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Sample records for bispecific antibody pretargeting

  1. Imaging small human prostate cancer xenografts after pretargeting with bispecific bombesin-antibody complexes and targeting with high specific radioactivity labeled polymer-drug conjugates

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    Patil, Vishwesh; Gada, Keyur; Panwar, Rajiv; Ferris, Craig; Khaw, Ban-An [Northeastern University, School of Pharmacy, Department of Pharmaceutical Sciences, Boston, MA (United States); Varvarigou, Alexandra [Institute of Radioisotopes and Radiodiagnostics, National Centre for Scientific Research ' ' Demokritos' ' , Athens (Greece); Majewski, Stan [West Virginia University, Department of Radiology, Nuclear Medicine Imaging Instrumentation Program, Center for Advanced Imaging, Morgantown, WV (United States); Weisenberger, Andrew [Jefferson LA, Thomas Jefferson National Accelerator Facility, Newport News, VA (United States); Tekabe, Yared [Columbia University Medical Center, New York, NY (United States)

    2012-05-15

    Pretargeting with bispecific monoclonal antibodies (bsMAb) for tumor imaging was developed to enhance target to background activity ratios. Visualization of tumors was achieved by the delivery of mono- and divalent radiolabeled haptens. To improve the ability to image tumors with bsMAb, we have combined the pretargeting approach with targeting of high specific activity radiotracer labeled negatively charged polymers. The tumor antigen-specific antibody was replaced with bombesin (Bom), a ligand that binds specifically to the growth receptors that are overexpressed by many tumors including prostate cancer. Bom-anti-diethylenetriaminepentaacetic acid (DTPA) bispecific antibody complexes were used to demonstrate pretargeting and imaging of very small human prostate cancer xenografts targeted with high specific activity {sup 111}In- or {sup 99m}Tc-labeled negatively charged polymers. Bispecific antibody complexes consisting of intact anti-DTPA antibody or Fab' linked to Bom via thioether bonds (Bom-bsCx or Bom-bsFCx, respectively) were used to pretarget PC-3 human prostate cancer xenografts in SCID mice. Negative control mice were pretargeted with Bom or anti-DTPA Ab. {sup 111}In-Labeled DTPA-succinyl polylysine (DSPL) was injected intravenously at 24 h (7.03 {+-} 1.74 or 6.88 {+-} 1.89 MBq {sup 111}In-DSPL) after Bom-bsCx or 50 {+-} 5.34 MBq of {sup 99m}Tc-DSPL after Bom-bsFCx pretargeting, respectively. Planar or single photon emission computed tomography (SPECT)/CT gamma images were obtained for up to 3 h and only planar images at 24 h. After imaging, all mice were killed and biodistribution of {sup 111}In or {sup 99m}Tc activities were determined by scintillation counting. Both planar and SPECT/CT imaging enabled detection of PC-3 prostate cancer lesions less than 1-2 mm in diameter in 1-3 h post {sup 111}In-DSPL injection. No lesions were visualized in Bom or anti-DTPA Ab pretargeted controls. {sup 111}In-DSPL activity in Bom-bsCx pretargeted tumors (1

  2. Comparison of IgG and F(ab'){sub 2} fragments of bispecific anti-RCC x anti-DTIn-1 antibody for pretargeting purposes

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    Schaijk, Frank G. van; Boerman, Otto C.; Soede, Annemieke C.; Corstens, Frans H.M. [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, P.O. Box 9101, Nijmegen (Netherlands); McBride, William J. [Immunomedics, Inc., Morris Plains, NJ (United States); Goldenberg, David M. [Immunomedics, Inc., Morris Plains, NJ (United States); Center for Molecular Medicine and Immunology, Garden State Cancer Center, Belleville, NJ (United States); Oosterwijk, Egbert [Radboud University Nijmegen Medical Center, Department of Urology, Nijmegen (Netherlands); Ludwig Institute for Cancer Research, New York, NY (United States)

    2005-09-01

    An effective pretargeting strategy was developed for renal cell carcinoma (RCC) based on a biologically produced bispecific monoclonal antibody: anti-RCC x anti-DTPA(In) (bsMAb: G250 x DTIn-1). Tumour uptake of a {sup 111}In-labelled bivalent peptide after pretargeting with bsMAb G250 x DTIn-1 was relatively high compared with that in other pretargeting systems using chemically coupled F(ab'){sub 2} fragments. Here, we investigated the effect of the bsMAb form in the pretargeting strategy. To determine the optimal interval between the administration of each of the bsMAb forms and the {sup 111}In-labelled bivalent peptide, the biodistribution of the radioiodinated bsMAb forms was studied in athymic mice with subcutaneous SK-RC-1 RCC tumours. Since tumour targeting of the radiolabelled peptide depends on the bsMAb form and dose, a bsMAb dose escalation study was carried out for both bsMAb forms. Under optimised conditions, the biodistribution of the {sup 111}In label in mice with pretargeted RCC was determined from 4 h up to 7 days p.i. The optimal interval between the two administrations was 72 h for the bsMAb IgG and 4 h for the bsMAb F(ab'){sub 2}. The optimal bsMAb dose for intact IgG was 67 pmol and the optimal bsMAb F(ab'){sub 2} dose was 200 pmol. Targeting of the pretargeted RCC with 4 pmol {sup 111}In-labelled bivalent peptide revealed high tumour uptake with both bsMAb forms. With the pretargeting strategy, using either bsMAb IgG or bsMAb F(ab'){sub 2}, very efficient peptide targeting of the tumour was obtained. Uptake and retention of the radiolabel in the tumour with the pretargeting approach are not affected by the bsMAb form used. (orig.)

  3. Bispecific antibody complex pre-targeting and targeted delivery of polymer drug conjugates for imaging and therapy in dual human mammary cancer xenografts. Targeted polymer drug conjugates for cancer diagnosis and therapy

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    Khaw, Ban-An; Gada, Keyur S.; Patil, Vishwesh; Panwar, Rajiv; Mandapati, Savitri [Northeastern University, Department of Pharmaceutical Sciences, Bouve College of Health Sciences, School of Pharmacy, Boston, MA (United States); Hatefi, Arash [Rutgers University, Department of Pharmaceutics, New Brunswick, NJ (United States); Majewski, Stan [West Virginia University, Department of Radiology, Morgantown, WV (United States); Weisenberger, Andrew [Thomas Jefferson National Accelerator Facility, Jefferson Lab, Newport News, VA (United States)

    2014-08-15

    Doxorubicin, a frontline chemotherapeutic agent, limited by its cardiotoxicity and other tissue toxicities, was conjugated to N-terminal DTPA-modified polyglutamic acid (D-Dox-PGA) to produce polymer pro-drug conjugates. D-Dox-PGA or Tc-99 m labeled DTPA-succinyl-polylysine polymers (DSPL) were targeted to HER2-positive human mammary carcinoma (BT-474) in a double xenografted SCID mouse model also hosting HER2-negative human mammary carcinoma (BT-20). After pretargeting with bispecific anti-HER2-affibody-anti-DTPA-Fab complexes (BAAC), anti-DTPA-Fab or only phosphate buffered saline, D-Dox-PGA or Tc-99 m DSPL were administered. Positive therapeutic control mice were injected with Dox alone at maximum tolerated dose (MTD). Only BT-474 lesions were visualized by gamma imaging with Tc-99 m-DSPL; BT-20 lesions were not. Therapeutic efficacy was equivalent in mice pretargeted with BAAC/targeted with D-Dox-PGA to mice treated only with doxorubicin. There was no total body weight (TBW) loss at three times the doxorubicin equivalent MTD with D-Dox-PGA, whereas mice treated with doxorubicin lost 10 % of TBW at 2 weeks and 16 % after the second MTD injection leading to death of all mice. Our cancer imaging and pretargeted therapeutic approaches are highly target specific, delivering very high specific activity reagents that may result in the development of a novel theranostic application. HER/2 neu specific affibody-anti-DTPA-Fab bispecific antibody pretargeting of HER2 positive human mammary xenografts enabled exquisite targeting of polymers loaded with radioisotopes for molecular imaging and doxorubicin for effective therapy without the associating non-tumor normal tissue toxicities. (orig.)

  4. Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

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    Orcutt, Kelly Davis; Slusarczyk, Adrian L. [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Cieslewicz, Maryelise [Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Ruiz-Yi, Benjamin [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Bhushan, Kumar R. [Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Frangioni, John V. [Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Department of Radiology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Wittrup, K. Dane, E-mail: wittrup@mit.ed [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States)

    2011-02-15

    Introduction: In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelates for use in PRIT applications. Methods: We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to DOTA, reformatted as a single chain variable fragment (scFv). Results: Modeling predicts that for high antigen density and saturating bsAb dose, a hapten-binding affinity of 100 pM is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nM to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2{+-}1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen, pretargeted high-affinity scFv results in significantly higher tumor retention of a {sup 111}In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions: We have engineered a versatile, high-affinity, DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals.

  5. Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

    International Nuclear Information System (INIS)

    Introduction: In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelates for use in PRIT applications. Methods: We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to DOTA, reformatted as a single chain variable fragment (scFv). Results: Modeling predicts that for high antigen density and saturating bsAb dose, a hapten-binding affinity of 100 pM is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nM to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2±1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen, pretargeted high-affinity scFv results in significantly higher tumor retention of a 111In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions: We have engineered a versatile, high-affinity, DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals.

  6. Taking up Cancer Immunotherapy Challenges: Bispecific Antibodies, the Path Forward?

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    Joanie Del Bano

    2015-12-01

    Full Text Available As evidenced by the recent approvals of Removab (EU, Trion Pharma in 2009 and of Blincyto (US, Amgen in 2014, the high potential of bispecific antibodies in the field of immuno-oncology is eliciting a renewed interest from pharmaceutical companies. Supported by rapid advances in antibody engineering and the development of several technological platforms such as Triomab or bispecific T cell engagers (BiTEs, the “bispecifics” market has increased significantly over the past decade and may occupy a pivotal space in the future. Over 30 bispecific molecules are currently in different stages of clinical trials and more than 70 in preclinical phase. This review focuses on the clinical potential of bispecific antibodies as immune effector cell engagers in the onco-immunotherapy field. We summarize current strategies targeting various immune cells and their clinical interests. Furthermore, perspectives of bispecific antibodies in future clinical developments are addressed.

  7. Bispecific antibodies and their use in applied research

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    Harshit Verma

    Full Text Available Bispecific antibodies (BsAb can, by virtue of combining two binding specificities, improve the selectivity and efficacy of antibody-based treatment of human disease. Antibodies with two distinct binding specificities have great potential for a wide range of clinical applications as targeting agents for in vitro and in vivo immunodiagnosis, therapy and for improving immunoassays. They have shown great promise for targeting cytotoxic effector cells, delivering radionuclides, toxins or cytotoxic drugs to specific targets, particularly tumour cells. The development of BsAb research goes through three main stages: chemical cross linking of murine-derived monoclonal antibody, hybrid hybridomas and engineered BsAb. This article is providing the potential applications of bispecific antibodies. [Vet World 2012; 5(12.000: 775-780

  8. A pretargeting system for tumor PET imaging and radioimmunotherapy

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    Françoise eKraeber-Bodéré

    2015-03-01

    Full Text Available Labeled antibodies, as well as their fragments and antibody-derived recombinant constructs, have long been proposed as general vectors to target radionuclides to tumor lesions for imaging and therapy. They have indeed shown promise in both imaging and therapeutic applications, but they have not fulfilled the original expectations of achieving sufficient image contrast for tumor detection or sufficient radiation dose delivered to tumors for therapy. Pretargeting was originally developed for tumor immunoscintigraphy. It was assumed that directly-radiolabled antibodies could be replaced by an unlabeled immunoconjugate capable of binding both a tumor-specific antigen and a small molecular weight molecule. The small molecular weight molecule would carry the radioactive payload and would be injected after the bispecific immunoconjugate. It has been demonstrated that this approach does allow for both antibody-specific recognition and fast clearance of the radioactive molecule, thus resulting in improved tumor-to-normal tissue contrast ratios. It was subsequently shown that pretargeting also held promise for tumor therapy, translating improved tumor-to-normal tissue contrast ratios into more specific delivery of absorbed radiation doses. Many technical approaches have been proposed to implement pretargeting, and two have been extensively documented. One is based on the avidin-biotin system, and the other on bispecific antibodies binding a tumor-specific antigen and a hapten. Both have been studied in preclinical models, as well as in several clinical studies, and have shown improved targeting efficiency. This article reviews the historical and recent preclinical and clinical advances in the use of bispecific-antibody-based pretargeting for radioimmunodetection and radioimmunotherapy of cancer. The results of recent evaluation of pretargeting in PET imaging also are discussed.

  9. A novel bispecific antibody, S-Fab, induces potent cancer cell killing.

    Science.gov (United States)

    Li, Li; He, Ping; Zhou, Changhua; Jing, Li; Dong, Bin; Chen, Siqi; Zhang, Ning; Liu, Yawei; Miao, Ji; Wang, Zhong; Li, Qing

    2015-01-01

    Bispecific antibodies that engage immune cells to kill cancer cells have been actively studied in cancer immunotherapy. In this study, we present a novel bispecific format, S-Fab, fabricated by linking a single-domain anti-carcinoembryonic antigen VHH to a conventional anti-CD3 Fab. In contrast to most bispecific antibodies, the S-Fab bispecific antibody can be efficiently expressed and purified from bacteria. The purified S-Fab is stable in serum and is able to recruit T cells to drive potent cancer cell killing. In xenograft models, the S-Fab antibody suppresses tumor growth in the presence of human immune cells. Our study suggested that the bispecific S-Fab format can be applied to a wide range of immunotherapies.

  10. Theranostic pretargeted radioimmunotherapy of colorectal cancer xenografts in mice using picomolar affinity 86Y- or 177Lu-DOTA-Bn binding scFv C825/GPA33 IgG bispecific immunoconjugates

    International Nuclear Information System (INIS)

    GPA33 is a colorectal cancer (CRC) antigen with unique retention properties after huA33-mediated tumor targeting. We tested a pretargeted radioimmunotherapy (PRIT) approach for CRC using a tetravalent bispecific antibody with dual specificity for GPA33 tumor antigen and DOTA-Bn-(radiolanthanide metal) complex. PRIT was optimized in vivo by titrating sequential intravenous doses of huA33-C825, the dextran-based clearing agent, and the C825 haptens 177Lu-or 86Y-DOTA-Bn in mice bearing the SW1222 subcutaneous (s.c.) CRC xenograft model. Using optimized PRIT, therapeutic indices (TIs) for tumor radiation-absorbed dose of 73 (tumor/blood) and 12 (tumor/kidney) were achieved. Estimated absorbed doses (cGy/MBq) to tumor, blood, liver, spleen, and kidney for single-cycle PRIT were 65.8, 0.9 (TI 73), 6.3 (TI 10), 6.6 (TI 10), and 5.3 (TI 12), respectively. Two cycles of PRIT (66.6 or 111 MBq 177Lu-DOTA-Bn) were safe and effective, with a complete response of established s.c. tumors (100 - 700 mm3) in nine of nine mice, with two mice alive without recurrence at >140 days. Tumor log kill in this model was estimated to be 2.1 - 3.0 based on time to 500-mm3 tumor recurrence. In addition, PRIT dosimetry/diagnosis was performed by PET imaging of the positron-emitting DOTA hapten 86Y-DOTA-Bn. We have developed anti-GPA33 PRIT as a triple-step theranostic strategy for preclinical detection, dosimetry, and safe targeted radiotherapy of established human colorectal mouse xenografts. (orig.)

  11. Theranostic pretargeted radioimmunotherapy of colorectal cancer xenografts in mice using picomolar affinity {sup 86}Y- or {sup 177}Lu-DOTA-Bn binding scFv C825/GPA33 IgG bispecific immunoconjugates

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    Cheal, Sarah M.; Lee, Sang-gyu; Punzalan, Blesida; Larson, Steven M. [Memorial Sloan Kettering Cancer Center, Department of Radiology, New York, NY (United States); Memorial Sloan Kettering Cancer Center, Molecular Pharmacology and Chemistry Program, New York, NY (United States); Xu, Hong; Guo, Hong-fen [Memorial Sloan Kettering Cancer Center, Department of Pediatrics, New York, NY (United States); Chalasani, Sandhya; Carrasquillo, Jorge A. [Memorial Sloan Kettering Cancer Center, Department of Radiology, New York, NY (United States); Fung, Edward K. [Memorial Sloan Kettering Cancer Center, Molecular Pharmacology and Chemistry Program, New York, NY (United States); Memorial Sloan Kettering Cancer Center, Department of Medical Physics, New York, NY (United States); Jungbluth, Achim [Memorial Sloan Kettering Cancer Center, Department of Pathology, New York, NY (United States); Zanzonico, Pat B.; O' Donoghue, Joseph [Memorial Sloan Kettering Cancer Center, Department of Medical Physics, New York, NY (United States); Smith-Jones, Peter M. [Stony Brook University, Department of Psychiatry and Behavioral Science, Stony Brook, NY (United States); Stony Brook University, Department of Radiology, Stony Brook, NY (United States); Wittrup, K.D. [Massachusetts Institute of Technology, Department of Chemical Engineering, Cambridge, MA (United States); Massachusetts Institute of Technology, Department of Biological Engineering, Cambridge, MA (United States); Massachusetts Institute of Technology, Koch Institute for Integrative Cancer Research, Cambridge, MA (United States); Cheung, Nai-Kong V. [Memorial Sloan Kettering Cancer Center, Molecular Pharmacology and Chemistry Program, New York, NY (United States); Memorial Sloan Kettering Cancer Center, Department of Pediatrics, New York, NY (United States)

    2016-05-15

    GPA33 is a colorectal cancer (CRC) antigen with unique retention properties after huA33-mediated tumor targeting. We tested a pretargeted radioimmunotherapy (PRIT) approach for CRC using a tetravalent bispecific antibody with dual specificity for GPA33 tumor antigen and DOTA-Bn-(radiolanthanide metal) complex. PRIT was optimized in vivo by titrating sequential intravenous doses of huA33-C825, the dextran-based clearing agent, and the C825 haptens {sup 177}Lu-or {sup 86}Y-DOTA-Bn in mice bearing the SW1222 subcutaneous (s.c.) CRC xenograft model. Using optimized PRIT, therapeutic indices (TIs) for tumor radiation-absorbed dose of 73 (tumor/blood) and 12 (tumor/kidney) were achieved. Estimated absorbed doses (cGy/MBq) to tumor, blood, liver, spleen, and kidney for single-cycle PRIT were 65.8, 0.9 (TI 73), 6.3 (TI 10), 6.6 (TI 10), and 5.3 (TI 12), respectively. Two cycles of PRIT (66.6 or 111 MBq {sup 177}Lu-DOTA-Bn) were safe and effective, with a complete response of established s.c. tumors (100 - 700 mm{sup 3}) in nine of nine mice, with two mice alive without recurrence at >140 days. Tumor log kill in this model was estimated to be 2.1 - 3.0 based on time to 500-mm{sup 3} tumor recurrence. In addition, PRIT dosimetry/diagnosis was performed by PET imaging of the positron-emitting DOTA hapten {sup 86}Y-DOTA-Bn. We have developed anti-GPA33 PRIT as a triple-step theranostic strategy for preclinical detection, dosimetry, and safe targeted radiotherapy of established human colorectal mouse xenografts. (orig.)

  12. Application of bispecific antibody against antigen and hapten for immunodetection and immunopurification

    OpenAIRE

    Kim, Hyori; Park, Sunyoung; Lee, Hwa Kyoung; Chung, Junho

    2013-01-01

    We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 × anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immu...

  13. Recombinant approaches to IgG-like bispecific antibodies

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    Jonathan S MARVIN; Zhenping ZHU

    2005-01-01

    One of the major obstacles in the development of bispecific antibodies (BsAb)has been the difficulty of producing the materials in sufficient quality and quantity by traditional technologies, such as the hybrid hybridoma and chemical conjugation methods. In contrast to the rapid and significant progress in the development of recombinant BsAb fragments (such as diabody and tandem single chain Fv), the successful design and production of full length IgG-like BsAb has been limited. Compared to smaller fragments, IgG-like BsAb have long serum halflife and are capable of supporting secondary immune functions, such as antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity. The development of IgG-like BsAb as therapeutic agents will depend heavily on our research progress in the design of recombinant BsAb constructs (or formats) and production efficiency. This review will focus on recent advances in various recombinant approaches to the engineering and production of IgG-like BsAb.

  14. Biodistribution Analyses of a Near-Infrared, Fluorescently Labeled, Bispecific Monoclonal Antibody Using Optical Imaging.

    Science.gov (United States)

    Peterson, Norman C; Wilson, George G; Huang, Qihui; Dimasi, Nazzareno; Sachsenmeier, Kris F

    2016-04-01

    In recent years, biodistribution analyses of pharmaceutical compounds in preclinical animal models have become an integral part of drug development. Here we report on the use of optical imaging biodistribution analyses in a mouse xenograft model to identify tissues that nonspecifically retained a bispecific antibody under development. Although our bispecific antibody bound both the epidermal growth factor receptor and insulin growth factor 1 receptor are expressed on H358, nonsmall-cell lung carcinoma cells, the fluorescence from labeled bispecific antibody was less intense than expected in xenografted tumors. Imaging analyses of live mice and major organs revealed that the majority of the Alexa Fluor 750 labeled bispecific antibody was sequestered in the liver within 2 h of injection. However, results varied depending on which near-infrared fluorophore was used, and fluorescence from the livers of mice injected with bispecific antibody labeled with Alexa Fluor 680 was less pronounced than those labeled with Alexa Fluor 750. The tissue distribution of control antibodies remained unaffected by label and suggests that the retention of fluorophores in the liver may differ. Given these precautions, these results support the incorporation of optical imaging biodistribution analyses in biotherapeutic development strategies.

  15. Biodistribution Analyses of a Near-Infrared, Fluorescently Labeled, Bispecific Monoclonal Antibody Using Optical Imaging.

    Science.gov (United States)

    Peterson, Norman C; Wilson, George G; Huang, Qihui; Dimasi, Nazzareno; Sachsenmeier, Kris F

    2016-04-01

    In recent years, biodistribution analyses of pharmaceutical compounds in preclinical animal models have become an integral part of drug development. Here we report on the use of optical imaging biodistribution analyses in a mouse xenograft model to identify tissues that nonspecifically retained a bispecific antibody under development. Although our bispecific antibody bound both the epidermal growth factor receptor and insulin growth factor 1 receptor are expressed on H358, nonsmall-cell lung carcinoma cells, the fluorescence from labeled bispecific antibody was less intense than expected in xenografted tumors. Imaging analyses of live mice and major organs revealed that the majority of the Alexa Fluor 750 labeled bispecific antibody was sequestered in the liver within 2 h of injection. However, results varied depending on which near-infrared fluorophore was used, and fluorescence from the livers of mice injected with bispecific antibody labeled with Alexa Fluor 680 was less pronounced than those labeled with Alexa Fluor 750. The tissue distribution of control antibodies remained unaffected by label and suggests that the retention of fluorophores in the liver may differ. Given these precautions, these results support the incorporation of optical imaging biodistribution analyses in biotherapeutic development strategies. PMID:27053562

  16. Pretargeting vs. direct targeting of human betalox5 islet cells subcutaneously implanted in mice using an anti-human islet cell antibody

    International Nuclear Information System (INIS)

    Introduction: We previously demonstrated MORF/cMORF pretargeting of human islets and betalox 5 cells (a human beta cell line) transplanted subcutaneously in mice with the anti-human islet antibody, HPi1. We now compare pretargeting with direct targeting in the beta cell transplant model to evaluate the degree to which target/non-target (T/NT) ratios may be improved by pretargeting. Methods: Specific binding of an anti-human islet antibody HPi1 to the beta cells transplanted subcutaneously in mice was examined against a negative control antibody. We then compared pretargeting by MORF-HPi1 plus 111In-labeled cMORF to direct targeting by 111In-labeled HPi1. Results: HPi1 binding to betalox5 human cells in the transplant was shown by immunofluorescence. Normal organ 111In backgrounds by pretargeting were always lower, although target accumulations were similar. More importantly, the transplant to pancreas and liver ratios was, respectively, 26 and 10 by pretargeting as compared to 9 and 0.6 by direct targeting. Conclusions: Pretargeting greatly improves the T/NT ratios, and based on the estimated endocrine to exocrine ratio within a pancreas, pretargeting may be approaching the sensitivity required for successful imaging of human islets within this organ.

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

    Science.gov (United States)

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

    1991-06-01

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

  18. Improvement of radioimmunotherapy using pretargeting

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    Eric eFrampas

    2013-06-01

    Full Text Available During the past two decades, considerable research has been devoted to radionuclide therapy using radiolabelled monoclonal antibodies and receptor binding agents. Conventional Radioimmunotherapy (RIT is now an established and important tool in the treatment of hematologic malignancies such as Non-Hodgkin lymphoma. For solid malignancies, the efficacy of RIT has not been as successful due to lower radiosensitivity, difficult penetration of the antibody into the tumor and potential excessive radiation to normal tissues. Innovative approaches have been developed in order to enhance tumor absorbed dose while limiting toxicity to overcome the different limitations due to the tumor and host characteristics.Pretargeting techniques (pRIT are a promising approach that consists of decoupling the delivery of a tumor monoclonal antibody (mAb from the delivery of the radionuclide. This results in a much higher tumor-to-normal tissue ratio and is favorable for therapy as well and imaging. This includes various strategies based on avidin/streptavidin-biotin, DNA-complementary DNA and bispecific antibody-hapten bindings. PRIT continuously evolves with the investigation of new molecular constructs and the development of radiochemistry. Pharmacokinetics improve dosimetry depending on the radionuclides used (alpha, beta and Auger emitters with prediction of tumor response and host toxicities. New constructs such as the Dock and Lock technology allow production of a variety of mABs directed against tumor-associated antigens. Survival benefit has already been shown in medullary thyroid carcinoma. Improvement in delivery of radioactivity to tumors with these pretargeting procedures associated with reduced hematologic toxicity will become the next generation of RIT. The following review addresses actual technical and clinical considerations and future development of pRIT.

  19. Overcoming the Constraints of Anti-HIV/CD89 Bispecific Antibodies That Limit Viral Inhibition

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    Xiaocong Yu

    2016-01-01

    Full Text Available Innovative strategies are necessary to maximize the clinical application of HIV neutralizing antibodies. To this end, bispecific constructs of human antibody F240, reactive with well-conserved gp41 epitope and antibody 14A8, reactive with the IgA receptor (CD89 on effector cells, were constructed. A F240 × 14A8 bispecific single chain variable region (scFv molecule was constructed by linking two scFvs using a conventional GGGGS linker. Despite immunoreactivity with HIV gp41 and neutrophils, this bispecific scFv failed to inhibit HIV infection. This is in sharp contrast to viral inhibition using a chemical conjugate of the Fab of these two antibodies. Therefore, we constructed two novel Fab-like bispecific antibody molecules centered on fusion of the IgG1 CH1 domain or CH1-hinge domain to the C-terminus of F240scFv and fusion of the kappa chain CL domain to the C-terminus of 14A8scFv. Both Bi-Fab antibodies showed significant ADCVI activity for multiple clade B and clade C isolates by arming the neutrophils to inhibit HIV infection. The approach presented in this study is unique for HIV immunotherapy in that the impetus of neutralization is to arm and mobilize PMN to destroy HIV and HIV infected cells.

  20. Overcoming the Constraints of Anti-HIV/CD89 Bispecific Antibodies That Limit Viral Inhibition.

    Science.gov (United States)

    Yu, Xiaocong; Duval, Mark; Gawron, Melissa; Posner, Marshall R; Cavacini, Lisa A

    2016-01-01

    Innovative strategies are necessary to maximize the clinical application of HIV neutralizing antibodies. To this end, bispecific constructs of human antibody F240, reactive with well-conserved gp41 epitope and antibody 14A8, reactive with the IgA receptor (CD89) on effector cells, were constructed. A F240 × 14A8 bispecific single chain variable region (scFv) molecule was constructed by linking two scFvs using a conventional GGGGS linker. Despite immunoreactivity with HIV gp41 and neutrophils, this bispecific scFv failed to inhibit HIV infection. This is in sharp contrast to viral inhibition using a chemical conjugate of the Fab of these two antibodies. Therefore, we constructed two novel Fab-like bispecific antibody molecules centered on fusion of the IgG1 CH1 domain or CH1-hinge domain to the C-terminus of F240scFv and fusion of the kappa chain CL domain to the C-terminus of 14A8scFv. Both Bi-Fab antibodies showed significant ADCVI activity for multiple clade B and clade C isolates by arming the neutrophils to inhibit HIV infection. The approach presented in this study is unique for HIV immunotherapy in that the impetus of neutralization is to arm and mobilize PMN to destroy HIV and HIV infected cells. PMID:27419146

  1. Prospective Design of Anti-Transferrin Receptor Bispecific Antibodies for Optimal Delivery into the Human Brain.

    Science.gov (United States)

    Kanodia, J S; Gadkar, K; Bumbaca, D; Zhang, Y; Tong, R K; Luk, W; Hoyte, K; Lu, Y; Wildsmith, K R; Couch, J A; Watts, R J; Dennis, M S; Ernst, J A; Scearce-Levie, K; Atwal, J K; Ramanujan, S; Joseph, S

    2016-05-01

    Anti-transferrin receptor (TfR)-based bispecific antibodies have shown promise for boosting antibody uptake in the brain. Nevertheless, there are limited data on the molecular properties, including affinity required for successful development of TfR-based therapeutics. A complex nonmonotonic relationship exists between affinity of the anti-TfR arm and brain uptake at therapeutically relevant doses. However, the quantitative nature of this relationship and its translatability to humans is heretofore unexplored. Therefore, we developed a mechanistic pharmacokinetic-pharmacodynamic (PK-PD) model for bispecific anti-TfR/BACE1 antibodies that accounts for antibody-TfR interactions at the blood-brain barrier (BBB) as well as the pharmacodynamic (PD) effect of anti-BACE1 arm. The calibrated model correctly predicted the optimal anti-TfR affinity required to maximize brain exposure of therapeutic antibodies in the cynomolgus monkey and was scaled to predict the optimal affinity of anti-TfR bispecifics in humans. Thus, this model provides a framework for testing critical translational predictions for anti-TfR bispecific antibodies, including choice of candidate molecule for clinical development. PMID:27299941

  2. Bispecific antibody and its clinical applications in cancer

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Bispecific antibody (BsAb) usually consists of two different antigen-binding arms, by which it is capable of simultaneously binding to target cells and effector cells, and can directly mediate the killing of target cells by retargeting and activating effector cells. The development of BsAb re-search goes through three main stages: chemical cross- linking of murine-derived monoclonal antibody, hybrid hy-bridomas and engineered BsAb. Among them, engineered BsAb has more formats than the other two, such as diabody, ScdHLX, ScZip, ScCH3, ScFab and BsIgG, etc. Compared with former murine-derived BsAbs, engineered BsAb has lower immunogenicity and stronger penetrating capacity, and currently, some of them appear suitable for clinical ap-plication in yields and qualities. Up to now, several phase Ⅰand phase Ⅱ clinical studies of BsAb, for instance, some (Fab')2 and Diabodies, have been performed. Among those BsAbs, anti-CD3/anti-tumor BsAbs is most common, they not only can activate T cell and induce CD3AK cytotoxic activity in in vitro experiment, and inhibit the growth of tumor on tumor-bearing mouse by retargeting T cells to lyse tumor cells, but also offer great promise in the therapy of some malignancies in clinic, especially of some advanced cancers as well as elimination of minimal residual tumors, indicated by increasing the tumor/blood ratio of antibody in patients and improving the natural killer cell (NK) anti-tumor activity in tumor sites, and also presenting of an in-crease level in TNF-a,INF-g, IL-6, IL-8, IL-10 and soluble CD25, etc. The responses are also shown via improving the quality of life and prolonging the survival of partial patients. The "Knobs into Holes" technology is a new strategy emerging during research on engineered BsAb, it is likely to be useful for heterodimerization and can improve the quan-tity, purity and stability of BsAb, it is also anticipated to increase the clinical potential of BsAb in the future.

  3. Characterization of a Bispecific FLT3 X CD3 Antibody in an Improved, Recombinant Format for the Treatment of Leukemia

    OpenAIRE

    Durben, Michael; Schmiedel, Dominik; Hofmann, Martin; Vogt, Fabian; Nübling, Tina; Pyz, Elwira; Bühring, Hans-Jörg; Rammensee, Hans-Georg; Salih, Helmut R.; Große-Hovest, Ludger; Jung, Gundram

    2015-01-01

    FLT3 is a receptor-tyrosine-kinase that is expressed on leukemic cells of the myeloid and lymphoid lineage rather specifically. We here report on the construction and selection of bispecific FLT3 X CD3 antibodies in a new recombinant format, termed Fabsc, that resembles the normal antibody structure more closely than the well-established bispecific single chain (bssc)-format. Our preferred antibody, which emerged from an initial selection procedure utilizing different FLT3- and CD3-antibodies...

  4. Modeling bispecific monoclonal antibody interaction with two cell membrane targets indicates the importance of surface diffusion.

    Science.gov (United States)

    Sengers, Bram G; McGinty, Sean; Nouri, Fatma Z; Argungu, Maryam; Hawkins, Emma; Hadji, Aymen; Weber, Andrew; Taylor, Adam; Sepp, Armin

    2016-07-01

    We have developed a mathematical framework for describing a bispecific monoclonal antibody interaction with two independent membrane-bound targets that are expressed on the same cell surface. The bispecific antibody in solution binds either of the two targets first, and then cross-links with the second one while on the cell surface, subject to rate-limiting lateral diffusion step within the lifetime of the monovalently engaged antibody-antigen complex. At experimental densities, only a small fraction of the free targets is expected to lie within the reach of the antibody binding sites at any time. Using ordinary differential equation and Monte Carlo simulation-based models, we validated this approach against an independently published anti-CD4/CD70 DuetMab experimental data set. As a result of dimensional reduction, the cell surface reaction is expected to be so rapid that, in agreement with the experimental data, no monovalently bound bispecific antibody binary complexes accumulate until cross-linking is complete. The dissociation of the bispecific antibody from the ternary cross-linked complex is expected to be significantly slower than that from either of the monovalently bound variants. We estimate that the effective affinity of the bivalently bound bispecific antibody is enhanced for about 4 orders of magnitude over that of the monovalently bound species. This avidity enhancement allows for the highly specific binding of anti-CD4/CD70 DuetMab to the cells that are positive for both target antigens over those that express only one or the other We suggest that the lateral diffusion of target antigens in the cell membrane also plays a key role in the avidity effect of natural antibodies and other bivalent ligands in their interactions with their respective cell surface receptors. PMID:27097222

  5. Biosensing made easy with PEG-targeted bi-specific antibodies.

    Science.gov (United States)

    Raftery, Lyndon J; Grewal, Yadveer S; Howard, Christopher B; Jones, Martina L; Shiddiky, Muhammad J A; Carrascosa, Laura G; Thurecht, Kristofer J; Mahler, Stephen M; Trau, Matt

    2016-04-28

    Whilst recent advances in nanotechnology have yielded many new biosensing capabilities, innovative biological attachment and detection modalities remain relatively underdeveloped. Bi-specific antibodies (bsAbs)--which exhibit binding capability for two separate targets--offer an inherent advantage over conventional antibody reagents by significantly simplifying sensor surface preparation. Herein, we report the deployment of bsAbs for simultaneous attachment to a polymer-coated transducer and label-free, electrochemical (EC) detection of target antigens. PMID:27045479

  6. Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface.

    Science.gov (United States)

    Lewis, Steven M; Wu, Xiufeng; Pustilnik, Anna; Sereno, Arlene; Huang, Flora; Rick, Heather L; Guntas, Gurkan; Leaver-Fay, Andrew; Smith, Eric M; Ho, Carolyn; Hansen-Estruch, Christophe; Chamberlain, Aaron K; Truhlar, Stephanie M; Conner, Elaine M; Atwell, Shane; Kuhlman, Brian; Demarest, Stephen J

    2014-02-01

    Robust generation of IgG bispecific antibodies has been a long-standing challenge. Existing methods require extensive engineering of each individual antibody, discovery of common light chains, or complex and laborious biochemical processing. Here we combine computational and rational design approaches with experimental structural validation to generate antibody heavy and light chains with orthogonal Fab interfaces. Parental monoclonal antibodies incorporating these interfaces, when simultaneously co-expressed, assemble into bispecific IgG with improved heavy chain-light chain pairing. Bispecific IgGs generated with this approach exhibit pharmacokinetic and other desirable properties of native IgG, but bind target antigens monovalently. As such, these bispecific reagents may be useful in many biotechnological applications.

  7. An efficient process of generating bispecific antibodies via controlled Fab-arm exchange using culture supernatants.

    Science.gov (United States)

    Paul, Suparna; Connor, Judy; Nesspor, Tom; Haytko, Peter; Boakye, Ken; Chiu, Mark L; Jiang, Haiyan

    2016-05-01

    Bispecific antibody generation is actively pursued for therapeutic and research antibody development. Although there are multiple strategies for generating bispecific antibodies (bsAbs); the common challenge is to develop a scalable method to prepare bsAbs with high purity and yield. The controlled Fab-arm exchange (cFAE) method combines two parental monoclonal antibodies (mAbs), each with a matched point mutation, F405L and K409R in the respective CH3 domains. The conventional process employs two steps: the purification of two parental mAbs from culture supernatants followed by cFAE. Following a reduction/oxidation reaction, the bispecific mAb is formed with greater than 95% heterodimerization efficiency. In this study, cFAE was initiated in culture supernatants expressing the two parental mAbs, thereby eliminating the need to first purify the parental mAbs. The bsAbs formed in culture supernatant was then purified using a Protein A affinity chromatography. The BsAbs generated in this manner had efficiency comparable to the conventional method using purified parental mAbs. BsAbs prepared by two different routes showed indistinguishable characteristics by SDS capillary electrophoresis, analytical size exclusion, and cation exchange chromatography. This alternative method significantly shortened timelines and reduced resources required for bsAb generation, providing an improved process with potential benefits in large-scale bsAb preparation, as well as for HTP small-scale bsAb matrix selection.

  8. Overcoming Instability of Antibody-Nanomaterial Conjugates: Next Generation Targeted Nanomedicines Using Bispecific Antibodies.

    Science.gov (United States)

    Howard, Christopher B; Fletcher, Nicholas; Houston, Zachary H; Fuchs, Adrian V; Boase, Nathan R B; Simpson, Joshua D; Raftery, Lyndon J; Ruder, Tim; Jones, Martina L; de Bakker, Christopher J; Mahler, Stephen M; Thurecht, Kristofer J

    2016-08-01

    Targeted nanomaterials promise improved therapeutic efficacy, however their application in nanomedicine is limited due to complexities associated with protein conjugations to synthetic nanocarriers. A facile method to generate actively targeted nanomaterials is developed and exemplified using polyethylene glycol (PEG)-functional nanostructures coupled to a bispecific antibody (BsAb) with dual specificity for methoxy PEG (mPEG) epitopes and cancer targets such as epidermal growth factor receptor (EGFR). The EGFR-mPEG BsAb binds with high affinity to recombinant EGFR (KD : 1 × 10(-9) m) and hyperbranched polymer (HBP) consisting of mPEG (KD : 10 × 10(-9) m) and demonstrates higher avidity for HBP compared to linear mPEG. The binding of BsAb-HBP bioconjugate to EGFR on MDA-MB-468 cancer cells is investigated in vitro using a fluorescently labeled polymer, and in in vivo xenograft models by small animal optical imaging. The antibody-targeted nanostructures show improved accumulation in tumor cells compared to non-targeted nanomaterials. This demonstrates a facile approach for tuning targeting ligand density on nanomaterials, by modulating surface functionality. Antibody fragments are tethered to the nanomaterial through simple mixing prior to administration to animals, overcoming the extensive procedures encountered for developing targeted nanomedicines.

  9. Effects of interlinker sequences on the biological properties of bispecific single-chain antibodies

    Institute of Scientific and Technical Information of China (English)

    FANG Min; JIANG Xin; YANG Zhi; YIN Changcheng; LI Hua; ZHAO Rui; ZHANG Zhong; LIN Qing; HUANG Hualiang

    2003-01-01

    Single-chain bispecific antibody (scBsAb) is one of the promising genetic engineering antibody formats for clinical application. But the effects of interlinker sequences on the biological properties of bispecific single-chain antibodies have not been studied in detail. Three interlinker sequences were designed and synthesized, and denominated as Fc, HSA, 205C′, respectively. Universal vectors with these different interlinker sequences for scBsAb expression in E. coli were constructed. A model scBsAb based on a reshaped single-chain antibody (scFv) against human CD3 and a scFv directed against human ovarian carcinoma were generated and expressed in E. coli. The results of SDS-PAGE and Western blot showed that the different interlinker sequences did not affect the expression levelof scBsAb. However, as demonstrated by ELISA and pharmacokinetics studies performed in mice, scBsAbs with different interlinker sequences had difference in the antigen-binding activities and terminal half-life time (T1/2β) in vivo, the interlinker HSA could remarkably prolong the retention time of scBsAb in blood. These results indicated that the peptide sequence of interlinker could affect important biological properties of scBsAb, such as antigen-binding properties and stability in vivo. So, selection of an appropriate interlinker sequence is very important for scBsAb construction. Optimal interlinker can bring scBsAb biologicalproperties more suitable for clinical application.

  10. Construction and expression of D-dimer and GPIIb/IIIa single-chain bispecific antibody

    OpenAIRE

    DAN, ZHAOKUI; Tan, Zui; Xia, Hongli; WU, GAN

    2013-01-01

    The aim of this study was to construct a plasmid expressing glycoprotein IIb-IIIa (GPIIb/IIIa) and D-dimer single-chain bispecific antibody for the targeted therapy of thrombosis. The phosphorylated gene encoding the anti-GPIIb/IIIa single-chain variable fragment (scFv) and the gene encoding the anti-D-dimer scFv were amplified by PCR and linked in tandem by blunt-end ligation. The recombinant plasmid was transfected into the competent cell line HB2151 and identified by PCR and DNA sequencing...

  11. Development of a bispecific monoclonal antibody to pesticide carbofuran and triazophos using hybrid hybridomas.

    Science.gov (United States)

    Jin, R Y; Guo, Y R; Wang, C M; Wu, J X; Zhu, G N

    2009-01-01

    A mouse hybrid hybridoma (tetradoma) was derived from fusing hybridomas producing monoclonal antibody to N-methylcarbamate pesticide carbofuran with hybridomas producing monoclonal antibody to organophosphorus pesticide Triazophos. The prepared tetradoma line (12C1 to 2H12) secreted hybrid immunoglobulin exhibiting parental and bispecific binding characteristics. The effect of relevant physicochemical factors on the immunoassay based on the 12C1 to 2H12 bispecific monoclonal antibody had been studied to optimize the ELISA performance. The developed immunoassay showed that the detection limit (I(20)) were 0.36 and 1.89 ng/mL for triazophos and carbofuran, respectively, without obvious cross-reactivity to other related compounds. Water samples spiked with triazophos at 0.5, 1, and 5 ng/mL or carbofuran at 5, 10, and 20 ng/mL were directly analyzed by the developed ELISA format. The mean recovery of triazophos and carbofuran were 108.1% and 107.5%, with variation coefficient of 15.9% and 17.7%, respectively.

