibrinogen. Furthermore, we have determined the amino-terminal sequence of the mature peptide directly on purified TN. The main differences with respect to the chicken TN molecule are that in the human there are 14 and half EGF-like repeats compared to 13 and half in the chicken and that, as previously reported, there are 15 FN-like repeats compared to 11 in the chicken. By Polymerase Chain Reaction (PCR) amplification we have also studied the different splicing patterns of the TN pre-mRNA in cultured cells. The results show the presence of at least four different isoforms containing different numbers of FN-like type III repeats. Using purified human TN as immunogen, we have obtained numerous monoclonal antibodies (Mabs) to TN. By screening a human melanoma cDNA library in the expression vector lambda gt11 with these Mabs and subsequently sequencing the insert of the positive clones, we have been able to localize, within the TN molecule, the epitopes recognized by two of these Mabs: BC-4, which recognizes an epitope within the EGF-like sequence and BC-2 which recognizes an epitope within the FN like type III repeats whose expression is regulated by alternative splicing of the TN pre-mRNA. Thus, while the Mab BC-4 may be useful in studies on TN distribution (since it recognizes all different TN isoforms) BC-2 may be useful in the study of the expression of particular TN isoforms generated by the alternative splicing of the TN primary transcript. Images PMID:1707164

  18. Monoclonal antibodies specific to human Δ42PD1: A novel immunoregulator potentially involved in HIV-1 and tumor pathogenesis

    PubMed Central

    Cheng, Lin; Tang, Xian; Liu, Li; Peng, Jie; Nishiura, Kenji; Cheung, Allen Ka Loon; Guo, Jia; Wu, Xilin; Tang, Hang Ying; An, Minghui; Zhou, Jingying; Cheung, Ka Wai; Wang, Hui; Guan, Xinyuan; Wu, Zhiwei; Chen, Zhiwei

    2015-01-01

    We recently reported the identification of Δ42PD1, a novel alternatively spliced isoform of human PD1 that induces the production of pro-inflammatory cytokines from human peripheral blood mononuclear cells and enhances HIV-specific CD8+ T cell immunity in mice when engineered in a fusion DNA vaccine. The detailed functional study of Δ42PD1, however, has been hampered due to the lack of a specific monoclonal antibody (mAb). In this study, we generated 2 high-affinity mAbs, clones CH34 (IgG2b) and CH101 (IgG1), from Δ42PD1-immunized mice. They recognize distinct domains of Δ42PD1 as determined by a yeast surface-displaying assay and ELISA. Moreover, they recognize native Δ42PD1 specifically, but not PD1, on cell surfaces by both flow cytometry and immunohistochemical assays. Δ42PD1 appeared to be expressed constitutively on healthy human CD14+ monocytes, but its level of expression was down-regulated significantly during chronic HIV-1 infection. Since the level of Δ42PD1 expression on CD14+ monocytes was negatively correlated with the CD4 count of untreated patients in a cross-sectional study, Δ42PD1 may play a role in HIV-1 pathogenesis. Lastly, when examining Δ42PD1 expression in human esophageal squamous-cell carcinoma tissues, we found high-level expression of Δ42PD1 on a subset of tumor-infiltrating T cells. Our study, therefore, resulted in 2 Δ42PD1-specific mAbs that can be used to further investigate Δ42PD1, a novel immune regulatory protein implicated in HIV-1 and tumor pathogenesis as well as other immune diseases. PMID:25692916

  19. A variety of human monoclonal antibodies against epidermal growth factor receptor isolated from a phage antibody library.

    PubMed

    Kurosawa, Gene; Kondo, Mariko; Kurosawa, Yoshikazu

    2016-11-04

    When the technology for constructing human antibody (Ab) libraries using a phage-display system was developed, many researchers in Ab-related fields anticipated that it would be widely applied to the development of pharmaceutical drugs against various diseases, including cancers. However, successful examples of such applications are very limited. Moreover, researchers who utilize phage-display technology now show divergent ways of thinking about phage Ab libraries. For example, there is debate about what should be the source of VH and VL genes for the construction of libraries to cover the whole repertoire of Abs present in the human body. In the immune system, the introduction of mutations into V genes followed by selection based on binding activity, termed Ab maturation, is required for the production of Abs exhibiting high affinity to the antigen (Ag). Therefore, introduction of mutations and selection are required for isolation of Abs with high affinity after isolation of clones from phage Ab libraries. We constructed a large human Ab library termed AIMS, developed a screening method termed ICOS, and succeeded in isolating many human monoclonal Abs (mAbs) that specifically and strongly bind to various tumor-associated Ags. Eight anti-EGFR mAbs were included, which we characterized. These mAbs showed various different activities against EGFR-expressing cancer cells. In this paper, we describe these data and discuss the possibility and necessity that the mAbs isolated from the AIMS library might be developed as therapeutic drugs against cancers without introduction of mutations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  20. A monoclonal antibody that specifically reacts with human embryonal carcinomas, spermatogonia and oocytes is able to induce human EC cell death.