  12. PHASE-I STUDY OF INTRAVENOUSLY APPLIED BISPECIFIC ANTIBODY IN RENAL-CELL CANCER-PATIENTS RECEIVING SUBCUTANEOUS INTERLEUKIN-2

    NARCIS (Netherlands)

    KROESEN, BJ; SLEIJFER, DT; JANSSEN, RAJ; VANDERGRAAF, WTA; THE, TH; DELEIJ, L; MULDER, NH

    1994-01-01

    In a phase I trial the toxicity and immunomodulatory effects of combined treatment with intravenous (i.v.) bispecific monoclonal antibody BIS-1 and subcutaneous (s.c.) interleukin 2 (IL-2) was studied in renal cell cancer patients. BIS-1 combines a specificity against CD3 on T lymphocytes with a spe

  13. An alternative chemical redox method for the production of bispecific antibodies: implication in rapid detection of food borne pathogens.

    Directory of Open Access Journals (Sweden)

    Mohammad Owais

    Full Text Available Bi-functional antibodies with the ability to bind two unrelated epitopes have remarkable potential in diagnostic and bio-sensing applications. In the present study, bispecific antibodies that recognize human red blood cell (RBC and the food borne pathogen Listeria monocytogenes (L. monocytogenes were engineered. The procedure involves initial reduction of a mixture of anti-RBC and anti-Listeria antibodies followed by gradual re-oxidation of the reduced disulphides. This facilitates association of the separated antibody chains and formation of hybrid immunoglobulins with affinity for the L. monocytogenes and human RBC. The bispecific antibodies caused the agglutination of the RBCs only in the presence of L. monocytogenes cells. The agglutination process necessitated the specific presence of L. monocytogenes and the red colored clumps formed were readily visible with naked eyes. The RBC agglutination assay described here provides a remarkably simple approach for the rapid and highly specific screening of various pathogens in their biological niches.

  14. A bispecific antibody effectively neutralizes all four serotypes of dengue virus by simultaneous blocking virus attachment and fusion.

    Science.gov (United States)

    Shi, Xin; Deng, Yongqiang; Wang, Huajing; Ji, Guanghui; Tan, Wenlong; Jiang, Tao; Li, Xiaofeng; Zhao, Hui; Xia, Tian; Meng, Yanchun; Wang, Chao; Yu, Xiaojie; Yang, Yang; Li, Bohua; Qin, E-De; Dai, Jianxin; Qin, Cheng-Feng; Guo, Yajun

    2016-01-01

    Although dengue virus (DENV) infection severely threatens the health of humans, no specific antiviral drugs are currently approved for clinical use against DENV infection. Attachment and fusion are 2 critical steps for the flavivirus infection, and the corresponding functional epitopes are located at E protein domain III (E-DIII) and domain II (E-DII), respectively. Here, we constructed a bispecific antibody (DVD-1A1D-2A10) based on the 2 well-characterized anti-DENV monoclonal antibodies 1A1D-2 (1A1D) and 2A10G6 (2A10). The 1A1D antibody binds E-DIII and can block the virus attaching to the cell surface, while the 2A10 antibody binds E-DII and is able to prevent the virus from fusing with the endosomal membrane. Our data showed that DVD-1A1D-2A10 retained the antigen-binding activity of both parental antibodies. Importantly, it was demonstrated to be significantly more effective at neutralizing DENV than its parental antibodies both in vitro and in vivo, even better than the combination of them. To eliminate the potential antibody-dependent enhancement (ADE) effect, this bispecific antibody was successfully engineered to prevent Fc-γ-R interaction. Overall, we generated a bispecific anti-DENV antibody targeting both attachment and fusion stages, and this bispecific antibody broadly neutralized all 4 serotypes of DENV without risk of ADE, suggesting that it has great potential as a novel antiviral strategy against DENV. PMID:26905804

  15. Fab-based bispecific antibody formats with robust biophysical properties and biological activity.

    Science.gov (United States)

    Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Lewis, Steven M; Lieu, Ricky L; Weldon, Caroline; Torres, Carina; Fine, Cody; Batt, Micheal A; Fitchett, Jonathan R; Glasebrook, Andrew L; Kuhlman, Brian; Demarest, Stephen J

    2015-01-01

    A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.

  16. Bispecific antibodies and retargeted cellular cytotoxicity: novel approaches to cancer therapy.

    Science.gov (United States)

    Wunderlich, J R; Mezzanzanica, D; Garrido, M A; Neblock, D S; Daddona, P E; Andrew, S M; Zurawski, V R; Canevari, S; Colnaghi, M I; Segal, D M

    1992-01-01

    We have used a relatively new technology to increase the number of human lymphocytes that will react with human ovarian carcinoma cells. This technology, often called "retargeting of the immune system," can temporarily redirect the activity of immune cells that were originally committed to react with foreign substances other than cancer cells. In the example presented here, the antitumor effects of retargeted human T lymphocytes, collected from normal donors, were tested in immunodeficient mice with a human ovarian carcinoma line growing intraperitoneally. We retargeted T cells in vitro with a bispecific antibody that reacted with the T cell receptor complex and with a cell-surface antigen expressed by the ovarian carcinoma cells. Retargeted lymphocytes, injected intraperitoneally into mice 4 days after intraperitoneal injection of the tumor cells, impeded tumor growth and doubled the host survival time. These findings provide support for the concept that treatment of ovarian cancer patients with retargeted T cells could prove beneficial. PMID:1633315

  17. Guiding the Killer and Bringing in Accomplices: Bispecific Antibody Treatment for Malignant Melanoma.

    Science.gov (United States)

    Szegezdi, Eva; Leverkus, Martin

    2016-02-01

    Discovery of oncogene and immune checkpoint targeting has transformed melanoma therapy in the last 5 years. However, treatment of primary or secondary drug-resistant melanoma remains a challenge. Agents designed to activate the cell death machinery directly, for example by activating the death receptors expressed by melanoma cells, could break drug resistance, and they may achieve long-lasting therapeutic success. He et al. report their studies of an MCSPxDR5 bispecific, tetravalent antibody that can simultaneously target death receptor 5 (DR5, TRAIL-R2) and melanoma-associated chondroitin sulfate proteoglycan (MCSP). This antibody can exert strong and selective DR5-dependent cytotoxic activity against MCSP-expressing melanoma cells. Crosslinking of the antibody with Fcγ-receptors increased the cytotoxic potential further, without compromising its selectivity. This approach offers a novel immunotherapeutic tool via coupling of three cooperating processes: delivering the death receptor agonist to the malignant cell population, potent activation of DR5-mediated cell death signaling, and recruitment of Fcγ-receptor-carrying immune cells that can mount an immune response against the tumor cells. PMID:26802233

  18. A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells.

    Science.gov (United States)

    Li, Aifen; Xing, Jieyu; Li, Li; Zhou, Changhua; Dong, Bin; He, Ping; Li, Qing; Wang, Zhong

    2016-12-01

    Her2, which is frequently overexpressed in breast cancer, is one of the most studied tumor-associated antigens for cancer therapy. Anti-HER2 monoclonal antibody, trastuzumab, has achieved significant clinical benefits in metastatic breast cancer. In this study, we describe a novel bispecific antibody Her2-S-Fab targeting Her2 by linking a single domain anti-CD16 VHH to the trastuzumab Fab. The Her2-S-Fab antibody can be efficiently expressed and purified from Escherichia coli, and drive potent cancer cell killing in HER2-overexpressing cancer cells. In xenograft model, the Her2-S-Fab suppresses tumor growth in the presence of human immune cells. Our results suggest that the bispecific Her2-S-Fab may provide a valid alternative to Her2 positive cancer therapy. PMID:27112931

  19. A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells.

    Science.gov (United States)

    Li, Aifen; Xing, Jieyu; Li, Li; Zhou, Changhua; Dong, Bin; He, Ping; Li, Qing; Wang, Zhong

    2016-12-01

    Her2, which is frequently overexpressed in breast cancer, is one of the most studied tumor-associated antigens for cancer therapy. Anti-HER2 monoclonal antibody, trastuzumab, has achieved significant clinical benefits in metastatic breast cancer. In this study, we describe a novel bispecific antibody Her2-S-Fab targeting Her2 by linking a single domain anti-CD16 VHH to the trastuzumab Fab. The Her2-S-Fab antibody can be efficiently expressed and purified from Escherichia coli, and drive potent cancer cell killing in HER2-overexpressing cancer cells. In xenograft model, the Her2-S-Fab suppresses tumor growth in the presence of human immune cells. Our results suggest that the bispecific Her2-S-Fab may provide a valid alternative to Her2 positive cancer therapy.

  20. Cancer T cell immunotherapy with bispecific antibodies and chimeric antigen receptors.

    Science.gov (United States)

    Lacher, Markus D; Provenzano, Maurizio

    2013-09-01

    Solid tumors contain several different types of malignant cells. This cellular heterogeneity complicates therapy for at least two reasons. First, each subpopulation may respond differently to a given treatment. Second, cancer cells are plastic, and thus may convert from a therapy-sensitive to a therapy-resistant cell type represented by another subpopulation. Therefore, successful therapies will have to target numerous malignant cell types, not just the rapidly proliferating cells as most standard treatments do. Immunotherapies with T cells engineered to recognize cancer cells via bispecific antibodies (bsAbs) or chimeric antigen receptors (CARs) are particularly promising approaches with potential to ablate both dividing and non/slow-dividing subpopulations of cancer cells. Here, we discuss several patents associated with exceptionally effective bsAbs of the tandem single-chain variable fragment (taFv) class and untangle a part of the complex network of patents directly or indirectly related to CARs. Furthermore, we speculate on the future of bsAbs and CARs for both treatment and prevention of solid tumors such as prostate cancer. PMID:23688207

  1. A Novel Bispecific Antibody against Human CD3 and Ephrin Receptor A10 for Breast Cancer Therapy

    OpenAIRE

    Taki, Shintaro; Kamada, Haruhiko; Inoue, Masaki; Nagano, Kazuya; Mukai, Yohei; Higashisaka, Kazuma; Yoshioka, Yasuo; Tsutsumi, Yasuo; Tsunoda, Shin-ichi

    2015-01-01

    Ephrin receptor A10 (EphA10), a transmembrane receptor that binds to ephrin, is a newly identified breast cancer marker protein that has also been detected in HER2-negative tissue. In this study, we report creation of a novel bispecific antibody (BsAb) binding both EphA10 and CD3, thereby forming a bridge between antigens expressed on both tumor and immune cells and promoting recognition of tumor cells by immune cells and redirection of cytotoxic T cells (CTL). This BsAb (EphA10/CD3) was expr...

  2. Blinatumomab: a bispecific T cell engager (BiTE) antibody against CD19/CD3 for refractory acute lymphoid leukemia

    OpenAIRE

    Wu, JingJing; Fu, Jiaping; Zhang, Mingzhi; Liu, Delong

    2015-01-01

    Targeted therapy has been the forefront of cancer treatment. Cancer immunotherapy is the most recent focus. In addition, novel immunotherapeutics targeting B cell receptor signaling (e.g., ibrutinib), T cell receptor ( e.g., CART19), and NK cells (e.g., AFM13) are being developed. This review summarized the new development in blinatumomab (MT103/MEDI-538), a first-in-class bispecific T engager (BiTE) antibody against CD19/CD3 in patients with relapsed/refractory precursor B cell acute lymphoi...

  3. Local but no systemic immunomodulation by intraperitoneal treatment of advanced ovarian cancer with autologous T lymphocytes re-targeted by a bi-specific monoclonal antibody

    NARCIS (Netherlands)

    C.H.J. Lamers (Cor); R.L.H. Bolhuis (Reinder); S.O. Warnaar (Sven); G. Stoter (Gerrit); J.W. Gratama (Jan-Willem)

    1997-01-01

    textabstractWe have reported a 27% overall anti-tumor response using i.p. immunotherapy of advanced ovarian carcinoma with autologous, ex vivo expanded, T lymphocytes re-targeted with bi-specific monoclonal antibody OC/TR, combined with soluble OC/TR and low-dose recombinant interleukin-2 (IL-2). Th

  4. Pretargeted radioimmunotherapy of colorectal cancer metastases: models and pharmacokinetics predict influence of the physical and radiochemical properties of the radionuclide

    Energy Technology Data Exchange (ETDEWEB)

    Frampas, Eric; Maurel, Catherine; Remaud-Le Saec, Patricia; Mauxion, Thibault; Faivre-Chauvet, Alain; Davodeau, Francois; Bardies, Manuel; Barbet, Jacques [Universite de Nantes, Inserm, UMR 892, Centre de Recherche en Cancerologie Nantes-Angers (CRCNA), Nantes (France); Goldenberg, David M. [Immunomedics, Inc., Morris Plains, NJ (United States); Center for Molecular Medicine and Immunology, Garden State Cancer Center, Morris Plains, NJ (United States)

    2011-12-15

    We investigated influences of pretargeting variables, tumor location, and radionuclides in pretargeted radioimmunotherapy (PRIT) as well as estimated tumor absorbed doses. LS-174T human colonic carcinoma cells expressing carcinoembryonic antigen (CEA) were inoculated in nude mice. Biodistribution of a bispecific anti-CEA x anti-hapten antibody, TF2, and of a TF2-pretargeted peptide was assessed and a multi-compartment pharmacokinetic model was devised. Tissue absorbed doses were calculated for {sup 131}I, {sup 177}Lu, {sup 90}Y, {sup 211}At, and {sup 213}Bi using realistic specific activities. Under conditions optimized for tumor imaging (10:1 TF2 to peptide molar ratio, interval time 15-24 h), tumor uptake reached {proportional_to}9 ID/g in subcutaneous tumors at 2 h with very low accretion in normal tissues (tumor to blood ratio >20:1 after 2 h). For a low dose of peptide (0.04 nmol), {sup 211}At is predicted to deliver a high absorbed dose to tumors [41.5 Gy considering a relative biologic effect (RBE) of 5], kidneys being dose-limiting. {sup 90}Y and {sup 213}Bi would also deliver high absorbed doses to tumor (18.6 for {sup 90}Y and 26.5 Gy for {sup 213}Bi, taking RBE into account, for 0.1 nmol) and acceptable absorbed doses to kidneys. With hepatic metastases, a twofold higher tumor absorbed dose is expected. Owing to the low activities measured in blood, the bone marrow absorbed dose is expected to be without significant toxicity. Pretargeting achieves high tumor uptake and higher tumor to background ratios compared to direct RIT. Short-lived radionuclides are predicted to deliver high tumor absorbed doses especially {sup 211}At, with kidneys being the dose-limiting organ. {sup 177}Lu and {sup 131}I should be considered for repeated injections. (orig.)

  5. MOR209/ES414, a Novel Bispecific Antibody Targeting PSMA for the Treatment of Metastatic Castration-Resistant Prostate Cancer.

    Science.gov (United States)

    Hernandez-Hoyos, Gabriela; Sewell, Toddy; Bader, Robert; Bannink, Jeannette; Chenault, Ruth A; Daugherty, Mollie; Dasovich, Maria; Fang, Hang; Gottschalk, Rebecca; Kumer, John; Miller, Robert E; Ravikumar, Padma; Wiens, Jennifer; Algate, Paul A; Bienvenue, David; McMahan, Catherine J; Natarajan, Sateesh K; Gross, Jane A; Blankenship, John W

    2016-09-01

    Treatment of metastatic, castration-resistant prostate cancer (mCRPC) remains a highly unmet medical need and current therapies ultimately result in disease progression. Immunotherapy is a rapidly growing approach for treatment of cancer but has shown limited success to date in the treatment of mCRPC. We have developed a novel humanized bispecific antibody, MOR209/ES414, built on the ADAPTIR (modular protein technology) platform, to redirect T-cell cytotoxicity toward prostate cancer cells by specifically targeting T cells through CD3ε to prostate cancer cells expressing PSMA (prostate-specific membrane antigen). In vitro cross-linking of T cells with PSMA-expressing tumor cells by MOR209/ES414 triggered potent target-dependent tumor lysis and induction of target-dependent T-cell activation and proliferation. This activity occurred at low picomolar concentrations of MOR209/ES414 and was effective at low T-effector to tumor target cell ratios. In addition, cytotoxic activity was equivalent over a wide range of PSMA expression on target cells, suggesting that as few as 3,700 PSMA receptors per cell are sufficient for tumor lysis. In addition to high sensitivity and in vitro activity, MOR209/ES414 induced limited production of cytokines compared with other bispecific antibody formats. Pharmacokinetic analysis of MOR209/ES414 demonstrated a serum elimination half-life in NOD/SCID γ (NSG) mice of 4 days. Administration of MOR209/ES414 in murine xenograft models of human prostate cancer significantly inhibited tumor growth, prolonged survival, and decreased serum prostate-specific antigen levels only in the presence of adoptively transferred human T cells. On the basis of these preclinical findings, MOR209/ES414 warrants further investigation as a potential therapeutic for the treatment of CRPC. Mol Cancer Ther; 15(9); 2155-65. ©2016 AACR. PMID:27406985

  6. Pharmacokinetics and Dosimetry Studies for Optimization of Pretargeted Radioimmunotherapy in CEA-Expressing Advanced Lung Cancer Patients

    Directory of Open Access Journals (Sweden)

    Caroline eBodet-Milin

    2015-11-01

    Full Text Available Objectives. A phase I pretargeted radioimmunotherapy trial (EudractCT 200800603096 was designed in patients with metastatic lung cancer expressing carcinoembryonic antigen (CEA to optimize bispecific antibody and labelled peptide doses, as well as the delay between their injections.Methods. Three cohorts of 3 patients received the anti-CEA x anti-histamine-succinyl-glycine (HSG humanized trivalent bispecific antibody (TF2 and the IMP288 bivalent HSG-peptide. Patients underwent a pre-therapeutic imaging session S1 (44 or 88 nmol/m2 of TF2 followed by 4.4 nmol/m2, 185 MBq, of 111In-labelled IMP288, and, 1-2 weeks later, a therapy session S2 (240 or 480 nmol/m2 of TF2 followed by 24 nmol/m2, 1.1 GBq/m2, 177Lu-labeled IMP288. The pretargeting delay was 24 or 48 hours. The dose schedule was defined based on pre-clinical TF2 pharmacokinetic studies, on our previous clinical data using the previous anti-CEA pretargeting system and on clinical results observed in the first patients injected using the same system in the Netherlands.Results. TF2 pharmacokinetics (PK was represented by a two-compartment model in which the central compartment volume was linearly dependent on the patient's surface area. PK were remarkably similar, with a clearance of 0.33 +/- 0.03 L/h per m2. 111In- and 177Lu-IMP288 PK were also well represented by a two-compartment model. IMP288 PK were faster (clearance 1.4 to 3.3 l/h. The central compartment volume was proportional to body surface area and IMP288clearance depended on the molar ratio of injected IMP288 to circulating TF2 at the time of IMP288 injection. Modelling of image quantification confirmed the dependence of IMP288 kinetics on circulating TF2, but tumour activity PK were variable. Organ absorbed doses were not significantly different in the 3 cohorts, but the tumour dose was significantly higher with the higher molar doses of TF2 (p < 0.002. S1 imaging predicted absorbed doses calculated in S2. Conclusion. The best

  7. Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle.

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    Katrin Spiesberger

    Full Text Available 30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested-mostly from the milk-of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4.With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody's activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M.Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma.

  8. XGFR*, a novel affinity-matured bispecific antibody targeting IGF-1R and EGFR with combined signaling inhibition and enhanced immune activation for the treatment of pancreatic cancer.

    Science.gov (United States)

    Schanzer, Juergen M; Wartha, Katharina; Moessner, Ekkehard; Hosse, Ralf J; Moser, Samuel; Croasdale, Rebecca; Trochanowska, Halina; Shao, Cuiying; Wang, Peng; Shi, Lei; Weinzierl, Tina; Rieder, Natascha; Bacac, Marina; Ries, Carola H; Kettenberger, Hubert; Schlothauer, Tilman; Friess, Thomas; Umana, Pablo; Klein, Christian

    2016-01-01

    The epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of IGF-1R and EGFR signaling in cancer therapy. We describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-IGF-1R/EGFR antibody XGFR*. XGFR* is based on the bispecific IgG antibody XGFR, which enabled heterodimerization of an IGF-1R binding scFab heavy chain with an EGFR-binding light and heavy chain by the "knobs-into-holes" technology. XGFR* is optimized for monovalent binding of human EGFR and IGF-1R with increased binding affinity for IGF-1R due to affinity maturation and highly improved protein stability to oxidative and thermal stress. It bears an afucosylated Fc-portion for optimal induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Stable Chinese hamster ovary cell clones with production yields of 2-3 g/L were generated, allowing for large scale production of the bispecific antibody. XGFR* potently inhibits EGFR- and IGF-1R-dependent receptor phosphorylation, reduces tumor cell proliferation in cells with heterogeneous levels of IGF-1R and EGFR receptor expression and induces strong ADCC in vitro. A comparison of pancreatic and colorectal cancer lines demonstrated superior responsiveness to XGFR*-mediated signaling and tumor growth inhibition in pancreatic cancers that frequently show a high degree of IGF-1R/EGFR co-expression. XGFR* showed potent anti-tumoral efficacy in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly complete tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a clinical development candidate for the treatment of pancreatic cancer. PMID:26984378

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

    Science.gov (United States)

    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

  10. TriFabs—Trivalent IgG-Shaped Bispecific Antibody Derivatives: Design, Generation, Characterization and Application for Targeted Payload Delivery

    Directory of Open Access Journals (Sweden)

    Klaus Mayer

    2015-11-01

    Full Text Available TriFabs are IgG-shaped bispecific antibodies (bsAbs composed of two regular Fab arms fused via flexible linker peptides to one asymmetric third Fab-sized binding module. This third module replaces the IgG Fc region and is composed of the variable region of the heavy chain (VH fused to CH3 with “knob”-mutations, and the variable region of the light chain (VL fused to CH3 with matching “holes”. The hinge region does not contain disulfides to facilitate antigen access to the third binding site. To compensate for the loss of hinge-disulfides between heavy chains, CH3 knob-hole heterodimers are linked by S354C-Y349C disulphides, and VH and VL of the stem region may be linked via VH44C-VL100C disulphides. TriFabs which bind one antigen bivalent in the same manner as IgGs and the second antigen monovalent “in between” these Fabs can be applied to simultaneously engage two antigens, or for targeted delivery of small and large (fluorescent or cytotoxic payloads.

  11. The Development of Bispecific Hexavalent Antibodies as a Novel Class of DOCK-AND-LOCKTM (DNLTM Complexes

    Directory of Open Access Journals (Sweden)

    Chien-Hsing Chang

    2013-05-01

    Full Text Available The DOCK-AND-LOCKTM (DNLTM method provides a modular approach to develop multivalent, multifunctional complexes of defined structures, of which bispecific hexavalent antibodies (bsHexAbs are prominent examples with potential applications in targeted therapy for malignant, autoimmune, and infectious diseases. Currently, bsHexAbs are constructed by derivatizing a divalent IgG, at the carboxyl termini of either the heavy chain (the CH3-format or the light chain (the Ck-format, to contain two stabilized dimers of Fab having a different specificity from the IgG. In this review, we briefly outline the features of the DNLTM method and describe key aspects of bsHexAbs examined with diverse preclinical studies, which include binding affinity to target cells, induction of signaling pathways, effector functions, serum stability, pharmacokinetics, and antitumor activity in human tumor xenograft models. Our findings favor the selection of the CK- over the CH3-format for further exploration of bsHexAbs in clinical trials.

  12. A Novel Bispecific Antibody against Human CD3 and Ephrin Receptor A10 for Breast Cancer Therapy.

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    Shintaro Taki

    Full Text Available Ephrin receptor A10 (EphA10, a transmembrane receptor that binds to ephrin, is a newly identified breast cancer marker protein that has also been detected in HER2-negative tissue. In this study, we report creation of a novel bispecific antibody (BsAb binding both EphA10 and CD3, thereby forming a bridge between antigens expressed on both tumor and immune cells and promoting recognition of tumor cells by immune cells and redirection of cytotoxic T cells (CTL. This BsAb (EphA10/CD3 was expressed in supernatants of BsAb gene-transfected cells as monomeric and dimeric molecules. Redirected T-cell lysis was observed when monomeric and dimeric BsAb were added to EphA10-overexpressing tumor cells in vitro. Furthermore, dimeric BsAb (EphA10/CD3 was more cytotoxic than monomeric BsAb, with efficient tumor cell lysis elicited by lower concentrations (≤10(-1 μg/mL and a lower effector to target (E/T cell ratio (E/T = 2.5. Dimeric BsAb (EphA10/CD3 also showed significant anti-tumor effects in human xenograft mouse models. Together, these results revealed opportunities to redirect the activity of CTL towards tumor cells that express EphA10 using the BsAb (EphA10/CD3, which could be tested in future clinical trials as a novel and potent therapeutic for breast cancer tumors.

  13. A Novel Bispecific Antibody against Human CD3 and Ephrin Receptor A10 for Breast Cancer Therapy.

    Science.gov (United States)

    Taki, Shintaro; Kamada, Haruhiko; Inoue, Masaki; Nagano, Kazuya; Mukai, Yohei; Higashisaka, Kazuma; Yoshioka, Yasuo; Tsutsumi, Yasuo; Tsunoda, Shin-ichi

    2015-01-01

    Ephrin receptor A10 (EphA10), a transmembrane receptor that binds to ephrin, is a newly identified breast cancer marker protein that has also been detected in HER2-negative tissue. In this study, we report creation of a novel bispecific antibody (BsAb) binding both EphA10 and CD3, thereby forming a bridge between antigens expressed on both tumor and immune cells and promoting recognition of tumor cells by immune cells and redirection of cytotoxic T cells (CTL). This BsAb (EphA10/CD3) was expressed in supernatants of BsAb gene-transfected cells as monomeric and dimeric molecules. Redirected T-cell lysis was observed when monomeric and dimeric BsAb were added to EphA10-overexpressing tumor cells in vitro. Furthermore, dimeric BsAb (EphA10/CD3) was more cytotoxic than monomeric BsAb, with efficient tumor cell lysis elicited by lower concentrations (≤10(-1) μg/mL) and a lower effector to target (E/T) cell ratio (E/T = 2.5). Dimeric BsAb (EphA10/CD3) also showed significant anti-tumor effects in human xenograft mouse models. Together, these results revealed opportunities to redirect the activity of CTL towards tumor cells that express EphA10 using the BsAb (EphA10/CD3), which could be tested in future clinical trials as a novel and potent therapeutic for breast cancer tumors. PMID:26678395

  14. Bispecific antibodies for treatment of cancer in experimental animal models and man

    NARCIS (Netherlands)

    Kroesen, Bart-Jan; Helfrich, Wijnand; Molema, Ingrid; de Leij, Lou

    1998-01-01

    Immunotherapy is a powerful anti-cancer treatment modality. However, despite numerous encouraging results obtained in pre-clinical studies, a definite breakthrough towards an established clinical treatment modality has as yet not occurred. Antibodies against tumor antigens have been shown to localis

  15. Rational Design and Characterization of the Novel, Broad and Potent Bispecific HIV-1 Neutralizing Antibody iMabm36

    Science.gov (United States)

    Sun, Ming; Pace, Craig S.; Yao, Xin; Yu, Faye; Padte, Neal N.; Huang, Yaoxing; Seaman, Michael S.; Li, Qihan; Ho, David D.

    2014-01-01

    While broadly neutralizing monoclonal antibodies (bNAbs) have always been considered potential therapeutic options for the prophylactic and treatment of HIV infection, their lack of breadth against all HIV variants has been one of the limiting factors. To provide sufficient neutralization breadth and potency against diverse viruses, including neutralization escape variants, strategies to combine different bNAbs have been explored recently. We rationally designed and engineered a novel bispecific HIV-1 neutralizing antibody (bibNAb), iMabm36, for high potency and breadth against HIV. iMabm36 is composed of the anti-CD4 Ab ibalizumab (iMab) linked to two copies of the single-domain Ab m36 which targets a highly conserved CD4-induced epitope. iMabm36 neutralizes a majority of a large, multi-clade panel of pseudoviruses (96%, n=118) at an IC50 concentration of less than 10 μg/mL, with 83% neutralized at an IC50 concentration of less than 0.1μg/ml. In addition, iMabm36 neutralizes six replication-competent transmitted-founder viruses to 100% inhibition at a concentration of less than 0.1μg/ml in a PBMC-based neutralizing assay. Mechanistically, improved antiviral activity of iMabm36 is dependent on both CD4 binding activity of iMab component and CD4i binding activity of the m36 component. After characterizing viral resistance to iMabm36 neutralization was due to mutations residing in the bridging sheet of gp120, an optimized m36 variant was engineered that, when fused to iMab, improved antiviral activity significantly. Together inter-dependency of this dual mechanism of action enables iMabm36 to potently inhibit HIV-1 entry. These results demonstrate that mechanistic-based design of bibNAbs could generate potential preventive and therapeutic candidates for HIV/AIDS. PMID:24853313

  16. Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/βKlotho Complex

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    Ganesh Kolumam

    2015-07-01

    Full Text Available Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR 1/βKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/βKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/βKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/βKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/βKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.

  17. Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/βKlotho Complex

    Science.gov (United States)

    Kolumam, Ganesh; Chen, Mark Z.; Tong, Raymond; Zavala-Solorio, Jose; Kates, Lance; van Bruggen, Nicholas; Ross, Jed; Wyatt, Shelby K.; Gandham, Vineela D.; Carano, Richard A.D.; Dunshee, Diana Ronai; Wu, Ai-Luen; Haley, Benjamin; Anderson, Keith; Warming, Søren; Rairdan, Xin Y.; Lewin-Koh, Nicholas; Zhang, Yingnan; Gutierrez, Johnny; Baruch, Amos; Gelzleichter, Thomas R.; Stevens, Dale; Rajan, Sharmila; Bainbridge, Travis W.; Vernes, Jean-Michel; Meng, Y. Gloria; Ziai, James; Soriano, Robert H.; Brauer, Matthew J.; Chen, Yongmei; Stawicki, Scott; Kim, Hok Seon; Comps-Agrar, Laëtitia; Luis, Elizabeth; Spiess, Christoph; Wu, Yan; Ernst, James A.; McGuinness, Owen P.; Peterson, Andrew S.; Sonoda, Junichiro

    2015-01-01

    Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/βKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/βKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/βKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/βKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/βKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin. PMID:26288846

  18. A first-in-human phase 1 study of ACE910, a novel factor VIII-mimetic bispecific antibody, in healthy subjects.

    Science.gov (United States)

    Uchida, Naoki; Sambe, Takehiko; Yoneyama, Koichiro; Fukazawa, Naoki; Kawanishi, Takehiko; Kobayashi, Shinichi; Shima, Midori

    2016-03-31

    ACE910 is a recombinant humanized bispecific antibody that binds to activated factor IX and factor X and mimics the cofactor function of factor VIII (FVIII). This first-in-human study examined the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ACE910 in healthy male adults. A total of 40 Japanese and 24 white subjects were randomized to receive a single subcutaneous injection of ACE910 (Japanese: 0.001, 0.01, 0.1, 0.3, or 1 mg/kg; white: 0.1, 0.3, or 1 mg/kg; n = 6 per dose group) or placebo (n = 2 per dose group). ACE910 exhibited a linear PK profile and had a half-life of ∼4 to 5 weeks. In FVIII-neutralized plasma, ACE910 shortened activated partial thromboplastin time and increased peak height of thrombin generation in a dose-dependent manner. All adverse events were nonserious and did not lead to any subject's withdrawal. Neither clinical findings nor laboratory abnormalities indicating hypercoagulability were observed. Two of 48 subjects receiving ACE910 (1 Japanese and 1 white) were positive for anti-ACE910 antibodies (anti-drug antibodies [ADAs]). One subject tested positive for ADAs both before and after ACE910 administration, whereas the other became ADA positive after receiving ACE910. The PK and PD profiles of ACE910 were similar in healthy Japanese and white subjects and suggest that ACE910 will be an effective and convenient prophylactic treatment of hemophilia A. This trial was registered at www.clinicaltrials.jp as #JapicCTI-121934. PMID:26626991

  19. Highly efficient elimination of colorectal tumor-initiating cells by an EpCAM/CD3-bispecific antibody engaging human T cells.

    Directory of Open Access Journals (Sweden)

    Ines Herrmann

    Full Text Available With their resistance to genotoxic and anti-proliferative drugs and potential to grow tumors and metastases from very few cells, cancer stem or tumor-initiating cells (TICs are a severe limitation for the treatment of cancer by conventional therapies. Here, we explored whether human T cells that are redirected via an EpCAM/CD3-bispecific antibody called MT110 can lyse colorectal TICs and prevent tumor growth from TICs. MT110 recognizes EpCAM, a cell adhesion molecule expressed on TICs from diverse human carcinoma, which was recently shown to promote tumor growth through engagement of elements of the wnt pathway. MT110 was highly potent in mediating complete redirected lysis of KRAS-, PI3 kinase- and BRAF-mutated colorectal TICs, as demonstrated in a soft agar assay. In immunodeficient mice, MT110 prevented growth of tumors from a 5,000-fold excess of a minimally tumorigenic TIC dose. T cells engaged by MT110 may provide a potent therapeutic means to eradicate TICs and bulk tumor cells derived thereof.

  20. Characterization of a novel single-chain bispecific antibody for retargeting of T cells to tumor cells via the TCR co-receptor CD8.

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    Irene Michalk

    Full Text Available There is currently growing interest in retargeting of effector T cells to tumor cells via bispecific antibodies (bsAbs. Usually, bsAbs are directed on the one hand to the CD3 complex of T cells and on the other hand to a molecule expressed on the surface of the target cell. A bsAb-mediated cross-linkage via CD3 leads to an activation of CD8+ T cells and consequently to killing of the target cells. In parallel, CD4+ T cells including TH1, TH2, TH17 cells and even regulatory T cells (Tregs will be activated as well. Cytokines produced by CD4+ T cells can contribute to severe side effects e. g. life-threatening cytokine storms and, thinking of the immunosupressive function of Tregs, can even be counterproductive. Therefore, we asked whether or not it is feasible to limit retargeting to CD8+ T cells e. g. via targeting of the co-receptor CD8 instead of CD3. In order to test for proof of concept, a novel bsAb with specificity for CD8 and a tumor-associated surface antigen was constructed. Interestingly, we found that pre-activated (but not freshly isolated CD8+ T cells can be retargeted via CD8-engaging bsAbs leading to an efficient lysis of target cells.

  1. Inhibitory Effect of Anti-HER-2 Anti-CD3 Bi-specific Antibody on the Growth of Gastric Carcinoma

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    To evaluate the effect of anti-HER-2 × anti-CD3 bi-specific antibody (BsAb) on the growth of HER-2/neu-expressing human gastric carcinoma in vitro and in vivo, an MTT assay was carried out to test the inhibitive rates of herceptin, anti-CD3 and BsAb antibodies on SGC-7901 gastric carcinoma cells. Immunocytochemistry methods were used to test the HER-2 level of SGC-7901. Nude mice models were employed to test the effect of HER-2 CD3 BsAb combined with effector cells( peripheral blood lymphatic cells of healthy human beings) on the growth of tumors in animals. Compared with that of the untreated control group, the tumor cell growth rates in vitro and in vivo will both be significantly inhibited when treated with effector cells combined with anti-CD3 McAb, herceptin or HER2 CD3 BsAb (p <0. 05), and the growth inhibition is the most remarkable in the group treated with HER2 CD3 BsAb combined with effector cells. The growth of tumor xenografts will also be significantly inhibited in the group treated with HER2CD3 BsAb combined with effector cells when compared with that in the group treated with anti-CD3 McAb or the group treated with herceptin combined with effector cells(p < 0. 05). We can conclude that HER-2/neu is possibly a useful target for immunotherapy of gastric carcinoma, and anti-HER2 × anti-CD3 BsAb has evident anti-tumor efficacy both, in vitro and in vivo.

  2. Immunotherapy of B-Cell Lymphoma with an Engineered Bispecific Antibody Targeting CD19 and CD5

    Directory of Open Access Journals (Sweden)

    Frank Breitling

    2013-05-01

    Full Text Available Using genetic engineering a humanized Fab fragment with specificity for CD19 was fused to a disulfide-stabilized single-chain antibody (dsFv recognizing CD5. This format should show reduced immunogenicity and improved tissue penetration. The specificity of bsAb FabCD19xdsFvCD5 binding to target cells was verified by flow cytometry on B and T lymphoma cell lines. Binding affinities of both arms were compared with the bivalent parental antibodies against CD19 and CD5 by binding competition assay. Redirected lysis of B lymphoma cells by preactivated PBMC from healthy donors was demonstrated in a chromium-release assay. A clear dose-response relationship could be established in the range from 1 ng/mL to 10 mg/mL bsAb. To evaluate the in vivo efficacy of bsAb FabCD19xdsFvCD5, NOD/SCID mice were intravenously injected with luciferase transfected Raji lymphoma cells together with pre-activated PBMC. Mice received five injections of therapeutic bsAb or control antibodies. While in the control groups all mice died within 40 to 50 days, 40% of bsAb treated animals survived longer than 60 days.

  3. 双特异性单抗对胃肠癌的体内杀伤作用%In vivo killing effect of bispecific single-chain antibody on gastric and colon cancer cells

    Institute of Scientific and Technical Information of China (English)

    刘洪一; 李荣; 刘巨超; 李力; 梁文涛; 钟苑; 徐迎新

    2012-01-01

    Objective To investigate the killing effect of CIK cells mediated by bispecific single - chain antibody, anti - CD3 and anti - CEA, on gastric and colon cancer cells in vivo. Methods Gastric and colon cancer models in nude mice were established after BGC823 cells from human poorly differentiated gastric adenocarcinoma and SW1116 cells from human moderately differentiated colon adenocarcinoma were planted into nude mice. The mice were divided into 3 groups, which were injected with normal saline, CIK cells and CIK cells plus bispecific antibody, respectively, once every 3 days for 6 times. Then the tumors were removed and weighed, and the inhibition rate in each group was calculated. Results Both the CIK cells and CIK cells plus bispecific single - chain antibody could inhibit tumor growth. Inhibition rates of gastric cancer were 29. 90% and 44. 33% respectively. Inhibition rates of colon cancer were 14. 93% and 38. 81% respectively. Compared with CIK cell group, the size and weight of the tumor were significantly smaller in CIK cells plus bispecific singlechain antibody group. Conclusion CIK cells alone can inhibit tumor cell growth and combination of CIK cells and the bispecific single - chain antibody, anti - CD3 and anti - CEA, is more effective in inhibiting tumor growth.%目的 探讨抗CD3抗CEA双特异性单链抗体介导CIK细胞在体内杀伤胃肠癌的作用.方法 分别将人胃低分化腺癌BGC823细胞和人结肠中分化腺癌SW1116细胞种植裸鼠后,建立裸鼠胃癌及肠癌模型,分为3组分别经尾静脉注入生理盐水、CIK细胞及CIK细胞+双特异性单抗,每3天治疗1次,共6次,取出肿瘤组织称重,并计算出各组的抑瘤率.结果 在体内,CIK组和CIK+双抗组均可抑制肿瘤生长,对胃癌的抑瘤率分别为29.90%和44.33%.对结肠癌的抑瘤率分别为14.93%和38.81%.CIK+双抗组:胃癌及结肠癌的瘤体积、瘤重明显小于CIK组.结论 CIK细胞本身可以抑制肿瘤

  4. Predictive patient-specific dosimetry and individualized dosing of pretargeted radioimmunotherapy in patients with advanced colorectal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Schoffelen, Rafke; Woliner-van der Weg, Wietske; Visser, Eric P.; Oyen, Wim J.G.; Boerman, Otto C. [Radboud University Medical Center, Department of Radiology and Nuclear Medicine, PO Box 9101, Nijmegen (Netherlands); Goldenberg, David M. [Garden State Cancer Center, Morris Plains, NJ (United States); Immunomedics, Inc., Morris Plains, NJ (United States); IBC Pharmaceuticals, Inc., Morris Plains, NJ (United States); Sharkey, Robert M.; McBride, William J.; Chang, Chien-Hsing [Immunomedics, Inc., Morris Plains, NJ (United States); Rossi, Edmund A. [IBC Pharmaceuticals, Inc., Morris Plains, NJ (United States); Graaf, Winette T.A. van der [Radboud University Medical Center, Department of Medical Oncology, Nijmegen (Netherlands)

    2014-08-15

    Pretargeted radioimmunotherapy (PRIT) with bispecific antibodies (bsMAb) and a radiolabeled peptide reduces the radiation dose to normal tissues. Here we report the accuracy of an {sup 111}In-labeled pretherapy test dose for personalized dosing of {sup 177}Lu-labeled IMP288 following pretargeting with the anti-CEA x anti-hapten bsMAb, TF2, in patients with metastatic colorectal cancer (CRC). In 20 patients bone marrow absorbed doses (BMD) and doses to the kidneys were predicted based on blood samples and scintigrams acquired after {sup 111}In-IMP288 injection for individualized dosing of PRIT with {sup 177}Lu-IMP288. Different dose schedules were studied, varying the interval between the bsMAb and peptide administration (5 days vs. 1 day), increasing the bsMAb dose (75 mg vs. 150 mg), and lowering the peptide dose (100 μg vs. 25 μg). TF2 and {sup 111}In/{sup 177}Lu-IMP288 clearance was highly variable. A strong correlation was observed between peptide residence times and individual TF2 blood concentrations at the time of peptide injection (Spearman's ρ = 0.94, P < 0.0001). PRIT with 7.4 GBq {sup 177}Lu-IMP288 resulted in low radiation doses to normal tissues (BMD <0.5 Gy, kidney dose <3 Gy). Predicted {sup 177}Lu-IMP288 BMD were in good agreement with the actual measured doses (mean ± SD difference -0.0026 ± 0.028 mGy/MBq). Hematological toxicity was mild in most patients, with only two (10 %) having grade 3-4 thrombocytopenia. A correlation was found between platelet toxicity and BMD (Spearman's ρ = 0.58, P = 0.008). No nonhematological toxicity was observed. These results show that individual high activity doses in PRIT in patients with CEA-expressing CRC could be safely administered by predicting the radiation dose to red marrow and kidneys, based on dosimetric analysis of a test dose of TF2 and {sup 111}In-IMP288. (orig.)

  5. Bispecific Antibody Conjugated Manganese-Based Magnetic Engineered Iron Oxide for Imaging of HER2/neu- and EGFR-Expressing Tumors

    Science.gov (United States)

    Wu, Shou-Cheng; Chen, Yu-Jen; Wang, Hsiang-Ching; Chou, Min-Yuan; Chang, Teng-Yuan; Yuan, Shyng-Shiou; Chen, Chiao-Yun; Hou, Ming-Feng; Hsu, John Tsu-An; Wang, Yun-Ming

    2016-01-01

    The overexpression of HER2/neu and EGFR receptors plays important roles in tumorigenesis and tumor progression. Targeting these two receptors simultaneously can have a more widespread application in early diagnosis of cancers. In this study, a new multifunctional nanoparticles (MnMEIO-CyTE777-(Bis)-mPEG NPs) comprising a manganese-doped iron oxide nanoparticle core (MnMEIO), a silane-amino functionalized poly(ethylene glycol) copolymer shell, a near infrared fluorescence dye (CyTE777), and a covalently conjugated anti-HER2/neu and anti-EGFR receptors bispecific antibody (Bis) were successfully developed. In vitro T2-weighted MR imaging studies in SKBR-3 and A431 tumor cells incubated with MnMEIO-CyTE777-(Bis)-mPEG NPs showed - 94.8 ± 3.8 and - 84.1 ± 2.8% negative contrast enhancement, respectively. Pharmacokinetics study showed that MnMEIO-CyTE777-(Bis)-mPEG NPs were eliminated from serum with the half-life of 21.3 mins. In vivo MR imaging showed that MnMEIO-CyTE777-(Bis)-mPEG NPs could specifically and effectively target to HER2/neu- and EGFR-expressing tumors in mice; the relative contrast enhancements were 11.8 (at 2 hrs post-injection) and 61.5 (at 24 hrs post-injection) fold higher in SKBR-3 tumors as compared to Colo-205 tumors. T2-weighted MR and optical imaging studies revealed that the new contrast agent (MnMEIO-CyTE777-(Bis)-mPEG NPs) could specifically and effectively target to HER2/neu- and/or EGFR-expressing tumors. Our results demonstrate that MnMEIO-CyTE777-(Bis)-mPEG NPs are able to recognize the tumors expressing both HER2/neu and/or EGFR, and may provide a novel molecular imaging tool for early diagnosis of cancers expressing HER2/neu and/or EGFR. PMID:26722378

  6. Bispecific engineered antibody domains (nanoantibodies that interact noncompetitively with an HIV-1 neutralizing epitope and FcRn.

    Directory of Open Access Journals (Sweden)

    Rui Gong

    Full Text Available Libraries based on an isolated human immunoglobulin G1 (IgG1 constant domain 2 (CH2 have been previously diversified by random mutagenesis. However, native isolated CH2 is not very stable and the generation of many mutations could lead to an increase in immunogenicity. Recently, we demonstrated that engineering an additional disulfide bond and removing seven N-terminal residues results in an engineered antibody domain (eAd (m01s with highly increased stability and enhanced binding to human neonatal Fc receptor (FcRn (Gong et al, JBC, 2009 and 2011. We and others have also previously shown that grafting of the heavy chain complementarity region 3 (CDR-H3 (H3 onto cognate positions of the variable domain leads to highly diversified libraries from which a number of binders to various antigens have been selected. However, grafting of H3s to non-cognate positions in constant domains results in additional residues at the junctions of H3s and the CH2 framework. Here we describe a new method based on multi-step PCR that allows the precise replacement of loop FG (no changes in its flanking sequences by human H3s from another library. Using this method and limited mutagenesis of loops BC and DE we generated an eAd phage-displayed library. Panning of this library against an HIV-1 gp41 MPER peptide resulted in selection of a binder, m2a1, which neutralized HIV-1 isolates from different clades with modest activity and retained the m01s capability of binding to FcRn. This result provides a proof of concept that CH2-based antigen binders that also mimic to certain extent other functions of full-size antibodies (binding to FcRn can be generated; we have previously hypothesized that such binders can be made and coined the term nanoantibodies (nAbs. Further studies in animal models and in humans will show how useful nAbs could be as therapeutics and diagnostics.

  7. Increase of PD-L1 expressing B-precursor ALL cells in a patient resistant to the CD19/CD3-bispecific T cell engager antibody blinatumomab.

    OpenAIRE

    Köhnke, Thomas; Krupka, Christina; Tischer, Johanna; Knösel, Thomas; Subklewe, Marion

    2015-01-01

    The bispecific T cell engager blinatumomab has shown encouraging clinical activity in B-precursor acute lymphoblastic leukemia (ALL). However, about half of relapsed/refractory patients do not respond to therapy. Here, we present the case of a 32-year-old male patient with refractory B-precursor ALL who was resistant to treatment with blinatumomab. Bone marrow immunohistochemistry revealed T cell infiltrates and an increase in programmed death-ligand 1 (PD-L1)-positive ALL cells as a potentia...

  8. Characterization of the first-in-class T-cell-engaging bispecific single-chain antibody for targeted immunotherapy of solid tumors expressing the oncofetal protein claudin 6

    Science.gov (United States)

    Stadler, Christiane R.; Bähr-Mahmud, Hayat; Plum, Laura M.; Schmoldt, Kathrin; Kölsch, Anne C.; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    abstract The fetal tight junction molecule claudin 6 (CLDN6) is virtually absent from any normal tissue, whereas it is aberrantly and frequently expressed in various cancers of high medical need. We engineered 6PHU3, a T-cell-engaging bispecific single chain molecule (bi-(scFv)2) with anti-CD3/anti-CLDN6 specificities, and characterized its pharmacodynamic properties. Our data show that upon engagement by 6PHU3, T cells strongly upregulate cytotoxicity and activation markers, proliferate and acquire an effector phenotype. 6PHU3 exerts potent killing of cancer cells in vitro with EC50 values in the pg/mL range. Subcutaneous xenograft tumors in NSG mice engrafted with human PBMCs are eradicated by 6PHU3 treatment and survival of mice is significantly prolonged. Tumors of 6PHU3-treated mice are strongly infiltrated with activated CD4+, CD8+ T cells and TEM type cells but not Tregs and display a general activation of a mostly inflammatory phenotype. These effects are only observed upon bispecific but not monospecific engagement of 6PHU3. Together with the exceptionally cancer cell selective expression of the oncofetal tumor marker CLDN6, this provides a safeguard with regard to toxicity. In summary, our data shows that the concept of T-cell redirection combined with that of highly selective targeting of CLDN6-positive solid tumors is effective. Thus, exploring 6PHU3 for clinical therapy is warranted. PMID:27141353

  9. (99m)Tc-bioorthogonal click chemistry reagent for in vivo pretargeted imaging.

    Science.gov (United States)

    García, María Fernanda; Zhang, Xiuli; Shah, Manankumar; Newton-Northup, Jessica; Cabral, Pablo; Cerecetto, Hugo; Quinn, Thomas

    2016-03-15

    Metal-free click chemistry has become an important tool for pretargeted approaches in the molecular imaging field. The application of bioorthogonal click chemistry between a pretargeted trans-cyclooctene (TCO) derivatized monoclonal antibody (mAb) and a (99m)Tc-modified 1,2,4,5-tetrazine for tumor imaging was examined in vitro and in vivo. The HYNIC tetrazine compound was synthesized and structurally characterized, confirming its identity. Radiolabeling studies demonstrated that the HYNIC tetrazine was labeled with (99m)Tc at an efficiency of >95% and was radiochemically stable. (99m)Tc-HYNIC tetrazine reacted with the TCO-CC49 mAb in vitro demonstrating its selective reactivity. In vivo biodistribution studies revealed non-specific liver and GI uptake due to the hydrophobic property of the compound, however pretargeted SPECT imaging studies demonstrated tumor visualization confirming the success of the cycloaddition reaction in vivo. These results demonstrated the potential of (99m)Tc-HYNIC-tetrazine for tumor imaging with pretargeted mAbs. PMID:26875936

  10. Rapid Detection of Hepatitis B Virus Surface Antigen by an Agglutination Assay Mediated by a Bispecific Diabody against Both Human Erythrocytes and Hepatitis B Virus Surface Antigen▿

    OpenAIRE

    Chen, Yu-Ping; Qiao, Yuan-Yuan; Zhao, Xiao-Hang; Chen, Hong-Song; Wang, Yan; Wang, Zhuozhi

    2007-01-01

    Bispecific antibodies have immense potential for use in clinical applications. In the present study, a bispecific diabody against human red blood cells (RBCs) and hepatitis B virus surface antigen (HBsAg) was used to detect HBsAg in blood specimens. The bispecific diabody was constructed by crossing over the variable region of the heavy chains and the light chains of anti-RBC and anti-HBsAg antibodies with a short linker, SRGGGS. In enzyme-linked immunosorbent assays, this bispecific diabody ...

  11. Increase of PD-L1 expressing B-precursor ALL cells in a patient resistant to the CD19/CD3-bispecific T cell engager antibody blinatumomab.

    Science.gov (United States)

    Köhnke, Thomas; Krupka, Christina; Tischer, Johanna; Knösel, Thomas; Subklewe, Marion

    2015-01-01

    The bispecific T cell engager blinatumomab has shown encouraging clinical activity in B-precursor acute lymphoblastic leukemia (ALL). However, about half of relapsed/refractory patients do not respond to therapy. Here, we present the case of a 32-year-old male patient with refractory B-precursor ALL who was resistant to treatment with blinatumomab. Bone marrow immunohistochemistry revealed T cell infiltrates and an increase in programmed death-ligand 1 (PD-L1)-positive ALL cells as a potential immune escape mechanism. We were able to recapitulate the clinical observation in vitro by showing that blinatumomab was not able to mediate cytotoxicity of CD19-positive ALL cells using autologous T cells. In contrast, the addition of healthy donor T cells led to lysis of ALL cells.These results strongly encourage further systematic evaluation of checkpoint molecules in cases of blinatumomab treatment failure and might highlight a possible mechanism to overcome resistance to this otherwise highly effective treatment. PMID:26449653

  12. Effect of bone marrow mesenchymal stem cells transplantation on fibroid myocardium with the aid of bispecific antibody%双特异抗体增效骨髓间充质干细胞干预心肌纤维化

    Institute of Scientific and Technical Information of China (English)

    邓玮; 陈庆伟; 李兴升; 刘灏; 牛司强; 周玥; 李桂琼; 柯大智; 莫显刚

    2011-01-01

    Objective: To investigate the effect of bone marrow mesenchymal stem cells ( BMSCs ) transplantation on fibroid myocardium with the aid of bispecific antibody ( BiAb ). Methods: BiAb [anti-CD29 × anti-myosin light chain antibody ( AMLCA )] was prepared and combined with isolated BMSCs from male mice and transfused into female mice with isoproterenol-induced myocardial fibrosis via tail vein. This study included five gronps: BMSCs+BiAb, BMSCs, BiAb, untreated, and control groups. Five weeks after treatment, expression levels of sex-determining region of Y-chromosome(SRY ), matrix metalloproteinases-9 (MMP-9), tissue inhibitor of metalloproteinase- 1 (TIMP-1) in myocardium were detected by fluorescent qRT-PCR. Collagen distribution was observed using sirius red staining. Results: Higher homing number of BMSCs was shown in the BMSCs+BiAb group than that of the BMSCs group (P<0.05). Compared with the untreated group, BMSCs+BiAb and BMSCs groups had decreased levels of MMP-9, TIMP-1 and collagen deposition (P<0.05).Decreased level of collagen content was significantly different in BMSCs+BiAb as compared to BMSCs group (P<0.05). Conclusion: Homing rate and repairing efficacy of BMSCs improve via treatment with combination of BiAb. BMSCs can improve MMP-TIMP expression in injured myocardium and interfere with myocardial fibrosis after homing.%目的:探讨在双特异抗体(Bispecific antibody,BiAb)的辅助下,骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)移植干预心肌纤维化的效果.方法:以anti-CD29(能识别间充质干细胞)和抗肌凝蛋白轻链抗体(Anti-myosin light chain antibody,AMLCA)(能特异性结合损伤心肌)制备BiAb(CD29×AMLCA).将此BiAb与雄性小鼠BMSCs经尾静脉输入异丙肾性心肌纤维化雌性小鼠(BMSCs+BiAb组).另设单纯BMSCs组、单纯BiAb组、正常对照组、未治疗组.5周后处死小鼠,荧光定量PCR检测心肌y染色体鉴别基因(Sex-determining region of Y-chromosome

  13. Passive immunization of pigs with bispecific llama single-domain antibody fragments against foot-and-mouth disease and porcine immunoglobulin

    NARCIS (Netherlands)

    Harmsen, M.M.; Fijten, H.P.D.; Dekker, A.; Eble, P.L.

    2008-01-01

    Foot-and-mouth disease (FMD) is a contagious viral disease of cloven-hoofed animals that occasionally causes outbreaks in Europe. We aim to develop an immunotherapy that confers rapid protection against FMD in outbreak situations. For this purpose, we previously isolated llama single-domain antibody

  14. Bispecific single-chain diabody-immunoliposomes targeting endoglin (CD105) and fibroblast activation protein (FAP) simultaneously.

    Science.gov (United States)

    Rabenhold, Markus; Steiniger, Frank; Fahr, Alfred; Kontermann, Roland E; Rüger, Ronny

    2015-03-10

    Liposomes are well-established drug delivery systems with cancer chemotherapy as main focus. To increase the cellular drug delivery, liposomes can be endowed with ligands, e.g. recombinant antibody fragments, which ensure specific cell interaction. Multispecific immunoliposomes can be prepared to improve the liposome to cell interaction by targeting multiple different targets at the same time, for instance by coupling two or more different ligands to the liposomal surface, resulting in a synergistic or additive increase in binding. An alternative approach is the use of bispecific ligands to address at least two different targets. For this purpose we cloned a single-chain diabody fragment (scDb`), a bispecific molecule targeting two antigens, endoglin (CD105) and fibroblast activation protein (FAP), expressed on cells of the tumor microenvironment. As model cell system, a human fibrosarcoma cell line was used expressing endoglin and FAP simultaneously. Monospecific immunoliposomes directed either against endoglin or FAP were compared in vitro for cell binding and cytotoxic activity with bispecific dual-targeted scFv`-IL (bispecific scFv`FAP/CD105-IL) and bispecific single-chain diabody`-IL (scDb`CD105/FAP-IL) targeting endoglin and FAP simultaneously. In the underlying study, bispecific scFv`FAP/CD105-IL interacted stronger with cells expressing FAP and endoglin (both targets simultaneously) compared to the monospecific immunoliposomes. Furthermore, bispecific scDb`-immunoliposomes increased the cell interaction massively and showed enhanced cytotoxicity against target cells using doxorubicin-loaded immunoliposomes. The use of recombinant bispecific ligands as scDb`-molecules facilitates the generation of bispecific immunoliposomes by using the established post-insertion technique, enabling an extension of the ligand specificity spectrum via genetic modification.

  15. Establishment and identification of bispecific monoclonal antibody against three kinds of β-agonists%抗3种β-激动剂双特异性单克隆抗体的建立及鉴定

    Institute of Scientific and Technical Information of China (English)

    余厚美; 崔廷婷; 冯才伟; 万宇平

    2012-01-01

    To prepare and identify bispecific monoclonal antibody (BsAb) against three kinds of β-agonists (clenbuterol, salbutamol, ractopamine ). Hybridoma cells secreting anti-clenbuterol and salbutamol mAb and hybridoma cells secreting anti-ractopamine mAb were naturalized respectively, making them sensitive to HAT. Then the two hybridoma cells sensitive to HAT were fused and hybrid-hybridoma cells secreting the BsAb were screened and identified. S hybrid-hybridoma cell lines were obtained. The sensitivities and specificities of BsAb were analyzed by ELISA, showed that the IC50 was 1 ng/mL,the liter of ascites were over 105. They can only react with clenbuterol, salbutamol and ractopamine. This method had higher sensitivity and simplicity, lower risk, and can be used widely.%制备抗克仑特罗、沙丁胺醇和莱克多巴胺双特异性抗体并初步鉴定.将分泌抗克仑特罗、沙丁胺醇抗体的细胞株和分泌抗莱克多巴胺抗体的细胞株分别驯化,使之成为HAT敏感株.将两者融合,筛选分泌双特异性单克隆抗体的杂交-杂交瘤株,然后对其进行初步鉴定.共获得5株杂交-杂交瘤细胞株,将其分泌的双特异性单克隆抗体(BsAb)经过酶联免疫吸附法( ELISA)初步检测,IC50可达1 ng/mL,腹水效价为105以上,用ELISA法证明BsAb只与克仑特罗、沙丁胺醇和莱克多巴胺有特异性反应.该方法制备简单、风险低、灵敏度高、周期短,具有较好的应用前景.

  16. A novel trifunctional IgG-like bispecific antibody to inhibit HIV-1 infection and enhance lysis of HIV by targeting activation of complement

    Directory of Open Access Journals (Sweden)

    Tomlinson Stephen

    2010-06-01

    Full Text Available Abstract Background The complement system is not only a key component of innate immunity but also provides a first line of defense against invading pathogens, especially for viral pathogens. Human immunodeficiency virus (HIV, however, possesses several mechanisms to evade complement-mediated lysis (CoML and exploit the complement system to enhance viral infectivity. Responsible for this intrinsic resistance against complement-mediated virolysis are complement regulatory membrane proteins derived from the host cell that inherently downregulates complement activation at several stages of the cascade. In addition, HIV is protected from complement-mediated lysis by binding soluble factor H (fH through the viral envelope proteins, gp120 and gp41. Whereas inhibition of complement activity is the desired outcome in the vast majority of therapeutic approaches, there is a broader potential for complement-mediated inhibition of HIV by complement local stimulation. Presentation of the hypothesis Our previous studies have proven that the complement-mediated antibody-dependent enhancement of HIV infection is mediated by the association of complement receptor type 2 bound to the C3 fragment and deposited on the surface of HIV virions. Thus, we hypothesize that another new activator of complement, consisting of two dsFv (against gp120 and against C3d respectively linked to a complement-activating human IgG1 Fc domain ((anti-gp120 × anti-C3d-Fc, can not only target and amplify complement activation on HIV virions for enhancing the efficiency of HIV lysis, but also reduce the infectivity of HIV through blocking the gp120 and C3d on the surface of HIV. Testing the hypothesis Our hypothesis was tested using cell-free HIV-1 virions cultivated in vitro and assessment of virus opsonization was performed by incubating appropriate dilutions of virus with medium containing normal human serum and purified (anti-gp120 × anti-C3d-Fc proteins. As a control group, viruses

  17. Pretargeting in tumored mice with radiolabeled morpholino oligomer showing low kidney uptake

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Guozheng; He, Jiang; Dou, Shuping; Gupta, Suresh; Vanderheyden, Jean-Luc; Rusckowski, Mary; Hnatowich, Donald J. [Division of Nuclear Medicine, Department of Radiology, University of Massachusetts Medical School, 55 Lake Avenue North, MA 01655-0243, Worcester (United States)

    2004-03-01

    We have recently shown that accumulation in mouse kidneys of technetium-99m labeled phosphorodiamidate morpholinos (MORFs) increases with the number of cytosines in the base sequence. To improve tumor/kidney ratios in tumored mice, pretargeting studies were performed with a cytosine-free MORF. An 18-mer MORF (5'-TCTTCTACTTCACAACTA) was conjugated to the anti-CEA antibody MN14 (Immunomedics) and administered to nude mice bearing LS174T tumors. Thereafter, the {sup 99m}Tc-labeled cytosine-free cMORF (5'-TAGTTGTGAAGTAGAAGA-amide-MAG{sub 3}) was administered. For comparison, the identical study was repeated but with our original pair of 18-mer MORFs (5'-GGGTGTACGTCACAACTA-conjugated MN14 and {sup 99m}Tc-labeled 5'-TAGTTGTGACGTACACCC-amide-MAG{sub 3}). Surface plasmon resonance was used to show that the hybridization affinities of the original and the modified pair of MORFs were essentially equal. Hybridization of the cytosine-free cMORF-{sup 99m}Tc to MN14-MORF was demonstrated in vitro by size-exclusion high-performance liquid chromatography. At 3 h, kidney levels in normal mice were 2.0%ID/organ for the modified cMORF vs. 4.1%ID/organ for the original cMORF sequence, while at 24 h, these values were 0.9% vs 1.8%ID/organ. Pretargeting studies in tumored mice receiving 25 {mu}g of conjugated antibody, 0.5 {mu}g of labeled cMORF 48 h later, followed by imaging and sacrifice at 3 h showed that kidney levels were reduced using the cytosine-free cMORF. Moreover, tumor accumulation was about 3.6%ID/g and was independent of sequence. The whole-body images clearly reflected the improved tumor to kidney ratios. By choosing a cytosine-free base sequence for pretargeting studies, kidney accumulation of cMORF-{sup 99m}Tc was reduced without adversely influencing tumor accumulation. The lowering of kidney radioactivity levels in this way may be important to reduce toxicity to this organ in connection with pretargeting radiotherapy studies. (orig.)

  18. Pretargeting in tumored mice with radiolabeled morpholino oligomer showing low kidney uptake.

    Science.gov (United States)

    Liu, Guozheng; He, Jiang; Dou, Shuping; Gupta, Suresh; Vanderheyden, Jean-Luc; Rusckowski, Mary; Hnatowich, Donald J

    2004-03-01

    We have recently shown that accumulation in mouse kidneys of technetium-99m labeled phosphorodiamidate morpholinos (MORFs) increases with the number of cytosines in the base sequence. To improve tumor/kidney ratios in tumored mice, pretargeting studies were performed with a cytosine-free MORF. An 18-mer MORF (5'-TCTTCTACTTCACAACTA) was conjugated to the anti-CEA antibody MN14 (Immunomedics) and administered to nude mice bearing LS174T tumors. Thereafter, the (99m)Tc-labeled cytosine-free cMORF (5'-TAGTTGTGAAGTAGAAGA-amide-MAG(3)) was administered. For comparison, the identical study was repeated but with our original pair of 18-mer MORFs (5'-GGGTGTACGTCACAACTA-conjugated MN14 and (99m)Tc-labeled 5'-TAGTTGTGACGTACACCC-amide-MAG(3)). Surface plasmon resonance was used to show that the hybridization affinities of the original and the modified pair of MORFs were essentially equal. Hybridization of the cytosine-free cMORF-(99m)Tc to MN14-MORF was demonstrated in vitro by size-exclusion high-performance liquid chromatography. At 3 h, kidney levels in normal mice were 2.0%ID/organ for the modified cMORF vs. 4.1%ID/organ for the original cMORF sequence, while at 24 h, these values were 0.9% vs 1.8%ID/organ. Pretargeting studies in tumored mice receiving 25 microg of conjugated antibody, 0.5 microg of labeled cMORF 48 h later, followed by imaging and sacrifice at 3 h showed that kidney levels were reduced using the cytosine-free cMORF. Moreover, tumor accumulation was about 3.6%ID/g and was independent of sequence. The whole-body images clearly reflected the improved tumor to kidney ratios. By choosing a cytosine-free base sequence for pretargeting studies, kidney accumulation of cMORF-(99m)Tc was reduced without adversely influencing tumor accumulation. The lowering of kidney radioactivity levels in this way may be important to reduce toxicity to this organ in connection with pretargeting radiotherapy studies. PMID:14691611

  19. Bifunctional phage-based pretargeted imaging of human prostate carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Newton-Northup, Jessica R. [Department of Biochemistry, University of Missouri, Columbia, MO 65211 (United States)], E-mail: newtonj@missouri.edu; Figueroa, Said D. [Department of Radiology, University of Missouri, Columbia, MO 65211 (United States); Quinn, Thomas P.; Deutscher, Susan L. [Department of Biochemistry, University of Missouri, Columbia, MO 65211 (United States); Research Service, Harry S. Truman Veterans Memorial Hospital, Columbia, MO 65201 (United States)

    2009-10-15

    Introduction: Two-step and three-step pretargeting systems utilizing biotinylated prostate tumor-homing bacteriophage (phage) and {sup 111}In-radiolabeled streptavidin or biotin were developed for use in cancer radioimaging. The in vivo selected prostate carcinoma-specific phage (G1) displaying up to five copies of the peptide IAGLATPGWSHWLAL was the focus of the present study. Methods: The ability of G1 phage to extravasate and target prostate tumor cells was investigated using immunohistochemistry. G1 phages were biotinylated, streptavidin was conjugated to diethylenetriaminepentaacetic acid (DTPA) and biotin was conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Biodistribution studies and single-photon emission computed tomography (SPECT)/CT imaging of xenografted PC-3 tumors via two-step pretargeted {sup 111}In-labeled streptavidin and three-step pretargeted {sup 111}In-labeled biotin were performed in SCID mice to determine the optimal pretargeting method. Results: The ability of G1 phage to extravasate the vasculature and bind directly to human PC-3 prostate carcinoma tumor cells in vivo was demonstrated via immunocytochemical analysis. Comparative biodistribution studies of the two-step and three-step pretargeting strategies indicated increased PC-3 human prostate carcinoma tumor uptake in SCID mice of 4.34{+-}0.26 %ID g{sup -1} at 0.5 h postinjection of {sup 111}In-radiolabeled biotin (utilized in a three-step protocol) compared to 0.67{+-}0.06 %ID g{sup -1} at 24 h postinjection of {sup 111}In radiolabeled streptavidin (employed in a two-step protocol). In vivo SPECT/CT imaging of xenografted PC-3 tumors in SCID mice with the three-step pretargeting method was superior to that of the two-step pretargeting method, and, importantly, blocking studies demonstrated specificity of tumor uptake of {sup 111}In-labeled biotin in the three-step pretargeting scheme. Conclusion: This study demonstrates the use of multivalent bifunctional

  20. Optimization of a Pretargeted Strategy for the PET Imaging of Colorectal Carcinoma via the Modulation of Radioligand Pharmacokinetics.

    Science.gov (United States)

    Zeglis, Brian M; Brand, Christian; Abdel-Atti, Dalya; Carnazza, Kathryn E; Cook, Brendon E; Carlin, Sean; Reiner, Thomas; Lewis, Jason S

    2015-10-01

    Pretargeted PET imaging has emerged as an effective strategy for merging the exquisite selectivity of antibody-based targeting vectors with the rapid pharmacokinetics of radiolabeled small molecules. We previously reported the development of a strategy for the pretargeted PET imaging of colorectal cancer based on the bioorthogonal inverse electron demand Diels-Alder reaction between a tetrazine-bearing radioligand and a transcyclooctene-modified huA33 immunoconjugate. Although this method effectively delineated tumor tissue, its clinical potential was limited by the somewhat sluggish clearance of the radioligand through the gastrointestinal tract. Herein, we report the development and in vivo validation of a pretargeted strategy for the PET imaging of colorectal carcinoma with dramatically improved pharmacokinetics. Two novel tetrazine constructs, Tz-PEG7-NOTA and Tz-SarAr, were synthesized, characterized, and radiolabeled with (64)Cu in high yield (>90%) and radiochemical purity (>99%). PET imaging and biodistribution experiments in healthy mice revealed that although (64)Cu-Tz-PEG7-NOTA is cleared via both the gastrointestinal and urinary tracts, (64)Cu-Tz-SarAr is rapidly excreted by the renal system alone. On this basis, (64)Cu-Tz-SarAr was selected for further in vivo evaluation. To this end, mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts were administered huA33-TCO, and the immunoconjugate was given 24 h to accumulate at the tumor and clear from the blood, after which (64)Cu-Tz-SarAr was administered via intravenous tail vein injection. PET imaging and biodistribution experiments revealed specific uptake of the radiotracer in the tumor at early time points (5.6 ± 0.7 %ID/g at 1 h p.i.), high tumor-to-background activity ratios, and rapid elimination of unclicked radioligand. Importantly, experiments with longer antibody accumulation intervals (48 and 120 h) yielded slight decreases in tumoral uptake but also concomitant

  1. Biotinylated magnetic nanoparticles for pretargeting: synthesis and characterization study

    OpenAIRE

    Chauhan, Ram Prakash; Singh, Gurjaspreet; Singh, Sweta; Bag, Narmada; Patra, Manoj; S. R. Vadera; Mishra, Anil K.; Mathur, Rashi

    2011-01-01

    In this paper, we have proposed a simple method to covalently conjugate biotin to magnetic nanoparticles, which can be targeted to the tumour sites by using pretargeting approach with avidin or streptavidin. Magnetic nanoparticles of manganese ferrite were synthesized by alkaline coprecipitation of ferric chloride hexahydrate, ferrous sulphate heptahydrate and manganese sulphate monohydrate using ammonium hydroxide. The synthesized magnetic nanoparticles were then successfully surface modifie...

  2. A novel platform for engineering blood-brain barrier-crossing bispecific biologics.

    Science.gov (United States)

    Farrington, Graham K; Caram-Salas, Nadia; Haqqani, Arsalan S; Brunette, Eric; Eldredge, John; Pepinsky, Blake; Antognetti, Giovanna; Baumann, Ewa; Ding, Wen; Garber, Ellen; Jiang, Susan; Delaney, Christie; Boileau, Eve; Sisk, William P; Stanimirovic, Danica B

    2014-11-01

    The blood-brain barrier (BBB) prevents the access of therapeutic antibodies to central nervous system (CNS) targets. The engineering of bispecific antibodies in which a therapeutic "arm" is combined with a BBB-transcytosing arm can significantly enhance their brain delivery. The BBB-permeable single-domain antibody FC5 was previously isolated by phenotypic panning of a naive llama single-domain antibody phage display library. In this study, FC5 was engineered as a mono- and bivalent fusion with the human Fc domain to optimize it as a modular brain delivery platform. In vitro studies demonstrated that the bivalent fusion of FC5 with Fc increased the rate of transcytosis (Papp) across brain endothelial monolayer by 25% compared with monovalent fusion. Up to a 30-fold enhanced apparent brain exposure (derived from serum and cerebrospinal fluid pharmacokinetic profiles) of FC5- compared with control domain antibody-Fc fusions after systemic dosing in rats was observed. Systemic pharmacological potency was evaluated in the Hargreaves model of inflammatory pain using the BBB-impermeable neuropeptides dalargin and neuropeptide Y chemically conjugated with FC5-Fc fusion proteins. Improved serum pharmacokinetics of Fc-fused FC5 contributed to a 60-fold increase in pharmacological potency compared with the single-domain version of FC5; bivalent and monovalent FC5 fusions with Fc exhibited similar systemic pharmacological potency. The study demonstrates that modular incorporation of FC5 as the BBB-carrier arm in bispecific antibodies or antibody-drug conjugates offers an avenue to develop pharmacologically active biotherapeutics for CNS indications. PMID:25070367

  3. Empowered Antibody Therapies - IBC conference.

    Science.gov (United States)

    Herold, Jens

    2010-10-01

    The Empowered Antibody Therapies conference, held in Burlingame, CA, USA, included topics covering new therapeutic developments in the field of multispecific antibodies. This conference report highlights selected presentations on DVD-Igs from Abbott Laboratories, ImmTACs from Immunocore, 'Dock-and-Lock' technology from Immunomedics, the bispecific BiTE antibody blinatumomab from Micromet, and Triomabs from TRION Pharma and Fresenius Biotech. PMID:20878591

  4. Immunotherapy of anti-CD3/anti-EGFR bispecific antibody on the mice borne human gastric cancer%抗EGFR/抗CD3双功能抗体对胃癌荷瘤小鼠的免疫治疗研究

    Institute of Scientific and Technical Information of China (English)

    张林; 侯艳红; 张健; 胡静; 张静

    2012-01-01

    目的 通过抗EGFR/抗CD3双功能抗体(EGFR/CD3 BsAb)治疗SGC7901胃癌细胞移植瘤小鼠模型,初步验证该抗体在体内对胃癌细胞的杀伤能力.方法 采取化学偶联法合成的EGFR/CD3 BsAb联合人外周血淋巴细胞(PBLS)经尾静脉给药注入荷5GC7901胃癌细胞移植瘤小鼠体内.实验裸鼠随机分为抗EGFR单抗联合PBLS组(抗EGFR组)、抗CD3单抗联合PBIS组(抗CD3组)、EGFR/CD3 BsAb联合PBLS组(EGFR/CD3 BsAb组)和空白对照组(生理盐水组).治疗后检测并比较加药各组对胃癌细胞的杀伤能力.结果 EGFR/CD3 BsAb成功制备,分子量为43kD.ECFR/CD3 BsAb组裸鼠的肿瘤抑制率为(57.2±8.6)%,显著高于抗EGFR组的(38.5±6.1)%和抗CD3组的(6.9±7.6)%(P<0.05);治疗结束时EGFR/CD3 BsAb组的瘤重为(517.1±45.4)mg,显著低于抗EGFR组的(737.4±54.3)mg和抗CD3组的(1097.9±167.7)mg(P<0.05).各组移植瘤组织EGFR免疫组化染色均为强阳性.EGFR/CD3 BsAb组裸鼠肿瘤组织CD3免疫组化染色可见部分细胞呈阳性.透射电镜观察显示,EGFR/CD3 BsAb组和抗EGFR组可见肿瘤坏死.结论 EGFR/CD3 BsAb在体内条件下可能对胃癌有治疗作用.%Objective To investigate the effect of the immunotherapy mediated by anti-CD3/anti-EGFR bispecific antibody ( BsAb) on the mice bome human gastric cancer and evaluate the effect of anti-CD3/anti-EGFR BsAb as a targeted therapeutic agent for gastric cancer. Methods The monoclonal antibody(mAb) of anti-CD3 and anti-EGFR were cross-linked to prepare the BsAb by chemical synthesis. The experimental therapy combined with PBLS as effector cells on the mice borne SGC7901 human gastric cancer was performed. Experimental mice were divided in to EGFR mAb group, CD3 mAb group, anti-CD3/anti-EGFR BsAb group and saline group. The comparisons of the curative activity among the anti-CD3/anti-EGFR BsAb, EGFR mAb and CD3 mAb groups were conducted in vivo. Results Anti-CD3/anti-EGFR BsAb was established successfully, and

  5. Bone marrow mesenchymal stem cells with support of ultrasound-mediated microbubbles and bispecific antibody prevent myocardial fibrosis via HSP-70%超声微泡联合双特异性抗体对骨髓间充质干细胞干预心肌纤维化的实验研究

    Institute of Scientific and Technical Information of China (English)

    邓玮; 陈庆伟; 王志刚; 李兴升; 刘灏; 周玥; 李桂琼; 柯大智; 莫显刚

    2011-01-01

    Objective To explore the effects of transplantated bone marrow mesenchymal stem cells (MSCs) on myocardial fibrosis with the aid of ultrasound-mediated microbubbles (MB) and bispecific antibody(BiAb) combination.Methods With the aid of MB,isolated MSCs from male mice and the BiAb were transfused into female mice with isoproterenol-induced myocardial fibrosis via tail vein (MSCs + BiAb + MB group).BiAb was producted with anti-CD29 which can recognize MSCs and anti-myosin light chain antibody (AMLCA) which can specifically bind to injured myocardium.There were six groups investigated:MSC + BiAb + MB,MSC,BiAb,MB,MSC + BiAb,untreated,and control groups.Five weeks after treatment administration,the expressions of sex-determining region of Y-chromosome (SRY) in myocardium were detected by fluorescent quantitative PCR.The distribution of collagen was observed by sirius red staining.Heat shock protein-70 (HSP-70) in myocardium was detected by immunohistochemistry.Results The highest homing number of MSCs was in the MSCs + BiAb + MB group,MSCs + BiAb group ranked the second,and lowest in MSCs group.Compared to the untreated group,the MSCs + BiAb + MB,MSCs + BiAb and MSCs groups had less collagen deposition (P <0.05),and decreased level of HSP-70.Compared to those of the MSCs group,the collagen deposition were decreased in MSCs + BiAb + MB group (P <0.05).Conclusions MB and BiAb can promote the homing number of MSCs in mice.MB can further the homing rate and the repairing efficacy of MSCs.The homing MSCs can prevent the process of myocardial fibrosis.And HSP-70 was involved in the internal mechanism.%目的 探讨骨髓间充质干细胞(MSCs)在超声辐照微泡(MB)和双特异抗体(BiAb)的辅助下干预心肌纤维化的效果及可能机制.方法 在MB的辅助下,将雄性小鼠MSCs与BiAb输入异丙肾性心肌纤维化雌性小鼠(MSCs+BiAb+MB组).BiAb由能识别间充质干细胞的anti-CD29和能特异性结合损伤心肌的抗肌凝蛋白轻链抗

  6. Comparative efficacy of 177Lu and 90Y for anti-CD20 pretargeted radioimmunotherapy in murine lymphoma xenograft models.

    Directory of Open Access Journals (Sweden)

    Sofia H L Frost

    Full Text Available Pretargeted radioimmunotherapy (PRIT is a multi-step method of selectively delivering high doses of radiotherapy to tumor cells while minimizing exposure to surrounding tissues. Yttrium-90 (90Y and lutetium-177 (177Lu are two of the most promising beta-particle emitting radionuclides used for radioimmunotherapy, which despite having similar chemistries differ distinctly in terms of radiophysical features. These differences may have important consequences for the absorbed dose to tumors and normal organs. Whereas 90Y has been successfully applied in a number of preclinical and clinical radioimmunotherapy settings, there have been few published pretargeting studies with 177Lu. We therefore compared the therapeutic potential of targeting either 90Y or 177Lu to human B-cell lymphoma xenografts in mice.Parallel experiments evaluating the biodistribution, imaging, dosimetry, therapeutic efficacy, and toxicity were performed in female athymic nude mice bearing either Ramos (Burkitt lymphoma or Granta (mantle cell lymphoma xenografts, utilizing an anti-CD20 antibody-streptavidin conjugate (1F5-SA and an 90Y- or 177Lu-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-biotin second step reagent.The two radionuclides displayed comparable biodistributions in tumors and normal organs; however, the absorbed radiation dose delivered to tumor was more than twice as high for 90Y (1.3 Gy/MBq as for 177Lu (0.6 Gy/MBq. More importantly, therapy with 90Y-DOTA-biotin was dramatically more effective than with 177Lu-DOTA-biotin, with 100% of Ramos xenograft-bearing mice cured with 37 MBq 90Y, whereas 0% were cured using identical amounts of 177Lu-DOTA-biotin. Similar results were observed in mice bearing Granta xenografts, with 80% of the mice cured with 90Y-PRIT and 0% cured with 177Lu-PRIT. Toxicities were comparable with both isotopes.90Y was therapeutically superior to 177Lu for streptavidin-biotin PRIT approaches in these human lymphoma

  7. Radioimmunotherapy with engineered antibody fragments

    International Nuclear Information System (INIS)

    Authors have developed and begun evaluating radiometal-chelated (213Bi) engineered antibody fragments as radioimmunotherapy agents that target the HER2/neu (c-erbB-2) antigen. The diabody format was found to have 40-fold greater affinity for HER2/neu and to be associated with significantly greater tumor localization than is achieved with scFv molecule. It is shown that short-lived isotopes like 213Bi would be most effective when used in conjunction with antibodies that targeted diffuse malignancies (leukemia or lymphoma) or when used for very rapid pretargeted radioimmunotherapy application in which the radioisotope is conjugated to a very small ligand

  8. Reducing toxicity of 4–1BB costimulation: targeting 4–1BB ligands to the tumor stroma with bi-specific aptamer conjugates

    Science.gov (United States)

    Schrand, B; Berezhnoy, A; Brenneman, R; Williams, A; Levay, A; Gilboa, E

    2015-01-01

    Systemic administration of immune modulatory antibodies to cancer patients is associated with autoimmune pathologies. We have developed a clinically feasible and broadly applicable approach to limit immune stimulation to disseminated tumor lesions using a bi-specific agonistic 4–1BB oligonucleotide aptamer targeted to a broadly expressed stromal product (e.g., VEGF or osteopontin). The stroma-targeted aptamer conjugates engendered potent antitumor immunity against unrelated tumors and exhibited a superior therapeutic index compared to non-targeted agonistic 4–1BB antibody. PMID:25949891

  9. Survival among patients with advanced renal cell carcinoma in the pretargeted versus targeted therapy eras.

    Science.gov (United States)

    Li, Pengxiang; Wong, Yu-Ning; Armstrong, Katrina; Haas, Naomi; Subedi, Prasun; Davis-Cerone, Margaret; Doshi, Jalpa A

    2016-02-01

    Between December 2005 and October 2009, FDA approved six targeted therapies shown to significantly extend survival for advanced renal cell carcinoma (RCC) patients in clinical trials. This study aimed to examine changes in survival between the pretargeted and targeted therapy periods in advanced RCC patients in a real-world setting. Utilizing the 2000-2010 SEER Research files, a pre-post study design with a contemporaneous comparison group was employed to examine differences in survival outcomes for patients diagnosed with advanced RCC (study group) or advanced prostate cancer (comparison group, for whom no significant treatment innovations happened during this period) across the pretargeted therapy era (2000-2005) and the targeted therapy era (2006-2010). RCC patients diagnosed in the targeted therapy era (N = 6439) showed improved survival compared to those diagnosed in the pretargeted therapy era (N = 7231, hazard ratio (HR) for all-cause death: 0.86, P < 0.01), while the change between the pre-post periods was not significant for advanced prostate cancer patients (HR: 0.97, P = 0.08). Advanced RCC patients had significantly larger improvements in overall survival compared to advanced prostate cancer patients (z = 4.31; P < 0.01). More detailed year-to-year analysis revealed greater survival improvements for RCC in the later years of the posttargeted period. Similar results were seen for cause-specific survival. Subgroup analyses by nephrectomy status, age, and gender showed consistent findings. Patients diagnosed with advanced RCC during the targeted therapy era had better survival outcomes than those diagnosed during the pretargeted therapy era. Future studies should examine the real-world survival improvements directly associated with targeted therapies. PMID:26645975

  10. An improved method for covalently conjugating morpholino oligomers to antitumor antibodies.

    Science.gov (United States)

    He, Jiang; Liu, Guozheng; Dou, Shuping; Gupta, Suresh; Rusckowski, Mary; Hnatowich, Donald

    2007-01-01

    Whether for conventional pretargeting, amplification pretargeting, or affinity enhancement pretargeting, it will be necessary to conjugate an antitumor antibody as the first injectate. This laboratory is investigating phosphorodiamidate morpholinos (MORFs) for pretargeting, and accordingly we are examining methods of attaching MORFs to antitumor antibodies that provide at least one group per molecule (gpm) without adversely influencing antibody properties. The aim of this investigation was to evaluate the commercial Hydralink for the conjugation of the anti-CEA MN14 antibody with an 18 mer amine-derivatized MORF. The conjugation was approached in both directions by first reacting MN14 with the NHS derivatives of 4-hydrozinonicotinate acetone hydrazone (SANH) or 4-formylbenzoate (SFB) and then combining with MORF that was previously reacted with SFB or SANH to yield MN14(SANH)-MORF and MN14(SFB)-MORF respectively. The storage stability, immunoreactive fraction, and the biodistribution in normal mice were compared for both conjugates. Thereafter, MN14(SANH)-MORF was used in a pretargeting study in tumored nude mice, and the results were compared to that obtained historically with MN14-MORF prepared by carbodiimide (EDC) coupling. Both new methods of conjugation provided between 1 and 2 gpm compared to 0.2 achieved previously by EDC. Furthermore, by repeat SE HPLC with and without CEA, both showed an unimpaired immunoreactive fraction. MN14(SANH)-MORF tolerated long-term storage best. More importantly, when labeled by hybridization with 99mTc-labeled complementary MORF (99mTc-cMORF), the biodistribution of MN14(SANH)-MORF was more favorable than that of MN14(SFB)-MORF in normal mice with lower liver (5.7 vs 9.4 %ID/g at 18 h) and spleen (3.5 vs 8.4 %ID/g) accumulations and higher blood levels (4.8 vs 3.4 %ID/g). Accordingly, only MN14(SANH)-MORF was used in a pretargeting study in tumored mice. When targeted with 99mTc-cMORF and at 2 days postinjection of antibody

  11. Adenovirus tumor targeting and hepatic untargeting by a coxsackie/adenovirus receptor ectodomain anti-carcinoembryonic antigen bispecific adapter.