    PubMed

    Nakano, T; Umezawa, A; Abe, H; Suzuki, N; Yamada, T; Nozawa, S; Hata, J

    1995-02-01

    We developed a mouse monoclonal antibody, 6E2 (IgG3), against a human embryonal carcinoma (EC) cell line, NCR-G3, that possesses totipotent differentiation capabilities. Culturing human EC cells in the presence of 6E2 causes their death. It has been shown that 6E2 kills EC cells dose dependently. In immunohistochemical examination with normal human germ cells, 6E2 reacted specifically with spermatogonia and oocytes. Among human germ cell tumor tissues on aceton-fixed frozen sections, 6E2 reacted with embryonal carcinomas, seminomas and dysgerminomas, but it did not react with choriocarcinomas or with yolk sac tumors. Consistently, in flow cytometric analysis of cultured human germ cell tumor cell lines, 6E2 reacted exclusively with EC cells including NCR-G3 cells. It was revealed, by preserving its antigenicity after treatment with periodic acid and tunicamycin and by radiolabeling cells followed by immunoprecipitation, that the molecule defined by 6E2 is a cell surface protein having a molecular weight of approximately 80 kDa. These data illustrate that the molecule defined by 6E2 links human germ cell tumors, especially embryonal carcinoma, seminoma and dysgerminoma, to their normal counterparts and that it may play a role in survival and proliferation of human EC cells.

  1. Monoclonal Antibodies in Diagnosis and Therapy

    NASA Astrophysics Data System (ADS)

    Waldmann, Thomas A.

    1991-06-01

    Monoclonal antibodies have been applied clinically to the diagnosis and therapy of an array of human disorders, including cancer and infectious diseases, and have been used for the modulation of immune responses. Effective therapy using unmodified monoclonal antibodies has, however, been elusive. Recently, monoclonal antibody-mediated therapy has been revolutionized by advances such as the definition of cell-surface structures on abnormal cells as targets for effective monoclonal antibody action, genetic engineering to create less immunogenic and more effective monoclonal antibodies, and the arming of such antibodies with toxins or radionuclides to enhance their effector function.

  2. Monoclonal antibodies to human glycophorin a and cell lines for the production thereof

    SciTech Connect

    Vanderlaan, M.; Bigbee, W.L.; Jensen, R.H.; Fong, S.S.N.; Langlois, R.G.

    1988-06-21

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that are highly specific to and exhibit high affinity for glycophorin A/sup N/ and differentiate between the M and N forms of human glycophorin A.

  3. Monoclonal antibodies to human glycophorin A and cell lines for the production thereof

    DOEpatents

    Vanderlaan, Martin; Bigbee, William L.; Jensen, Ronald H.; Fong, Stella S. N.; Langlois, Richard G.

    1988-01-01

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that are highly specific to and exhibit high affinity for glycophorin A.sup.N and differentiate between the M and N forms of human glycophorin A.

  4. Serological analysis of human anti-human antibody responses in colon cancer patients treated with repeated doses of humanized monoclonal antibody A33.

    PubMed

    Ritter, G; Cohen, L S; Williams, C; Richards, E C; Old, L J; Welt, S

    2001-09-15

    Mouse monoclonal antibody A33 (mAb A33) recognizes a M(r) 43,000 cell surface glycoprotein (designated A33) expressed in human colonic epithelium and colon cancer but absent from most other normal tissues. In patients, mAb A33 localizes with high specificity to colon cancer and is retained for up to 6 weeks in the cancer but cleared rapidly from normal colon (5-6 days). As a carrier of (125)I or (131)I, mAb A33 has shown antitumor activity. Induction of strong human anti-mouse antibody (immunoglobulin; HAMA) responses in patients, however, limits the use of the murine mAb A33 to very few injections. A humanized version of this antibody (huAb A33) has been prepared for Phase I and II clinical studies in patients with colon cancer. In those studies, immunogenicity of huAb A33 has been monitored using a novel, highly sensitive BIACORE method, which allows measurement of human anti-human antibodies (HAHAs) without the use of secondary reagents. We found that 63% (26 of 41) of the patients treated with repeated doses of huAb A33 developed HAHAs against a conformational antigenic determinant located in the V(L) and V(H) regions of huAb A33. Detailed serological analysis showed two distinct types of HAHAs. HAHA of type I (49% of patients) was characterized by an early onset with peak HAHA levels after 2 weeks of treatment, which declined with ongoing huAb A33 treatment. HAHA of type II (17% of patients) was characterized by a typically later onset of HAHA than in type I and by progressively increasing HAHA levels with each subsequent huAb A33 administration. Colon cancer patients with type I HAHAs did not develop infusion-related adverse events. In contrast, HAHA of type II was indicative of infusion-related adverse events. By using this new method, we were able to distinguish these two types of HAHAs in patients while on antibody treatment, allowing patients to be removed from study prior to the onset of severe infusion-related adverse events.

  5. A murine monoclonal anti-idiotypic antibody detects a common idiotope on human, mouse and rabbit antibodies to allergen Lol p IV.

    PubMed

    Zhou, E M; Dzuba-Fischer, J M; Rector, E S; Sehon, A H; Kisil, F T

    1991-09-01

    A syngeneic mouse monoclonal anti-idiotypic antibody (anti-Id), designated as B1/1, was generated against a monoclonal antibody (MoAb 91) specific for Ryegrass pollen allergen Lol p IV. This anti-Id recognized an idiotope (Id) that was also present on other monoclonal antibodies with the same specificity as MoAb 91. Observations that (i) the anti-Id inhibited the binding of MoAb 91 to Lol p IV and (ii) the Id-anti-Id interaction could be inhibited by Lol p IV indicated that the Id was located within or near the antigen combining site. These properties served to characterize B1/1 as an internal image anti-Id. Evidence that an immune response in different species to Lol p IV elicits the formation of antibodies which express a common Id was provided by the observations that (i) the Id-anti-Id interactions could be inhibited by mouse, human and rabbit antisera to Lol p IV and (ii) the binding of these antisera to Lol p IV could be inhibited by the anti-Id. Interestingly, the internal image anti-Id B1/1 also recognized an Id on a monoclonal antibody which was directed to an epitope of Lol p IV, different from that recognized by MoAb 91.

  6. Efficacy and safety of AVP-21D9, an anthrax monoclonal antibody, in animal models and humans.

    PubMed

    Malkevich, Nina V; Hopkins, Robert J; Bernton, Edward; Meister, Gabriel T; Vela, Eric M; Atiee, George; Johnson, Virginia; Nabors, Gary S; Aimes, Ronald T; Ionin, Boris; Skiadopoulos, Mario H

    2014-07-01

    Anthrax is an acute infectious disease caused by the spore-forming bacterium Bacillus anthracis. Timely administration of antibiotics approved for the treatment of anthrax disease may prevent associated morbidity and mortality. However, any delay in initiating antimicrobial therapy may result in increased mortality, as inhalational anthrax progresses rapidly to the toxemic phase of disease. An anthrax antitoxin, AVP-21D9, also known as Thravixa (fully human anthrax monoclonal antibody), is being developed as a therapeutic agent against anthrax toxemia. The efficacy of AVP-21D9 in B. anthracis-infected New Zealand White rabbits and in cynomolgus macaques was evaluated, and its safety and pharmacokinetics were assessed in healthy human volunteers. The estimated mean elimination half-life values of AVP-21D9 in surviving anthrax-challenged rabbits and nonhuman primates (NHPs) ranged from approximately 2 to 4 days and 6 to 11 days, respectively. In healthy humans, the mean elimination half-life was in the range of 20 to 27 days. Dose proportionality was observed for the maximum serum concentration (Cmax) of AVP-21D9 and the area under the concentration-time curve (AUC). In therapeutic efficacy animal models, treatment with AVP-21D9 resulted in survival of up to 92% of the rabbits and up to 67% of the macaques. Single infusions of AVP-21D9 were well tolerated in healthy adult volunteers across all doses evaluated, and no serious adverse events were reported. (This study has been registered at ClinicalTrials.gov under registration no. NCT01202695.). Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  7. Human multiple organ-reactive monoclonal autoantibody recognizes growth hormone and a 35,000-molecular weight protein.

    PubMed Central

    Satoh, J; Essani, K; McClintock, P R; Notkins, A L

    1984-01-01

    By fusing peripheral leukocytes from a patient with insulin-dependent diabetes with mouse myeloma cells, a heterohybridoma was isolated that, for over one year, has secreted a human monoclonal autoantibody, designated MOR-h1 (multiple organ-reactive human 1). This antibody reacts with antigens in several endocrine organs including the pituitary, thyroid, stomach, and pancreas. By double immunofluorescence, MOR-h1 was found to react specifically with growth hormone (GH)-containing cells in the anterior pituitary and, by enzyme-linked immunosorbent assay, MOR-h1 was shown to react with both natural and biosynthetic GH. Absorption experiments revealed that GH could remove the capacity of MOR-h1 to react not only with cells in the anterior pituitary, but also with cells in the thyroid, stomach, and pancreas. The demonstration with hyperimmune serum that these organs do not contain GH indicated that MOR-h1 was reacting with a different molecule(s) in these organs. By passing extracts of pituitary, thyroid, and stomach through an MOR-h1 affinity column and analyzing the eluted antigens by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a 35,000-mol wt polypeptide was isolated from each of these organs. In addition, a 21,500-mol wt polypeptide with an electrophoretic mobility identical to purified human GH was isolated from the pituitary, but not the other organs. It is concluded that MOR-h1 reacts with a 35,000-mol wt polypeptide present in the pituitary, thyroid, and stomach and that this antibody also recognizes a determinant on GH. Images PMID:6384271

  8. Description and partial characterization of a nucleolar RNA-associated autoantigen defined by a human monoclonal antibody

    PubMed Central

    1987-01-01

    B lymphocytes from a patient with systemic lupus erythematosus (SLE) and several circulating autoantibodies (including antinucleolar antibodies) were immortalized by fusion with a hypoxanthine/guanine phosphoribosyl transferase (HGPRT)-deficient human B cell line. Multiple human monoclonal antibodies (mAb) were obtained which, in solid-phase enzyme immunoassay, were reactive with DNA. One mAb was of special interest because it reacted strongly with both single-stranded DNA and an extractable nuclear antigen found in rabbit thymus extract (RTE). In an immunofluorescent assay using fixed human cells, the latter mAb also bound predominantly to cell nucleoli. A combination of enzyme digestion and metabolic inhibitor studies of the target cells in this immunofluorescent assay suggested that the antigen(s) bound by the mAb was an RNA-associated protein or a ribonucleoprotein that is distinct from intact RNA polymerase I and not associated with the transcriptional units of the nucleolus. In other experiments, using fractions of RTE isolated by ion-exchange chromatography, the antigens bound by the mAb were shown to be highly negatively charged molecules. Immunoprecipitation and SDS-PAGE analyses of labeled cell extracts bound by the mAb revealed a doublet of 17 and 18 kD. Since the original patient's serum autoantibodies also bound to both an RNase-sensitive, acidic, extractable nuclear antigen and to nucleoli, and immunoprecipitated proteins of similar molecular masses in SDS-PAGE, it appears that the described mAb is a product of an immortalized autoantibody-producing B cell clone from the SLE patient's peripheral blood. This mAb probably defines a novel RNA-associated autoantigen residing predominantly in the nucleolus or, less likely, a variant of either RNA polymerase I or the ribosomal autoantigens (P proteins). PMID:2435834

  9. Monoclonal antibody to a subset of human monocytes found only in the peripheral blood and inflammatory tissues

    SciTech Connect

    Zwadlo, G.; Schlegel, R.; Sorg, C.