    Science.gov (United States)

    Li, Hua-Jung; Everts, Maaike; Pereboeva, Larisa; Komarova, Svetlana; Idan, Anat; Curiel, David T; Herschman, Harvey R

    2007-06-01

    Adenovirus vectors have a number of advantages for gene therapy. However, because of their lack of tumor tropism and their preference for liver infection following systemic administration, they cannot be used for systemic attack on metastatic disease. Many epithelial tumors (e.g., colon, lung, and breast) express carcinoembryonic antigen (CEA). To block the natural hepatic tropism of adenovirus and to "retarget" the virus to CEA-expressing tumors, we used a bispecific adapter protein (sCAR-MFE), which fuses the ectodomain of the coxsackie/adenovirus receptor (sCAR) with a single-chain anti-CEA antibody (MFE-23). sCAR-MFE untargets adenovirus-directed luciferase transgene expression in the liver by >90% following systemic vector administration. Moreover, sCAR-MFE can "retarget" adenovirus to CEA-positive epithelial tumor cells in cell culture, in s.c. tumor grafts, and in hepatic tumor grafts. The sCAR-MFE bispecific adapter should, therefore, be a powerful agent to retarget adenovirus vectors to epithelial tumor metastases.

  12. Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society December 7-10, 2015, San Diego, CA, USA.

    Science.gov (United States)

    Pauthner, Matthias; Yeung, Jenny; Ullman, Chris; Bakker, Joost; Wurch, Thierry; Reichert, Janice M; Lund-Johansen, Fridtjof; Bradbury, Andrew R M; Carter, Paul J; Melis, Joost P M

    2016-01-01

    The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6-10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on "Antibodies to watch" in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries. PMID:26909869

  13. Potential for bispecific T-cell engagers: role of blinatumomab in acute lymphoblastic leukemia.

    Science.gov (United States)

    Le Jeune, Caroline; Thomas, Xavier

    2016-01-01

    Patients with relapsed/refractory (R/R) B-precursor acute lymphoblastic leukemia (ALL) and patients whose minimal residual disease persists during treatment have a poor leukemia-free survival. Despite improvements in front-line therapy, the outcome in these patients remains poor, especially after relapse. As there are no standard chemotherapeutic regimens for the treatment of patients with R/R B-precursor ALL, T-cell-based therapeutic approaches have recently come to the forefront in ALL therapy. Recently, monoclonal antibodies have been developed to target specific antigens expressed in B-lineage blast cells. In this setting, CD19 is of great interest as this antigen is expressed in B-lineage cells. Therefore, it has been selected as the target antigen for blinatumomab, a new bi-specific T-cell engager antibody. This sophisticated antibody binds sites for both CD19 and CD3, leading to T-cell proliferation and activation and B-cell apoptosis. Owing to its short serum half-life, blinatumomab has been administrated by continuous intravenous infusion with a favorable safety profile. The most significant toxicities were central nervous system events and the cytokine release syndrome. This new therapeutic approach using blinatumomab has been shown to be effective in patients with positive minimal residual disease and in patients with R/R B-precursor ALL leading to a recent approval by the US Food and Drug Administration after an accelerated review process. This review focuses on the profile of blinatumomab and its efficacy and safety.

  14. A bispecific peptide based near-infrared probe for in vivo tumor diagnosis

    Science.gov (United States)

    Ding, Li; Chen, Wei R.; Gu, Yueqing

    2013-02-01

    The epidermal growth factor receptor EGFR and HER2 are members of recepeter tyrosine kinase family. Overexpression of EGFR and HER2 has been observed in a variety of human tumors, making these receptors promising targets for tumor diagnosis. An affibody targeting HER2 and a nanobody targeting EGFR were reported before. In this Manuscript, we described an bispecific peptide combined with an affibody and a nanonbody through a linker―(G4S)3 . And the bispecific peptide was labeled with near-infrared (NIR) fluorochrome ICG-Der-02 for in vivo tumor EGFR and HER2 targeting. Afterwards, the EGFR and HER2 specificity of the fluorescent probe was tested in vitro for receptor binding assay and fluorescence microscopy and in vivo for subcutaneous MDA-MB-231 tumor targeting. The results indicated that the bispecific peptide had a high affinity to EGFR and HER2. Besides, in vitro and in vivo tumor targeting experiment indicated that the ICG-Der-02-( bispecific peptide) showed excellent tumor activity accumulation. Noninvasive NIR fluorescence imaging is able to detect tumor EGFR and HER2 expression based upon the highly potent bispecific peptide probe.

  15. Crystal structures of mono- and bi-specific diabodies and reduction of their structural flexibility by introduction of disulfide bridges at the Fv interface

    Science.gov (United States)

    Kim, Jin Hong; Song, Dong Hyun; Youn, Suk-Jun; Kim, Ji Won; Cho, Geunyoung; Kim, Sun Chang; Lee, Hayyoung; Jin, Mi Sun; Lee, Jie-Oh

    2016-01-01

    Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Diabodies are engineered antibody fragments that are composed of two Fv domains connected by short peptide linkers. They are attractive candidates for mediators in assembling protein nano-structures because they can simultaneously bind to two different proteins and are rigid enough to be crystallized. However, comparison of previous crystal structures demonstrates that there is substantial structural diversity in the Fv interface region of diabodies and, therefore, reliable prediction of its structure is not trivial. Here, we present the crystal structures of ten mono- and bi-specific diabodies. We found that changing an arginine residue in the Fv interface to threonine greatly reduced the structural diversity of diabodies. We also found that one of the bispecific diabodies underwent an unexpected process of chain swapping yielding a non-functional monospecific diabody. In order to further reduce structural flexibility and prevent chain shuffling, we introduced disulfide bridges in the Fv interface regions. The disulfide-bridged diabodies have rigid and predictable structures and may have applications in crystallizing proteins, analyzing cryo-electron microscopic images and building protein nano-assemblies. PMID:27682821

  16. Development of bispecific molecules for the in situ detection of protein-protein interactions and protein phosphorylation.

    Science.gov (United States)

    van Dieck, Jan; Schmid, Volker; Heindl, Dieter; Dziadek, Sebastian; Schraeml, Michael; Gerg, Michael; Massoner, Petra; Engel, Alfred M; Tiefenthaler, Georg; Vural, Serhat; Stritt, Simon; Tetzlaff, Fabian; Soukupova, Monika; Kopetzki, Erhard; Bossenmaier, Birgit; Thomas, Marlene; Klein, Christian; Mertens, Alfred; Heller, Astrid; Tacke, Michael

    2014-03-20

    Investigation of protein-protein interactions (PPIs) and protein phosphorylation in clinical tissue samples can offer valuable information about the activation status and function of proteins involved in disease progression. However, existing antibody-based methods for phosphorylation detection have been found to lack specificity, and methods developed for examining PPIs in vitro cannot be easily adapted for tissues samples. In this study, we eliminated some of these limitations by developing a specific immunohistochemical staining method that uses "dual binders" (DBs), which are bispecific detection agents consisting of two Fab fragment molecules joined by a flexible linker, to detect PPIs and protein phosphorylation. We engineered DBs by selecting Fab fragments with fast off-rate kinetics, which allowed us to demonstrate that stable target binding was achieved only upon simultaneous, cooperative binding to both epitopes. We show that DBs specifically detect the activated HER2/HER3 complex in formalin-fixed, paraffin-embedded cancer cells and exhibit superior detection specificity for phospho-HER3 compared to the corresponding monoclonal antibody. Overall, the performance of DBs makes them attractive tools for future development for clinical applications. PMID:24529991

  17. Introduction of new derivatives of biotin and DTPA for labeling of antibodies with 111 In ti detect malignant tumors

    International Nuclear Information System (INIS)

    Radiolabeled monoclonal antibodies, have created new innovations in diagnosis, research, and therapy of diseases in last 2 decades. One of the serious limitations of applications of radiolabeled antibodies in vivo is relatively low target to background activity. Various strategies have been proposed to solve this problem including pre-targeting methods that was suggested in 1989. Regarding importance of monoclonal antibodies and radioisotopes, based on pre-targeting strategy, we have introduced new derivative of biotin and DTPA to decrease background activity. DTPA-bio and new derivative (DTPA-bio-1 OX) were labeled with 111 In, labeled compounds and injected through tail veins into Balb/c mice, and percent of injected dose per gram of blood (% ID/g of blood ) was determined at 15, 30, 60, 120, 180 and 240 min after injection. Based on results, 111 In-DTPA-bio rapidly cleared from serum, indicating activity not bound to the target. While in the case of new derivative, by attaching 10 Adenine base (IOX) molecular weight of label is increased causing delayed clearance from serum. Therefore, there is enough time for label to accumulate in the target tissues. With advent of second generation of monoclonal antibodies and antibody engineering, pre targeting methods have changed greatly. It seems that derivatives we introduced will have and important role in new pre-targeting methods

  18. Antibody engineering & therapeutics, the annual meeting of the antibody society December 7–10, 2015, San Diego, CA, USA

    Science.gov (United States)

    Pauthner, Matthias; Yeung, Jenny; Ullman, Chris; Bakker, Joost; Wurch, Thierry; Reichert, Janice M.; Lund-Johansen, Fridtjof; Bradbury, Andrew R.M.; Carter, Paul J.; Melis, Joost P.M.

    2016-01-01

    ABSTRACT The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6–10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on “Antibodies to watch” in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries. PMID:26909869

  19. Design, Synthesis, and Evaluation of a Low-Molecular-Weight (11)C-Labeled Tetrazine for Pretargeted PET Imaging Applying Bioorthogonal in Vivo Click Chemistry.

    Science.gov (United States)

    Denk, Christoph; Svatunek, Dennis; Mairinger, Severin; Stanek, Johann; Filip, Thomas; Matscheko, Dominik; Kuntner, Claudia; Wanek, Thomas; Mikula, Hannes

    2016-07-20

    A low-molecular-weight tetrazine labeled with the short-lived positron emitter carbon-11 was developed as a bioorthogonal PET probe for pretargeted imaging. A method for efficient and fast synthesis of this imaging agent is presented using radiolabeling of a readily available precursor. High reactivity with trans-cyclooctenes was observed and in vivo investigations including PET/MR scanning showed homogeneous biodistribution, good metabolic stability, and rapid excretion in naive mice. These properties are key to the success of bioorthogonal (11)C-PET imaging, which has been shown in a simple pretargeting experiment using TCO-modified mesoporous silica nanoparticles. Overall, this (11)C-labeled tetrazine represents a highly versatile and advantageous chemical tool for bioorthogonal PET imaging and enables pretargeting approaches using carbon-11 for the first time. PMID:27308894

  20. AZ17: a new bispecific drug targeting IL-6 and IL-23 with potential clinical use-improves psoriasis in a human xenograft transplantation model

    DEFF Research Database (Denmark)

    Stenderup, Karin; Kjeldsen, Cecilia Rosada; Shanebeck, K;

    2015-01-01

    ; widely accepted to be associated with psoriasis. To meet the need for new therapeutics, we aimed to create a clinically relevant bispecific drug, by combining the inhibitory properties of anti-IL-6 and anti-IL-23 antibodies, exhibiting high affinity, high stability and the ability to be produced in high...... variables that were synthesized separately in Escherichia coli. To improve stability and extend pharmacokinetics, a flexible poly-ethylene glycol molecule was used as linker. In preclinical psoriasis models, AZ17 reduced IL-23-induced ear inflammation and improved psoriasis in a xenograft transplantation...... model where psoriasis skin is transplanted onto immune-deficient mice. The data presented here suggest AZ17 to be a promising drug candidate in psoriasis and other inflammatory diseases associated with Th17 cell development....

  1. Pretargeted Positron Emission Tomography Imaging That Employs Supramolecular Nanoparticles with in Vivo Bioorthogonal Chemistry.

    Science.gov (United States)

    Hou, Shuang; Choi, Jin-Sil; Garcia, Mitch Andre; Xing, Yan; Chen, Kuan-Ju; Chen, Yi-Ming; Jiang, Ziyue K; Ro, Tracy; Wu, Lily; Stout, David B; Tomlinson, James S; Wang, Hao; Chen, Kai; Tseng, Hsian-Rong; Lin, Wei-Yu

    2016-01-26

    A pretargeted oncologic positron emission tomography (PET) imaging that leverages the power of supramolecular nanoparticles with in vivo bioorthogonal chemistry was demonstrated for the clinically relevant problem of tumor imaging. The advantages of this approach are that (i) the pharmacokinetics (PKs) of tumor-targeting and imaging agents can be independently altered via chemical alteration to achieve the desired in vivo performance and (ii) the interplay between the two PKs and other controllable variables confers a second layer of control toward improved PET imaging. In brief, we utilized supramolecular chemistry to synthesize tumor-targeting nanoparticles containing transcyclooctene (TCO, a bioorthogonal reactive motif), called TCO⊂SNPs. After the intravenous injection and subsequent concentration of the TCO⊂SNPs in the tumors of living mice, a small molecule containing both the complementary bioorthogonal motif (tetrazine, Tz) and a positron-emitting radioisotope ((64)Cu) was injected to react selectively and irreversibly to TCO. High-contrast PET imaging of the tumor mass was accomplished after the rapid clearance of the unreacted (64)Cu-Tz probe. Our nanoparticle approach encompasses a wider gamut of tumor types due to the use of EPR effects, which is a universal phenomenon for most solid tumors.

  2. Anti-CD45 Pretargeted Radioimmunotherapy using Bismuth-213: High Rates of Complete Remission and Long-Term Survival in a Mouse Myeloid Leukemia Xenograft Model

    Energy Technology Data Exchange (ETDEWEB)

    Pagel, John M; Kenoyer, Aimee L; Back, Tom; Hamlin, Donald K; Wilbur, D Scott; Fisher, Darrell R; Park, Steven I; Frayo, Shani; Axtman, Amanda; Orgun, Nural; Orozoco, Johnnie; Shenoi, Jaideep; Lin, Yukang; Gopal, Ajay K; Green, Damian J; Appelbaum, Frederick R; Press, Oliver W

    2011-07-21

    Pretargeted radioimmunotherapy (PRIT) using an anti-CD45 antibody (Ab)-streptavidin (SA) conjugate and DOTA-biotin labeled with β-emitting radionuclides has been explored as a strategy to decrease relapse and toxicity. α-emitting radionuclides exhibit high cytotoxicity coupled with a short path-length, potentially increasing the therapeutic index and making them an attractive alternative to β-emitting radionuclides for patients with Acute Myeloid Leukemia (AML). Accordingly, we have used 213Bi in mice with human leukemia xenografts. Results demonstrated excellent localization of 213Bi-DOTA-biotin to tumors with minimal uptake into normal organs. After 10 minutes, 4.5 ± 1.1% of the injected dose of 213Bi was delivered per gram of tumor. α imaging demonstrated uniform radionuclide distribution within tumor tissue 45 minutes after 213Bi-DOTA-biotin injection. Radiation absorbed doses were similar to those observed using a β-emitting radionuclide (90Y) in the same model. We conducted therapy experiments in a xenograft model using a single-dose of 213Bi-DOTA-biotin given 24 hours after anti-CD45 Ab-SA conjugate. Among mice treated with anti-CD45 Ab-SA conjugate followed by 800 μCi of 213Bi- or 90Y-DOTA-biotin, 80% and 20%, respectively, survived leukemia-free for >100 days with minimal toxicity. These data suggest that anti-CD45 PRIT using an α-emitting radionuclide may be highly effective and minimally toxic for treatment of AML.

  3. Characterization of poultry egg-white avidins and their potential as a tool in pretargeting cancer treatment.

    Science.gov (United States)

    Hytönen, Vesa P; Laitinen, Olli H; Grapputo, Alessandro; Kettunen, Anu; Savolainen, Janne; Kalkkinen, Nisse; Marttila, Ari T; Nordlund, Henri R; Nyholm, Thomas K M; Paganelli, Giovanni; Kulomaa, Markku S

    2003-01-01

    Chicken avidin and bacterial streptavidin are proteins used in a wide variety of applications in the life sciences due to their strong affinity for biotin. A new and promising use for them is in medical pretargeting cancer treatments. However, their pharmacokinetics and immunological properties are not always optimal, thereby limiting their use in these applications. To search for potentially beneficial new candidates, we screened egg white from four different poultry species for avidin. Avidin proteins, isolated from the duck, goose, ostrich and turkey, showed a similar tetrameric structure, similar glycosylation and stability against both temperature and proteolytic activity of proteinase K as chicken avidin. Biotin-binding properties of these avidins, measured using IAsys optical biosensor, were similar to those found in avidin from the chicken. Three of these novel avidins, however, showed different immunological cross-reactivities when compared with chicken avidin. The patient sera responses to duck, goose and ostrich avidins were also lower than those observed for chicken and turkey avidins. Our findings suggest that the use of these proteins offers advantages over chicken avidin and bacterial streptavidin in pretargeting applications. PMID:12558501

  4. Monoclonal antibodies

    International Nuclear Information System (INIS)

    kits available commercially. There must, however, be a realistic pricing structure and some identification of the needs of developing countries to balance the costs of the kits, which are generally far too expensive. There is extensive literature concerning the preparation, characterization and application of MAbs. Areas of their use as therapeutics, the prophylactic prevention of diseases, or the development of anti-idiotypic vaccines are not examined. There is also no mention of engineered MAbs, in which recombinant technologies are used to produce bispecific antibodies and antibody fragments. These areas are being exploited in the human fields where, in terms of process biotechnology, kilograms of MAbs are routinely produced. It is reasonable to consider such exploitation in the veterinary sphere, whereby animals are protected by designer antibodies against defined epitopes. The main danger in the use of MAbs is that the research base necessary to understand the biology of host/pathogen relationships and the epidemiology of disease is being constantly eroded. (author)

  5. Targeting non-human coronaviruses to human cancer cells using a bispecific single-chain antibody

    NARCIS (Netherlands)

    Würdinger, T; Verheije, M H; Raaben, M; Bosch, B J; de Haan, C A M; van Beusechem, V W; Rottier, P J M; Gerritsen, W R

    2005-01-01

    To explore the potential of using non-human coronaviruses for cancer therapy, we first established their ability to kill human tumor cells. We found that the feline infectious peritonitis virus (FIPV) and a felinized murine hepatitis virus (fMHV), both normally incapable of infecting human cells, co

  6. The maturation of antibody technology for the HIV epidemic.

    Science.gov (United States)

    Winnall, Wendy R; Beasley, Matthew D; Center, Rob J; Parsons, Matthew S; Kiefel, Ben R; Kent, Stephen J

    2014-08-01

    Antibodies are one of our most useful biological tools. Indeed, improvements in antibody-based technologies have ushered in a new era of antibody-based therapeutics, research and diagnostic tools. Although improved technologies have led to the development of therapeutic antibodies for treatment of malignancies and inflammatory conditions, the use of advanced antibody technology in the therapy of viral infections is in its infancy. Non-human primate studies have demonstrated that antibodies against the HIV envelope can both prevent viral infection and control viremia. Despite the obvious potential of antibody therapies against HIV, there remain limitations in production and purification capacity that require further research. Recent advances in recombinant antibody technology have led to the development of a range of novel antibody fragments, such as single-domain nanobodies and bispecific antibodies, that are capable of targeting cancer cells to cytotoxic T cells. Novel antibody production techniques have also been designed, allowing antibodies to be obtained from non-mammalian cells, bovine colostrum and the periplasm and cytoplasm of bacteria. These advances may allow large-scale production of HIV antibodies that are capable of protecting against HIV infection or serving as therapeutics that reduce the need for life-long antiretroviral treatment. This review summarises recent advances in antibody-based technologies and discusses the possibilities and challenges of using these advances to design prophylactics and therapeutics against HIV. PMID:24797582

  7. Rapid detection of hypoxia-inducible factor-1-active tumours: pretargeted imaging with a protein degrading in a mechanism similar to hypoxia-inducible factor-1{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Ueda, Masashi [Kyoto University, Radioisotopes Research Laboratory, Kyoto University Hospital, Faculty of Medicine, Kyoto (Japan); Kyoto University, Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto (Japan); Kudo, Takashi; Konishi, Hiroaki; Miyano, Azusa; Ono, Masahiro; Saji, Hideo [Kyoto University, Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto (Japan); Kuge, Yuji [Kyoto University, Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto (Japan); Hokkaido University, Central Institute of Isotope Science, Sapporo (Japan); Mukai, Takahiro [Kyushu University, Department of Biomolecular Recognition Chemistry, Graduate School of Pharmaceutical Sciences, Fukuoka (Japan); Tanaka, Shotaro; Kizaka-Kondoh, Shinae; Hiraoka, Masahiro [Kyoto University, Department of Radiation Oncology and Image-applied Therapy, Graduate School of Medicine, Kyoto (Japan)

    2010-08-15

    Hypoxia-inducible factor-1 (HIF-1) plays an important role in malignant tumour progression. For the imaging of HIF-1-active tumours, we previously developed a protein, POS, which is effectively delivered to and selectively stabilized in HIF-1-active cells, and a radioiodinated biotin derivative, (3-{sup 123}I-iodobenzoyl)norbiotinamide ({sup 123}I-IBB), which can bind to the streptavidin moiety of POS. In this study, we aimed to investigate the feasibility of the pretargeting method using POS and {sup 123}I-IBB for rapid imaging of HIF-1-active tumours. Tumour-implanted mice were pretargeted with POS. After 24 h, {sup 125}I-IBB was administered and subsequently, the biodistribution of radioactivity was investigated at several time points. In vivo planar imaging, comparison between {sup 125}I-IBB accumulation and HIF-1 transcriptional activity, and autoradiography were performed at 6 h after the administration of {sup 125}I-IBB. The same sections that were used in autoradiographic analysis were subjected to HIF-1{alpha} immunohistochemistry. {sup 125}I-IBB accumulation was observed in tumours of mice pretargeted with POS (1.6%ID/g at 6 h). This result is comparable to the data derived from {sup 125}I-IBB-conjugated POS-treated mice (1.4%ID/g at 24 h). In vivo planar imaging provided clear tumour images. The tumoral accumulation of {sup 125}I-IBB significantly correlated with HIF-1-dependent luciferase bioluminescence (R=0.84, p<0.01). The intratumoral distribution of {sup 125}I-IBB was heterogeneous and was significantly correlated with HIF-1{alpha}-positive regions (R=0.58, p<0.0001). POS pretargeting with {sup 123}I-IBB is a useful technique in the rapid imaging and detection of HIF-1-active regions in tumours. (orig.)

  8. Natural Mosquito-Pathogen Hybrid IgG4 Antibodies in Vector-Borne Diseases: A Hypothesis

    Science.gov (United States)

    Londono-Renteria, Berlin; Cardenas, Jenny C.; Troupin, Andrea; Colpitts, Tonya M.

    2016-01-01

    Chronic exposure to antigens may favor the production of IgG4 antibodies over other antibody types. Recent studies have shown that up to a 30% of normal human IgG4 is bi-specific and is able to recognize two antigens of different nature. A requirement for this specificity is the presence of both eliciting antigens in the same time and at the same place where the immune response is induced. During transmission of most vector-borne diseases, the pathogen is delivered to the vertebrate host along with the arthropod saliva during blood feeding and previous studies have shown the existence of IgG4 antibodies against mosquito salivary allergens. However, there is very little ongoing research or information available regarding IgG4 bi-specificity with regard to infectious disease, particularly during immune responses to vector-borne diseases, such as malaria, filariasis, or dengue virus infection. Here, we provide background information and present our hypothesis that IgG4 may not only be a useful tool to measure exposure to infected mosquito bites, but that these bi-specific antibodies may also play an important role in modulation of the immune response against malaria and other vector-borne diseases in endemic settings. PMID:27746778

  9. In vivo evaluation of 18F-labeled TCO for pre-targeted PET imaging in the brain

    International Nuclear Information System (INIS)

    Introduction: The tetrazine-trans-cylooctene cycloaddition using radiolabeled tetrazine or radiolabeled trans-cyclooctene (TCO) has been reported to be a very fast, selective and bioorthogonal reaction that could be useful for in vivo radiolabeling of molecules. We wanted to evaluate the in vivo biodistribution profile and brain uptake of 18F-labeled TCO ([18F]TCO) to assess its potential for pre-targeted imaging in the brain. Methods: We evaluated the in vivo behavior of [18F]TCO via an ex vivo biodistribution study complemented by in vivo μPET imaging at 5, 30, 60, 90, 120 and 240 min post tracer injection. An in vivo metabolite study was performed at 5 min, 30 min and 120 min post [18F]TCO injection by RP-HPLC analysis of plasma and brain extracts. Incubation with human liver microsomes was performed to further evaluate the metabolite profile of the tracer. Results: μPET imaging and ex-vivo biodistribution revealed an high initial brain uptake of [18F]TCO (3.8%ID/g at 5 min pi) followed by a washout to 3.0%ID/g at 30 min pi. Subsequently the brain uptake increased again to 3.7%ID/g at 120 min pi followed by a slow washout until 240 min pi (2.9%ID/g). Autoradiography confirmed homogenous brain uptake. On the μPET images bone uptake became gradually visible after 120 min pi and was clearly visible at 240 min pi. The metabolite study revealed a fast metabolization of [18F]TCO in plasma and brain into three main polar radiometabolites. Conclusions: Although [18F]TCO has previously been described to be a useful tracer for radiolabeling of tetrazine modified targeting molecules, our study indicates that its utility for in vivo chemistry and pre-targeted imaging will be limited. Although [18F]TCO clearly enters the brain, it is quickly metabolized with a non-specific accumulation of radioactivity in the brain and bone

  10. Comparative efficacy of 177Lu and 90Y for Anti-CD20 Pretargeted Radioimmunotherapy in Murine Lymphoma Xenograft Models

    International Nuclear Information System (INIS)

    Purpose Pretargeted radioimmunotherapy (PRIT) is a multi-step method of selectively delivering high doses of radiotherapy to tumor cells while minimizing exposure to surrounding tissues. Yttrium-90 (90Y) and lutetium-177 (177Lu) are two of the most promising beta-particle emitting radionuclides used for radioimmunotherapy, which despite having similar chemistries differ distinctly in terms of radiophysical features. These differences may have important consequences for the absorbed dose to tumors and normal organs. Whereas 90Y has been successfully applied in a number of preclinical and clinical radioimmunotherapy settings, there have been few published pretargeting studies with 177Lu. We therefore compared the therapeutic potential of targeting either 90Y or 177Lu to human B-cell lymphoma xenografts in mice. Methods Parallel experiments evaluating the biodistribution, imaging, dosimetry, therapeutic efficacy, and toxicity were performed in female athymic nude mice bearing either Ramos (Burkitt lymphoma) or Granta (mantle cell lymphoma) xenografts, utilizing an anti-CD20 antibodystreptavidin conjugate (1F5-SA) and an 90Y- or 177Lu-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-biotin second step reagent. Results The two radionuclides displayed comparable biodistributions in tumors and normal organs; however, the absorbed radiation dose delivered to tumor was more than twice as high for 90Y (1.3 Gy/MBq) as for 177Lu (0.6 Gy/MBq). More importantly, therapy with 90Y-DOTAbiotin was dramatically more effective than with 177Lu-DOTA-biotin, with 100% of Ramos xenograft-bearing mice cured with 37 MBq 90Y, whereas 0% were cured using identical amounts of 177Lu-DOTA-biotin. Similar results were observed in mice bearing Granta xenografts, with 80% of the mice cured with 90Y-PRIT and 0% cured with 177Lu-PRIT. Toxicities were comparable with both isotopes. Conclusion 90Y was therapeutically superior to 177Lu for streptavidin-biotin PRIT approaches in

  11. New Antibody Conjugates in Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Serengulam V. Govindan

    2010-01-01

    Full Text Available Targeting of radiation, drugs, and protein toxins to cancers selectively with monoclonal antibodies (MAbs has been a topic of considerable interest and an area of continued development. Radioimmunotherapy (RAIT of lymphoma using directly labeled MAbs is of current interest after approval of two radiolabeled anti-CD20 MAbs, as illustrated with the near 100% overall response rate obtained in a recent clinical trial using an investigational radiolabeled anti-CD22 MAb, 90Y-epratuzumab. The advantage of pretargeted RAIT over directly labeled MAbs is continuing to be validated in preclinical models of lymphoma and solid tumors. Importantly, the advantages of combining RAIT with radiation sensitizers, with immunotherapy, or a drug conjugate targeting a different antigen are being studied clinically and preclinically. The area of drug-conjugated antibodies is progressing with encouraging data published for the trastuzumab-DM1 conjugate in a phase I clinical trial in HER2-positive breast cancer. The Dock-and-Lock platform technology has contributed to the design and the evaluation of complex antibody-cytokine and antibody-toxin conjugates. This review describes the advances made in these areas, with illustrations taken from advances made in the authors' institutions.

  12. Conventional and Pretargeted Radioimmunotherapy Using Bismuth-213 to Target and Treat Non-Hodgkin Lymphomas Expressing CD20: A Preclinical Model toward Optimal Consolidation Therapy to Eradicate Minimal Residual Disease.

    Energy Technology Data Exchange (ETDEWEB)

    Park, Steven I.; Shenoi, Jaideep; Pagel, John M.; Hamlin, Donald K.; Wilbur, D. Scott; Orgun, Nural; Kenoyer, Aimee L.; Frayo, Shani; Axtman, Amanda; Back, Tom; Lin, Yukang; Fisher, Darrell R.; Gopal, Ajay K.; Green, Damian J.; Press, Oliver W.

    2010-11-18

    Radioimmunotherapy (RIT) with α-emitting radionuclides is an attractive approach for the treatment of minimal residual disease (MRD) because the short path lengths and high energies of α-particles produce optimal cytotoxicity at small target sites while minimizing damage to surrounding normal tissues. Pretargeted RIT (PRIT) using antibody-streptavidin (Ab-SA) constructs and radiolabeled biotin allows rapid, specific localization of radioactivity at tumor sites, making it an optimal method to target α-emitters with short half-lives, such as bismuth-213 (213Bi). Athymic mice bearing Ramos lymphoma xenografts received anti-CD20 1F5(scFv)4SA fusion protein (FP), followed by a dendrimeric clearing agent and [213Bi]DOTA-biotin. After 90 min, tumor uptake for 1F5(scFv)4SA was 16.5 ± 7.0 % injected dose per gram (ID/g) compared with 2.3 ± 0.9 % ID/g for the control FP. Mice treated with anti-CD20 PRIT and 600 µCi [213Bi]DOTA-biotin exhibited marked tumor growth delays compared to controls (mean tumor volume 0.01 ± 0.02 vs. 203.38 ± 83.03 mm3 after 19 days, respectively). The median survival for the 1F5(scFv)4SA group was 90 days compared to 23 days for the control FP (p<0.0001). Treatment was well tolerated, with no treatment-related mortalities. This study demonstrates the favorable biodistribution profile and excellent therapeutic efficacy attainable with 213Bi-labeled anti-CD20 PRIT.

  13. A mechanistic compartmental model for total antibody uptake in tumors.

    Science.gov (United States)

    Thurber, Greg M; Dane Wittrup, K

    2012-12-01

    Antibodies are under development to treat a variety of cancers, such as lymphomas, colon, and breast cancer. A major limitation to greater efficacy for this class of drugs is poor distribution in vivo. Localization of antibodies occurs slowly, often in insufficient therapeutic amounts, and distributes heterogeneously throughout the tumor. While the microdistribution around individual vessels is important for many therapies, the total amount of antibody localized in the tumor is paramount for many applications such as imaging, determining the therapeutic index with antibody drug conjugates, and dosing in radioimmunotherapy. With imaging and pretargeted therapeutic strategies, the time course of uptake is critical in determining when to take an image or deliver a secondary reagent. We present here a simple mechanistic model of antibody uptake and retention that captures the major rates that determine the time course of antibody concentration within a tumor including dose, affinity, plasma clearance, target expression, internalization, permeability, and vascularization. Since many of the parameters are known or can be estimated in vitro, this model can approximate the time course of antibody concentration in tumors to aid in experimental design, data interpretation, and strategies to improve localization. PMID:22974563

  14. Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice.

    Science.gov (United States)

    Moayeri, Mahtab; Tremblay, Jacqueline M; Debatis, Michelle; Dmitriev, Igor P; Kashentseva, Elena A; Yeh, Anthony J; Cheung, Gordon Y C; Curiel, David T; Leppla, Stephen; Shoemaker, Charles B

    2016-03-01

    Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 μg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 μg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 μg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors. PMID:26740390

  15. Macrophages are critical effectors of antibody therapies for cancer.

    Science.gov (United States)

    Weiskopf, Kipp; Weissman, Irving L

    2015-01-01

    Macrophages are innate immune cells that derive from circulating monocytes, reside in all tissues, and participate in many states of pathology. Macrophages play a dichotomous role in cancer, where they promote tumor growth but also serve as critical immune effectors of therapeutic antibodies. Macrophages express all classes of Fcγ receptors, and they have immense potential to destroy tumors via the process of antibody-dependent phagocytosis. A number of studies have demonstrated that macrophage phagocytosis is a major mechanism of action of many antibodies approved to treat cancer. Consequently, a number of approaches to augment macrophage responses to therapeutic antibodies are under investigation, including the exploration of new targets and development of antibodies with enhanced functions. For example, the interaction of CD47 with signal-regulatory protein α (SIRPα) serves as a myeloid-specific immune checkpoint that limits the response of macrophages to antibody therapies, and CD47-blocking agents overcome this barrier to augment phagocytosis. The response of macrophages to antibody therapies can also be enhanced with engineered Fc variants, bispecific antibodies, or antibody-drug conjugates. Macrophages have demonstrated success as effectors of cancer immunotherapy, and further investigation will unlock their full potential for the benefit of patients. PMID:25667985

  16. Natural Killer (NK- and T-Cell Engaging Antibody-Derived Therapeutics

    Directory of Open Access Journals (Sweden)

    Christoph Stein

    2012-06-01

    Full Text Available Unmodified antibodies (abs have been successful in the treatment of hematologic malignancies, but less so for the treatment of solid tumors. They trigger anti-tumor effects through their Fc-domains, and one way to improve their efficacy is to optimize their interaction with the effectors through Fc-engineering. Another way to empower abs is the design of bispecific abs and related fusion proteins allowing a narrower choice of effector cells. Here we review frequently chosen classes of effector cells, as well as common trigger molecules. Natural Killer (NK- and T-cells are the most investigated populations in therapeutical approaches with bispecific agents until now. Catumaxomab, the first bispecific ab to receive drug approval, targets the tumor antigen Epithelial Cell Adhesion Molecule (EpCAM and recruits T-cells via a binding site for the cell surface protein CD3. The next generation of recombinant ab-derivatives replaces the broadly reactive Fc-domain by a binding domain for a single selected trigger. Blinatumomab is the first clinically successful member of this class, targeting cancer cells via CD19 and engaging T-cells by CD3. Other investigators have developed related recombinant fusion proteins to recruit effectors, such as NK-cells and macrophages. The first such agents currently in preclinical and clinical development will be discussed.

  17. {sup 18}F-FDG PET predicts survival after pretargeted radioimmunotherapy in patients with progressive metastatic medullary thyroid carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Salaun, Pierre-Yves; Robin, Philippe [University Hospital, Nuclear Medicine Department, Brest (France); Campion, Loic [ICO-Gauducheau Cancer Institute, Statistical Department, Nantes (France); Ansquer, Catherine; Mathieu, Cedric [University Hospital, Nuclear Medicine Department, Nantes (France); Frampas, Eric [University Hospital, Radiology Department, Nantes (France); Universite de Nantes, Nantes-Angers Cancer Research Center, Inserm, U 892, CNRS, UMR 6299, Nantes (France); Bournaud, Claire [University Hospital, Nuclear Medicine Department, Lyon (France); Vuillez, Jean-Philippe [University Hospital, Nuclear Medicine Department, Grenoble (France); Taieb, David [University Hospital, Nuclear Medicine Department, Marseille (France); Rousseau, Caroline [Universite de Nantes, Nantes-Angers Cancer Research Center, Inserm, U 892, CNRS, UMR 6299, Nantes (France); ICO-Rene Gauducheau, Nuclear Medicine Department, Nantes (France); Drui, Delphine [University Hospital, Endocrinology Department, Nantes (France); Mirallie, Eric [University Hospital, Surgery Department, Nantes (France); Borson-Chazot, Francoise [University Hospital, Endocrinology Department, Lyon (France); Goldenberg, David M. [IBC Pharmaceuticals, Inc., and Immunomedics, Inc., Morris Plains, NJ (United States); Center for Molecular Medicine and Immunology, Garden State Cancer Center, Morris Plains, NJ (United States); Chatal, Jean-Francois [GIP ARRONAX, Saint-Herblain (France); Barbet, Jacques [Universite de Nantes, Nantes-Angers Cancer Research Center, Inserm, U 892, CNRS, UMR 6299, Nantes (France); GIP ARRONAX, Saint-Herblain (France); Kraeber-Bodere, Francoise [University Hospital, Nuclear Medicine Department, Nantes (France); Universite de Nantes, Nantes-Angers Cancer Research Center, Inserm, U 892, CNRS, UMR 6299, Nantes (France); ICO-Rene Gauducheau, Nuclear Medicine Department, Nantes (France); Hotel Dieu University Hospital, Nuclear Medicine Department, Nantes (France)

    2014-08-15

    PET is a powerful tool for assessing targeted therapy. Since {sup 18}F-FDG shows a potential prognostic value in medullary thyroid carcinoma (MTC), this study evaluated {sup 18}F-FDG PET alone and combined with morphological and biomarker evaluations as a surrogate marker of overall survival (OS) in patients with progressive metastatic MTC treated with pretargeted anti-CEA radioimmunotherapy (pRAIT) in a phase II clinical trial. Patients underwent PET associated with morphological imaging (CT and MRI) and biomarker evaluations, before and 3 and 6 months, and then every 6 months, after pRAIT for 36 months. A combined evaluation was performed using anatomic, metabolic and biomarker methods. The prognostic value of the PET response was compared with demographic parameters at inclusion including age, sex, RET mutation, time from initial diagnosis, calcitonin and CEA concentrations and doubling times (DT), SUV{sub max}, location of disease and bone marrow involvement, and with response using RECIST, biomarker concentration variation, impact on DT, and combined methods. Enrolled in the study were 25 men and 17 women with disease progression. The median OS from pRAIT was 3.7 years (0.2 to 6.5 years) and from MTC diagnosis 10.9 years (1.7 to 31.5 years). After pRAIT, PET/CT showed 1 patient with a complete response, 4 with a partial response and 24 with disease stabilization. The combined evaluation showed 20 responses. For OS from pRAIT, univariate analysis showed the prognostic value of biomarker DT (P = 0.011) and SUV{sub max} (P = 0.038) calculated before pRAIT and impact on DT (P = 0.034), RECIST (P = 0.009), PET (P = 0.009), and combined response (P = 0.004) measured after pRAIT. PET had the highest predictive value with the lowest Akaike information criterion (AIC 74.26) as compared to RECIST (AIC 78.06), biomarker variation (AIC 81.94) and impact on DT (AIC 79.22). No benefit was obtained by combining the methods (AIC 78.75). This result was confirmed by the

  18. Thyroid Antibodies

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? Thyroid Antibodies Share this page: Was this page helpful? Also known as: Thyroid Autoantibodies; Antithyroid Antibodies; Antimicrosomal Antibody; Thyroid Microsomal Antibody; ...

  19. A novel bispecific immunotoxin delivered by human bone marrow-derived mesenchymal stem cells to target blood vessels and vasculogenic mimicry of malignant gliomas

    Directory of Open Access Journals (Sweden)

    Zhang Y

    2015-06-01

    Full Text Available Yonghong Zhang,1,2 Xinlin Sun,1 Min Huang,1 Yiquan Ke,1 Jihui Wang,1 Xiao Liu1 1National Key Clinic Specialty, Neurosurgery Institute of Guangdong Province, Guangdong Provincial Key Laboratory on Brain Function Repair and Regeneration, Department of Neurosurgery, Zhujiang Hospital, Southern Medical University, Guangzhou, 2Department of Neurosurgery, First Hospital of Lanzhou University, Lanzhou, People’s Republic of China Background: In previous years, immunotoxins have been shown to be a greatly promising therapeutic tool for brain malignancies, such as gliomas. Human mesenchymal stem cells (hMSCs exhibit tropism to tumor tissue. However, the effect of bispecific immunotoxins in malignant gliomas is still unknown. The aim of this study was to investigate the function of bispecific immunotoxins in human malignant gliomas.Materials and methods: In the present study, the bispecific immunotoxin VEGF165-ephrin A1-PE38KDEL was established using deoxyribonucleic acid shuffling and cloning techniques. The VEGF165-ephrin A1-PE38KDEL was delivered by hMSCs to mouse malignant gliomas. The effects of the bispecific immunotoxins on glioma-derived blood vessels and vasculogenic mimicry to elucidate the molecular mechanisms underlying the antitumorigenic effects of immunotoxins were examined in vivo.Results: In vitro, transfected hMSCs significantly inhibited the cell viability of gliomas cell lines U87 and U251 in a dose-dependent manner compared with untransfected hMSCs (P<0.01. In vivo, the intratumoral injection of engineered hMSCs was effective at inhibiting tumor growth in a malignant glioma tumor model.Conclusion: The bispecific immunotoxin secreted from hMSCs acts as a novel strategy for improving treatment options for malignant gliomas in the clinic. Keywords: bispecific immunotoxin, human mesenchymal stem cells, ephrin A1, VEGF165, malignant glioma

  20. Lack of Widespread BBB Disruption in Alzheimer's Disease Models: Focus on Therapeutic Antibodies.

    Science.gov (United States)

    Bien-Ly, Nga; Boswell, C Andrew; Jeet, Surinder; Beach, Thomas G; Hoyte, Kwame; Luk, Wilman; Shihadeh, Vera; Ulufatu, Sheila; Foreman, Oded; Lu, Yanmei; DeVoss, Jason; van der Brug, Marcel; Watts, Ryan J

    2015-10-21

    The blood-brain barrier (BBB) limits brain uptake of therapeutic antibodies. It is believed that the BBB is disrupted in Alzheimer's disease (AD), potentially increasing drug permeability de facto. Here we compared active versus passive brain uptake of systemically dosed antibodies (anti-transferrin receptor [TfR] bispecific versus control antibody) in mouse models of AD. We first confirmed BBB disruption in a mouse model of multiple sclerosis as a positive control. Importantly, we found that BBB permeability was vastly spared in mouse models of AD, including PS2-APP, Tau transgenics, and APOE4 knockin mice. Brain levels of TfR in mouse models or in human cases of AD resembled controls, suggesting target engagement of TfR bispecific is not limited. Furthermore, infarcts from human AD brain showed similar occurrences compared to age-matched controls. These results question the widely held view that the BBB is largely disrupted in AD, raising concern about assumptions of drug permeability in disease. PMID:26494278

  1. BISPECIFIC ANTIBODY-MEDIATED TARGET CELL-SPECIFIC COSTIMULATION OF RESTING T-CELLS VIA CD5 AND CD28

    NARCIS (Netherlands)

    KROESEN, BJ; BAKKER, A; VANLIER, RAW; THE, HT; DELEIJ, L

    1995-01-01

    Induction of T-cell activation requires multiple signals provided by cell surface receptor interactions and/or cytokines. T-cell stimulation via the T-cell receptor/CD3 complex provides an important initial activation event which, when combined with the proper costimulatory signals, results in an ac

  2. IBC's 23rd Antibody Engineering and 10th Antibody Therapeutics Conferences and the Annual Meeting of The Antibody Society: December 2-6, 2012, San Diego, CA.

    Science.gov (United States)

    Marquardt, John; Begent, Richard H J; Chester, Kerry; Huston, James S; Bradbury, Andrew; Scott, Jamie K; Thorpe, Philip E; Veldman, Trudi; Reichert, Janice M; Weiner, Louis M

    2012-01-01

    Now in its 23rd and 10th years, respectively, the Antibody Engineering and Antibody Therapeutics conferences are the Annual Meeting of The Antibody Society. The scientific program covers the full spectrum of challenges in antibody research and development from basic science through clinical development. In this preview of the conferences, the chairs provide their thoughts on sessions that will allow participants to track emerging trends in (1) the development of next-generation immunomodulatory antibodies; (2) the complexity of the environment in which antibodies must function; (3) antibody-targeted central nervous system (CNS) therapies that cross the blood brain barrier; (4) the extension of antibody half-life for improved efficacy and pharmacokinetics (PK)/pharmacodynamics (PD); and (5) the application of next generation DNA sequencing to accelerate antibody research. A pre-conference workshop on Sunday, December 2, 2012 will update participants on recent intellectual property (IP) law changes that affect antibody research, including biosimilar legislation, the America Invents Act and recent court cases. Keynote presentations will be given by Andreas Plückthun (University of Zürich), who will speak on engineering receptor ligands with powerful cellular responses; Gregory Friberg (Amgen Inc.), who will provide clinical updates of bispecific antibodies; James D. Marks (University of California, San Francisco), who will discuss a systems approach to generating tumor targeting antibodies; Dario Neri (Swiss Federal Institute of Technology Zürich), who will speak about delivering immune modulators at the sites of disease; William M. Pardridge (University of California, Los Angeles), who will discuss delivery across the blood-brain barrier; and Peter Senter (Seattle Genetics, Inc.), who will present his vision for the future of antibody-drug conjugates. For more information on these meetings or to register to attend, please visit www.IBCLifeSciences.com/Antibody

  3. Country report: Germany. Preclinical evaluation of Y-90 labelled Rituximab and ERIC-1, two antibodies for tumor therapy

    International Nuclear Information System (INIS)

    This project focuses on harnessing the great potential of radionuclide therapy, using various different vehicles to transport radionuclides into tumor tissues. A central aim of the project will be to manufacture specific vehicle molecules whose tumor affinity and suitability for radioactive coupling have already been proven through laboratory trials on animals and cell cultures at the Department of Nuclear Medicine, University of Cologne and to label it with Y- 90. The vectors to be used to transport radionuclides into tumor tissue for treatment are antibodies against lymphomas and neuroblastomas. The technology applied for coupling Y-90 to various antibodies has been developed to a high level in Cologne and is now ready to be transferred and adapted to GMP (Good Manufacturing Practice) conditions. The antibody against NHL can be acquired commercially and must then be modified for binding to the therapeutically active nuclide Y-90. Similarly, the antibody against neuroblastoma must also be modified to bind to Y-90 but is produced in Cologne. To improve the therapeutic value of antibodies we tried to introduce the pretargeting method

  4. Improved homing of endothelial progenitor cells by the bispecific protein GPVI-CD133

    OpenAIRE

    Schönberger, T.; H. Langer; Gauß, A; Hafner, R; von der Ruhr, J; Schumm, M.; Bühring, HJ; van Zandvoort, M; Jung, G; Skutella, T.; Gawaz, M

    2011-01-01

    We designed a bifunctional protein, capable of capturing circulating endothelial progenitor cells to collagen-rich vascular lesions. The protein consists of the soluble platelet collagen receptor glycoprotein VI and an antibody to CD133. This construct substantially mediates EPC homing to vascular lesions. Furthermore, it augments reendothelialization of vascular lesions and reduces the extent of myocardial infarction. Therefore, this bifunctional protein could be a potential new therapeutic ...

  5. Improved anti-leukemia activities of adoptively transferred T cells expressing bispecific T-cell engager in mice.

    Science.gov (United States)

    Liu, X; Barrett, D M; Jiang, S; Fang, C; Kalos, M; Grupp, S A; June, C H; Zhao, Y

    2016-01-01

    Despite the impressive clinical efficacy of T cells engineered to express chimeric antigen receptors (CAR-Ts), the current applications of CAR-T cell therapy are limited by major treatment-related toxicity. Thus, safer yet effective alternative approaches must be developed. In this study, we compared CD19 bispecific T-cell engager (BiTE)-transferred T cells that had been transfected by RNA electroporation with CD19 CAR RNA-transferred T cells both in vitro and in an aggressive Nalm6 leukemia mouse model. BiTEs were secreted from the transferred T cells and enabled both the transferred and bystander T cells to specifically recognize CD19(+) cell lines, with increased tumor killing ability, prolonged functional persistence, increased cytokine production and potent proliferation compared with the CAR-T cells. More interestingly, in comparison with CD3/CD28 bead-stimulated T cells, T cells that were expanded by a rapid T-cell expansion protocol (REP) showed enhanced anti-tumor activities for both CAR and BiTE RNA-electroporated T cells both in vitro and in a Nalm6 mouse model (P<0.01). Furthermore, the REP T cells with BiTE RNAs showed greater efficacy in the Nalm6 leukemia model compared with REP T cells with CAR RNA (P<0.05) and resulted in complete leukemia remission. PMID:27258611

  6. The use of novel monoclonal antibodies in the treatment of acute lymphoblastic leukemia.

    Science.gov (United States)

    DeAngelo, Daniel J

    2015-01-01

    Regardless of age, patients with relapsed or refractory acute lymphoblastic leukemia (ALL) have extremely poor outcomes. The success of reinduction chemotherapy remains dismal, because complete remission rates are low and seldom durable. Clearly, new and novel strategies are needed to improve the outcome of patients with relapsed or refractory ALL. Patients with early relapse, especially those still receiving chemotherapy, tend to have a much poorer outcome and are often chemotherapy resistant. Although high-dose approaches may overcome chemotherapy resistance, long-term disease-free and overall survival remains limited. Several approaches have been used to target antigens, including cluster of differentiation (CD) 19, CD20, CD22, and CD52, on the surface of the malignant lymphoblast with striking efficacy. This review will focus on the clinical application of the major classes of antibodies, including naked antibodies, drug-antibody conjugates, immunotoxins, and T cell-engaging bispecific antibodies. Hopefully, these novel monoclonal antibodies will result in a significant improvement in the outcome of patients with relapsed or refractory ALL.

  7. Thermally induced degradation pathways of three different antibody-based drug development candidates.

    Science.gov (United States)

    Fincke, Anja; Winter, Jonas; Bunte, Thomas; Olbrich, Carsten

    2014-10-01

    Protein-based medicinal products are prone to undergo a variety of chemical and physical degradation pathways. One of the most important exogenous stress condition to consider during manufacturing, transport and storage processes is temperature, because antibody-based therapeutics are only stable in a limited temperature range. In this study, three different formats of antibody-based molecules (IgG1, a bispecific scFv and a fab fragment) were exposed to thermal stress conditions occurring during transport and storage. For evaluation, an analytical platform was developed for the detection and characterization of relevant degradation pathways of different antibody-based therapeutics. The effect of thermal stress conditions on the stability of the three antibody-based formats was therefore investigated using visual inspection, different spectroscopic measurements, dynamic light scattering (DLS), differential scanning calorimetry (DSC), electrophoresis, asymmetric flow field-flow fractionation (AF4) and surface plasmon resonance technology (SPR). In summary, thermal stress led to heterogeneous chemical and physical degradation pathways of all three antibody-based formats used. In addition, identical exogenous stress conditions resulted in different kinds and levels of aggregates and fragmentation products. This knowledge is fundamental for a systematic and successful stabilization of protein-based therapeutics by the use of formulation additives.

  8. A bispecific protein capable of engaging CTLA-4 and MHCII protects non-obese diabetic mice from autoimmune diabetes.

    Directory of Open Access Journals (Sweden)

    Hongmei Zhao

    Full Text Available Crosslinking ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4 to the T cell receptor (TCR with a bispecific fusion protein (BsB comprised of a mutant mouse CD80 and lymphocyte activation antigen-3 (LAG-3 has been shown to attenuate TCR signaling and to direct T-cell differentiation toward Foxp3(+ regulatory T cells (Tregs in an allogenic mixed lymphocyte reaction (MLR. Here, we show that antigen-specific Tregs can also be induced in an antigen-specific setting in vitro. Treatment of non-obese diabetic (NOD female mice between 9-12 weeks of age with a short course of BsB elicited a transient increase of Tregs in the blood and moderately delayed the onset of autoimmune type 1 diabetes (T1D. However, a longer course of treatment (10 weeks of 4-13 weeks-old female NOD animals with BsB significantly delayed the onset of disease or protected animals from developing diabetes, with only 13% of treated animals developing diabetes by 35 weeks of age compared to 80% of the animals in the control group. Histopathological analysis of the pancreata of the BsB-treated mice that remained non-diabetic revealed the preservation of insulin-producing β-cells despite the presence of different degrees of insulitis. Thus, a bifunctional protein capable of engaging CTLA-4 and MHCII and indirectly co-ligating CTLA-4 to the TCR protected NOD mice from developing T1D.

  9. An EGFR/HER2-Bispecific and enediyne-energized fusion protein shows high efficacy against esophageal cancer.

    Directory of Open Access Journals (Sweden)

    Xiao-Fang Guo

    Full Text Available Esophageal cancer is one of the most common cancers, and the 5-year survival rate is less than 10% due to lack of effective therapeutic agents. This study was to evaluate antitumor activity of Ec-LDP-Hr-AE, a recently developed bispecific enediyne-energized fusion protein targeting both epidermal growth factor receptor (EGFR and epidermal growth factor receptor 2 (HER2, on esophageal cancer. The fusion protein Ec-LDP-Hr-AE consists of two oligopeptide ligands and an enediyne antibiotic lidamycin (LDM for receptor binding and cell killing, respectively. The current study demonstrated that Ec-LDP-Hr had high affinity to bind to esophageal squamous cell carcinoma (ESCC cells, and enediyne-energized fusion protein Ec-LDP-Hr-AE showed potent cytotoxicity to ESCC cells with differential expression of EGFR and HER2. Ec-LDP-Hr-AE could cause significant G2-M arrest in EC9706 and KYSE150 cells, and it also induced apoptosis in ESCC cells in a dosage-dependent manner. Western blot assays showed that Ec-LDP-Hr-AE promoted caspase-3 and caspase-7 activities as well as PARP cleavage. Moreover, Ec-LDP-Hr-AE inhibited cell proliferation via decreasing phosphorylation of EGFR and HER2, and further exerted inhibition of the activation of their downstream signaling molecules. In vivo, at a tolerated dose, Ec-LDP-Hr-AE inhibited tumor growth by 88% when it was administered to nude mice bearing human ESCC cell KYSE150 xenografts. These results indicated that Ec-LDP-Hr-AE exhibited potent anti-caner efficacy on ESCC, suggesting it could be a promising candidate for targeted therapy of esophageal cancer.

  10. Mechanistic Projection of First-in-Human Dose for Bispecific Immunomodulatory P-Cadherin LP-DART: An Integrated PK/PD Modeling Approach.

    Science.gov (United States)

    Chen, X; Haddish-Berhane, N; Moore, P; Clark, T; Yang, Y; Li, H; Xuan, D; Barton, H A; Betts, A M; Barletta, F

    2016-09-01

    A bispecific immunomodulatory biotherapeutic molecule (P-cadherin LP-DART) based on the Dual Affinity Re-Targeting (DART) scaffold has been developed as a potential antitumor treatment showing efficacy in preclinical testing. A minimal anticipated biological effect level (MABEL) approach was applied to project the first-in-human (FIH) dose, because of its immune agonistic properties following target engagement. The pharmacological activity of P-cadherin LP-DART is driven by binding to both P-cadherin on the tumor cells and CD3 on T cells. Therefore, the concentration of the tri-molecular synapse formed between drug, T cell, and tumor cell, rather than drug concentration, is responsible for efficacy. A mechanistic pharmacokinetic/pharmacodynamic (PK/PD)-driven approach was explored to understand the exposure-response relationship based on the synapse concentration to project the MABEL dose. Orthogonal approaches including PK-driven and receptor occupancy calculations were also investigated. This study showcases the application of PK/PD modeling in immune-oncology, and could potentially be implemented for other bispecific biotherapeutics. PMID:27170541

  11. Generation and characterization of a bispecific diabody targeting both EPH receptor A10 and CD3.

    Science.gov (United States)

    Kamada, Haruhiko; Taki, Shintaro; Nagano, Kazuya; Inoue, Masaki; Ando, Daisuke; Mukai, Yohei; Higashisaka, Kazuma; Yoshioka, Yasuo; Tsutsumi, Yasuo; Tsunoda, Shin-ichi

    2015-01-24

    The EPH receptor A10 (EphA10) is up-regulated in breast cancer but is not normally expressed in healthy tissue, thus it has been suggested that EphA10 may be a useful target for cancer therapy. This study reports a diabody, an antibody derivative binding two different target molecules, EphA10 expressed in tumor cells and CD3 expressed in T cells, which showed T cell dependent-cytotoxicity. The diabody, which has His-tagged and FLAG-tagged chains, was expressed in Escherichia coli and purified in both heterodimer (Db-1) and homodimer (Db-2) formulations by liquid chromatography. Flow cytometry analysis using EphA10-expressing cells showed that binding activity of heterodimers was stronger than that of homodimers. Addition of diabodies to PBMC cultures resulted in T-cell mediated redirected lysis, and the bioactivity was consistent with the stronger binding activity of heterodimeric diabody formulations. Our results indicate that diabodies recognizing both EphA10 and CD3 could have a range of potential applications in cancer therapy, such as breast cancers that express the EPH receptor A10, especially triple negative breast cancer. PMID:25528586

  12. From aspecific to bispecific immunotherapy : immunomodulatory effects of interleukin-2 and T cell directed monoclonal antibodies in renal cell carcinoma patients

    NARCIS (Netherlands)

    Janssen, Richard Antonius Jozef

    1994-01-01

    The aim of this study was to get more insight into the immunological effects of rIL-2 therapy in renal cell carcinoma patients. Special attention was given to the activation of T cells, since these cells appear to play an important role in the immune response against tumour cells. In addition, the i

  13. CD40-targeted adenoviral gene transfer to dendritic cells through the use of a novel bispecific single-chain Fv antibody enhances cytotoxic T cell activation

    NARCIS (Netherlands)

    Brandao, JG; Scheper, RJ; Lougheed, SM; Curiel, DT; Tillman, BW; Gerritsen, WR; van den Eertwegh, AJM; Pinedo, HM; Haisma, HJ; de Gruijl, TD

    2003-01-01

    Adenoviral (Ad) transduction of dendritic cells (DC) is a promising vaccination strategy. However, clinical applicability of Ad vectors is hampered by the necessity to use high titers of infectious Ad particles for efficient DC transduction. Here, we report on the production of a bacterially express

  14. Advances in cancer immunotherapy by using bispecific antibody%双特异性抗体治疗恶性肿瘤的研究进展

    Institute of Scientific and Technical Information of China (English)

    张辛燕; 李小平

    2002-01-01

    双特异性抗体(BsAb),一方面结合免疫效应细胞上的激活分子或偶联毒素,另一方面利用抗体的特异性与肿瘤相关抗原(TAA)结合。通过双结合,激活效应细胞如细胞毒性T细胞和NK细胞,或通过免疫毒素发挥靶向抗肿瘤效应,是一种很有潜力的恶性肿瘤免疫治疗方式。本文对双特异性抗体的构建方法、作用机制及其相关研究加以概述。

  15. Monoclonal antibodies.

    Science.gov (United States)

    2009-01-01

    The ability to produce and exploit monoclonal antibodies (mAbs) has revolutionized many areas of biological sciences. The unique property of an mAb is that it is a single species of immunoglobulin (IG) molecule. This means that the specificity of the interaction of the paratopes on the IG, with the epitopes on an antigenic target, is the same on every molecule. This property can be used to great benefit in immunoassays to provide tests of defined specificity and sensitivity, which improve the possibilities of standardization. The performance of assays can often be determined relating the actual weight of antibody (hence the number of molecules) to the activity. Often the production of an mAb against a specific epitope is the only way that biological entities can be differentiated. This chapter outlines the areas involving the development of assays based on mAbs. The problems involved address include the physical aspects of mAbs and how they may affect assay design and also the implications of results based on monospecific reagents. Often these are not fully understood, leading to assays that are less than satisfactory, which does not justify the relatively high cost of preparing and screening of mAbs. There are many textbooks and reviews dealing with the preparation of mAbs, the principles involved, and various purification and manipulative methods for the preparation of fragments and conjugation. There has been little general information attempting to summarize the best approaches to assay design using mAbs. Much time can be wasted through bad planning, and this is particularly relevant to mAbs. A proper understanding of some basic principles is essential. It is beyond the scope of this chapter to discuss all aspects, but major areas are highlighted. PMID:19219589

  16. Blockade of the PD-1/PD-L1 axis augments lysis of AML cells by the CD33/CD3 BiTE antibody construct AMG 330: reversing a T-cell-induced immune escape mechanism.

    Science.gov (United States)

    Krupka, C; Kufer, P; Kischel, R; Zugmaier, G; Lichtenegger, F S; Köhnke, T; Vick, B; Jeremias, I; Metzeler, K H; Altmann, T; Schneider, S; Fiegl, M; Spiekermann, K; Bauerle, P A; Hiddemann, W; Riethmüller, G; Subklewe, M

    2016-02-01

    Bispecific T-cell engagers (BiTEs) are very effective in recruiting and activating T cells. We tested the cytotoxicity of the CD33/CD3 BiTE antibody construct AMG 330 on primary acute myeloid leukemia (AML) cells ex vivo and characterized parameters contributing to antileukemic cytolytic activity. The E:T ratio and the CD33 expression level significantly influenced lysis kinetics in long-term cultures of primary AML cells (n=38). AMG 330 induced T-cell-mediated proinflammatory conditions, favoring the upregulation of immune checkpoints on target and effector cells. Although not constitutively expressed at the time of primary diagnosis (n=123), PD-L1 was strongly upregulated on primary AML cells upon AMG 330 addition to ex vivo cultures (n=27, Pcheckpoint molecules in upcoming clinical trials with AMG 330 to enhance BiTE antibody construct-mediated cytotoxicity. PMID:26239198

  17. Bispecific designed ankyrin repeat proteins (DARPins) targeting epidermal growth factor receptor inhibit A431 cell proliferation and receptor recycling

    NARCIS (Netherlands)

    Boersma, Ykelien L; Chao, Ginger; Steiner, Daniel; Wittrup, K Dane; Plückthun, Andreas

    2011-01-01

    The EGF receptor (EGFR) has been implicated in the development and progression of many tumors. Although monoclonal antibodies directed against EGFR have been approved for the treatment of cancer in combination with chemotherapy, there are limitations in their clinical efficacy, necessitating the sea

  18. [Antinuclear antibodies].

    Science.gov (United States)

    Cabiedes, Javier; Núñez-Álvarez, Carlos A

    2010-01-01

    Anti-nuclear antibodies (ANA) are immunoglobulin directed against autologous cell nuclear and cytoplasmic components. Besides the autoimmune ANA there are other ANA that can be detected in circulation, like natural and infectious ANA. Because of its high sensibility, detection of the ANA must be done by indirect immunofluorescence (IIF) as screening test and all of those positive samples are convenient to confirm its specificity by ELISA, western blot or other techniques. Positive ANA detected by IIF must be evaluated taking in to account the pattern and titer. The following recommended step is the specificity characterization (reactivity against extractable nuclear antigens [ENA], dsDNA, etc.) which is useful for the diagnosis and follow up of patients with autoimmune diseases, and by such reasoning, its detection must be performed in an orderly and reasonable way using guides or strategies focused to the good use and interpretation of the autoantibodies. The objective of this review is to present a compilation of the literature and our experience in the detection and study of the ANA.

  19. Use of Heteropolymeric Monoclonal Antibodies to Attach Antigens to the C3b Receptor of Human Erythrocytes: A Potential Therapeutic Treatment

    Science.gov (United States)

    Taylor, Ronald P.; Sutherland, William M.; Reist, Craig J.; Webb, Donna J.; Wright, Eleanor L.; Labuguen, Ronald H.

    1991-04-01

    We have prepared bispecific, cross-linked monoclonal antibodies (heteropolymers) with specificity for both targeted antigens and the human erythrocyte (RBC) complement receptor. These heteropolymers facilitate binding of target antigens (human IgG and dinitrophenylated bovine γ globulin) to human RBCs under conditions that either allow or preclude complement activation. Quantitative analyses of this binding agree well with the number of complement receptors per RBC. In vitro "whole-blood" model experiments indicate heteropolymer-facilitated binding of antigens to RBCs is rapid and stable at 37^circC. It may be possible to extend these prototype experiments to the in vivo situation and use heteropolymer-attached RBCs for the safe and rapid binding, neutralization, and removal from the circulation of pathogenic antigens associated with infectious disease.

  20. Bi-specific splice-switching PMO oligonucleotides conjugated via a single peptide active in a mouse model of Duchenne muscular dystrophy.

    Science.gov (United States)

    Shabanpoor, Fazel; McClorey, Graham; Saleh, Amer F; Järver, Peter; Wood, Matthew J A; Gait, Michael J

    2015-01-01

    The potential for therapeutic application of splice-switching oligonucleotides (SSOs) to modulate pre-mRNA splicing is increasingly evident in a number of diseases. However, the primary drawback of this approach is poor cell and in vivo oligonucleotide uptake efficacy. Biological activities can be significantly enhanced through the use of synthetically conjugated cationic cell penetrating peptides (CPPs). Studies to date have focused on the delivery of a single SSO conjugated to a CPP, but here we describe the conjugation of two phosphorodiamidate morpholino oligonucleotide (PMO) SSOs to a single CPP for simultaneous delivery and pre-mRNA targeting of two separate genes, exon 23 of the Dmd gene and exon 5 of the Acvr2b gene, in a mouse model of Duchenne muscular dystrophy. Conjugations of PMOs to a single CPP were carried out through an amide bond in one case and through a triazole linkage ('click chemistry') in the other. The most active bi-specific CPP-PMOs demonstrated comparable exon skipping levels for both pre-mRNA targets when compared to individual CPP-PMO conjugates both in cell culture and in vivo in the mdx mouse model. Thus, two SSOs with different target sequences conjugated to a single CPP are biologically effective and potentially suitable for future therapeutic exploitation. PMID:25468897

  1. Comprehensive Analysis of the Therapeutic IgG4 Antibody Pembrolizumab: Hinge Modification Blocks Half Molecule Exchange In Vitro and In Vivo.

    Science.gov (United States)

    Yang, Xiaoyu; Wang, Fengqiang; Zhang, Ying; Wang, Larry; Antonenko, Svetlana; Zhang, Shuli; Zhang, Yi Wei; Tabrizifard, Mohammad; Ermakov, Grigori; Wiswell, Derek; Beaumont, Maribel; Liu, Liming; Richardson, Daisy; Shameem, Mohammed; Ambrogelly, Alexandre

    2015-12-01

    IgG4 antibodies are evolving as an important class of cancer immunotherapies. However, human IgG4 can undergo Fab arm (half molecule) exchange with other IgG4 molecules in vivo. The hinge modification by a point mutation (S228P) prevents half molecule exchange of IgG4. However, the experimental confirmation is still expected by regulatory agencies. Here, we report for the first time the extensive analysis of half molecule exchange for a hinge-modified therapeutic IgG4 molecule, pembrolizumab (Keytruda) targeting programmed death 1 (PD1) receptor that was approved for advanced melanoma. Studies were performed in buffer or human serum using multiple exchange partners including natalizumab (Tysabri) and human IgG4 pool. Formation of bispecific antibodies was monitored by fluorescence resonance energy transfer, exchange with Fc fragments, mixed mode chromatography, immunoassays, and liquid chromatography-mass spectrometry. The half molecule exchange was also examined in vivo in SCID (severe combined immunodeficiency) mice. Both in vitro and in vivo results indicate that the hinge modification in pembrolizumab prevented half molecule exchange, whereas the unmodified counterpart anti-PD1 wt showed active exchange activity with other IgG4 antibodies or self-exchange activity with its own molecules. Our work, as an example expected for meeting regulatory requirements, contributes to establish without ambiguity that hinge-modified IgG4 antibodies are suitable for biotherapeutic applications.

  2. T-cell ligands modulate the cytolytic activity of the CD33/CD3 BiTE antibody construct, AMG 330

    International Nuclear Information System (INIS)

    Preclinical and emerging clinical studies demonstrate that bispecific T-cell engaging (BiTE) antibody constructs can potently lyse targeted tumor cells, but the determinants for their activity remain incompletely understood. Using human acute myeloid leukemia (AML) cell lines engineered to overexpress individual T-cell ligands, we found that expression of the inhibitory ligands, PD-L1 and PD-L2, reduced the cytolytic activity of the BiTE antibody construct targeting CD33, AMG 330; conversely, expression of the activating ligands, CD80 and CD86, augmented the cytotoxic activity of AMG 330. Consistent with these findings, treatment with an activating antibody directed at the co-stimulatory T-cell receptor, CD28, significantly increased AMG 330-induced cytotoxicity in human AML cell lines. Using specimens from 12 patients with newly diagnosed or relapsed/refractory AML, we found that activation of CD28 also increased the activity of AMG 330 in primary human AML cells (P=0.023). Together, our findings indicate that T-cell ligands and co-receptors modulate the anti-tumor activity of the CD33/CD3 BiTE antibody construct, AMG 330. These findings suggest that such ligands/co-receptors could serve as biomarkers of response and that co-treatment strategies with pharmacological modulators of T-cell receptor signaling could be utilized to further enhance the activity of this targeted therapeutic

  3. Monoclonal antibodies and cancer

    International Nuclear Information System (INIS)

    The usefulness of radiolabeled monoclonal antibodies for imaging and treatment of human (ovarian) cancer was investigated. A review of tumor imaging with monoclonal antibodies is presented. Special attention is given to factors that influence the localization of the antibodies in tumors, isotope choice and methods of radiolabeling of the monoclonal antibodies. Two monoclonal antibodies, OC125 and OV-TL3, with high specificity for human epithelial ovarian cancer are characterized. A simple radio-iodination technique was developed for clinical application of the monoclonal antibodies. The behavior of monoclonal antibodies in human tumor xenograft systems and in man are described. Imaging of tumors is complicated because of high background levels of radioactivity in other sites than the tumor, especially in the bloodpool. A technique was developed to improve imaging of human tumor xenographs in nude mice, using subtraction of a specific and a non-specific antibody, radiolabeled with 111In, 67Ga and 131I. To investigate the capability of the two monoclonal antibodies, to specifically localize in human ovarian carcinomas, distribution studies in mice bearing human ovarian carcinoma xenografts were performed. One of the antibodies, OC125, was used for distribution studies in ovarian cancer patients. OC125 was used because of availability and approval to use this antibody in patients. The same antibody was used to investigate the usefulness of radioimmunoimaging in ovarian cancer patients. The interaction of injected radiolabeled antibody OC125 with circulating antigen and an assay to measure the antibody response in ovarian cancer patients after injection of the antibody is described. 265 refs.; 30 figs.; 19 tabs

  4. [VGKC-complex antibodies].

    Science.gov (United States)

    Watanabe, Osamu

    2013-04-01

    Various antibodies are associated with voltage-gated potassium channels (VGKCs). Representative antibodies to VGKCs were first identified by radioimmunoassays using radioisotope-labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were detected only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in patients with Morvan's syndrome and in those with a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins (for example LGI-1 and CASPR-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now commonly known as VGKC-complex antibodies. In general, LGI-1 antibodies are most commonly detected in patients with limbic encephalitis with syndrome of inappropriate secretion of antidiuretic hormone. CASPR-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability. Furthermore, VGKC-complex antibodies are tightly associated with chronic idiopathic pain. Hyperexcitability of nociceptive pathways has also been implicated. These antibodies may be detected in sera of some patients with neurodegenerative diseases (for example, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease).

  5. Genetically engineered multivalent single chain antibody constructs for cancer therapy

    Energy Technology Data Exchange (ETDEWEB)

    Surinder Batra, Ph D

    2006-02-27

    its tumor: normal tissue ratio for improved therapeutic index, we engineered a variety antibody constructs. These constructs were evaluated using novel approaches like special radionuclides, pretargeting and optimization. Due to the smaller size, the engineered antibody molecules should penetrate better throughout a tumor mass, with less dose heterogeneity, than is the case with intact IgG. Multivalent scFvs with an appropriate radionuclide, therefore, hold promising prospects for cancer therapy and clinical imaging in MAb-based radiopharmaceuticals. In addition, the human anti-mouse antibodies (HAMA) responses in patients against antibody-based therapy are usually directed against the immunoglobulin constant regions; however, anti-idiotypic responses can also be detected. The HAMA responses reduce the efficacy of treatment by removing the circulating antibody molecules, fragments, and possibly scFvs by altering the pharmacokinetic properties of the antibody. HAMA responses against divalent IgG, divalent Ig fragments, and possibly multimeric scFvs could cause immune complex formation with hypersensitivity or allergic reactions that could be harmful to patients. The use of small molecules, such as scFvs (monomeric as well as multimeric), with their shorter biological half-lives and the lack of the constant regions and humanized variable (binding regions) performed in our studies should reduce the development of HAMA. The generation of humanized and fully human scFvs should further reduce the development of HAMA. Specific accomplishments on the project are the production of large amounts of recombinant antibodies as they are required in large amounts for cancer diagnosis and therapy. A variety of single-chain Fv (scFv) constructs were engineered for the desired pharmacokinetic properties. Tetrameric and dimeric scFvs showed a two-fold advantage: (1) there was a considerable gain in avidity as compared to smaller fragments, and (2) the biological half-life was more

  6. Genetically engineered multivalent single chain antibody constructs for cancer therapy

    International Nuclear Information System (INIS)

    increase its tumor: normal tissue ratio for improved therapeutic index, we engineered a variety antibody constructs. These constructs were evaluated using novel approaches like special radionuclides, pretargeting and optimization. Due to the smaller size, the engineered antibody molecules should penetrate better throughout a tumor mass, with less dose heterogeneity, than is the case with intact IgG. Multivalent scFvs with an appropriate radionuclide, therefore, hold promising prospects for cancer therapy and clinical imaging in MAb-based radiopharmaceuticals. In addition, the human anti-mouse antibodies (HAMA) responses in patients against antibody-based therapy are usually directed against the immunoglobulin constant regions; however, anti-idiotypic responses can also be detected. The HAMA responses reduce the efficacy of treatment by removing the circulating antibody molecules, fragments, and possibly scFvs by altering the pharmacokinetic properties of the antibody. HAMA responses against divalent IgG, divalent Ig fragments, and possibly multimeric scFvs could cause immune complex formation with hypersensitivity or allergic reactions that could be harmful to patients. The use of small molecules, such as scFvs (monomeric as well as multimeric), with their shorter biological half-lives and the lack of the constant regions and humanized variable (binding regions) performed in our studies should reduce the development of HAMA. The generation of humanized and fully human scFvs should further reduce the development of HAMA. Specific accomplishments on the project are the production of large amounts of recombinant antibodies as they are required in large amounts for cancer diagnosis and therapy. A variety of single-chain Fv (scFv) constructs were engineered for the desired pharmacokinetic properties. Tetrameric and dimeric scFvs showed a two-fold advantage: (1) there was a considerable gain in avidity as compared to smaller fragments, and (2) the biological half-life was more

  7. Heavy chain only antibodies

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud;

    2013-01-01

    Unlike conventional antibodies, heavy chain only antibodies derived from camel contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHHs possess unique properties that enable them to excel conventional therapeutic antibodies and their smaller antigen-...... for combating HER2+ breast cancer. © 2013 by Tabriz University of Medical Sciences.......Unlike conventional antibodies, heavy chain only antibodies derived from camel contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHHs possess unique properties that enable them to excel conventional therapeutic antibodies and their smaller antigen......-binding fragments in cancer targeting and therapy. VHHs express low immunogenicity, are highly robust and easy to manufacture and have the ability to recognize hidden or uncommon epitopes. We highlight the utility of VHH in design of new molecular, multifunctional particulate and immune cell-based systems...

  8. Engineering antibody therapeutics.

    Science.gov (United States)

    Chiu, Mark L; Gilliland, Gary L

    2016-06-01

    The successful introduction of antibody-based protein therapeutics into the arsenal of treatments for patients has within a few decades fostered intense innovation in the production and engineering of antibodies. Reviewed here are the methods currently used to produce antibodies along with how our knowledge of the structural and functional characterization of immunoglobulins has resulted in the engineering of antibodies to produce protein therapeutics with unique properties, both biological and biophysical, that are leading to novel therapeutic approaches. Antibody engineering includes the introduction of the antibody combining site (variable regions) into a host of architectures including bi and multi-specific formats that further impact the therapeutic properties leading to further advantages and successes in patient treatment. PMID:27525816

  9. RBC Antibody Screen

    Science.gov (United States)

    ... the baby is Rh-positive and the mother's antibody status is negative for anti-D, the mother is given additional RhIG. This test also may be used to help diagnose autoimmune-related hemolytic anemia ... when a person produces antibodies against his or her own RBC antigens. This ...

  10. γδ T cell-mediated antibody-dependent cellular cytotoxicity with CD19 antibodies assessed by an impedance-based label-free real-time cytotoxicity assay

    Directory of Open Access Journals (Sweden)

    Ursula Jördis Eva Seidel

    2014-12-01

    Full Text Available γδ T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation (SCT strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC with monoclonal antibodies (mAbs. These features make γδ T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL. To evaluate combination of human γδ T cells with CD19 antibodies for immunotherapy of B-lineage ALL, γδ T cells were expanded after a GMP-compliant protocol and ADCC of both primary and expanded γδ T cells with an Fc optimized CD19 antibody (4G7SDIE and a bispecific antibody with the specificities CD19 and CD16 (N19-C16 was evaluated in CD107a degranulation assays and intracellular cytokine staining (ICS. CD107a, TNF-α and IFN-γ expression of primary γδ T cells were significantly increased and correlated with CD16-expression of γδ T cells. γδ T cells highly expressed CD107a after expansion and no further increased expression by 4G7SDIE and N19-C16 was measured. Cytotoxicity of purified expanded γδ T cells targeting CD19-expressing cells was assessed in both europium-TDA release and in an impedance-based label-free method (using the xCELLigence system measuring γδ T cell lysis in real-time. Albeit in the 2 h end-point europium-TDA release assay no increased lysis was observed, in real-time xCELLigence assays both significant antibody-independent cytotoxicity and ADCC of γδ T cells were observed. The xCELLigence system outperformed the end-point europium-TDA release assay in sensitivity and allows drawing of conclusions to lysis kinetics of γδ T cells over prolonged periods of time periods. Combination of CD19 antibodies with primary as well as expanded γδ T cells exhibits an promising approach, which may enhance clinical outcome of patients with pediatric B-lineage ALL and

  11. γδ T Cell-Mediated Antibody-Dependent Cellular Cytotoxicity with CD19 Antibodies Assessed by an Impedance-Based Label-Free Real-Time Cytotoxicity Assay.

    Science.gov (United States)

    Seidel, Ursula Jördis Eva; Vogt, Fabian; Grosse-Hovest, Ludger; Jung, Gundram; Handgretinger, Rupert; Lang, Peter

    2014-01-01

    γδ T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) with monoclonal antibodies (mAbs). These features make γδ T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL). To evaluate combination of human γδ T cells with CD19 antibodies for immunotherapy of B-lineage ALL, γδ T cells were expanded after a GMP-compliant protocol and ADCC of both primary and expanded γδ T cells with an Fc-optimized CD19 antibody (4G7SDIE) and a bi-specific antibody with the specificities CD19 and CD16 (N19-C16) was evaluated in CD107a-degranulation assays and intracellular cytokine staining. CD107a, TNFα, and IFNγ expression of primary γδ T cells were significantly increased and correlated with CD16-expression of γδ T cells. γδ T cells highly expressed CD107a after expansion and no further increased expression by 4G7SDIE and N19-C16 was measured. Cytotoxicity of purified expanded γδ T cells targeting CD19-expressing cells was assessed in both europium-TDA release and in an impedance-based label-free method (using the xCELLigence system) measuring γδ T cell lysis in real-time. Albeit in the 2 h end-point europium-TDA release assay no increased lysis was observed, in real-time xCELLigence assays both significant antibody-independent cytotoxicity and ADCC of γδ T cells were observed. The xCELLigence system outperformed the end-point europium-TDA release assay in sensitivity and allows drawing of conclusions to lysis kinetics of γδ T cells over prolonged periods of time periods. Combination of CD19 antibodies with primary as well as expanded γδ T cells exhibits a promising approach, which may enhance clinical outcome of patients with pediatric B-lineage ALL and requires clinical

  12. Selection of Recombinant Human Antibodies.

    Science.gov (United States)

    Tomszak, Florian; Weber, Susanne; Zantow, Jonas; Schirrmann, Thomas; Hust, Michael; Frenzel, André

    2016-01-01

    Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies. PMID:27236551

  13. Anti-insulin antibody test

    Science.gov (United States)

    Insulin antibodies - serum; Insulin Ab test; Insulin resistance - insulin antibodies; Diabetes - insulin antibodies ... You appear to have an allergic response to insulin Insulin no longer seems to control your diabetes

  14. HIV Antibody Test

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? HIV Antibody and HIV Antigen (p24) Share this page: Was this page helpful? Also known as: HIV Screening Tests; AIDS Test; AIDS Screen; HIV Serology; ...

  15. Antinuclear antibody panel

    Science.gov (United States)

    ... blood may be due to: Chronic liver disease Collagen vascular disease Drug-induced lupus erythematosus Myositis (inflammatory muscle disease) ... Saunders; 2011:chap 51. Read More Antibody Arthritis Collagen vascular disease Drug-induced lupus erythematosus Liver disease Scleroderma Systemic ...

  16. Anti-cartilage antibody.

    Science.gov (United States)

    Greenbury, C L; Skingle, J

    1979-08-01

    Antibody to cartilage has been demonstrated by indirect immunofluorescence on rat trachea in the serum of about 3% of 1126 patients with rheumatoid arthritis. Titres ranged from 1:20 to 1:640. The antibody was not found in 284 patients with primary or secondary osteoarthritis or in 1825 blood donors, nor, with the exception of two weak reactors, in 1314 paraplegic patients. In most cases the antibody appears to be specific for native type II collagen. Using this as an antigen in a haemagglutination test 94% of anti-cartilage sera were positive, whereas among 100 rheumatoid control sera there were only three weak positives. More than 80% of patients with antibody had some erosion of articular cartilage, but there was no correlation with age, sex, duration of disease, nor any recognisable clinical event or change.

  17. Antibody tumor penetration

    Science.gov (United States)

    Thurber, Greg M.; Schmidt, Michael M.; Wittrup, K. Dane

    2009-01-01

    Antibodies have proven to be effective agents in cancer imaging and therapy. One of the major challenges still facing the field is the heterogeneous distribution of these agents in tumors when administered systemically. Large regions of untargeted cells can therefore escape therapy and potentially select for more resistant cells. We present here a summary of theoretical and experimental approaches to analyze and improve antibody penetration in tumor tissue. PMID:18541331

  18. Expression of Recombinant Antibodies

    OpenAIRE

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transg...

  19. Antibody informatics for drug discovery

    DEFF Research Database (Denmark)

    Shirai, Hiroki; Prades, Catherine; Vita, Randi;

    2014-01-01

    for antibody rational design using computational approaches to affinity and stability improvement, as well as ab-initio and homology-based antibody modeling; (ii) resources for antibody sequences, structures, and immune epitopes and open drug discovery resources for development of antibody drugs; and (iii...