    1986-07-15

    A monoclonal antibody is described that was generated by immunizing mice with cultured human blood monocytes. The antibody (27E10) belongs to the IgG1 subclass and detects a surface antigen at M/sub r/ 17,000 that is found on 20% of peripheral blood monocytes. The antigen is increasingly expressed upon culture of monocytes, reaching a maximum between days 2 and 3. Stimulation of monocytes with interferon-..gamma.. (IFN-..gamma..), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and lipopolysaccharide (LPS) Ylalanine (fMLP) increased the 27E10 antigen density. The amount of 27E10-positive cells is not or is only weakly affected. The antigen is absent from platelets, lymphotyces, and all tested human cell lines, yet it cross-reacts with 15% of freshly isolated granulocytes. By using the indirect immunoperoxidase technique, the antibody is found to be negative on cryostat sections of normal human tissue (skin, lung, and colon) and positive on only a few monocyte-like cells in liver and on part of the cells of the splenic red pulp. In inflammatory tissue, however, the antibody is positive on monocytes/macrophages and sometimes on endothelial cells and epidermal cells, depending on the stage and type of inflammation, e.g., BCG ranulomas are negative, whereas psoriasis vulgaris, atopic dermatitis, erythrodermia, pressure urticaria, and periodontitis contain positively staining cells. In contact eczemas at different times after elicitation (6 hr, 24 hr, and 72 hr), the 27E10 antigen is seen first after 24 hr on a few infiltrating monocytes/macrophages, which increase in numbers after 72 hr.

  10. Production and Characterization of Mouse Monoclonal Antibodies Recognizing Human Pan-IgG Specific Conformational or Linear Epitopes

    PubMed Central

    Hajighasemi, Fatemeh; Khoshnoodi, Jalal; Shokri, Fazel

    2012-01-01

    Background Pan-IgG specific monoclonal antibodies (MAbs) are essential tools for assessment of humoral immunity, immune deficiency and gammopathy. In this study, four hybridoma clones producing MAbs with different specificities for human IgG isotypes were established. Methods Splenocytes from Balb/c mice immunized with Fc fractions of human IgG were fused with SP2/0 myeloma cells. Hybridoma cells were selected in HAT selective medium and cloned by limiting dilution assay. Antibody-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed using a panel of purified myeloma IgG proteins by ELISA and immunoblotting. Cross-reactivity to immunoglobulins (Igs) of other species was studied by indirect ELISA using serum samples collected from 9 animals. Results Immunoblotting studies revealed recognition of either linear or conformational epitopes by these MAbs. The most abundant cross-reactivity (100%) was observed with monkey Igs, while no cross-reactivity was detected with rabbit, guinea pig, sheep, goat, cat and hen sera. Two of the MAbs cross-reacted with either dog or horse sera. The affinity constant of two MAbs was measured by ELISA and found to be 0.74×108M−1 and 0.96×107M−1. Conclusion Our results indicate that these pan-IgG specific MAbs recognize restricted linear or conformational epitopes located on all human IgG subclasses with no cross-reactivity to IgG from most species studied. These MAbs are potentially useful tools for quantification of total or antigen-specific IgG levels. PMID:23408136

  11. Colocalization of elastase and myeloperoxidase in human blood and bone marrow neutrophils using a monoclonal antibody and immunogold.

    PubMed Central

    Cramer, E. M.; Beesley, J. E.; Pulford, K. A.; Breton-Gorius, J.; Mason, D. Y.

    1989-01-01

    The authors have localized elastase in human blood and bone marrow neutrophils by immunoelectron microscopy using a monoclonal anti-human elastase antibody (NP 57) and compared its distribution with myeloperoxidase (MPO) and lactoferrin (LF), which mark primary and secondary neutrophil granule, respectively. Human bone marrow and blood polymorphonuclear leukocytes (PMN), either unstimulated or after phagocytosis of latex microbeads, were fixed in 4% paraformaldehyde. Ultrathin frozen sections were immunolabeled with NP 57, followed by an immunogold probe. In bone marrow granulocyte precursors elastase appeared simultaneously in the immature first granules of myeloblasts with MPO. As these granules became denser with maturation, labeling for both enzymes became weaker and sometimes negative (possibly due to masking of immunoreactivity). The ellipsoidal primary granules were strongly labeled by NP57. LF positive granules appeared later, at the myelocyte stage, and contained neither MPO nor elastase. In mature neutrophils, immunolabeling for elastase was found together with MPO in the large electron-dense primary granules and in a different granule population from the LF-positive secondary granules. Double labeling with two different-sized gold particles was used to compare the kinetics of degranulation of secondary and primary granules. The observation and the analysis of single phagosome content was made possible by this new technique. In conclusion, immunoelectron microscopy was used to show elastase in the primary granules of neutrophils, where it appears simultaneously with MPO. This technique has also allowed comparison of the kinetics of degranulation of both types of granules, and could be applied to different experimental and pathologic conditions. Images Figure 2 Figure 1 Figure 3 Figure 4 Figure 5 PMID:2547320

  12. A human monoclonal antibody targeting the conserved staphylococcal antigen IsaA protects mice against Staphylococcus aureus bacteremia.

    PubMed

    van den Berg, Sanne; Bonarius, Hendrik P J; van Kessel, Kok P M; Elsinga, Goffe S; Kooi, Neeltje; Westra, Hans; Bosma, Tjibbe; van der Kooi-Pol, Magdalena M; Koedijk, Danny G A M; Groen, Herman; van Dijl, Jan Maarten; Buist, Girbe; Bakker-Woudenberg, Irma A J M

    2015-01-01

    Due to substantial therapy failure and the emergence of antibiotic-resistant Staphylococcus aureus strains, alternatives for antibiotic treatment of S. aureus infections are urgently needed. Passive immunization using S. aureus-specific monoclonal antibodies (mAb) could be such an alternative to prevent and treat severe S. aureus infections. The invariantly expressed immunodominant staphylococcal antigen A (IsaA) is a promising target for passive immunization. Here we report the development of the human anti-IsaA IgG1 mAb 1D9, which was shown to bind to all 26 S. aureus isolates tested. These included both methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA, respectively). Immune complexes consisting of IsaA and 1D9 stimulated human as well as murine neutrophils to generate an oxidative burst. In a murine bacteremia model, the prophylactic treatment with a single dose of 5 mg/kg 1D9 improved the survival of mice challenged with S. aureus isolate P (MSSA) significantly, while therapeutic treatment with the same dose did not influence animal survival. Neither prophylactic nor therapeutic treatment with 5 mg/kg 1D9 resulted in improved survival of mice with S. aureus USA300 (MRSA) bacteremia. Importantly, our studies show that healthy S. aureus carriers elicit an immune response which is sufficient to generate protective mAbs against invariant staphylococcal surface antigens. Human mAb 1D9, possibly conjugated to for example another antibody, antibiotics, cytokines or chemokines, may be valuable to fight S. aureus infections in patients.

  13. Targeted cancer immunotherapy with oncolytic adenovirus coding for a fully human monoclonal antibody specific for CTLA-4.

    PubMed

    Dias, J D; Hemminki, O; Diaconu, I; Hirvinen, M; Bonetti, A; Guse, K; Escutenaire, S; Kanerva, A; Pesonen, S; Löskog, A; Cerullo, V; Hemminki, A

    2012-10-01

    Promising clinical results have been achieved with monoclonal antibodies (mAbs) such as ipilimumab and tremelimumab that block cytotoxic T lymphocyte-associated antigen-4 (CTLA-4, CD152). However, systemic administration of these agents also has the potential for severe immune-related adverse events. Thus, local production might allow higher concentrations at the target while reducing systemic side effects. We generated a transductionally and transcriptionally targeted oncolytic adenovirus Ad5/3-Δ24aCTLA4 expressing complete human mAb specific for CTLA-4 and tested it in vitro, in vivo and in peripheral blood mononuclear cells (PBMCs) of normal donors and patients with advanced solid tumors. mAb expression was confirmed by western blotting and immunohistochemistry. Biological functionality was determined in a T-cell line and in PBMCs from cancer patients. T cells of patients, but not those of healthy donors, were activated by an anti-CTLA4mAb produced by Ad5/3-Δ24aCTLA4. In addition to immunological effects, a direct anti-CTLA-4-mediated pro-apoptotic effect was observed in vitro and in vivo. Local production resulted in 43-fold higher (P<0.05) tumor versus plasma anti-CTLA4mAb concentration. Plasma levels in mice remained below what has been reported safe in humans. Replication-competent Ad5/3-Δ24aCTLA4 resulted in 81-fold higher (P<0.05) tumor mAb levels as compared with a replication-deficient control. This is the first report of an oncolytic adenovirus producing a full-length human mAb. High mAb concentrations were seen at tumors with lower systemic levels. Stimulation of T cells of cancer patients by Ad5/3-Δ24aCTLA4 suggests feasibility of testing the approach in clinical trials.

  14. Immunoscintigraphy of human pancreatic carcinoma in nude mice with I-131-F(ab')/sub 2/-fragments of monoclonal antibodies

    SciTech Connect

    Senekowitsch, R.; Maul, F.D.; Wenisch, H.J.C.; Kriegel, H.; Hor, G.

    1985-05-01

    In the present study radioiodinated F(ab')/sub 2/-fragments of CA19-9 and antibody that reacts specifically with human gastrointestinal cancer were examined for their ability to detect human pancreatic carcinoma hosted in nude mice. Tumor-bearing mice received 80..mu..Ci of I-131-F(ab')/sub 2/ with a specific activity of 1.8..mu..Ci/..mu..g. All mice were imaged after the injection and every 24hr up to 6 days. The retained radioactivity was also registered with a whole-body counter immediately after imaging. As a control F(ab's)/sub 2/ of a nonspecific antibody were administered in parallel to another group of animals bearing the same tumor. Three animals of each group were killed at 1,2,4 and 8 days for determination of the distribution of both labeled antibody-fragments. On scintigraphic images obtained with the CA19-9-F(ab')/sub 2/ the tumors could be visualized 24hr after injection, the best dilineation however was achieved 96hr p.i.. The biodistribution data exhibited a more rapid blood clearance for the specific fragments compared to that for the unspecific ones. Tumors showed an increase in uptake up to 48hr reaching 1.7% of the injected dose per gram, declining to values of 0.08%/g at day 6 p.i.. The highest tumor-to-blood ratios were found after 96h. They were 7 for the CA19-9-fragments compared to 1.5 for the unspecific fragments. The whole body counting revealed a more rapid excretion for the fragments of the specific monoclonal antibodies than for the unspecific ones. In summary the authors were able to show that CA19-9-F(ab')/sub 2/-fragments can be used for immunodetection of human pancreatic carcinoma hosted in nude mice.

  15. FG-3019, a Human Monoclonal Antibody Recognizing Connective Tissue Growth Factor, is Subject to Target-Mediated Drug Disposition.