  20. Checkpoint Antibodies but not T Cell-Recruiting Diabodies Effectively Synergize with TIL-Inducing γ-Irradiation.

    Science.gov (United States)

    Hettich, Michael; Lahoti, Jayashree; Prasad, Shruthi; Niedermann, Gabriele

    2016-08-15

    T cell-recruiting bispecific antibodies (bsAb) show promise in hematologic malignancies and are also being evaluated in solid tumors. In this study, we investigated whether T cell-recruiting bsAbs synergize with hypofractionated tumor radiotherapy (hRT) and/or blockade of the programmed death-1 (PD-1) immune checkpoint, both of which can increase tumor-infiltrating lymphocyte (TIL) numbers. Unexpectedly, large melanomas treated with hRT plus bsAb (AC133×CD3) relapsed faster than those treated with hRT alone, accompanied by massive TIL apoptosis. This fast relapse was delayed by the further addition of anti-PD-1. Mechanistic investigations revealed restimulation-induced cell death mediated by BIM and FAS as an additional cause of bsAb-mediated TIL depletion. In contrast, the double combination of hRT and anti-PD-1 strongly increased TIL numbers, and even very large tumors were completely eradicated. Our study reveals the risk that CD3-engaging bsAbs can induce apoptotic TIL depletion followed by rapid tumor regrowth, reminiscent of tolerance induction by CD3 mAb-mediated T-cell depletion, warranting caution in their use for the treatment of solid tumors. Our findings also argue that combining radiotherapy and anti-PD-1 can be quite potent, including against very large tumors. Cancer Res; 76(16); 4673-83. ©2016 AACR. PMID:27302161

  1. Engineering antibodies for cancer therapy.

    Science.gov (United States)

    Boder, Eric T; Jiang, Wei

    2011-01-01

    The advent of modern antibody engineering has led to numerous successes in the application of these proteins for cancer therapy in the 13 years since the first Food and Drug Administration approval, which has stimulated active interest in developing more and better drugs based on these molecules. A wide range of tools for discovering and engineering antibodies has been brought to bear on this challenge in the past two decades. Here, we summarize mechanisms of monoclonal antibody therapeutic activity, challenges to effective antibody-based treatment, existing technologies for antibody engineering, and current concepts for engineering new antibody formats and antibody alternatives as next generation biopharmaceuticals for cancer treatment.

  2. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    linker for yeast surface display of scFv and scFab fragments, we compared a series of different Gly-Ser-based linkers in display and antigen binding proficiency. We show that these formats of the model antibody can accommodate linkers of different lengths and that introduction of alanine or glutamate......-2. Based on the presented data we suggest that affinity maturation of the model antibody proceeds through multiple incremental steps of subtle improvements. We moreover conclude that the best affinity improved candidates are likely to be obtained through optimization of both the antigen...... fragments by in vivo homologous recombination large combinatorial antibody libraries can easily be generated. We have optimized ordered assembly of three CDR fragments into a gapped vector and observed increased transformation efficiency in a yeast strain carrying a deletion of the SGS1 helicase...

  3. Antithyroglobulin Antibodies and Antimicrosomal Antibodies in Various Thyroid Diseases

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Gwon Jun; Hong, Key Sak; Choi, Kang Won; Lee, Kyu; Koh, Chang Soon; Lee, Mun Ho; Park, Sung Hoe; Chi, Je Geun; Lee, Sang Kook [Seoul National University College of Medicine, Seoul (Korea, Republic of)

    1979-03-15

    The authors investigated the incidence of antithyroglobulin antibodies and antibodies and antimicrosomal antibodies measured by tanned red cell hemagglutination method in subjects suffering from various thyroid disorders. 1) In 15 normal patients, neither suffering from any thyroid diseases nor from any other autoimmune disorders, the antithyroglobulin antibodies were all negative, but the antimicrosomal antibody was positive only in one patient (6.7%). 2) The antithyroglobulin antibodies were positive in 31.5% (34 patients) of 108 patients with various thyroid diseases, and the antimicrosomal antibodies were positive in 37.0% (40 patients). 3) of the 25 patients with Graves' diseases, 7 patients (28.0%) showed positive for the antithyroglobulin antibodies, and 9 (36.0%) for the antimicrosomal antibodies. There was no definite differences in clinical and thyroid functions between the groups with positive and negative results. 4) Both antibodies were positive in 16 (88.9%) and 17 (94.4%) patients respectively among 18 patients with Hashimoto's thyroiditis, all of them were diagnosed histologically. 5) Three out of 33 patients with thyroid adenoma showed positive antibodies, and 3 of 16 patients with thyroid carcinoma revealed positive antibodies. 6) TRCH antibodies demonstrated negative results in 2 patients with subacute thyroiditis, but positive in one patient with idiopathic primary myxedema. 7) The number of patients with high titers(>l:802) was 16 for antithyroglobulin antibody, and 62.5% (10 patients) of which was Hashimoto's thyroiditis. Thirteen (65.0) of 20 patients with high titers (>l:802) for antimicrosomal antibody was Hashimoto's thyroiditis. TRCH test is a simple, sensitive method, and has high reliability and reproducibility. The incidences and titers of antithyroglobulin antibody and antimicrosomal antibody are especially high in Hashimoto's thyroiditis.

  4. Monoclonal antibodies in myeloma

    DEFF Research Database (Denmark)

    Sondergeld, P.; van de Donk, N. W. C. J.; Richardson, P. G.;

    2015-01-01

    The development of monoclonal antibodies (mAbs) for the treatment of disease goes back to the vision of Paul Ehrlich in the late 19th century; however, the first successful treatment with a mAb was not until 1982, in a lymphoma patient. In multiple myeloma, mAbs are a very recent and exciting add...

  5. Prediction of Antibody Epitopes

    DEFF Research Database (Denmark)

    Nielsen, Morten; Marcatili, Paolo

    2015-01-01

    self-proteins. Given the sequence or the structure of a protein of interest, several methods exploit such features to predict the residues that are more likely to be recognized by an immunoglobulin.Here, we present two methods (BepiPred and DiscoTope) to predict linear and discontinuous antibody...

  6. Lupus anticoagulants and antiphospholipid antibodies

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/000547.htm Lupus anticoagulants and antiphospholipid antibodies To use the sharing features on this page, please enable JavaScript. Lupus anticoagulants are antibodies against substances in the lining ...

  7. Recombinant antibodies and tumor targeting

    OpenAIRE

    Sheikholvaezin, Ali

    2006-01-01

    Different antibody derived constructs are rapidly advancing as putative tools for treatment of malignant diseases. Antibody engineering has added significant new technologies to modify size, affinities, solubility, stability and biodistribution properties for immunoconjugates. In the present thesis, the aim was to increase our knowledge on how new recombinant antibodies could be tailored to optimize localization to experimental tumors in mice. One hybridoma, producing the monoclonal antibody ...

  8. Production and function evaluation of a kind of bispecific antibody crosslinked with PLGA nanoparticles in vitro%PLGA纳米颗粒偶联的双特异性抗体制备及功能研究

    Institute of Scientific and Technical Information of China (English)

    李正成; 何成思; 喻新建; 于浩洋

    2016-01-01

    目的 制备一种能提高T细胞对癌细胞杀伤能力的双特异性抗体.方法 利用生物可降解材料PLGA(聚乳酸-羟基乙酸共聚物)制备成纳米颗粒,然后利用化学方法在纳米颗粒表面偶联能靶向结合T细胞的CD3抗体和癌细胞标志物MUC1的抗体,利用T细胞对癌细胞的杀伤实验来检测其功能.结果 成功制备出直径200 nm的PLGA纳米颗粒并偶联上CD3和MUC1抗体,功能检测得出制备的双特异性抗体能显著提高T细胞对癌细胞的杀伤率.结论 制备出一种能显著提高T细胞对癌细胞杀伤率的双特异性抗体.

  9. Cytokine induction of VCAM-1 but not IL13Rα2 on glioma cells: a tale of two antibodies.

    Directory of Open Access Journals (Sweden)

    Vaidehi Mahadev

    Full Text Available The interleukin-13 receptor alpha2 (IL13Rα2 is a cell surface receptor that is over-expressed by a subset of high-grade gliomas, but not expressed at significant levels by normal brain tissue. For both malignant and non-malignant cells, IL13Rα2 surface expression is reported to be induced by various cytokines such as IL-4 or IL-13 and tumor necrosis factor (TNF. Our group has developed a therapeutic platform to target IL13Rα2-positive brain tumors by engineering human cytotoxic T lymphocytes (CTLs to express the IL13-zetakine chimeric antigen receptor. We therefore sought to investigate the potential of cytokine stimulation to induce IL13Rα2 cell surface expression, and thereby increase susceptibility to IL13Rα2-specific T cell killing. In the course of these experiments, we unexpectedly found that the commercially available putative IL13Rα2-specific monoclonal antibody B-D13 recognizes cytokine-induced VCAM-1 on glioblastoma. We provide evidence that the induced receptor is not IL13Rα2, because its expression does not consistently correlate with IL13Rα2 mRNA levels, it does not bind IL-13, and it is not recognized by IL13-zetakine CTL. Instead we demonstrate by immunoprecipitation experiments and mass spectrometry that the antigen recognized by the B-D13 antibody following cytokine stimulation is VCAM-1, and that VCAM-1, but not IL13Rα2, is induced on glioma cells by TNF alone or in combination with IL-13 or IL-4. Further evaluation of several commercial B-D13 antibodies revealed that B-D13 is bi-specific, recognizing both IL13Rα2 and VCAM-1. This binding is non-overlapping based on soluble receptor competition experiments, and mass spectrometry identifies two distinct heavy and light chain species, providing evidence that the B-D13 reagent is di-clonal. PE-conjugation of the B-D13 antibody appears to disrupt IL13Rα2 recognition, while maintaining VCAM-1 specificity. While this work calls into question previous studies that have used

  10. Antibody mimetics: promising complementary agents to animal-sourced antibodies.

    Science.gov (United States)

    Baloch, Abdul Rasheed; Baloch, Abdul Wahid; Sutton, Brian J; Zhang, Xiaoying

    2016-01-01

    Despite their wide use as therapeutic, diagnostic and detection agents, the limitations of polyclonal and monoclonal antibodies have inspired scientists to design the next generation biomedical agents, so-called antibody mimetics that offer many advantages over conventional antibodies. Antibody mimetics can be constructed by protein-directed evolution or fusion of complementarity-determining regions through intervening framework regions. Substantial progress in exploiting human, butterfly (Pieris brassicae) and bacterial systems to design and select mimetics using display technologies has been made in the past 10 years, and one of these mimetics [Kalbitor® (Dyax)] has made its way to market. Many challenges lie ahead to develop mimetics for various biomedical applications, especially those for which conventional antibodies are ineffective, and this review describes the current characteristics, construction and applications of antibody mimetics compared to animal-sourced antibodies. The possible limitations of mimetics and future perspectives are also discussed. PMID:25264572

  11. A monoclonal antibody against leptin.

    Science.gov (United States)

    Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Bayat, Ali Ahmad; Mahmoudi, Ahmad Reza; Vojgani, Yasaman; Tavangar, Banafsheh; Hadavi, Reza; Zarei, Saeed

    2012-10-01

    Leptin is an important protein that regulates energy storage and homeostasis in humans and animals. Leptin deficiency results in various abnormalities such as diabetes, obesity, and infertility. Producing a high affinity monoclonal antibody against human leptin provides an important tool to monitor and trace leptin function in different biological fluids. In this study, recombinant human leptin was conjugated to KLH and injected into mice. After immunization, mouse myeloma SP2/0 cells were fused with murine splenocytes followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, a high affinity antibody was selected and purified by affinity chromatography. The affinity constant of the antibody was measured by ELISA. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibody. The anti-leptin antibody had a high affinity (around 1.13 × 10(-9) M) for its antigen. The saturation of the antibody with leptin (20 moles leptin per 1 mole antibody) in Western blot analysis proved that the antibody had specific binding to its antigen. Immunocytochemistry and flow cytometry on JEG-3 (human placental choriocarcinoma cell) cells revealed that the anti-leptin antibody recognized intracellular leptin. In conclusion, we report here the production and characterization of a murine anti-leptin antibody with high affinity for human leptin. PMID:23098305

  12. Engineering antibodies by yeast display.

    Science.gov (United States)

    Boder, Eric T; Raeeszadeh-Sarmazdeh, Maryam; Price, J Vincent

    2012-10-15

    Since its first application to antibody engineering 15 years ago, yeast display technology has been developed into a highly potent tool for both affinity maturing lead molecules and isolating novel antibodies and antibody-like species. Robust approaches to the creation of diversity, construction of yeast libraries, and library screening or selection have been elaborated, improving the quality of engineered molecules and certainty of success in an antibody engineering campaign and positioning yeast display as one of the premier antibody engineering technologies currently in use. Here, we summarize the history of antibody engineering by yeast surface display, approaches used in its application, and a number of examples highlighting the utility of this method for antibody engineering.

  13. Antiphospholipid Antibody and Antiphospholipid Syndrome

    Institute of Scientific and Technical Information of China (English)

    吴竞生

    2008-01-01

    @@ Antiphospholipid antibodies (APA) APA is a big category for all kinds of negative charge phospholipid or lecithin - a protein complex autoantibodies or the same antibody, through its recognition of antigen (target protein) different, and phospholipids or lecithin - protein complex combination of various rely on the interference Phospholipid clotting and anti-coagulation factor, and promote endothelial cells, platelets, complement activation and play a role. APA including lupus anticoagulant(LA) and anticardiolipin antibody (ACA), In addition, there are anti-β2 glycoprotein-I (β2-GPI) antibody, anti-prothrombin (a- PT) antibody, anti-lysophosphatidic acid antibody and anti-phosphatidylserine antibody, and so on. APA as the main target of phospholipid-binding protein, including β2-GPI, prothrombin, annexin, protein C (PC) and protein S (PS), plasminogen, and so on.

  14. The Broad Anti-AML Activity of the CD33/CD3 BiTE Antibody Construct, AMG 330, Is Impacted by Disease Stage and Risk.

    Science.gov (United States)

    Harrington, Kimberly H; Gudgeon, Chelsea J; Laszlo, George S; Newhall, Kathryn J; Sinclair, Angus M; Frankel, Stanley R; Kischel, Roman; Chen, Guang; Walter, Roland B

    2015-01-01

    The CD33/CD3-bispecific T-cell engaging (BiTE) antibody construct, AMG 330, potently lyses CD33+ leukemic cells in vitro. Using specimens from 41 patients with acute myeloid leukemia (AML), we studied the factors that might contribute to clinical response or resistance. For this purpose, thawed aliquots of primary AML samples were immunophenotypically characterized and subjected to various doses of AMG 330 in the presence or absence of healthy donor T-cells. After 48 hours, drug-specific cytotoxicity was quantified and correlated with CD33 expression levels, amounts of T-cells present, and other disease characteristics. AMG 330 caused modest cytotoxicity that was correlated with the amount of autologous T-cells (P = 0.0001) but not CD33 expression, as AMG 330 exerted marked cytotoxic effects in several specimens with minimal CD33 expression. With healthy donor T-cells added, AMG 330 cytotoxicity depended on the drug dose and effector:target (E:T) cell ratio. High cytotoxic activity was observed even with minimal CD33 expression, and AMG 330 cytotoxicity and CD33 expression correlated only at high E:T cell ratio and high AMG 330 doses (Pspectrum, while suggesting the presence of yet undefined, CD33-independent, relative resistance mechanisms in specific patient subsets.

  15. The antibody Hijikata Tatsumi

    Directory of Open Access Journals (Sweden)

    Éden Peretta

    2012-11-01

    Full Text Available Considered one of the most influential modern dance representatives in Japan, Tatsumi Hijikata’s work was a milestone in the Japanese post-war experimental artistic scene. Heretic son of his time, he staged a fertile mix of artistic and cultural influences, overlapping subversive elements of European arts and philosophy with radical references from pre-modern Japanese culture. In this way he built the foundations of its unstable antibody, its political-artistic project of dissolution of a organism, both physical and social.

  16. VIRAL ANTIBODIES IN PRESCHOOL CHILDREN

    Directory of Open Access Journals (Sweden)

    S. Saidi

    1974-08-01

    Full Text Available One hundred sera from children 1 - 6 years of age, representative of a large serum collection, were tested for the prevalence of antibodies against different viruses. Hemagglutination-inhibition (HI antibodies were found in 68% for measles; 61 % for rubella; 75'% for influenza A2/Hong Kong/68, 16% for influenza B/Md./59, 0% for group A arboviruses, 10% for group B arboviruses, 3% for phlebotomus fever group and 4% for Congo-Crimean hemorrhagic fever (C-CHF group of arboviruses Poliomyelitis-neutralizing antibodies for type 1, 2 and 3 were 90%; 85% and 84%~ respectively. Antibody to EH virus was detected in 84% of the sera by immuno-fluorescence. None of the sera were positive for hepatitis-B antigen or antibody by immuno-precipitation test. The prevalence of some viral antibodies found in this survey are compared with results obtained from surveys in other parts of the country.

  17. Metrics for antibody therapeutics development

    OpenAIRE

    Reichert, Janice M

    2010-01-01

    A wide variety of full-size monoclonal antibodies (mAbs) and therapeutics derived from alternative antibody formats can be produced through genetic and biological engineering techniques. These molecules are now filling the preclinical and clinical pipelines of every major pharmaceutical company and many biotechnology firms. Metrics for the development of antibody therapeutics, including averages for the number of candidates entering clinical study and development phase lengths for mAbs approv...

  18. Monoclonal antibodies for treating cancer

    International Nuclear Information System (INIS)

    The purpose of this study is to assess the current status of in-vivo use of monoclonal antibodies for treating cancer. Publications appearing between 1980 and 1988 were identified by computer searches using MEDLINE and CANCERLIT, by reviewing the table of contents of recently published journals, and by searching bibliographies of identified books and articles. More than 700 articles, including peer-reviewed articles and book chapters, were identified and selected for analysis. The literature was reviewed and 235 articles were selected as relevant and representative of the current issues and future applications for in-vivo monoclonal antibodies for cancer therapy and of the toxicity and efficacy which has been associated with clinical trials. Approaches include using antibody alone (interacting with complement or effector cells or binding directly with certain cell receptors) and immunoconjugates (antibody coupled to radioisotopes, drugs, toxins, or other biologicals). Most experience has been with murine antibodies. Trials of antibody alone and radiolabeled antibodies have confirmed the feasibility of this approach and the in-vivo trafficking of antibodies to tumor cells. However, tumor cell heterogeneity, lack of cytotoxicity, and the development of human antimouse antibodies have limited clinical efficacy. Although the immunoconjugates are very promising, heterogeneity and the antimouse immune response have hampered this approach as has the additional challenge of chemically or genetically coupling antibody to cytotoxic agents. As a therapeutic modality, monoclonal antibodies are still promising but their general use will be delayed for several years. New approaches using human antibodies and reducing the human antiglobulin response should facilitate treatment. 235 references

  19. Antineutrophil cytoplasmic antibodies

    Directory of Open Access Journals (Sweden)

    G.D. Sebastiani

    2011-06-01

    Full Text Available Antineutrophil cytoplasmic antibodies (ANCA are predominantly IgG autoantibodies directed against constituents of primary granules of neutrophils and monocytes’ lysosomes. Although several antigenic targets have been identified, those ANCA directed to proteinase 3 or myeloperoxidase are clinically relevant, whereas the importance of other ANCA remains unknown. Both are strongly associated with small vessel vasculitides, the ANCA-associated vasculitides, which include Wegener’s granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome, and the localised forms of these diseases (eg, pauci-immune necrotising and crescentic glomerulonephritis. ANCA is a useful serological test to assist in diagnosis of small-vessel vasculitides. 85-95% of patients with Wegener’s granulomatosis, microscopic polyangiitis, and pauci-immune necrotising and crescentic glomerulonephritis have serum ANCA. ANCA directed to either proteinase 3 or myeloperoxidase are clinically relevant, yet the relevance of other ANCA remains unknown. Besides their diagnostic potential, ANCA might be valuable in disease monitoring. In addition, data seem to confirm the long-disputed pathogenic role of these antibodies. There is increasing evidence that myeloperoxidase- ANCA are directly involved in the pathogenesis of necrotizing vasculitis. This is less clear for proteinase 3-ANCA, markers for Wegener’s granulomatosis. With respect to proteinase 3-ANCA, complementary proteinase 3, a peptide translated from the antisense DNA strand of proteinase 3 and homologous to several microbial peptides, may be involved in induction of proteinase 3-antineutrophil cytoplasmic autoantibodies.

  20. 针对HER2靶点的抗体药物研究与肿瘤靶向治疗%Development of antibody drugs targeting against HER2 for cancer therapy

    Institute of Scientific and Technical Information of China (English)

    汤沁; 丁倩; 林莉; 张珍珍; 代争; 詹金彪

    2012-01-01

    Human epidermal growth factor receptor 2 (HER2) belongs to the transmembrane glycoprotein receptor family. Overexpression of HER2 could directly lead to tumorigenesis and metastasis. This phenomenon could be observed in the breast cancer, ovarian cancer, gastric cancer, lung cancer and prostate cancer. Compared with the conventional chemotherapy, the targeted treatment of antibody is more specific and has lower side effects. This review describes the current status of monotherapy and combination therapies of anti-HER2 antibodies, trastuzumab and pertuzumab, with chemotherapeutic drugs. The development trends of new formats of anti-HER2 antibody drugs such as bispecific antibody, immunotoxin are also discussed.%人类表皮生长因子受体2(HER2)属于跨膜酪氨酸激酶受体家族的成员,在肿瘤细胞中存在过表达.研究显示在乳腺癌、卵巢癌、胃癌、肺癌、前列腺癌中均存在不同程度的HER2过表达.抗体靶向治疗与传统化疗相比,特异性强,毒副作用小.本文介绍了曲妥珠单抗和帕妥珠单抗的单药治疗效果和与化疗药物、激素治疗、疫苗的联合治疗效果,以及偶联药物策略,阐述了其他新型抗HER2抗体药物,特别是双特异抗体、免疫毒素以及抗体融合蛋白等研究近况,为相应的HER2抗体药物开发和临床应用提供参考.

  1. Targeting of Antibodies using Aptamers

    OpenAIRE

    Missailidis, Sotiris

    2003-01-01

    The chapter presents a methodology for the rapid selection of aptamers against antibody targets. It is a detailed account of the various methodological steps that describe the selection of aptamers, including PCR steps, buffers to be used, target immobilisation, partitioning and amplification of aptamers, clonning and sequencing, to results in high affinity and specificity ligands for the chosen target antibody.

  2. Structural Characterization of Peptide Antibodies

    DEFF Research Database (Denmark)

    Chailyan, Anna; Marcatili, Paolo

    2015-01-01

    The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptide...

  3. Pathogenic role of antiphospholipid antibodies

    NARCIS (Netherlands)

    Salmon, J. E.; de Groot, P. G.

    2008-01-01

    The antiphospholipid antibody syndrome (APS) is characterized by recurrent arterial and venous thrombosis and/or pregnancy in association with antiphospholipid (aPL) antibodies. The pathogenic mechanisms in APS that lead to in vivo injury are incompletely understood. Recent evidence suggests that AP

  4. Nonmitogenic Anti-CD3 Monoclonal Antibodies Deliver a Partial T Cell Receptor Signal and Induce Clonal Anergy

    Science.gov (United States)

    Smith, Judith A.; Tso, J. Yun; Clark, Marcus R.; Cole, Michael S.; Bluestone, Jeffrey A.

    1997-01-01

    Anti-CD3 monoclonal antibodies (mAbs) are potent immunosuppressive agents used in clinical transplantation. However, the activation-related adverse side effects associated with these mAbs have prompted the development of less toxic nonmitogenic anti-CD3 mAb therapies. At present, the functional and biochemical consequences of T cell exposure to nonmitogenic anti-CD3 is unclear. In this study, we have examined the early signaling events triggered by a nonmitogenic anti-CD3 mAb. Like the mitogenic anti-CD3 mAb, nonmitogenic anti-CD3 triggered changes in the T cell receptor (TCR) complex, including ζ chain tyrosine phosphorylation and ZAP-70 association. However, unlike the mitogenic anti-CD3 stimulation, nonmitogenic anti-CD3 was ineffective at inducing the highly phosphorylated form of ζ (p23) and tyrosine phosphorylation of the associated ZAP-70 tyrosine kinase. This proximal signaling deficiency correlated with minimal phospholipase Cγ-1 phosphorylation and failure to mobilize detectable Ca2+. Not only did biochemical signals delivered by nonmitogenic anti-CD3 resemble altered peptide ligand signaling, but exposure of Th1 clones to nonmitogenic anti-CD3 also resulted in functional anergy. Finally, a bispecific anti-CD3 × anti-CD4 F(ab)′2 reconstituted early signal transduction events and induced proliferation, suggesting that defective association of lck with the TCR complex may underlie the observed signaling differences between the mitogenic and nonmitogenic anti-CD3. PMID:9126922

  5. Metrics for antibody therapeutics development.

    Science.gov (United States)

    Reichert, Janice M

    2010-01-01

    A wide variety of full-size monoclonal antibodies (mAbs) and therapeutics derived from alternative antibody formats can be produced through genetic and biological engineering techniques. These molecules are now filling the preclinical and clinical pipelines of every major pharmaceutical company and many biotechnology firms. Metrics for the development of antibody therapeutics, including averages for the number of candidates entering clinical study and development phase lengths for mAbs approved in the United States, were derived from analysis of a dataset of over 600 therapeutic mAbs that entered clinical study sponsored, at least in part, by commercial firms. The results presented provide an overview of the field and context for the evaluation of on-going and prospective mAb development programs. The expansion of therapeutic antibody use through supplemental marketing approvals and the increase in the study of therapeutics derived from alternative antibody formats are discussed. PMID:20930555

  6. Metrics for antibody therapeutics development.

    Science.gov (United States)

    Reichert, Janice M

    2010-01-01

    A wide variety of full-size monoclonal antibodies (mAbs) and therapeutics derived from alternative antibody formats can be produced through genetic and biological engineering techniques. These molecules are now filling the preclinical and clinical pipelines of every major pharmaceutical company and many biotechnology firms. Metrics for the development of antibody therapeutics, including averages for the number of candidates entering clinical study and development phase lengths for mAbs approved in the United States, were derived from analysis of a dataset of over 600 therapeutic mAbs that entered clinical study sponsored, at least in part, by commercial firms. The results presented provide an overview of the field and context for the evaluation of on-going and prospective mAb development programs. The expansion of therapeutic antibody use through supplemental marketing approvals and the increase in the study of therapeutics derived from alternative antibody formats are discussed.

  7. A bispecific protein rG7S-MICA recruits natural killer cells and enhances NKG2D-mediated immunosurveillance against hepatocellular carcinoma.

    Science.gov (United States)

    Wang, Tong; Sun, Fumou; Xie, Wei; Tang, Mingying; He, Hua; Jia, Xuelian; Tian, Xuemei; Wang, Min; Zhang, Juan

    2016-03-28

    MHC class I-related chain A (MICA) is a principal immunoligand of the natural killer (NK) cell receptor NK group 2, member D (NKG2D) and plays a key role in NK cell-mediated immune recognition. Shedding of MICA from tumor cells leads to immunosuppression. To reconstitute the immunosurveilance function of NK cells, we constructed a fusion protein rG7S-MICA and explored its potential anti-tumor activity against hepatocellular carcinoma (HCC). rG7S-MICA consists of human MICA and a single-chain antibody fragment (scFv) targeting the tumor-associated antigen cluster of differentiation 24 (CD24). In vitro, rG7S-MICA engaged both NK cells and CD24(+) human HCC cells, and triggered NK cell-mediated cytolysis. Furthermore, in CD24(+) HCC-bearing nude mice, rG7S-MICA specifically targeted to the tumor tissue, where it effectively recruited NK cells and induced the release of cytokines, and showed superior anti-tumor activity. In conclusion, rG7S-MICA provides a new approach for HCC-targeting immunotherapy and has attracting potentials for clinical applications.

  8. The antineutrophil antibody in uveitis.

    OpenAIRE

    Young, D W

    1991-01-01

    Ninety eight patients with uveitis of various types were tested for the presence of the antineutrophil antibody or ANCA by an indirect immunofluorescence method. This antibody is found in patients with diseases associated with small vessel vasculitis, including Wegener's granulomatosis and microscopic polyarteritis. Eleven true positive cases were found. A positive test was not associated with the anatomical site of the uveitis but was related to the time course of the disease. In particular ...

  9. Functional effects of anticardiolipin antibodies.

    Science.gov (United States)

    Harris, E N; Pierangeli, S S

    1996-10-01

    The 'lupus anticoagulant' phenomenon is the best documented functional effect of antiphospholipid (aPL) antibodies, occurring either by inhibition of the prothrombinase and/or Factor X activation reactions. Understanding the mechanism by which aPL antibodies inhibit phospholipid dependent coagulation reactions may yield important clues about their 'thrombogenic effects' in vivo. We conducted a series of studies to determine the specificity, diversity, and mechanism by which aPL antibodies inhibit phospholipid dependent reactions. Results showed that purified immunoglobulins with lupus anticoagulant and anti-cardiolipin activities were absorbed by negatively charged phospholipids and both activities were recovered from the phospholipid-antibody precipitate. Purified aPL antibodies inhibited the prothrombinase reaction in a plasma free system in which beta 2-glycoprotein 1 (beta 2-GP1) was absent. Affinity purified aPL antibodies had 25-50 times the inhibitory activity of immunoglobulin preparations. The phospholipid binding proteins, beta 2-GPI and placental anticoagulant protein I (PAP I), independently inhibited the prothrombinase reaction, and when these proteins were combined with aPL, inhibition of the prothrombinase reaction was additive. Antibodies of syphilis had no inhibitory effect, partially accounted for by lack of specificity for phosphotidylserine (PS). Although aPL antibodies inhibited the protein C activation reaction, there was no correlation of these activities with inhibition of the prothrombinase reaction. Together, these results show that aPL exert their effects by interaction with negatively charged phospholipids, in particular phosphotidylserine, but lack of correlation between inhibition of the prothrombinase and protein C activation reactions, suggests that the nature of the coagulation protein is also important. PMID:8902763

  10. Antibodies to watch in 2014.

    Science.gov (United States)

    Reichert, Janice M

    2014-01-01

    Since 2010, mAbs has documented the biopharmaceutical industry's progress in transitioning antibody therapeutics to first Phase 3 clinical studies and regulatory review, and its success at gaining first marketing approvals for antibody-based products. This installment of the "Antibodies to watch" series outlines events anticipated to occur between December 2013 and the end of 2014, including first regulatory actions on marketing applications for vedolizumab, siltuximab, and ramucirumab, as well as the Fc fusion proteins Factor IX-Fc and Factor VIII-Fc; and the submission of first marketing applications for up to five therapeutics (secukinumab, ch14.18, onartuzumab, necitumumab, gevokizumab). Antibody therapeutics in Phase 3 studies are described, with an emphasis on those with study completion dates in 2014, including antibodies targeting interleukin-17a or the interleukin-17a receptor (secukinumab, ixekizumab, brodalumab), proprotein convertase subtilisin/kexin type 9 (alirocumab, evolocumab, bococizumab), and programmed death 1 receptor (lambrolizumab, nivolumab). Five antibodies with US Food and Drug Administration's Breakthrough Therapy designation (obinutuzumab, ofatumumab, lambrolizumab, bimagrumab, daratumumab) are also discussed. PMID:24284914

  11. Radiolabeled monoclonal antibodies: a review

    International Nuclear Information System (INIS)

    Since the description by Kohler and Milstein 1975 of their technique for producing monoclonal antibodies of predefined specificity, it has become a mainstay in most laboratories that utilize immunochemical techniques to study problems in basic, applied or clinical research. Paradoxically, the very success of monoclonal antibodies has generated a literature which is now so vast and scattered that it has become difficult to obtain a perspective. This brief review represents the distillation of many publications relating to the production and use of monoclonaal antibodies as radiopharmaceuticals. Significant advances were made possible in the last few years by combined developments in the fields of tumor-associated antigens and of monoclonal antibodies. In fact monoclonal antibodies against some well defined tumor-associated antigens, has led to significantly greater practical possibilities for producing highly specific radiolabeled antibodies as radiopharmaceuticals for diagnosis and therapy of human tumors. One of the main requirements of this methodology is the availability of stable radiopharmaceutical reagents which after labeling in vivo injection retain the capacity of specific interaction with the defined antigen and their molecular integrity. Since injection into human is the objetive of this kind of study all the specifications of radiopharmaceutical have to be fulfilled e.g. sterility, apirogenicity and absence of toxicity. (author)

  12. Radioimmunoguided surgery using monoclonal antibody

    International Nuclear Information System (INIS)

    The potential proficiency of radioimmunoguided surgery in the intraoperative detection of tumors was assessed using labeled monoclonal antibody B72.3 in 66 patients with tissue-proved tumor. Monoclonal antibody B72.3 was injected 5 to 42 days preoperatively, and the hand-held gamma-detecting probe was used intraoperatively to detect the presence of tumor. Intraoperative probe counts of less than 20 every 2 seconds, or tumor-to-adjacent normal tissue ratios less than 2:1 were considered negative (system failure). Positive probe counts were detected in 5 of 6 patients with primary colon cancer (83 percent), in 31 of 39 patients with recurrent colon cancer (79 percent), in 4 of 5 patients with gastric cancer (80 percent), in 3 of 8 patients with breast cancer (37.5 percent), and in 4 of 8 patients with ovarian cancer (50 percent) undergoing second-look procedures. Additional patients in each group were scored as borderline positive. Overall, radioimmunoguided surgery using B72.3 identified tumors in 47 patients (71.2 percent), bordered on positive in 6 patients (9.1 percent), and failed to identify tumor in 13 patients (19.7 percent). Improved selection of patients for antigen-positive tumors, the use of higher affinity second-generation antibodies, alternate routes of antibody administration, alternate radionuclides, and more sophisticatedly bioengineered antibodies and antibody combinations should all lead to improvements in radioimmunoguided surgery

  13. Tabhu: tools for antibody humanization.

    KAUST Repository

    Olimpieri, Pier Paolo

    2014-10-09

    SUMMARY: Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity into a suitable human template. Unfortunately, this procedure may results in a partial or complete loss of affinity of the grafted molecule that can be restored by back-mutating some of the residues of human origin to the corresponding murine ones. This trial-and-error procedure is hard and involves expensive and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps of the humanization experiment protocol. AVAILABILITY: http://www.biocomputing.it/tabhu CONTACT: anna.tramontano@uniroma1.it, pierpaolo.olimpieri@uniroma1.it SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

  14. Avian Diagnostic and Therapeutic Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Bradley, David Sherman [UND SMHS

    2012-12-31

    A number of infectious agents have the potential of causing significant clinical symptomology and even death, but dispite this, the number of incidence remain below the level that supports producing a vaccine. Therapeutic antibodies provide a viable treatment option for many of these diseases. We proposed that antibodies derived from West Nile Virus (WNV) immunized geese would be able to treat WNV infection in mammals and potential humans. We demonstrated that WNV specific goose antibodies are indeed successful in treating WNV infection both prophylactically and therapeutically in a golden hamster model. We demonstrated that the goose derived antibodies are non-reactogenic, i.e. do not cause an inflammatory response with multiple exposures in mammals. We also developed both a specific pathogen free facility to house the geese during the antibody production phase and a patent-pending purification process to purify the antibodies to greater than 99% purity. Therefore, the success of these study will allow a cost effective rapidly producible therapeutic toward clinical testing with the necessary infrastructure and processes developed and in place.

  15. Production and characterization of peptide antibodies

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Hansen, Paul Robert; Houen, Gunnar

    2012-01-01

    Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies are...

  16. Immunotherapy with the trifunctional anti-CD20 x anti-CD3 antibody FBTA05 (Lymphomun) in paediatric high-risk patients with recurrent CD20-positive B cell malignancies.

    Science.gov (United States)

    Schuster, Friedhelm R; Stanglmaier, Michael; Woessmann, Wilhelm; Winkler, Beate; Siepermann, Meinolf; Meisel, Roland; Schlegel, Paul G; Hess, Jürgen; Lindhofer, Horst; Borkhardt, Arndt; Buhmann, Raymund

    2015-04-01

    Children with B cell malignancies refractory to standard therapy are known to have a poor prognosis and very limited treatment options. Here, we report on the treatment and follow-up of ten patients diagnosed with relapsed or refractory mature B-cell Non Hodgkin Lymphoma (B-NHL), Burkitt leukaemia (B-AL) or pre B-acute lymphoblastic leukaemia (pre B-ALL). All children were treated with FBTA05 (now designated Lymphomun), an anti-CD3 x anti-CD20 trifunctional bispecific antibody (trAb) in compassionate use. Within individual treatment schedules, Lymphomun was applied (a) after allogeneic stem cell transplantation (allo-SCT, n = 6) to induce sustained long-term remission, or (b) stand alone prior to subsequent chemotherapy to eradicate residual disease before allo-SCT (n = 4). Nine of ten children displayed a clinical response: three stable diseases (SD), one partial remission (PR) and five induced or sustained complete remissions (CR). Five of these nine responders died during follow-up. The other patients still maintain CR with a current overall survival of 874-1424 days (median: 1150 days). In conclusion, despite the dismal clinical prognosis of children refractory to standard therapy, immunotherapy with Lymphomun resulted in a favourable clinical outcome in this cohort of refractory paediatric patients. PMID:25495919

  17. Production of recombinant antibodies using bacteriophages

    OpenAIRE

    Shukra, A. M.; Sridevi, N. V.; Dev Chandran,; Kapil Maithal,

    2014-01-01

    Recombinant antibody fragments such as Fab, scFv, diabodies, triabodies, single domain antibodies and minibodies have recently emerged as potential alternatives to monoclonal antibodies, which can be engineered using phage display technology. These antibodies match the strengths of conventionally produced monoclonal antibodies and offer advantages for the development of immunodiagnostic kits and assays. These fragments not only retain the specificity of the whole monoclonal ...

  18. Antibody-Directed Phototherapy (ADP

    Directory of Open Access Journals (Sweden)

    M. Adil Butt

    2013-04-01

    Full Text Available Photodynamic therapy (PDT is a clinically-approved but rather under-exploited treatment modality for cancer and pre-cancerous superficial lesions. It utilises a cold laser or LED to activate a photochemical reaction between a light activated drug (photosensitiser-drug and oxygen to generate cytotoxic oxygen species. These free radical species damage cellular components leading to cell death. Despite its benefits, the complexity, limited potency and side effects of PDT have led to poor general usage. However, the research area is very active with an increasing understanding of PDT-related cell biology, photophysics and significant progress in molecular targeting of disease. Monoclonal antibody therapy is maturing and the next wave of antibody therapies includes antibody-drug conjugates (ADCs, which promise to be more potent and curable. These developments could lift antibody-directed phototherapy (ADP to success. ADP promises to increase specificity and potency and improve drug pharmacokinetics, thus delivering better PDT drugs whilst retaining its other benefits. Whole antibody conjugates with first generation ADP-drugs displayed problems with aggregation, poor pharmacokinetics and loss of immuno-reactivity. However, these early ADP-drugs still showed improved selectivity and potency. Improved PS-drug chemistry and a variety of conjugation strategies have led to improved ADP-drugs with retained antibody and PS-drug function. More recently, recombinant antibody fragments have been used to deliver ADP-drugs with superior drug loading, more favourable pharmacokinetics, enhanced potency and target cell selectivity. These improvements offer a promise of better quality PDT drugs.

  19. Antibodies: an alternative for antibiotics?

    Science.gov (United States)

    Berghman, L R; Abi-Ghanem, D; Waghela, S D; Ricke, S C

    2005-04-01

    In 1967, the success of vaccination programs, combined with the seemingly unstoppable triumph of antibiotics, prompted the US Surgeon General to declare that "it was time to close the books on infectious diseases." We now know that the prediction was overly optimistic and that the fight against infectious diseases is here to stay. During the last 20 yr, infectious diseases have indeed made a staggering comeback for a variety of reasons, including resistance against existing antibiotics. As a consequence, several alternatives to antibiotics are currently being considered or reconsidered. Passive immunization (i.e., the administration of more or less pathogen-specific antibodies to the patient) prior to or after exposure to the disease-causing agent is one of those alternative strategies that was almost entirely abandoned with the introduction of chemical antibiotics but that is now gaining interest again. This review will discuss the early successes and limitations of passive immunization, formerly referred to as "serum therapy," the current use of antibody administration for prophylaxis or treatment of infectious diseases in agriculture, and, finally, recent developments in the field of antibody engineering and "molecular farming" of antibodies in various expression systems. Especially the potential of producing therapeutic antibodies in crops that are routine dietary components of farm animals, such as corn and soy beans, seems to hold promise for future application in the fight against infectious diseases. PMID:15844826

  20. Antibodies to watch in 2016.

    Science.gov (United States)

    Reichert, Janice M

    2016-01-01

    The number of novel antibody therapeutics that received first marketing approvals in 2015 met expectations, with 6 (alirocumab (Praluent®), evolocumab (Repatha®), daratumumab (Darzalex®), dinutuximab (Unituxin®), idarucizumab (Praxbind®), mepolizumab (Nucala®)) granted first approvals as of mid-November*. Seven novel antibody therapeutics (begelomab, brodalumab, elotuzumab, ixekizumab, necitumumab, obiltoxaximab, reslizumab) are in regulatory review, and thus a similar number, if not more, are projected to gain first approvals in 2016. Commercial late-stage antibody therapeutics development exceeded expectations by increasing from 39 candidates in Phase 3 studies as of late 2014 to 53 as of late 2015. Of the 53 candidates, transitions to regulatory review by the end of 2016 are projected for 8 (atezolizumab, benralizumab, bimagrumab, durvalumab, inotuzumab ozogamicin, lebrikizumab, ocrelizumab, tremelimumab). Other "antibodies to watch" include 15 candidates (bavituximab, bococizumab, dupilumab, fasinumab, fulranumab, gevokizumab, guselkumab, ibalizumab, LY2951742, onartuzumab, REGN2222, roledumab, romosozumab, sirukumab, Xilonix) undergoing evaluation in Phase 3 studies that have estimated primary completion dates in 2016. As evidenced by the antibody therapeutics discussed in this perspective, the biopharmaceutical industry has a highly active late-stage clinical pipeline that may deliver numerous new products to the global market in the near future. *See Note added in proof for updates through December 31, 2015. PMID:26651519

  1. Epigenetics of the antibody response.

    Science.gov (United States)

    Li, Guideng; Zan, Hong; Xu, Zhenming; Casali, Paolo

    2013-09-01

    Epigenetic marks, such as DNA methylation, histone post-translational modifications and miRNAs, are induced in B cells by the same stimuli that drive the antibody response. They play major roles in regulating somatic hypermutation (SHM), class switch DNA recombination (CSR), and differentiation to plasma cells or long-lived memory B cells. Histone modifications target the CSR and, possibly, SHM machinery to the immunoglobulin locus; they together with DNA methylation and miRNAs modulate the expression of critical elements of that machinery, such as activation-induced cytidine deaminase (AID), as well as factors central to plasma cell differentiation, such as B lymphocyte-induced maturation protein-1 (Blimp-1). These inducible B cell-intrinsic epigenetic marks instruct the maturation of antibody responses. Their dysregulation plays an important role in aberrant antibody responses to foreign antigens, such as those of microbial pathogens, and self-antigens, such as those targeted in autoimmunity, and B cell neoplasia.