    PubMed

    Brenner, Mitchell C; Krzyzanski, Wojciech; Chou, James Z; Signore, Pierre E; Fung, Cyra K; Guzman, David; Li, Dongxia; Zhang, Weihua; Olsen, David R; Nguyen, Viet-Tam L; Koo, Carolyn W; Sternlicht, Mark D; Lipson, Kenneth E

    2016-08-01

    To evaluate and model the pharmacokinetic and pharmacodynamic behavior in rats of FG-3019, a human monoclonal antibody targeting connective tissue growth factor (CTGF). FG-3019, human CTGF (rhCTGF), or the N-terminal domain of rhCTGF were administered intravenously to rats and concentrations of these proteins as well as endogenous CTGF were determined by immunoassays. FG-3019, or (125)I-labeled FG-3019, and human CTGF (rhCTGF) were co-administered to assess the impact of CTGF on the elimination rate and tissue localization of FG-3019, which was further characterized by immunohistochemical analysis. A PK/PD model for target-mediated elimination of FG-3019 was developed to fit the kinetic data. FG-3019 exhibited non-linear pharmacokinetics in rats. Circulating concentrations of the N-terminal half of CTGF increased after dosing with FG-3019, reached maximal levels after 1-5 days, and returned toward baseline levels as FG-3019 cleared from the circulation, whereas the concentration of intact CTGF was unaffected by administration of FG-3019. Co-administration of rhCTGF dramatically enhanced the rate of FG-3019 elimination, redistributing the majority of (125)I-labeled FG-3019 from the blood to the liver, kidney, spleen and adrenal gland. FG-3019 co-administered with CTGF was found along the sinusoids of the liver and adrenal glands, the capillaries of the kidney glomeruli and in the spleen. A pharmacokinetic model for target-mediated elimination of FG-3019 was used to fit the time courses of FG-3019 and endogenous CTGF plasma concentrations, as well as time courses of rhCTGF and rhCTGF N-fragment after intravenous administration of these species. FG-3019 is subject to target mediated elimination in rats.

  16. Characterization of two distinct antigens expressed on either resting or activated human B cells as defined by monoclonal antibodies.

    PubMed Central

    Kokai, Y; Ishii, Y; Kikuchi, K

    1986-01-01

    Two antigen systems (L29 & L30) expressed on two distinct human B cell subpopulations were identified by using BL1-4D6 and TB3-7D5 monoclonal antibodies, respectively. L29 was expressed on approximately one-third of B cells in human lymphoid tissues. These B cells associated with L29 were large activated B cells located in the germinal centres of lymphoid follicles. L30, on the other hand, existed on approximately two-thirds of B cells mainly located in the mantle zone of lymphoid follicles, most of which also expressed IgM and IgD on their cell membrane. In addition, L30 was shared on mature granulocytes. With the use of polyclonal activators such as pokeweek mitogen (PWM) and protein A-bearing staphylococci (SAC), L29 antigen was inducible on PWM- or SAC-stimulated B cells in correspondence with the emergence of Tac and T10 antigens of these B cells. In contrast, L30 antigen on the B cells stimulated by the polyclonal activators was decreased in its expression and was finally lost from these B cells. Although none of L29 and L30 was expressed on normal, non-activated human thymus and peripheral T cells, L29 but not L30 was expressed on concanavalin A-activated T cells. Immunochemical studies showed that L30 consist of a single polypeptide with mol. wt of 40,000. L29 antigen is presently under study. Images Fig. 2 Fig. 4 PMID:3527505

  17. Structural, functional, and tissue distribution analysis of human transferrin receptor-2 by murine monoclonal antibodies and a polyclonal antiserum.

    PubMed

    Deaglio, Silvia; Capobianco, Andrea; Calì, Angelita; Bellora, Francesca; Alberti, Federica; Righi, Luisella; Sapino, Anna; Camaschella, Clara; Malavasi, Fabio

    2002-11-15

    Human transferrin receptor-2 (TFR-2) is a protein highly homologous to TFR-1/CD71 and is endowed with the ability to bind transferrin (TF) with low affinity. High levels of TFR-2 mRNA were found in the liver and in erythroid precursors. Mutations affecting the TFR-2 gene led to hemochromatosis type 3, a form of inherited iron overload. Several issues on distribution and function of the receptor were answered by raising a panel of 9 monoclonal antibodies specific for TFR-2 by immunizing mice with murine fibroblasts transfected with the human TFR-2 cDNA. A polyclonal antiserum was also produced in mice immunized with 3 peptides derived from the TFR-2 sequence, exploiting an innovative technique. The specificity of all the reagents produced was confirmed by reactivity with TFR-2(+) target cells and simultaneous negativity with TFR-1(+) cells. Western blot analyses showed a dominant chain of approximately 90 kDa in TFR-2 transfectants and HepG2 cell line. Analysis of distribution in normal tissues and in representative cell lines revealed that TFR-2 displays a restricted expression pattern--it is present at high levels in hepatocytes and in the epithelial cells of the small intestine, including the duodenal crypts. Exposure of human TFR-2(+) cells to TF-bound iron is followed by a significant up-regulation and relocalization of membrane TFR-2. The tissue distribution pattern, the behavior following exposure to iron-loaded TF, and the features of the disease resulting from TFR-2 inactivation support the hypothesis that TFR-2 contributes to body iron sensing.

  18. A Recombinant Humanized Anti-Cocaine Monoclonal Antibody Inhibits the Distribution of Cocaine to the Brain in Rats

    PubMed Central

    Gooden, Felicia C. T.; Tabet, Michael R.; Ball, William J.

    2014-01-01

    The monoclonal antibody (mAb), h2E2, is a humanized version of the chimeric human/murine anti-cocaine mAb 2E2. The recombinant h2E2 protein was produced in vitro from a transfected mammalian cell line and retained high affinity (4 nM Kd) and specificity for cocaine over its inactive metabolites benzoylecgonine (BE) and ecgonine methyl ester. In rats, pharmacokinetic studies of h2E2 (120 mg/kg i.v.) showed a long terminal elimination half-life of 9.0 days and a low volume of distribution at steady state (Vdss) of 0.3 l/kg. Pretreatment with h2E2 produced a dramatic 8.8-fold increase in the area under the plasma cocaine concentration-time curve (AUC) and in brain a concomitant decrease of 68% of cocaine’s AUC following an i.v. injection of an equimolar cocaine dose. Sequestration of cocaine in plasma by h2E2, shown via reduction of cocaine’s Vdss, indicates potential clinical efficacy. Although the binding of cocaine to h2E2 in plasma should inhibit distribution and metabolism, the elimination of cocaine remained multicompartmental and was still rapidly eliminated from plasma despite the presence of h2E2. BE was the major cocaine metabolite, and brain BE concentrations were sixfold higher than in plasma, indicating that cocaine is normally metabolized in the brain. In the presence of h2E2, brain BE concentrations were decreased and plasma BE was increased, consistent with the observed h2E2-induced changes in cocaine disposition. The inhibition of cocaine distribution to the brain confirms the humanized mAb, h2E2, as a lead candidate for development as an immunotherapy for cocaine abuse. PMID:24733787

  19. Human epidermal growth factor receptor 2 positive (HER2+) metastatic breast cancer: how the latest results are improving therapeutic options.

    PubMed

    Jiang, Hanfang; Rugo, Hope S

    2015-11-01

    Human epidermal growth factor receptor 2 positive (HER2+) metastatic breast cancer (MBC) remains an incurable disease, and approximately 25% of patients with HER2+ early breast cancer still relapse after adjuvant trastuzumab-based treatment. HER2 is a validated therapeutic target that remains relevant throughout the disease process. Recently, a number of novel HER2 targeted agents have become available, including lapatinib (a small molecule tyrosine kinase inhibitor of both HER2 and the epidermal growth factor receptor), pertuzumab (a new anti-HER2 monoclonal antibody) and ado-trastuzumab emtansine (T-DM1, a novel antibody-drug conjugate), which provide additional treatment options for patients with HER2+ MBC. The latest clinical trials have demonstrated improved outcome with treatment including pertuzumab or T-DM1 compared with standard HER2 targeted therapy. Here we review the clinical development of approved and investigational targeted agents for the treatment of HER2+ MBC, summarize the latest results of important clinical trials supporting use of these agents in the treatment of HER2+ MBC, and discuss how these results impact therapeutic options in clinical practice.

  20. Generation and characterization of a panel of monoclonal antibodies specific for human fibroblast growth factor receptor 4 (FGFR4).

    PubMed

    Chen, Chaoyuan; Patel, Sima; Corisdeo, Susanne; Liu, Xiangdong; Micolochick, Holly; Xue, Jiyang; Yang, Qifeng; Lei, Ying; Wang, Baiyang; Soltis, Daniel

    2005-06-01

    Fibroblast growth factor receptor 4 (FGFR4) is a member of the FGFR family of receptor tyrosine kinases, and plays important roles in a variety of biological functions such as cell proliferation, differentiation, migration, angiogenesis, tissue repair, and tumorigenesis. The human FGFRs share a high degree of sequence homology between themselves, as well as with their murine homologs. Consequently, it has been suggested that it may be difficult to prepare monoclonal antibodies (MAbs) that are specific for the individual receptor types. In this communication, we report on the development and characterization of a panel of anti-human FGFR4 MAbs that were generated in mice using a rapid immunization protocol. Using a modified rapid immunization at multiple sites (RIMMS) protocol with the soluble extracellular domain of human FGFR4 (FGFR4-ECD), the immunized mice developed high levels of polyclonal IgG to the immunogen within 13 days of the first immunization. The lymph node cells isolated from the immunized animals were then fused with mouse myeloma cells for hybridoma generation. Use of an efficient hybridoma cloning protocol in combination with an ELISA screening procedure allowed for early identification of stable hybridomas secreting antihuman FGFR4 IgG. Several identified MAbs specifically reacted with the FGFR4 protein without binding to the other human isoforms (FGFR1, FGFR2, and FGFR3). As evaluated by BIAcore analysis, most anti-FGFR4 MAbs displayed high affinities (8.6 x 10(8) approximately 3.9 x 10(10) M) to FGFR4. Furthermore, these MAbs were able to bind to FGFR4 expressed on human breast tumor cell lines MDA-MB-361 and MDA-MB-453. Taken together, the results demonstrate that the RIMMS strategy is an effective approach for generating class-switched, high-affinity MAbs in mice to evolutionarily conserved proteins such as human FGFR4. These MAbs may be useful tools for further investigation of the biological functions and pathological roles of human FGFR4.

  1. Human Monoclonal Antibody Fragments Binding to Insulin-like Growth Factors 1 and 2 with Picomolar Affinity

    PubMed Central

    Zhao, Qi; Feng, Yang; Zhu, Zhongyu; Dimitrov, Dimiter S.