  2. Molecular-specific urokinase antibodies

    Science.gov (United States)

    Atassi, M. Zouhair (Inventor); Morrison, Dennis R. (Inventor)

    2009-01-01

    Antibodies have been developed against the different molecular forms of urokinase using synthetic peptides as immunogens. The peptides were synthesized specifically to represent those regions of the urokinase molecules which are exposed in the three-dimensional configuration of the molecule and are uniquely homologous to urokinase. Antibodies are directed against the lysine 158-isoleucine 159 peptide bond which is cleaved during activation from the single-chain (ScuPA) form to the bioactive double chain (54 KDa and 33 KDa) forms of urokinase and against the lysine 135 lysine 136 bond that is cleaved in the process of removing the alpha-chain from the 54 KDa form to produce the 33 KDa form of urokinase. These antibodies enable the direct measurement of the different molecular forms of urokinase from small samples of conditioned medium harvested from cell cultures.

  3. Tabhu: tools for antibody humanization

    DEFF Research Database (Denmark)

    Olimpieri, Pier Paolo; Marcatili, Paolo; Tramontano, Anna

    2015-01-01

    into a suitable human template. Unfortunately, this procedure may results in a partial or complete loss of affinity of the grafted molecule that can be restored by back-mutating some of the residues of human origin to the corresponding murine ones. This trial-and-error procedure is hard and involves expensive......Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can...... elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity...

  4. Autologous antibodies that bind neuroblastoma cells.

    Science.gov (United States)

    Sun, Yujing; Sholler, Giselle S; Shukla, Girja S; Pero, Stephanie C; Carman, Chelsea L; Zhao, Ping; Krag, David N

    2015-11-01

    Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies.

  5. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S.

    2013-02-26

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  6. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A; Thompson, Vicki S

    2013-02-26

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  7. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S

    2010-04-13

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  8. Antibody profiling sensitivity through increased reporter antibody layering

    International Nuclear Information System (INIS)

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  9. The Broad Anti-AML Activity of the CD33/CD3 BiTE Antibody Construct, AMG 330, Is Impacted by Disease Stage and Risk.

    Directory of Open Access Journals (Sweden)

    Kimberly H Harrington

    Full Text Available The CD33/CD3-bispecific T-cell engaging (BiTE antibody construct, AMG 330, potently lyses CD33+ leukemic cells in vitro. Using specimens from 41 patients with acute myeloid leukemia (AML, we studied the factors that might contribute to clinical response or resistance. For this purpose, thawed aliquots of primary AML samples were immunophenotypically characterized and subjected to various doses of AMG 330 in the presence or absence of healthy donor T-cells. After 48 hours, drug-specific cytotoxicity was quantified and correlated with CD33 expression levels, amounts of T-cells present, and other disease characteristics. AMG 330 caused modest cytotoxicity that was correlated with the amount of autologous T-cells (P = 0.0001 but not CD33 expression, as AMG 330 exerted marked cytotoxic effects in several specimens with minimal CD33 expression. With healthy donor T-cells added, AMG 330 cytotoxicity depended on the drug dose and effector:target (E:T cell ratio. High cytotoxic activity was observed even with minimal CD33 expression, and AMG 330 cytotoxicity and CD33 expression correlated only at high E:T cell ratio and high AMG 330 doses (P<0.003. AMG 330 resulted in significantly higher cytotoxicity in specimens from patients with newly diagnosed AML than those with relapsed/refractory disease despite similar levels of CD33 on myeloblasts. AMG 330 cytotoxicity also appeared greater in specimens from patients with favorable-risk disease as compared to other specimens. Together, our data demonstrate that AMG 330 is highly active in primary AML specimens across the entire disease spectrum, while suggesting the presence of yet undefined, CD33-independent, relative resistance mechanisms in specific patient subsets.

  10. Pharmacokinetics interactions of monoclonal antibodies.

    Science.gov (United States)

    Ferri, Nicola; Bellosta, Stefano; Baldessin, Ludovico; Boccia, Donatella; Racagni, Giorgi; Corsini, Alberto

    2016-09-01

    The clearance of therapeutic monoclonal antibodies (mAbs) typically does not involve cytochrome P450 (CYP450)-mediated metabolism or interaction with cell membrane transporters, therefore the pharmacokinetics interactions of mAbs and small molecule drugs are limited. However, a drug may affect the clearance of mAbs through the modulation of immune response (e.g., methotrexate reduces the clearance of infliximab, adalimumab, and golimumab, possibly due to methotrexate's inhibitory effect on the formation of antibodies against the mAbs). In addition, mAbs that are cytokine modulators may modify the metabolism of drugs through their effects on P450 enzymes expression. For example, cytokine modulators such as tocilizumab (anti-IL-6 receptor antibody) may reverse the "inhibitory" effect of IL-6 on CYP substrates, resulting in a "normalization" of CYP activities. Finally, a drug may alter the clearance of mAbs by either increasing or reducing the levels of expression of targets of mAbs on the cell surface. For instance, statins and fibrates induce PCSK9 expression and therefore increase cellular uptake and clearance of alirocumab and evolocumab, anti-PCSK9 antibodies. In the present review, we will provide an overview on the pharmacokinetics properties of mAbs as related to the most relevant examples of mAbs-small molecule drug interaction.

  11. Localization of 131I-labelled monoclonal antibody ERIC1 in a subcutaneous xenograft model of neuroblastoma in SCID mice

    International Nuclear Information System (INIS)

    Our purpose was to evaluate a novel strategy of immunolocalization of human neuroblastoma by targeting the neural cell adhesion molecule (NCAM), which is over-expressed on neuroblastoma. Neuroblastoma is the most frequent solid extracranial childhood tumour and also the most common neoplasm in the first year of life. The most frequent metastatic sites are bone, bone marrow and liver. The neural cell adhesion molecule (NCAM) is constitutively expressed on nearly 100% of all neuroblastomas. NCAM has been successfully used as a target for immunotoxines and bi-specific antibodies in multiple myeloma, small cell lung cancer, glioma or neuroblastoma, in vitro and in vivo. NCAM expression was quantified on established neuroblastoma cells by real time PCR and NCAM expression on the cell surface shown by flow cytometry. A SCID mouse model using IMR5-75 neuroblastoma cells to induce subcutaneous tumour growth was established. 131I was used to label monoclonal NCAM specific ERIC1 antibodies generating the 131I-ERIC1 antibody, which shows a high affinity to NCAM also after labelling (KD= 9 x 10-8). Groups of five to six mice received intravenous injections of 3-9 MBq of 131I-ERIC1 on day 0 into the tail vein. At time points 2.5 h, 24 h, 48 h, 72 h and 96 h post injection mice were sacrificed by cervical dislocation. Blood, liver, spleen, kidneys, muscle, femur, thyroid, intestines, lung, tumour and urine were removed and weighed. Organ-specific radioactivity was measured in a well-counter as well as that of the tail and remnant cadavers. Data were calculated as dose per gram of tissue (%ID/g) and tumour to non-tumour ratios (T/NT ratio). For blocking experiments mice were pre-injected with 140μg of unlabelled ERIC1 into the tail vein 24 h prior 131I-ERIC1 application. Mice from these blocking experiments were measured after 72 h. Measurement of organ-specific radioactivity showed low organ-specific uptake (5.33 %ID/g after 72 h), which continuously decreased over the 96 hour

  12. Alternative affinity tools: more attractive than antibodies?

    NARCIS (Netherlands)

    Ruigrok, V.J.B.; Levisson, M.; Eppink, M.H.M.; Smidt, H.; Oost, van der J.

    2011-01-01

    Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids

  13. Detection of Campylobacter species using monoclonal antibodies

    Science.gov (United States)

    Young, Colin R.; Lee, Alice; Stanker, Larry H.

    1999-01-01

    A panel of species specific monoclonal antibodies were raised to Campylobacter coli, Campylobacter jejuni and Campylobacter lari. The isotypes, and cross-reactivity profiles of each monoclonal antibody against an extensive panel of micro- organisms, were determined.

  14. [Neuroimmunological diseases associated with VGKC complex antibodies].

    Science.gov (United States)

    Watanabe, Osamu

    2013-05-01

    Antibodies to voltage-gated potassium channels(VGKC) were first identified by radioimmunoassay of radioisotope labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were found only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in Morvan's syndrome and in a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins(for example LGI-1, Caspr-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now usually known as VGKC-complex antibodies. In general, LGI-1 antibodies are most common in limbic encephalitis with SIADH. Caspr-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability.

  15. Production and Screening of Monoclonal Peptide Antibodies.

    Science.gov (United States)

    Trier, Nicole Hartwig; Mortensen, Anne; Schiolborg, Annette; Friis, Tina

    2015-01-01

    Hybridoma technology is a remarkable and indispensable tool for generating high-quality monoclonal antibodies. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents, but have also emerged as the most rapidly expanding class of therapeutic biologicals. In this chapter, an overview of hybridoma technology and the laboratory procedures used routinely for hybridoma production and antibody screening are presented, including characterization of peptide antibodies.

  16. Virus Strain Discrimination Using Recombinant Antibodies

    OpenAIRE

    Boonham, N.; Barker, I.

    2002-01-01

    Most routine testing for plant viruses is currently carried out using monoclonal and polyclonal antibodies. Traditional methods of antibody production however can be time consuming and require the use of expensive cell culture facilities. Recombinant antibody technology however is starting to make an impact in this area, enabling the selection of antibody fragments in a few weeks compared with the many months associated with traditional methods and requires only basic microbiological faciliti...

  17. Sequence and structural analysis of antibodies

    OpenAIRE

    Raghavan, A. K.

    2009-01-01

    The work presented in this thesis focusses on the sequence and structural analysis of antibodies and has fallen into three main areas. First I developed a method to assess how typical an antibody sequence is of the expressed human antibody repertoire. My hypothesis was that the more \\humanlike" an antibody sequence is (in other words how typical it is of the expressed human repertoire), the less likely it is to elicit an immune response when used in vivo in humans. In practi...

  18. Immunoglobulin G4: an odd antibody

    NARCIS (Netherlands)

    R.C. Aalberse; S.O. Stapel; J. Schuurman; T. Rispens

    2009-01-01

    Despite its well-known association with IgE-mediated allergy, IgG4 antibodies still have several poorly understood characteristics. IgG4 is a very dynamic antibody: the antibody is involved in a continuous process of half-molecules (i.e. a heavy and attached light-chain) exchange. This process, also

  19. Anti-DNA antibodies in SLE

    Energy Technology Data Exchange (ETDEWEB)

    Voss, E.W.

    1988-01-01

    This book contains 8 chapters. Some of the titles are: Anti-DNA Antibodies in SLE: Historical Perspective; Specificity of Anti-DNA Antibodies in Systemic Lupus Erythematosus; Monoclonial Autoimmune Anti-DNA Antibodies; and Structure--Function Analyses of Anti-DNA Autoantibodies.

  20. The role of antibodies in myasthenia gravis.

    Science.gov (United States)

    De Baets, M; Stassen, M H W

    2002-10-15

    Myasthenia gravis is an autoimmune disease associated with antibodies directed to the postsynaptic acetylcholine receptor. These antibodies reduce the number of receptors. Autoantibodies against AChR and other muscle antigens can be used for the diagnosis of myasthenia gravis and related disorders. The origin and the role of these antibodies in the disease are discussed. Experimental autoimmune myasthenia gravis, an experimental model closely mimicking the disease, has provided answers to many questions about the role of antibodies, complement macrophages and AChR anchor proteins. Genetically modified anti-AChR antibodies may also be used in the future to treat myasthenia. PMID:12220686

  1. Production of Monoclonal Antibody against Human Nestin.

    Science.gov (United States)

    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. PMID:23407796

  2. Production of Monoclonal Antibody against Human Nestin

    OpenAIRE

    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 a...

  3. Monoclonal Antibody Therapies against Anthrax

    OpenAIRE

    Zhaochun Chen; Mahtab Moayeri; Robert Purcell

    2011-01-01

    Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. It not only causes natural infection in humans but also poses a great threat as an emerging bioterror agent. The lethality of anthrax is primarily attributed to the two major virulence factors: toxins and capsule. An extensive effort has been made to generate therapeutically useful monoclonal antibodies to each of the virulence components: protective antigen (PA), lethal factor (LF) and ede...

  4. Antibody Peptide Based Antifungal Immunotherapy

    OpenAIRE

    Magliani, Walter; Conti, Stefania; Giovati, Laura; Zanello, Pier Paolo; Sperindè, Martina; Ciociola, Tecla; Polonelli, Luciano

    2012-01-01

    Fungal infections still represent relevant human illnesses worldwide and some are accompanied by unacceptably high mortality rates. The limited current availability of effective and safe antifungal agents makes the development of new drugs and approaches of antifungal vaccination/immunotherapy every day more needed. Among them, small antibody(Ab)-derived peptides are arousing great expectations as new potential antifungal agents. In this topic, the search path from the study of the yeast kill...

  5. Application of Monoclonal Antibodies in Veterinary Parasitology

    Directory of Open Access Journals (Sweden)

    Gupta A.

    2011-08-01

    Full Text Available The discovery of hybridoma technology by Kohler and Milstein in 1975, heralded a new era in antibody research. Mouse hybridomas were the first reliable source of monoclonal antibodies. The generation of monoclonal antibodies from species other than rats and mice, has developed slowly over the last 30 years. The advent of antibody engineering and realization of the advantages of non murine antibodies has increased their relevance recently. However, in the area of veterinary parasitology, monoclonal antibodies are just beginning to fulfill the promises inherent in their great specificity for recognizing and selectively binding to antigens. This review describes the recent advances in the application of monoclonal antibodies for immunodiagnosis / prophylaxis and immunotherapy of parasitic diseases. [Vet. World 2011; 4(4.000: 183-188

  6. Advances in monoclonal antibody application in myocarditis

    Institute of Scientific and Technical Information of China (English)

    Li-na HAN; Shuang HE; Yu-tang WANG; Li-ming YANG; Si-yu LIU; Ting ZHANG

    2013-01-01

    Monoclonal antibodies have become a part of daily preparation technologies in many laboratories.Attempts have been made to apply monoclonal antibodies to open a new train of thought for clinical treatments of autoimmune diseases,inflammatory diseases,cancer,and other immune-associated diseases.This paper is a prospective review to anticipate that monoclonal antibody application in the treatment of myocarditis,an inflammatory disease of the heart,could be a novel approach in the future.In order to better understand the current state of the art in monoclonal antibody techniques and advance applications in myocarditis,we,through a significant amount of literature research both domestic and abroad,developed a systematic elaboration of monoclonal antibodies,pathogenesis of myocarditis,and application of monoclonal antibodies in myocarditis.This paper presents review of the literature of some therapeutic aspects of monoclonal antibodies in myocarditis and dilated cardiomyopathy to demonstrate the advance of monoclonal antibody application in myocarditis and a strong anticipation that monoclonal antibody application may supply an effective therapeutic approach to relieve the severity of myocarditis in the future.Under conventional therapy,myocarditis is typically associated with congestive heart failure as a progressive outcome,indicating the need for alternative therapeutic strategies to improve long-term results.Reviewing some therapeutic aspects of monoclonal antibodies in myocarditis,we recently found that monoclonal antibodies with high purity and strong specificity can accurately act on target and achieve definite progress in the treatment of viral myocarditis in rat model and may meet the need above.However,several issues remain.The technology on howto make a higher homologous and weak immunogenic humanized or human source antibody and the treatment mechanism of monoclonal antibodies may provide solutions for these open issues.If we are to further stimulate

  7. Controlled delivery of antibodies from injectable hydrogels.

    Science.gov (United States)

    Fletcher, Nathan A; Babcock, Lyndsey R; Murray, Ellen A; Krebs, Melissa D

    2016-02-01

    Therapeutic antibodies are currently used for the treatment of various diseases, but large doses delivered systemically are typically required. Localized controlled delivery techniques would afford major benefits such as decreasing side effects and required doses. Injectable biopolymer systems are an attractive solution due to their minimally invasive potential for controlled release in a localized area. Here, alginate-chitosan hydrogels are demonstrated to provide controlled delivery of IgG model antibodies and also of Fab antibody fragments. Also, an alternate delivery system comprised of poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with antibodies and encapsulated in alginate was shown to successfully provide another level of control over release. These biopolymer systems that offer controlled delivery for antibodies and antibody fragments will be promising for many applications in drug delivery and regenerative medicine.

  8. Factors determining antibody distribution in tumors

    Science.gov (United States)

    Thurber, Greg M.; Schmidt, Michael M.; Wittrup, K. Dane

    2009-01-01

    The development of antibody therapies for cancer is increasing rapidly, primarily owing to their specificity. Antibody distribution in tumors is often extremely uneven, however, leading to some malignant cells being exposed to saturating concentrations of antibody, whereas others are completely untargeted. This is detrimental because large regions of cells escape therapy, whereas other regions might be exposed to suboptimal concentrations that promote a selection of resistant mutants. The distribution of antibody depends on a variety of factors, including dose, affinity, antigens per cell and molecular size. Because these parameters are often known or easily estimated, a quick calculation based on simple modeling considerations can predict the uniformity of targeting within a tumor. Such analyses should enable experimental researchers to identify in a straightforward way the limitations in achieving evenly distributed antibody, and design and test improved antibody therapeutics more rationally. PMID:18179828

  9. Stabilization of antibody fragments in adverse environments.

    Science.gov (United States)

    Dooley, H; Grant, S D; Harris, W J; Porter, A J

    1998-08-01

    Antibody fragments have the potential to be used as sensitive and specific binding agents in a broad range of industrial applications. Genetic manipulation has been used to design a series of antibody fragment configurations with a flexible linker and/or a disulphide bond between the heavy chain and light chain of an antibody fragment against the herbicide atrazine. The thermostability and stability to a range of denaturants, polar and non-polar solvents, surfactants and proteases have been compared. It has been found that a novel antibody fragment construct (STAB: stabilized antibody) containing both a flexible linker and a disulphide bond can be effectively produced and shows greatly improved stability in these diverse environments. These STABs should be useful in environmental diagnostics and remediation, and may provide a generic approach for stabilizing antibody fragments in formulations containing detergents and penetrants for topical application in the pharmaceutical and cosmetic industries.

  10. Monoclonal antibodies as diagnostics; an appraisal

    Directory of Open Access Journals (Sweden)

    Siddiqui M

    2010-01-01

    Full Text Available Ever since the development of Hybridoma Technology in 1975 by Kohler and Milstein, our vision for antibodies as tools for research for prevention, detection and treatment of diseases, vaccine production, antigenic characterization of pathogens and in the study of genetic regulation of immune responses and disease susceptibility has been revolutionized. The monoclonal antibodies being directed against single epitopes are homogeneous, highly specific and can be produced in unlimited quantities. In animal disease diagnosis, they are very useful for identification and antigenic characterization of pathogens. Monoclonal antibodies have tremendous applications in the field of diagnostics, therapeutics and targeted drug delivery systems, not only for infectious diseases caused by bacteria, viruses and protozoa but also for cancer, metabolic and hormonal disorders. They are also used in the diagnosis of lymphoid and myeloid malignancies, tissue typing, enzyme linked immunosorbent assay, radio immunoassay, serotyping of microorganisms, immunological intervention with passive antibody, antiidiotype inhibition, or magic bullet therapy with cytotoxic agents coupled with anti mouse specific antibody. Recombinant deoxyribonucleic acid technology through genetic engineering has successfully led to the possibility of reconstruction of monoclonal antibodies viz. chimeric antibodies, humanized antibodies and complementarily determining region grafted antibodies and their enormous therapeutic use.

  11. Snake venom antibodies in Ecuadorian Indians.

    Science.gov (United States)

    Theakston, R D; Reid, H A; Larrick, J W; Kaplan, J; Yost, J A

    1981-10-01

    Serum samples from 223 Waorani Indians, a tribe in eastern Ecuador, were investigated by enzyme-linked immunosorbent assay for antibodies to snake venom. Seventy-eight per cent were positive, confirming the highest incidence and mortality from snake bite poisoning yet recorded in the world. Most samples were positive for more than one venom antibody. Antibodies were found to venoms of Bothrops viper in 60% of positive cases, of Micrurus coral snake in 21%, and of the bushmaster, Lachesis muta, in 18%. Further studies are needed to determine whether high venom-antibody levels afford protection against further snake envenoming. PMID:7299877

  12. Sequence and structual analysis of antibodies.

    OpenAIRE

    Raghavan, A. K.

    2008-01-01

    The work presented in this thesis focuses on the sequence and structural analysis of antibodies and has fallen into three main areas. First I developed a method to assess how typical an antibody sequence is of the expressed human antibody repertoire. My hypothesis was that the more "human like" an antibody sequence is (in other words how typical it is of the expressed human repertoire), the less likely it is to elicit an immune response when used in vivo in humans. In practice, I found that, ...

  13. Antiphospholipid Antibodies and Systemic Scleroderma

    Directory of Open Access Journals (Sweden)

    Awa Oumar Touré

    2013-03-01

    Full Text Available Objective: Antiphospholipid antibodies (APLs could be associated with an increased risk of vascular pathologies in systemic scleroderma. The aim of our study was to search for APLs in patients affected by systemic scleroderma and to evaluate their involvement in the clinical manifestations of this disease. Materials and Methods: We conducted a cross-sectional descriptive study, from January 2009 until August 2010, with patients received at the Department of Dermatology (Dakar, Senegal. Blood samples were taken at the hematology laboratory and were analyzed for the presence of APLs. Results: Forty patients were recruited. Various types of either isolated or associated APLs were found in 23 patients, i.e. 57.5% of the study population. The most frequently encountered antibody was IgG anti-β2 GPI (37.5% of the patients, followed by anticardiolipins (17.5% and lupus anticoagulants (5%. No statistically significant association of positive antiphospholipid-related tests to any of the scleroderma complications could be demonstrated. Conclusion: A high proportion of patients showing association of systemic scleroderma and APLs suggests the presence of a morbid correlation between these 2 pathologies. It would be useful to follow a cohort of patients affected by systemic scleroderma in order to monitor vascular complications following confirmation of the presence of antiphospholipid syndrome.

  14. Antibodies to human fetal erythroid cells from a nonimmune phage antibody library

    OpenAIRE

    Huie, Michael A.; Cheung, Mei-Chi; Muench, Marcus O.; Becerril, Baltazar; Kan, Yuet W.; Marks, James D.

    2001-01-01

    The ability to isolate fetal nucleated red blood cells (NRBCs) from the maternal circulation makes possible prenatal genetic analysis without the need for diagnostic procedures that are invasive for the fetus. Such isolation requires antibodies specific to fetal NRBCs. To generate a panel of antibodies to antigens present on fetal NRBCs, a new type of nonimmune phage antibody library was generated in which multiple copies of antibody fragments are displayed on each pha...

  15. High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells

    OpenAIRE

    Jäger, Volker; Büssow, Konrad; Wagner, Andreas; Weber, Susanne; Hust, Michael; Frenzel, André; Schirrmann, Thomas

    2013-01-01

    Background The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable...

  16. The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

    NARCIS (Netherlands)

    Joosten, V.; Lokman, C.; Hondel, C.A.M.J.J. van den; Punt, P.J.

    2003-01-01

    In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications

  17. Anti-influenza M2e antibody

    Energy Technology Data Exchange (ETDEWEB)

    Bradbury, Andrew M.

    2013-04-16

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

  18. Anti-influenza M2e antibody

    Energy Technology Data Exchange (ETDEWEB)

    Bradbury, Andrew M. (Santa Fe, NM)

    2011-12-20

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

  19. "Unconventional" Neutralizing Activity of Antibodies Against HIV

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However, only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes, such as high sequence diversity, heavy glycosylation, and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohemagglutinin (PHA)-stimulated human PBMCs as target cells. Thus, in essence the assay evaluates HIV-1 replication in CD4+ T cells. Recently, several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera, although they do not have neutralizing activity when tested by the "conventional" neutralization assay, do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications, through opsonization of virus particles into macrophages and immature dendritic cells (iDCs), or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.

  20. Photonic crystal fiber based antibody detection

    DEFF Research Database (Denmark)

    Duval, A; Lhoutellier, M; Jensen, J B;

    2004-01-01

    An original approach for detecting labeled antibodies based on strong penetration photonic crystal fibers is introduced. The target antibody is immobilized inside the air-holes of a photonic crystal fiber and the detection is realized by the means of evanescent-wave fluorescence spectroscopy...... and the use of a transversal illumination setup....

  1. Monoclonal antibodies reactive with hairy cell leukemia

    NARCIS (Netherlands)

    Visser, L; Shaw, A; Slupsky, J; Vos, H; Poppema, S

    1989-01-01

    Monoclonal antibodies reactive with hairy cell leukemia were developed to aid in the diagnosis of this subtype of B cell chronic lymphocytic leukemia and to gain better insight into the origin of hairy cells. Three antibodies were found to be of value in the diagnosis of hairy cell leukemia. Antibod

  2. Synthetic Antibodies for Reversible Cell Recognition

    Science.gov (United States)

    Zhou, Jing Zhou

    2011-12-01

    Antibody-mediated cell recognition plays a critical role in various biological and biomedical applications. However, strong antibody-cell interactions can lead to the difficulty of separating antibodies from the bound cells in a simple and non-destructive manner, which is often necessary to numerous applications such as cell sorting or separation. Thus, this thesis research is aimed to create an antibody-like nanomaterial with the function of reversible cell recognition It was hypothesized that nucleic acid aptamer and dendrimer could be used as fundamental structural components to develop an antibody-like nanomaterial. The aptamer functions as the binding site of an antibody; the dendrimer is used as a robust, defined nano-scaffold to support the aptamer and to carry small molecules (e.g., fluorophores). To test this hypothesis, a novel method was first developed to discover the essential nucleotides of full-length aptamers to mimic the binding sites of antibodies. The essential nucleotides were further conjugated with a dendrimer to synthesize a monovalent aptamer-dendrimer nanomaterial. The results clearly showed that the essential nucleotides could maintain high affinity and specificity after tethered on dendrimer surface. To further test the hypothesis that antibody-like nanomaterials can be rationally designed to acquire the capability of reversible cell recognition, an aptamer that was selected at 0 °C was used as a model to synthesize a "Y-shaped" nanomaterial by conjugating two aptamers to the same dendrimer. The results showed that the nanomaterial-cell interaction could be affected by the distance between two binding aptamers. In addition, the "Y-shaped" antibody-like nanomaterial could bind target cells more strongly than its monovalent control. Importantly, the strong cell-nanomaterial interaction could be rapidly reversed when the temperature was shifted from 0 °C to 37 °C. In summary, we developed a synthetic antibody that can not only mimic the

  3. Trends in Malignant Glioma Monoclonal Antibody Therapy

    Science.gov (United States)

    Chekhonin, Ivan; Gurina, Olga

    2015-01-01

    Although new passive and active immunotherapy methods are emerging, unconjugated monoclonal antibodies remain the only kind of biological preparations approved for high-grade glioma therapy in clinical practice. In this review, we combine clinical and experimental data discussion. As antiangiogenic therapy is the standard of care for recurrent glioblastoma multiforme (GBM), we analyze major clinical trials and possible therapeutic combinations of bevacizumab, the most common monoclonal antibody to vascular endothelial growth factor (VEGF). Another humanized antibody to gain recognition in GBM is epidermal growth factor (EGFR) antagonist nimotuzumab. Other antigens (VEGF receptor, platelet-derived growth factor receptor, hepatocyte growth factor and c-Met system) showed significance in gliomas and were used to create monoclonal antibodies applied in different malignant tumors. We assess the role of genetic markers (isocitrate dehydrogenase, O6-methylguanine-DNA methyltransnsferase) in GBM treatment outcome prediction. Besides antibodies studied in clinical trials, we focus on perspective targets and briefly list other means of passive immunotherapy.

  4. Antiphospholipid antibody: laboratory, pathogenesis and clinical manifestations

    Directory of Open Access Journals (Sweden)

    T. Ziglioli

    2011-06-01

    Full Text Available Antiphospholipid antibodies (aPL represent a heterogeneous group of antibodies that recognize various antigenic targets including beta2 glycoprotein I (β2GPI, prothrombin (PT, activated protein C, tissue plasminogen activator, plasmin and annexin A2. The most commonly used tests to detect aPL are: lupus anticoagulant (LAC, a functional coagulation assay, anticardiolipin antibody (aCL and anti-β2GPI antibody (anti-β2GPI, which are enzyme-linked immunoassay (ELISA. Clinically aPL are associated with thrombosis and/or with pregnancy morbidity. Apparently aPL alone are unable to induce thrombotic manifestations, but they increase the risk of vascular events that can occur in the presence of another thrombophilic condition; on the other hand obstetrical manifestations were shown to be associated not only to thrombosis but mainly to a direct antibody effect on the trophoblast.

  5. Vector-Mediated In Vivo Antibody Expression.

    Science.gov (United States)

    Schnepp, Bruce C; Johnson, Philip R

    2014-08-01

    This article focuses on a novel vaccine strategy known as vector-mediated antibody gene transfer, with a particular focus on human immunodeficiency virus (HIV). This strategy provides a solution to the problem of current vaccines that fail to generate neutralizing antibodies to prevent HIV-1 infection and AIDS. Antibody gene transfer allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein. This approach uses recombinant adeno-associated viral (rAAV) vectors, which have been shown to transduce muscle with high efficiency and direct the long-term expression of a variety of transgenes, to deliver the gene encoding a broadly neutralizing antibody into the muscle. Following rAAV vector gene delivery, the broadly neutralizing antibodies are endogenously synthesized in myofibers and passively distributed to the circulatory system. This is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. Vector-mediated gene transfer studies in mice and monkeys with anti-HIV and simian immunodeficiency virus (SIV)-neutralizing antibodies demonstrated long-lasting neutralizing activity in serum with complete protection against intravenous challenge with virulent HIV and SIV. These results indicate that existing potent anti-HIV antibodies can be rapidly moved into the clinic. However, this methodology need not be confined to HIV. The general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets such as hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis. PMID:26104192

  6. Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization

    International Nuclear Information System (INIS)

    Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization at low temperatures was studied. The antibody activity of antibody was not affected by irradiation at an irradiation dose of below 8 MR and low temperatures. Immobilization of peroxidase-labeled anti-rabbit IgG goat IgG, anti-peroxidase, peroxidase, and anti-alpha-fetoprotein was carried out with hydrophilic and hydrophobic monomers. The activity of the immobilized enzyme-labeled antibody membranes varied with the thickness of the membranes and increased with decreasing membrane thickness. The activity of the immobilized antibody particles was varied by particle size. Immobilized anti-alpha-fetoprotein particles and membranes can be used for the assay of alpha-fetoprotein by the antigen-antibody reaction, such as a solid-phase sandwich method with high sensitivity

  7. Radiohalogenated half-antibodies and maleimide intermediate therefor

    Science.gov (United States)

    Kassis, A.I.; Khawli, L.A.

    1991-02-19

    N-(m-radiohalophenyl) maleimide can be conjugated with a reduced antibody having a mercapto group to provide a radiolabeled half-antibody having immunological specific binding characteristics of whole antibody. No Drawings

  8. Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality

    DEFF Research Database (Denmark)

    Kibat, Janek; Schirrmann, Thomas; Knape, Matthias J;

    2016-01-01

    Many diagnostic and therapeutic concepts require antibodies of high specificity. Recombinant binder libraries and related selection approaches allow the efficient isolation of antibodies against almost every target of interest. Nevertheless, it cannot be guaranteed that selected antibodies perfor...

  9. Combinatorial antibody libraries: new advances, new immunological insights.

    Science.gov (United States)

    Lerner, Richard A

    2016-08-01

    Immunochemists have become quite proficient in engineering existing antibody molecules to control their pharmacological properties. However, in terms of generating new antibodies, the combinatorial antibody library has become a central feature of modern immunochemistry. These libraries are essentially an immune system in a test tube and enable the selection of antibodies without the constraints of whole animal or cell-based systems. This Review provides an overview of how antibody libraries are constructed and discusses what can be learnt from these synthetic systems. In particular, the Review focuses on new biological insights from antibody libraries - such as the concept of 'SOS antibodies' - and the growing use of intracellular antibodies to perturb cellular functions.

  10. Antiphospholipid Antibodies in Lupus Nephritis.

    Science.gov (United States)

    Parodis, Ioannis; Arnaud, Laurent; Gerhardsson, Jakob; Zickert, Agneta; Sundelin, Birgitta; Malmström, Vivianne; Svenungsson, Elisabet; Gunnarsson, Iva

    2016-01-01

    Lupus nephritis (LN) is a major manifestation of systemic lupus erythematosus (SLE). It remains unclear whether antiphospholipid antibodies (aPL) alter the course of LN. We thus investigated the impact of aPL on short-term and long-term renal outcomes in patients with LN. We assessed levels of aPL cross-sectionally in SLE patients diagnosed with (n = 204) or without (n = 294) LN, and prospectively in 64 patients with active biopsy-proven LN (52 proliferative, 12 membranous), before and after induction treatment (short-term outcomes). Long-term renal outcome in the prospective LN cohort was determined by the estimated glomerular filtration rate (eGFR) and the Chronic Kidney Disease (CKD) stage, after a median follow-up of 11.3 years (range: 3.3-18.8). Cross-sectional analysis revealed no association between LN and IgG/IgM anticardiolipin or anti-β2-glycoprotein I antibodies, or lupus anticoagulant. Both aPL positivity and levels were similar in patients with active LN and non-renal SLE. Following induction treatment for LN, serum IgG/IgM aPL levels decreased in responders (p<0.005 for all), but not in non-responders. Both at active LN and post-treatment, patients with IgG, but not IgM, aPL had higher creatinine levels compared with patients without IgG aPL. Neither aPL positivity nor levels were associated with changes in eGFR from either baseline or post-treatment through long-term follow-up. Moreover, aPL positivity and levels both at baseline and post-treatment were similar in patients with a CKD stage ≥3 versus 1-2 at the last follow-up. In conclusion, neither aPL positivity nor levels were found to be associated with the occurrence of LN in SLE patients. However, IgG aPL positivity in LN patients was associated with a short-term impairment of the renal function while no effect on long-term renal outcome was observed. Furthermore, IgG and IgM aPL levels decreased following induction treatment only in responders, indicating that aPL levels are affected by

  11. Antiphospholipid Antibodies in Lupus Nephritis.

    Directory of Open Access Journals (Sweden)

    Ioannis Parodis

    Full Text Available Lupus nephritis (LN is a major manifestation of systemic lupus erythematosus (SLE. It remains unclear whether antiphospholipid antibodies (aPL alter the course of LN. We thus investigated the impact of aPL on short-term and long-term renal outcomes in patients with LN. We assessed levels of aPL cross-sectionally in SLE patients diagnosed with (n = 204 or without (n = 294 LN, and prospectively in 64 patients with active biopsy-proven LN (52 proliferative, 12 membranous, before and after induction treatment (short-term outcomes. Long-term renal outcome in the prospective LN cohort was determined by the estimated glomerular filtration rate (eGFR and the Chronic Kidney Disease (CKD stage, after a median follow-up of 11.3 years (range: 3.3-18.8. Cross-sectional analysis revealed no association between LN and IgG/IgM anticardiolipin or anti-β2-glycoprotein I antibodies, or lupus anticoagulant. Both aPL positivity and levels were similar in patients with active LN and non-renal SLE. Following induction treatment for LN, serum IgG/IgM aPL levels decreased in responders (p<0.005 for all, but not in non-responders. Both at active LN and post-treatment, patients with IgG, but not IgM, aPL had higher creatinine levels compared with patients without IgG aPL. Neither aPL positivity nor levels were associated with changes in eGFR from either baseline or post-treatment through long-term follow-up. Moreover, aPL positivity and levels both at baseline and post-treatment were similar in patients with a CKD stage ≥3 versus 1-2 at the last follow-up. In conclusion, neither aPL positivity nor levels were found to be associated with the occurrence of LN in SLE patients. However, IgG aPL positivity in LN patients was associated with a short-term impairment of the renal function while no effect on long-term renal outcome was observed. Furthermore, IgG and IgM aPL levels decreased following induction treatment only in responders, indicating that aPL levels are

  12. Structure Based Antibody-Like Peptidomimetics

    Directory of Open Access Journals (Sweden)

    Mark I. Greene

    2012-02-01

    Full Text Available Biologics such as monoclonal antibodies (mAb and soluble receptors represent new classes of therapeutic agents for treatment of several diseases. High affinity and high specificity biologics can be utilized for variety of clinical purposes. Monoclonal antibodies have been used as diagnostic agents when coupled with radionuclide, immune modulatory agents or in the treatment of cancers. Among other limitations of using large molecules for therapy the actual cost of biologics has become an issue. There is an effort among chemists and biologists to reduce the size of biologics which includes monoclonal antibodies and receptors without a reduction of biological efficacy. Single chain antibody, camel antibodies, Fv fragments are examples of this type of deconstructive process. Small high-affinity peptides have been identified using phage screening. Our laboratory used a structure-based approach to develop small-size peptidomimetics from the three-dimensional structure of proteins with immunoglobulin folds as exemplified by CD4 and antibodies. Peptides derived either from the receptor or their cognate ligand mimics the functions of the parental macromolecule. These constrained peptides not only provide a platform for developing small molecule drugs, but also provide insight into the atomic features of protein-protein interactions. A general overview of the reduction of monoclonal antibodies to small exocyclic peptide and its prospects as a useful diagnostic and as a drug in the treatment of cancer are discussed.

  13. Next generation of antibody therapy for cancer

    Institute of Scientific and Technical Information of China (English)

    Zhenping Zhu; Li Yan

    2011-01-01

    Monoclonal antibodies (mAbs) have become a major class of therapeutic agents providing effective altematives to treating various human diseases. To date, 15 mAbs have been approved by regulatory agencies in the world for clinical use in oncology indications. The selectivity and specificity, the unique pharmacokinetics, and the ability to engage and activate the host immune system differentiate these biologics from traditional small molecule anticancer drugs. mAb-basod regimens have brought clinical benefits, including improvements in overall survival, to patients with a variety of cancers. Many challenges still remain, however, to fully realize the potential of these new medicines. With our further understanding of cancer biology, mechanism of antibody action, and advancement of antibody engineering technologies, many novel antibody formats or antibody-derived molecules are emerging as promising new generation therapeutics. Carefully designed and engineered, they retain the advantage of specificity and selectivity of original antibodies, but in the meantime acquire additional special features such as improved pharmacokinetics, increased selectivity, and enhanced anticancer efficacy. Promising clinical results are being generated with these newly improved antibody-based therapeutics.

  14. Baculovirus display of functional antibody Fab fragments.

    Science.gov (United States)

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  15. Studies on Purification of Methamidophos Monoclonal Antibodies and Comoarative Immunoactivity of Purified Antibodies

    Institute of Scientific and Technical Information of China (English)

    SU-QING ZHAO; YUAN-MING SUN; CHUN-YAN ZHANG; XIAO-YU HUANG; HOU-RUI ZHANG; ZHEN-YU ZHU

    2003-01-01

    Objective To purify Methamidophos (Met) monoclonal antibodies with two methods andcompare immune activity of purified antibodies. Method Caprylic acid ammonium sulphateprecipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method wereused to purify Met monoclonal antibodies, UV spectrum scanning was used to determine proteincontent and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gelelectrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linkedimmunosorbent assay (ELISA) was used to determine immune activity of purified antibodies.Results Antibody protein content and recovery rate with CAASP method were 7.62 mg/mL and8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mLand 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASPmethod. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was181.26 μg/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01μg/mL and 1.03 μg/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82μg/mL, and the linear working range and LOD were 10.91-11412.29 ug/mL and 3.42 μg/mL,respectively. Conclusion Antibodies purified by SPA method are better than those by CAASPmethod, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelledtesting paper for analyzing Met residue in vegetable and drink water.

  16. Immunocytochemical and Immunohistochemical Staining with Peptide Antibodies.

    Science.gov (United States)

    Friis, Tina; Pedersen, Klaus Boberg; Hougaard, David; Houen, Gunnar

    2015-01-01

    Peptide antibodies are particularly useful for immunocytochemistry (ICC) and immunohistochemistry (IHC), where antigens may denature due to fixation of tissues and cells. Peptide antibodies can be made to any defined sequence, including unknown putative proteins and posttranslationally modified sequences. Moreover, the availability of large amounts of the antigen (peptide) allows inhibition/adsorption controls, which are important in ICC/IHC, due to the many possibilities for false-positive reactions caused by immunoglobulin Fc receptors, nonspecific reactions, and cross-reactivity of primary and secondary antibodies with other antigens and endogenous immunoglobulins, respectively. Here, simple protocols for ICC and IHC are described together with recommendations for appropriate controls.

  17. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1992-12-31

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

  18. The antibody approach of labeling blood cells

    International Nuclear Information System (INIS)

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

  19. Reshaping Human Antibodies: Grafting an Antilysozyme Activity

    Science.gov (United States)

    Verhoeyen, Martine; Milstein, Cesar; Winter, Greg

    1988-03-01

    The production of therapeutic human monoclonal antibodies by hybridoma technology has proved difficult, and this has prompted the ``humanizing'' of mouse monoclonal antibodies by recombinant DNA techniques. It was shown previously that the binding site for a small hapten could be grafted from the heavy-chain variable domain of a mouse antibody to that of a human myeloma protein by transplanting the hypervariable loops. It is now shown that a large binding site for a protein antigen (lysozyme) can also be transplanted from mouse to human heavy chain. The success of such constructions may be facilitated by an induced-fit mechanism.

  20. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1991-01-01

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

  1. The antibody approach of labeling blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.

    1991-12-31

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

  2. Modern trends in radioimmunotherapy of cancer. Pre targeting strategies for the treatment of ovarian cancer

    Energy Technology Data Exchange (ETDEWEB)

    Mcquarrie, S.A.; Xiao, Z.; Mercer, J. R.; Suresh, M. R. [Edmonton Univ. of Alberta, Edmonton, AL (Canada). Faculty of Pharmacy and Pharmaceutical Sciences; Miller, G. G. [University of Alberta, Noujaim Institute for Pharmaceutical Oncology Research, Edmonton, AL (Canada)

    2001-06-01

    A review of published data on some of the problems associated in treating cancer using radioimmunotherapy is presented. Potential improvements for this type of therapy using pretargeting strategies are discussed and preliminary results on a novel multistep regimen to treat human ovarian cancer are presented. A pretargeting strategy using ovarian cancer are presented. A pretargeting strategy using a biotinylated, anti-CA 125 monoclonal antibody (MAb) to attract biotinylated long-circulating liposomes to the surface of CA 125-expressing ovarian cancer cells, was employed. Confocal laser scanning microscopy and fluorescent labels were used to establish the biodistribution patterns in NIH:OVCAR-3 (CA-125 positive) and SK-OV-3 (CA-125 negative) human ovarian cancer cells. Shedding kinetics of the pretargeted stage were measured using {sup 125}I labeled MAbs. No significant internalization of the MAb used in the pretargeting step was observed by 4 hrs. The antibody was gradually internalized starting at 6 hrs, and most of the labelled MAb was detected in cytoplasm by 24 hrs. Shedding and exocytosis of the antigen-MAb complex was not significant for up to 6-hours following administration of the iodinated MAb. Biotinylated liposomes were shown to specifically target the biotinylated MAb/streptavidin complex on the cell surface. It has been demonstrated that by a three-step pretargeting approach, biotinylated liposomes can be specifically delivered to cells pretargeted with biotinylated MAb/SAv complex. The slow internalization and shedding properties of the two MAbs are ideal for multistep pretargeting methods. A successful multistep linkage was established with the biotinylated MAb B27.1, streptavidin and biotinylated liposomes to OVCAR-3 cells, but not to SK-OV-3 cells.