    2011-01-01

    The type 1 insulin-like growth factor receptor (IGF1R) and its ligands (IGF1 and IGF2) have been implicated in a variety of physiological processes and in diseases such as cancer. In addition to IGF1R, IGF2 also activates the insulin receptor (IR) isoform A and therefore antibodies against IGF2 can inhibit cell proliferation mediated by the signaling through both IGF1R and IR triggered by IGF2. We identified a new human monoclonal antibody (mAb), m708.2, which bound to IGF1 and IGF2 but not to insulin. m708.2 potently inhibited signal transduction mediated by the interaction of IGF1 or IGF2 with the IGF1R and IGF2 with the IR. It also inhibited the growth of the breast cancer cell line MCF-7. An affinity-matured derivative of m708.2, m708.5, bound to IGF1 with equilibrium dissociation constant, KD = 200 pM and to IGF2 with KD = 60 pM. m708.5 inhibited signal transduction mediated by IGF1 and IGF2 and cancer cell growth more potently than m708.2. These results suggest that m708.5 could have potential as a candidate therapeutic for cancers driven by the IGF1,2 interactions with IGF1R and IR. PMID:21750218

  2. Ligation of human Fc receptor like-2 by monoclonal antibodies down-regulates B-cell receptor-mediated signalling

    PubMed Central

    Shabani, Mahdi; Bayat, Ali Ahmad; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Hojjat-Farsangi, Mohammad; Ulivieri, Cristina; Amirghofran, Zahra; Baldari, Cosima Tatiana; Shokri, Fazel

    2014-01-01

    B-cell antigen receptor (BCR) signalling and its regulation through negative and positive regulators are critical for balancing B-cell response and function. Human Fc receptor like-2 (FCRL2), a member of the newly identified FCRL family, could influence B-cell signalling due to possession of both immunoreceptor tyrosine-based activation and inhibitory motifs (ITAM and ITIM). Since the natural ligand of FCRL2 has not been identified, we generated FCRL2-specific monoclonal antibodies (mAbs) and employed them to investigate the influence of FCRL2 stimulation on BCR signalling in an FCRL2-expressing B-cell line. Two anti-FCRL2 mAb-producing hybridoma clones (5A7-E7 and 3D8-G8) were selected. None of the mAbs displayed any cross-reactivity with the other members of the FCRL family including recombinant FCRL1, -3, -4 and -5, as tested by FACS and ELISA techniques. Engagement of the FCRL2 by these mAbs resulted in significant inhibition of BCR signalling mediators such as calcium mobilization and phosphorylation of the mitogen-activated protein kinases Erk, p38 and Jnk. These findings indicate that the FCRL2 ITIM motifs are functional and the anti-FCRL2 mAbs may mimic the natural ligand of FCRL2 by induction of inhibitory signals in B cells. PMID:24797767

  3. Monoclonal Antibody against Angiotensin-Converting Enzyme: Its Use as a Marker for Murine, Bovine, and Human Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Auerbach, R.; Alby, L.; Grieves, J.; Joseph, J.; Lindgren, C.; Morrissey, L. W.; Sidky, Y. A.; Tu, M.; Watt, S. L.

    1982-12-01

    A monoclonal antibody has been prepared against rat angiotensin-converting enzyme (ACE). By selection for antibody binding to endothelial cells of bovine rather than rat origin we have obtained a reagent that has broad cross-species binding properties and that can at the same time serve as a useful marker for the surface of endothelial cells. The IgM-producing clone that we have established, α -ACE 3.1.1, has been grown in ascites form to yield ascites fluid that binds selectively to immobilized ACE at a >1:10,000 dilution. By use of enzyme-linked immunosorbent assays, immunofluorescence histology, and flow cytometry, we have demonstrated the presence of ACE on endothelial cells of murine, bovine, and human origin. By means of a fluorescence-activated cell sorter (FACS-IV) we have been able to selectively isolate viable endothelial cells from a mixture of endothelial cells and fibroblasts. We believe the antibody will be useful not only for the selection and in vitro cultivation of endothelial cells but also as a tool for the identification and pharmacological study of ACE.

  4. A procedure for the rapid purification in high yield of human glucocerebrosidase using immunoaffinity chromatography with monoclonal antibodies.

    PubMed

    Aerts, J M; Donker-Koopman, W E; Murray, G J; Barranger, J A; Tager, J M; Schram, A W

    1986-05-01

    A novel chromatographic immunoaffinity procedure is described for the purification of Form I glucocerebrosidase (see J. M. F. G. Aerts, W. E. Donker-Koopman, M. K. Van der Vliet, L. M. V. Jonsson, E. I. Ginns, G. J. Murray, J. A. Barranger, J. M. Tager, and A. W. Schram, 1985, Eur. J. Biochem. 150, 565-574) from extracts of human tissues. The affinity support consists of two monoclonal anti-(glucocerebrosidase) antibodies immobilized by covalent coupling to CNBr-activated Sepharose 4B. After adsorption of the enzyme from a crude detergent extract, the column is washed successively with 30% ethylene glycol in citrate buffer (pH 6), 1% Triton X-100 in citrate phosphate buffer (pH 5.2), and 50% ethylene glycol in citrate buffer. The enzyme is eluted with 90% ethylene glycol in citrate buffer. After dilution to 30% ethylene glycol, the immunoaffinity purification is repeated. The procedure can be completed within less than 18 h. The final preparations have a high specific activity (50 U/mg protein (n = 4) for the placental enzyme) and contain no detectable impurities after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The yield is high (81 +/- 8% for the placental enzyme). The immunoaffinity column has a high capacity, can be regenerated easily, and can be utilized repeatedly without loss of activity.