  3. Radioimmunological proof of thyroglobulin antibodies in humans by the use of a double antibody method

    International Nuclear Information System (INIS)

    Thyroid antibodies, especially thyroglobulin antibodies, allow themselves to be proven with the double antibody method, in competitive radio binding assays and with the solid phase technique. These methods offer advantages relative to sensitivity and quantifiability. In this work a sensitive radioimmunoassay as a double antibody method was worked out whereby a 125 I-thyroglobulin/thyroglobulin antibody immune complex was precipitated out using anti-human immunoglobulin. The measured results from the radioimmunoassay show a good correlation with the results of the immune histological findings. A high to very high Tg antibody level occurs with autoimmune thyroiditis (80%), primary hypothyroidism (74%) and hyperthyroidism (70%). The control values with healthy people came to less than 5% specific binding. In correlation with the results of other authors this method is advantageous relative to test start and evaluation procedures. (orig.)

  4. Chemical biology: How to minimalize antibodies

    Science.gov (United States)

    Rader, Christoph

    2015-02-01

    The success of antibodies as pharmaceuticals has triggered interest in crafting much smaller mimics. A crucial step forward has been taken with the chemical synthesis of small molecules that recruit immune cells to attack cancer cells.

  5. Patient-Derived Antibody Targets Tumor Cells

    Science.gov (United States)

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

  6. Localization of tumors by radiolabelled antibodies

    International Nuclear Information System (INIS)

    A method of utilizing radiolabelled antibodies to carcinoembryonic antigens for determining the site of tumors which produce or are associated with carcinoembryonic antigen is disclosed. 3 claims, no drawings

  7. Quantitative Assessment of Antibody Internalization with Novel Monoclonal Antibodies against Alexa Fluorophores

    OpenAIRE

    Liao-Chan, Sindy; Daine-Matsuoka, Barbara; Heald, Nathan; Wong, Tiffany; Lin, Tracey; Cai, Allen G.; Lai, Michelle; D’Alessio, Joseph A.; Theunissen, Jan-Willem

    2015-01-01

    Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of inte...

  8. Influenza-Specific Antibody-Dependent Phagocytosis

    OpenAIRE

    Ana-Sosa-Batiz, Fernanda; Vanderven, Hillary; Jegaskanda, Sinthujan; Johnston, Angus; Rockman, Steven; Laurie, Karen; Barr, Ian; Reading, Patrick; Lichtfuss, Marit; Stephen J Kent

    2016-01-01

    Background Immunity to human influenza A virus (IAV) infection is only partially understood. Broadly non-neutralizing antibodies may assist in reducing disease but have not been well characterized. Methods We measured internalization of opsonized, influenza protein-coated fluorescent beads and live IAV into a monocytic cell line to study antibody-dependent phagocytosis (ADP) against multiple influenza hemagglutinin (HA) subtypes. We analyzed influenza HA-specific ADP in healthy human donors, ...

  9. Single-domain antibodies for brain targeting

    OpenAIRE

    Lalatsa, Katerina; Moreira Leite, Diana

    2014-01-01

    Smaller recombinant antibody fragments as single-domain antibodies (sdAbs) are emerging as credible alternatives because of their target specificity, high affinity, and cost-effective recombinant production. sdAbs have been forged into multivalent and multispecif ic therapeutics, or targeting moieties, that are able to shuttle their linked therapeutic cargo (i.e., drugs, nanoparticles, toxins, enzymes, and radionuclides) to the receptor of interest. Their ability to permeate across the blood ...

  10. Therapeutic monoclonal antibody for Sporotrichosis

    Directory of Open Access Journals (Sweden)

    Sandro eAlmeida

    2012-11-01

    Full Text Available Sporotrichosis is a chronic subcutaneous mycosis that affects either humans or animals and occurs worldwide. This subcutaneous mycosis had been attributed to a single etiological agent, Sporothrix schenckii. S. schenckii exhibits a considerable genetic variability, where recently, was suggesting that this taxon consists of a complex of species. Sporotrichosis is caused by traumatic inoculation of the fungus, which is a ubiquitous environmental saprophyte that can be isolated from soil and plant debris. The infection is limited to the cutaneous forms but, recently, occurrences of more severe clinical forms of this mycosis were described, especially among immunocompromized individuals. The immunological mechanisms involved in prevention and control of sporotrichosis are still not very well understood. Some works suggest that cell-mediated immunity plays an important role in protecting the host against S. schenckii. In contrast, the role of the humoral immune response in protection against this fungus have not been studied in detail. In a previous study, we showed that antigens secreted by S. schenckii induce a specific humoral response in infected animals, mainly against the 70-kDa molecules, indicating a possible participation of specific antibodies to this molecule in infection control. In an other work of the our group, we produced a mAb against a 70-kDa glycoprotein of S. schenckii in order to better understand the effect of passive immunization of mice infected with S. schenckii. Results showed a significant reduction in the number of CFU in organs of mice when the mAb was injected before and during S. schenckii infection. Similar results were observed when T-cell deficient mice were used. Drugs of choice in the treatment of sporothrichosis require long periods and frequently relapses are observed, mainly in immunocompromized patients. The strong protection induced by mAb against a 70-kDa glycoprotein makes it a strong candidate for a

  11. IMPORTANCE OF RESEARCH HLA ANTIBODIES CLASS I AND II, AND MICA ANTIBODIES IN KIDNEY TRANSPLANTATION

    Directory of Open Access Journals (Sweden)

    M. Sh. Khubutia

    2011-01-01

    Full Text Available The purpose of this study was to investigate the occurrence of HLA and MICA antibodies in patients from the waiting list for kidney transplantation and their influence on the course of post-transplant period. Determination of HLA antibodies class I and II, and MICA antibodies was performed on a platform of Luminex (xMAP-tech- nology using sets LABScreen ONE LAMBDA (U.S.. A total of 156 patients from the waiting list for kidney transplantation. Revealed the presence of HLA and MICA antibodies in the serum of 31.4% of patients. Regraf- ted patients increased the content of antibodies to the antigens of HLA system was noted in 88.2% of cases, 47% met the combination of antibodies to the I, II classes and MICA. In patients awaiting first kidney transplantation, HLA and MICA antibodies were determined in 23.7% of cases. The presence of pretransplant HLA and MICA antibodies had a significant influence on the course of post-transplant period. Patients with the presence of HLA and MICA in 50% of cases delayed graft function. Sessions of plasmapheresis can reduce the concentration of HLA and MICA antibodies on average by 61.1%. 

  12. Antibody-Conjugated Nanoparticles for Biomedical Applications

    Directory of Open Access Journals (Sweden)

    Manuel Arruebo

    2009-01-01

    Full Text Available Nanoscience and Nanotechnology have found their way into the fields of Biotechnology and Medicine. Nanoparticles by themselves offer specific physicochemical properties that they do not exhibit in bulk form, where materials show constant physical properties regardless of size. Antibodies are nanosize biological products that are part of the specific immune system. In addition to their own properties as pathogens or toxin neutralizers, as well as in the recruitment of immune elements (complement, improving phagocytosis, cytotoxicity antibody dependent by natural killer cells, etc., they could carry several elements (toxins, drugs, fluorochroms, or even nanoparticles, etc. and be used in several diagnostic procedures, or even in therapy to destroy a specific target. The conjugation of antibodies to nanoparticles can generate a product that combines the properties of both. For example, they can combine the small size of nanoparticles and their special thermal, imaging, drug carrier, or magnetic characteristics with the abilities of antibodies, such as specific and selective recognition. The hybrid product will show versatility and specificity. In this review, we analyse both antibodies and nanoparticles, focusing especially on the recent developments for antibody-conjugated nanoparticles, offering the researcher an overview of the different applications and possibilities of these hybrid carriers.

  13. Standardization of anti-DNA antibody assays.

    Science.gov (United States)

    Pisetsky, David S

    2013-07-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and represent important biomarkers for clinical and research purposes. These antibodies are part of a family of antibodies to nucleosomes and bind to conserved sites widely present on DNA. While the value of anti-DNA as a biomarker is well established, the assay for these antibodies has involved a variety of DNA sources and systems to detect DNA-anti-DNA interactions. The influence of these variations on antibody detection has complicated assay standardization. As an antigen, DNA has unique features since it is a highly charged polymer that has structural heterogeneity. This heterogeneity can affect antigenicity which can vary on the basis of DNA origin, size, conformation and mobility. In addition, as a polymer, DNA can promote patterns of antibody binding based on monogamous or bivalent interaction which require an extended polynucleotide structure. Understanding the nature of DNA as an antigen can facilitate interpretation of serological tests and underpin efforts at better standardization.

  14. Quality control of antibodies for assay development.

    Science.gov (United States)

    Schumacher, Sarah; Seitz, Harald

    2016-09-25

    Antibodies are used as powerful tools in basic research, for example, in biomarker identification, and in various forms for diagnostics, for example, identification of allergies or autoimmune diseases. Due to their robustness and ease of handling, immunoassays are favourite methods for investigation of various biological or medical questions. Nevertheless in many cases, additional analyses such as mass spectrometry are used to validate or confirm the results of immunoassays. To minimize the workload and to increase confidence in immunoassays, there are urgent needs for antibodies which are both highly specific and well validated. Unfortunately many commercially available antibodies are neither well characterized nor fully tested for cross-reactivities. Adequate quality control and validation of an antibody is time-consuming and can be frustrating. Such validation needs to be performed for every assay/application. However, where an antibody validation is successful, a highly specific and stable reagent will be on hand. This article describes the validation processes of antibodies, including some often neglected factors, as well as unspecific binding to other sample compounds in a multiparameter diagnostic assay. The validation consists of different immunological methods, with important assay controls, and is performed in relation to the development of a diagnostic test. PMID:26873787

  15. Avian Diagnostic and Therapeutic Antibodies to Viral Emerging Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    David Bradley

    2011-03-31

    During the current period the following key objectives were achieved: demonstration of high titer antibody production by geese following immunization with inactived H1N1 virus; completion of the epitope mapping of West Nile Virus-specific goose antibodies and initiation of epitope mapping of H1N1 flu-specific goose antibodies; advancement in scalable purification of goose antibodies.

  16. A multi-Fc-species system for recombinant antibody production

    OpenAIRE

    Nizak Clément; Vielemeyer Ole; El Marjou Ahmed; Moutel Sandrine; Benaroch Philippe; Dübel Stefan; Perez Franck

    2009-01-01

    Abstract Background Genomic, transcriptomic and proteomic projects often suffer from a lack of functional validation creating a strong demand for specific and versatile antibodies. Antibody phage display represents an attractive approach to select rapidly in vitro the equivalent of monoclonal antibodies, like single chain Fv antibodies, in an inexpensive and animal free way. However, so far, recombinant antibodies have not managed to impose themselves as efficient alternatives to natural anti...

  17. Targeted immunotherapy in Hodgkin lymphoma

    DEFF Research Database (Denmark)

    Hutchings, Martin

    2015-01-01

    In this issue of Blood, Rothe et al introduce a new principle of targeted Hodgkin lymphoma (HL) immunotherapy in their report from a phase 1 study of the bispecific anti-CD30/CD16A antibody construct AFM13.......In this issue of Blood, Rothe et al introduce a new principle of targeted Hodgkin lymphoma (HL) immunotherapy in their report from a phase 1 study of the bispecific anti-CD30/CD16A antibody construct AFM13....

  18. A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments

    NARCIS (Netherlands)

    Huls, GA; Heijnen, IAFM; Cuomo, ME; Koningsberger, JC; Boel, E; de Vries, ARV; Loyson, SAJ; Helfrich, W; Henegouwen, GPV; van Meijer, M; de Kruif, J; Logtenberg, T

    1999-01-01

    A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab), The purified huMab had an affinity of 5 nM and effectively mediated tumor cell kil

  19. Thermodynamics of antibody-antigen interaction revealed by mutation analysis of antibody variable regions.

    Science.gov (United States)

    Akiba, Hiroki; Tsumoto, Kouhei

    2015-07-01

    Antibodies (immunoglobulins) bind specific molecules (i.e. antigens) with high affinity and specificity. In order to understand their mechanisms of recognition, interaction analysis based on thermodynamic and kinetic parameters, as well as structure determination is crucial. In this review, we focus on mutational analysis which gives information about the role of each amino acid residue in antibody-antigen interaction. Taking anti-hen egg lysozyme antibodies and several anti-small molecule antibodies, the energetic contribution of hot-spot and non-hot-spot residues is discussed in terms of thermodynamics. Here, thermodynamics of the contribution from aromatic, charged and hydrogen bond-forming amino acids are discussed, and their different characteristics have been elucidated. The information gives fundamental understanding of the antibody-antigen interaction. Furthermore, the consequences of antibody engineering are analysed from thermodynamic viewpoints: humanization to reduce immunogenicity and rational design to improve affinity. Amino acid residues outside hot-spots in the interface play important roles in these cases, and thus thermodynamic and kinetic parameters give much information about the antigen recognition. Thermodynamic analysis of mutant antibodies thus should lead to advanced strategies to design and select antibodies with high affinity.

  20. The antibody mining toolbox: an open source tool for the rapid analysis of antibody repertoires.

    Science.gov (United States)

    D'Angelo, Sara; Glanville, Jacob; Ferrara, Fortunato; Naranjo, Leslie; Gleasner, Cheryl D; Shen, Xiaohong; Bradbury, Andrew R M; Kiss, Csaba

    2014-01-01

    In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1-2 million reads can be accomplished in 10-15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries. PMID:24423623

  1. Passive antibody transfer in chickens to model maternal antibody after avian influenza vaccination.

    Science.gov (United States)

    Faulkner, Olivia B; Estevez, Carlos; Yu, Qingzhong; Suarez, David L

    2013-04-15

    Birds transfer maternal antibodies (MAb) to their offspring through the egg yolk where the antibody is absorbed and enters the circulatory system. Maternal antibodies provide early protection from disease, but may interfere with the vaccination efficacy in the chick. MAb are thought to interfere with vaccine antigen processing that reduces the subsequent immune response. Once MAb titers are depleted, the chick will respond to vaccination, but they are also susceptible to viral infection. This study examines the effect of MAb on seroconversion to different viral-vectored avian influenza virus (AIV) vaccines. Chicks were given passively transferred antibodies (PTA) using AIV hyperimmunized serum, and subsequently vaccinated with a fowlpox-AIV recombinant vaccine (FPr) or a Newcastle disease virus-AIV recombinant vaccine (NDVr). Our results indicate that passively transferred antibodies led to significant reduction of seroconversion and clinical protection from virulent challenge in recombinant virus vaccinated chicks thus demonstrating maternal antibody interference to vaccination. The passive antibody transfer model system provides an important tool to evaluate maternal antibody interference to vaccination. PMID:23398721

  2. Antibodies Produced in Response to Cryptococcus neoformans Pulmonary Infection in Mice Have Characteristics of Nonprotective Antibodies

    OpenAIRE

    Zaragoza, Oscar; Casadevall, Arturo

    2004-01-01

    Murine cryptocococcal pulmonary infection elicited serum immunoglobulin M (IgM) and IgG to the capsular polysaccharide, but only IgG stained yeast cells in alveoli. Both isotypes produced punctuate immunofluorescence patterns on yeast cells like those of nonprotective antibodies. The difficulties involved in associating humoral immunity with protection in murine cryptocococcal infection could reflect nonprotective antibody responses.

  3. A study on associations between antiprothrombin antibodies, antiplasminogen antibodies and thrombosis

    NARCIS (Netherlands)

    Simmelink, MJA; De Groot, PG; Derksen, RHWM

    2003-01-01

    Anti-prothrombin antibodies area frequent cause of lupus anticoagulant (LAC), a thrombotic risk factor. Prothrombin shares structural homology with plasminogen, a kringle protein with an important role in fibrinolysis. Cross-reactivity between antiprothrombin antibodies and plasminogen has been desc

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

    Science.gov (United States)

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

    2016-08-01

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

  5. Platform for high-throughput antibody selection using synthetically-designed antibody libraries.

    Science.gov (United States)

    Batonick, Melissa; Holland, Erika G; Busygina, Valeria; Alderman, Dawn; Kay, Brian K; Weiner, Michael P; Kiss, Margaret M

    2016-09-25

    Synthetic humanized antibody libraries are frequently generated by random incorporation of changes at multiple positions in the antibody hypervariable regions. Although these libraries have very large theoretical diversities (>10(20)), the practical diversity that can be achieved by transformation of Escherichia coli is limited to about 10(10). To constrain the practical diversity to sequences that more closely mimic the diversity of natural human antibodies, we generated a scFv phage library using entirely pre-defined complementarity determining regions (CDR). We have used this library to select for novel antibodies against four human protein targets and demonstrate that identification of enriched sequences at each of the six CDRs in early selection rounds can be used to reconstruct a consensus antibody with selectivity for the target. PMID:26607994

  6. Antibody engineering using phage display with a coiled-coil heterodimeric Fv antibody fragment.

    Directory of Open Access Journals (Sweden)

    Xinwei Wang

    Full Text Available A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to V(H and V(L for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of V(H frameworks and V(H-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering.

  7. Adaptive responses to antibody based therapy.

    Science.gov (United States)

    Rodems, Tamara S; Iida, Mari; Brand, Toni M; Pearson, Hannah E; Orbuch, Rachel A; Flanigan, Bailey G; Wheeler, Deric L

    2016-02-01

    Receptor tyrosine kinases (RTKs) represent a large class of protein kinases that span the cellular membrane. There are 58 human RTKs identified which are grouped into 20 distinct families based upon their ligand binding, sequence homology and structure. They are controlled by ligand binding which activates intrinsic tyrosine-kinase activity. This activity leads to the phosphorylation of distinct tyrosines on the cytoplasmic tail, leading to the activation of cell signaling cascades. These signaling cascades ultimately regulate cellular proliferation, apoptosis, migration, survival and homeostasis of the cell. The vast majority of RTKs have been directly tied to the etiology and progression of cancer. Thus, using antibodies to target RTKs as a cancer therapeutic strategy has been intensely pursued. Although antibodies against the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) have shown promise in the clinical arena, the development of both intrinsic and acquired resistance to antibody-based therapies is now well appreciated. In this review we provide an overview of the RTK family, the biology of EGFR and HER2, as well as an in-depth review of the adaptive responses undertaken by cells in response to antibody based therapies directed against these receptors. A greater understanding of these mechanisms and their relevance in human models will lead to molecular insights in overcoming and circumventing resistance to antibody based therapy. PMID:26808665

  8. Cytolytic antibodies to melanocytes in vitiligo.

    Science.gov (United States)

    Cui, J; Arita, Y; Bystryn, J C

    1993-06-01

    Patients with vitiligo have been found to have circulating antibodies to pigment cells. To evaluate the functional activity of these antibodies, a highly sensitive europium release assay was used to compare complement-mediated cytolysis of human melanocytes by sera of 56 patients with vitiligo (20 with active disease, 25 with inactive disease, 11 with unidentified disease activity) and 47 control individuals. Significant melanocyte lysis was mediated by 32 (57%) of the patients with vitiligo but by only three (6%) of the control sera (p < 0.001), and by 17 (85%) of 20 patients with active vitiligo versus 11 (44%) of 25 patients with inactive disease (p < 0.025). Mean melanocyte lysis by vitiligo sera was 24% versus 6% by control sera (p < 0.0001). A subset of 12 vitiligo sera with high titers of cytolytic antibodies to melanocytes (34% mean cytolysis) reacted minimally (< 2% mean cytolysis) to a panel of control cells that included human and murine melanomas, human fibroblasts, lung carcinoma, and rhabdomyosarcoma. These findings indicate that antibodies present in patients with vitiligo have the functional ability to selectively kill melanocytes and are more common in active disease. These observations support, but do not prove, the hypothesis that vitiligo is an autoimmune disease and that anti-pigment cell antibodies have a role in inducing the disease. PMID:8496621

  9. Antigen-Antibody Interaction Database (AgAbDb): a compendium of antigen-antibody interactions.

    Science.gov (United States)

    Kulkarni-Kale, Urmila; Raskar-Renuse, Snehal; Natekar-Kalantre, Girija; Saxena, Smita A

    2014-01-01

    Antigen-Antibody Interaction Database (AgAbDb) is an immunoinformatics resource developed at the Bioinformatics Centre, University of Pune, and is available online at http://bioinfo.net.in/AgAbDb.htm. Antigen-antibody interactions are a special class of protein-protein interactions that are characterized by high affinity and strict specificity of antibodies towards their antigens. Several co-crystal structures of antigen-antibody complexes have been solved and are available in the Protein Data Bank (PDB). AgAbDb is a derived knowledgebase developed with an objective to compile, curate, and analyze determinants of interactions between the respective antigen-antibody molecules. AgAbDb lists not only the residues of binding sites of antigens and antibodies, but also interacting residue pairs. It also helps in the identification of interacting residues and buried residues that constitute antibody-binding sites of protein and peptide antigens. The Antigen-Antibody Interaction Finder (AAIF), a program developed in-house, is used to compile the molecular interactions, viz. van der Waals interactions, salt bridges, and hydrogen bonds. A module for curating water-mediated interactions has also been developed. In addition, various residue-level features, viz. accessible surface area, data on epitope segment, and secondary structural state of binding site residues, are also compiled. Apart from the PDB numbering, Wu-Kabat numbering and explicit definitions of complementarity-determining regions are provided for residues of antibodies. The molecular interactions can be visualized using the program Jmol. AgAbDb can be used as a benchmark dataset to validate algorithms for prediction of B-cell epitopes. It can as well be used to improve accuracy of existing algorithms and to design new algorithms. AgAbDb can also be used to design mimotopes representing antigens as well as aid in designing processes leading to humanization of antibodies. PMID:25048123

  10. Antibody-based biological toxin detection

    Energy Technology Data Exchange (ETDEWEB)

    Menking, D.E.; Goode, M.T. [Army Edgewood Research, Development and Engineering Center, Aberdeen Proving Ground, MD (United States)

    1995-12-01

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

  11. Origin and pathogenesis of antiphospholipid antibodies

    Directory of Open Access Journals (Sweden)

    C.M. Celli

    1998-06-01

    Full Text Available Antiphospholipid antibodies (aPL are a heterogeneous group of antibodies that are detected in the serum of patients with a variety of conditions, including autoimmune (systemic lupus erythematosus, infectious (syphilis, AIDS and lymphoproliferative disorders (paraproteinemia, myeloma, lymphocytic leukemias. Thrombosis, thrombocytopenia, recurrent fetal loss and other clinical complications are currently associated with a subgroup of aPL designating the antiphospholipid syndrome. In contrast, aPL from patients with infectious disorders are not associated with any clinical manifestation. These findings led to increased interest in the origin and pathogenesis of aPL. Here we present the clinical features of the antiphospholipid syndrome and review the origin of aPL, the characteristics of experimentally induced aPL and their historical background. Within this context, we discuss the most probable pathogenic mechanisms induced by these antibodies.

  12. Imaging spectrum of primary antiphospholipid antibody syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Kwon Ha; Won, Jong Jin [Wonkwang University Hospital, Iksan (Korea, Republic of); Ha, Hyun Kwon; Kim, Jung Hoon; Kim, Jeong Gon; Ki, Won Woo; Kim, Pyo Nyun; Lee, Moon Gyu; Auh, Yong Ho [Asan Medical Center, Seoul (Korea, Republic of)

    1998-04-01

    Antiphospholipid antibody syndrome is recognized as one of the most important causes of hypercoagulability. It can be clinically diagnosed if patients have experienced unexplained recurrent venous or arterial thrombosis, recurrent fetal loss, or thrombocytopenia in the presence of circulating autoantibodies to phospholipids, such as anticardiolipin antibody or lupus anticoagulant. Approximately half of all patients with this syndrome do not have associated systemic disease, and their condition is described as primary antiphospholipid antibody syndrome (PAPS). In the remainder, the syndrome is accompanied by systemic lupus erythematosus or other connective tissue diseases, and is known as secondary antiphospholipid syndrome (1). The purpose of this paper is to illustrate the systemic manifestation of PAPS, focusing on the radiological findings of CT, MR and angiography in clinically proven patients. (author). 8 refs., 10 figs.

  13. Back to the future: recombinant polyclonal antibody therapeutics.

    Science.gov (United States)

    Wang, Xian-Zhe; Coljee, Vincent W; Maynard, Jennifer A

    2013-11-01

    Antibody therapeutics are one of the fastest growing classes of pharmaceuticals, with an annual US market over $20 billion, developed to treat a variety of diseases including cancer, auto-immune and infectious diseases. Most are currently administered as a single molecule to treat a single disease, however there is mounting evidence that cocktails of multiple antibodies, each with a unique binding specificity and protective mechanism, may improve clinical efficacy. Here, we review progress in the development of oligoclonal combinations of antibodies to treat disease, focusing on identification of synergistic antibodies. We then discuss the application of modern antibody engineering technologies to produce highly potent antibody preparations, including oligoclonal antibody cocktails and truly recombinant polyclonal antibodies. Specific examples illustrating the synergy conferred by multiple antibodies will be provided for diseases caused by botulinum toxin, cancer and immune thrombocytopenia. The bioprocessing and regulatory options for these preparations will be discussed. PMID:24443710

  14. Characterization of monoclonal antibodies directed against human thyroid stimulating hormone

    International Nuclear Information System (INIS)

    Monoclonal antibodies directed against human thyroid stimulating hormone (TSH) were obtained from hybrid myelomas, following fusion of mouse NSI myeloma cells with mouse spleen cells. Ten different antibodies were obtained from 4 separate fusions. Eight antibodies were of the IgG1 subclass. Affinities of antibodies for TSH were in the range 2 x 108-5 x 1010 M-1. Five of the antibodies were specific for TSH and did not react with LH, FSH or hCG. The remaining antibodies reacted with all these hormones and were assumed to recognise their common (α) subunit. The 5 specific antibodies fell into 3 subgroups recognising distinct antigenic determinants, whereas the 5 non-specific antibodies recognised a single determinant or closely related set of sites. It is concluded that these antibodies should be valuable reagents for use in sensitive and specific two-site immunoradiometric assays. (Auth.)

  15. Anti-DNA antibodies--quintessential biomarkers of SLE.

    Science.gov (United States)

    Pisetsky, David S

    2016-02-01

    Antibodies that recognize and bind to DNA (anti-DNA antibodies) are serological hallmarks of systemic lupus erythematosus (SLE) and key markers for diagnosis and disease activity. In addition to common use in the clinic, anti-DNA antibody testing now also determines eligibility for clinical trials, raising important questions about the nature of the antibody-antigen interaction. At present, no 'gold standard' for serological assessment exists, and anti-DNA antibody binding can be measured with a variety of assay formats, which differ in the nature of the DNA substrates and in the conditions for binding and detection of antibodies. A mechanism called monogamous bivalency--in which high avidity results from simultaneous interaction of IgG Fab sites with a single polynucleotide chain--determines anti-DNA antibody binding; this mechanism might affect antibody detection in different assay formats. Although anti-DNA antibodies can promote pathogenesis by depositing in the kidney or driving cytokine production, they are not all alike, pathologically, and anti-DNA antibody expression does not necessarily correlate with active disease. Levels of anti-DNA antibodies in patients with SLE can vary over time, distinguishing anti-DNA antibodies from other pathogenic antinuclear antibodies. Elucidation of the binding specificities and the pathogenic roles of anti-DNA antibodies in SLE should enable improvements in the design of informative assays for both clinical and research purposes.

  16. Utility of feline coronavirus antibody tests.

    Science.gov (United States)

    Addie, Diane D; le Poder, Sophie; Burr, Paul; Decaro, Nicola; Graham, Elizabeth; Hofmann-Lehmann, Regina; Jarrett, Oswald; McDonald, Michael; Meli, Marina L

    2015-02-01

    Eight different tests for antibodies to feline coronavirus (FCoV) were evaluated for attributes that are important in situations in veterinary practice. We compared four indirect immunofluorescent antibody tests (IFAT), one enzyme-linked immunosorbent assay (ELISA) (FCoV Immunocomb; Biogal) and three rapid immunochromatographic (RIM) tests against a panel of samples designated by consensus as positive or negative. Specificity was 100% for all but the two IFATs based on transmissible gastroenteritis virus (TGEV), at 83.3% and 97.5%. The IFAT and ELISA tests were best for obtaining an antibody titre and for working in the presence of virus. The RIM tests were the best for obtaining a result quickly (10-15 mins); of these, the Speed F-Corona was the most sensitive, at 92.4%, followed by FASTest feline infectious peritonitis (FIP; 84.6%) and Anigen Rapid FCoV antibody test (64.1%). Sensitivity was 100% for the ELISA, one FCoV IFAT and one TGEV IFAT; and 98.2% for a second TGEV IFA and 96.1% for a second FCoV IFAT. All tests worked with effusions, even when only blood products were stipulated in the instruction manual. The ELISA and Anigen RIM tests were best for small quantities of sample. The most appropriate FCoV antibody test to use depends on the reason for testing: in excluding a diagnosis of FIP, sensitivity, specificity, small sample quantity, rapidity and ability to work in the presence of virus all matter. For FCoV screening, speed and sensitivity are important, and for FCoV elimination antibody titre is essential. PMID:24966245

  17. Fully human IgG and IgM antibodies directed against the carcinoembryonic antigen (CEA Gold 4 epitope and designed for radioimmunotherapy (RIT of colorectal cancers

    Directory of Open Access Journals (Sweden)

    Pugnière Martine

    2004-10-01

    VG-IgM biodistribution was not possible in this mouse model in which IgM displays a very short half-life due to poly-Ig receptor expression in the liver. Conclusion Our human anti-CEA IgG2κ is a promising candidate for radioimmunotherapy in intact form, as F(ab'2 fragments, or as a bispecific antibody.

  18. Antibodies for detecting and quantifying anticoagulant agents

    OpenAIRE

    Salvador, Juan Pablo; Marco, María Pilar

    2012-01-01

    [EN] The present invention relates to the design of haptens that are structurally related to coumarin oral anticoagulant compounds (COAC), to be used for the production of specific antibodies against said type of substances and the subsequent use thereof for the development of diagnosis tools for use in laboratories or in point-of-care (PoC) devices. In particular, the produced antibodies have been used to develop a diagnosis tool that enables the plasma levels of COAC to be quantified in pat...

  19. Basic immunology of antibody targeted radiotherapy

    International Nuclear Information System (INIS)

    Antibody targeted radiotherapy brings an important new treatment modality to Radiation oncology clinic. Radiation dose to tumor and normal tissues are determined by a complex interplay of antibody, antigen, tumor, radionuclide, and host-related factors. A basic understanding of these immunologic and physiologic factors is important to optimally utilize this therapy in the clinic. Preclinical and clinical studies need to be continued to broaden our understanding and to develop new strategies to further improve the efficacy of this promising form of targeted therapy

  20. Radiolabelling of monoclonal antibodies for radiotherapy. Thailand

    International Nuclear Information System (INIS)

    Nuclear medicine is now playing a great role not only in diagnostic application but also in therapy of cancer patients. Under the concept of targeted radiotherapy, a number of radiopharmaceuticals based on radiolabelled biomolecules had been evaluated for treatment of cancer by many investigators. Of these, monoclonal antibodies and some small specific peptides labelled with beta emitting radiometals such as Sm-153, Re-186, Re-188 or Y-90, are being introduced into clinical trials. The objective of this project is to develop laboratory procedures to label monoclonal antibodies, peptide or other proteins with beta emitting radionuclides to prepare radiopharmaceuticals for therapeutic purpose

  1. Radiolabelling of monoclonal antibodies for radiotherapy

    International Nuclear Information System (INIS)

    Nuclear medicine is now playing a great role not only in diagnostic application but also in therapy of cancer patients. Under the concept of targeted radiotherapy, a number of radiopharmaceuticals based on radiolabelled biomolecules had been evaluated for treatment of cancer by many investigators. Of these, monoclonal antibodies and some small specific peptides labelled with beta emitting radiometals such as Sm-153, Re-186, Re-188 or Y-90, are being introduced into clinical trials. The objective of this project is to develop laboratory procedures to label monoclonal antibodies, peptide or other proteins with beta emitting radionuclides to prepare radiopharmaceuticals for therapeutic purpose

  2. Modeling single cell antibody excretion on a biosensor.

    Science.gov (United States)

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

    2016-07-01

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

  3. A New Lyophilized Kit for Rapid Radiofluorination of Peptides

    Science.gov (United States)

    McBride, William J.; D'Souza, Christopher A.; Karacay, Habibe; Sharkey, Robert M.; Goldenberg, David M.

    2012-01-01

    Radiolabeling compounds with positron-emitting radionuclides often involves a time-consuming, customized process. Herein, we report a simple lyophilized kit formulation for labeling peptides with 18F, based on the aluminum-fluoride procedure. The prototype kit contains IMP485, a NODA (1,4,7-triazacyclononane-1,4-diacetate)-MPAA (methyl phenylacetic acid)-di-HSG (histamine-succinyl-glycine) hapten-peptide, [NODA-MPAA-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2], used for pretargeting, but we also examined a similar kit formulation for a somatostatin-binding peptide [IMP466, NOTA-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Throl] bearing a NOTA ligand to determine if the benefits of using a kit can be extended to other AlF-binding peptides. The NODA-MPAA ligand forms a single stable complex with (AlF)2+ in high yields. In order to establish suitable conditions for a facile kit, the formulation was optimized for pH, peptide to Al3+ ratio, bulking agent, radioprotectant, and the buffer. For optimal labeling, the kit was reconstituted with an aqueous solution of 18F− and ethanol (1:1), heated at 100–110 °C for 15 min, and then simply and rapidly purified using one of two equally effective solid-phase extraction (SPE) methods. Al18F-IMP485 was isolated as a single isomer complex, in high yield (45–97%) and high specific activity (up to 223 GBq/μmol), within 20 min. The labeled product was stable in human serum at 37 °C for 4 h and in vivo, urine samples showed the intact product was eliminated. Tumor targeting of the Al18F-IMP485 in nude mice bearing human colon cancer xenografts pretargeted with an anti-CEACAM5 bispecific antibody showed very low uptake (0.06% ± 0.02 ID/g) in bone, further illustrating its stability. At 1 h, pretargeted animals had high Al18F-IMP485 tumor uptake (28.1% ± 4.5 ID/g), with ratios of 9 ± 4, 123 ± 38, 110 ± 43 and 120 ± 108 for kidney, liver, blood and bone, respectively. Tumor uptake remained high at 3 h post-injection, with increased tumor

  4. Antibody-mediated immune suppression is improved when blends of anti-RBC monoclonal antibodies are used in mice.

    Science.gov (United States)

    Bernardo, Lidice; Amash, Alaa; Marjoram, Danielle; Lazarus, Alan H

    2016-08-25

    Although the prevention of hemolytic disease of the fetus and newborn is highly effective using polyclonal anti-D, a recombinant alternative is long overdue. Unfortunately, anti-D monoclonal antibodies have been, at best, disappointing. To determine the primary attribute defining an optimal antibody, we assessed suppression of murine red blood cell (RBC) immunization by single-monoclonal antibodies vs defined blends of subtype-matched antibodies. Allogeneic RBCs expressing the HOD antigen (hen egg lysozyme [HEL]-ovalbumin-human transmembrane Duffy(b)) were transfused into naïve mice alone or together with selected combinations of HEL-specific antibodies, and the resulting suppressive effect was assessed by evaluating the antibody response. Polyclonal HEL antibodies dramatically inhibited the antibody response to the HOD antigen, whereas single-monoclonal HEL antibodies were less effective despite the use of saturating doses. A blend of monoclonal HEL-specific antibodies reactive with different HEL epitopes significantly increased the suppressive effect, whereas a blend of monoclonal antibodies that block each other's binding to the HEL protein did not increase suppression. In conclusion, these data show that polyclonal antibodies are superior to monoclonal antibodies at suppressing the immune response to the HOD cells, a feature that can be completely recapitulated using monoclonal antibodies to different epitopes. PMID:27330002

  5. Treatment of leukemia with radiolabeled monoclonal antibodies.

    Science.gov (United States)

    Sgouros, G; Scheinberg, D A

    1993-01-01

    In contrast to radioimmunotherapy of solid disease, wherein the primary obstacle to success is access of radiolabeled antibody to antigen-positive cells, in the treatment of leukemia delivering a lethal absorbed dose to the isolated cell appears to be the primary obstacle. The isolated cell is defined as one that is exposed only to self-irradiation (from internalized or surface-bound radiolabeled antibody) and to irradiation from free antibody in the blood. It is isolated in the sense that the particulate (beta, electron, alpha) emissions from its nearest neighboring antigen-positive cell do not contribute to its absorbed dose. Disease in the bone marrow and other tissues, since it is confined to a smaller volume, is more easily eradicated because the absorbed dose to a given cell nucleus is enhanced by emissions from adjacent cells (a smaller fraction of the emission energy is 'wasted'). The optimization simulations presented above for the M195 antibody suggest that the optimum dose of antibody that should be administered is that required to yield a concentration within the distribution volume of the antibody that is approximately equal to the concentration of antigen sites as determined by the tumor burden. Although not specifically considered in the modeling example presented above, antibody internalization and catabolism may be expected to play an important role in radioimmunotherapy treatment planning of leukemia. Depending upon the kinetics of internalization and catabolism, the absorbed dose to the red marrow and to antigen-positive cells may be reduced considerably, since catabolism, assuming that it is followed by rapid extrusion of the radioactive label, would decrease the cells' exposure time considerably. The recently demonstrated effectiveness of radioimmunotherapy in certain cases of B-cell lymphoma and in reducing tumor burden in acute myelogenous leukemia suggests that radioimmunotherapy is beginning to fulfill the promise held when it was initially

  6. Anti Rh Hemolytic Disease due to Anti C Antibody: Is Testing for Anti D Antibodies Enough?

    OpenAIRE

    Negi, Gita; Singh, Gaur Dushyant

    2011-01-01

    Rh blood group system is a complex blood group system. Rh antibodies are produced in Rh negative individuals following exposure to foreign RBCs after transfusion or pregnancy. Anti C is a rare cause of hemolytic disease of newborn and is very scarcely reported in the literature. The aim of the present case report of Hemolytic disease caused by Anti C antibody is to bring out the fact that antibodies other than anti D should be considered in cases that give a suggestive history but no evidence...

  7. Phase Transitions in Antibody Solutions: from Pharmaceuticals to Human Disease

    Science.gov (United States)

    Wang, Ying; Lomakin, Aleksey; Benedek, George; Dana Farber Cancer Institute Collaboration; Amgen Inc. Collaboration

    2014-03-01

    Antibodies are very important proteins. Natural antibodies play essential role in the immune system of human body. Pharmaceutical antibodies are used as drugs. Antibodies are also indispensable tools in biomedical research and diagnostics. Recently, a number of observations of phase transitions of pharmaceutical antibodies have been reported. These phase transitions are undesirable from the perspective of colloid stability of drug solutions in processing and storage, but can be used for protein purification, X-ray crystallography, and improving pharmokinetics of drugs. Phase transitions of antibodies can also take place in human body, particularly in multiple myeloma patients who overproduce monoclonal antibodies. These antibodies, in some cases, crystallize at body temperature and cause severe complications called cryoglobulinemia. I will present the results of our current studies on phase transitions of both pharmaceutical antibodies and cryoglobulinemia-associated antibodies. These studies have shown that different antibodies have different propensity to undergo phase transitions, but their phase behavior has universal features which are remarkably different from those of spherical proteins. I will discuss how studies of phase behavior can be useful in assessing colloid stability of pharmaceutical antibodies and in early diagnostics of cryoglobulinemia, as well as general implications of the fact that some antibodies can precipitate at physiological conditions.

  8. IgA Antibodies in Rett Syndrome

    Science.gov (United States)

    Reichelt, K. L.; Skjeldal, O.

    2006-01-01

    The level of IgA antibodies to gluten and gliadin proteins found in grains and to casein found in milk, as well as the level of IgG to gluten and gliadin, have been examined in 23 girls with Rett syndrome and 53 controls. Highly statistically significant increases were found for the Rett population compared to the controls. The reason for this…

  9. Antibody-based imaging strategies for cancer

    NARCIS (Netherlands)

    Warram, Jason M.; de Boer, Esther; Sorace, Anna G.; Chung, Thomas K.; Kim, Hyunki; Pleijhuis, Rick G.; van Dam, Gooitzen M.; Rosenthal, Eben L.

    2014-01-01

    Although mainly developed for preclinical research and therapeutic use, antibodies have high antigen specificity, which can be used as a courier to selectively deliver a diagnostic probe or therapeutic agent to cancer. It is generally accepted that the optimal antigen for imaging will depend on both

  10. Bone marrow dosimetry for monoclonal antibody therapy

    International Nuclear Information System (INIS)

    Immunoglobulins must permeate through the basement membrane of capillaries in order to enter the extracellular space (ECS) of tissue. Since the process is quite slow, the blood plasma activity in various organs contributes considerably to the radiation dose of the dose-limiting tissues. In bone marrow the basement membrane is absent and the blood circulation is functionally open. Therefore, blood plasma and marrow ECS maintain equal concentrations of labeled immunoglobulins. A combination of factors including intravenous administration, slow absorption into most tissues, slow breakdown and elimination of labeled immunoglobulin, and rapid entry into bone marrow ECS as well as known radiosensitivity of marrow led the authors to expect this tissue would prove to be the primary tissue at risk for systemic monoclonal antibody therapy. They have developed and applied in a Phase I clinical study of 131I labeled CEA antibody a procedure for estimation of radiation dose to red bone marrow. Serieal measurements of blood plasma and total body retention are carried out. Binding of labeled antibody to the cellular components of blood is verified to be very low. They have observed bone marrow depression at doses greater than 400 rad. If no special procedures are used to reconstitute marrow after radiation treatment, this level represents a much greater than generally recognized limitation to radiolabeled monoclonal antibody therapy. 25 references, 4 tables

  11. JDIP Genomics, Antibodies, and Proteomics Core Update

    Science.gov (United States)

    The JDIP Genomics, Proteomics, and Antibodies Core has developed several resources that are available for use by JDIP researchers. Five tasks have been completed or are in progress: Task 1 – Transposon mutants: Nearly 24,000 gene disruption M. paratuberculosis mutants are now available for JDIP re...

  12. Developing recombinant antibodies for biomarker detection

    Energy Technology Data Exchange (ETDEWEB)

    Baird, Cheryl L.; Fischer, Christopher J.; Pefaur, Noah B.; Miller, Keith D.; Kagen, Jacob; Srivastava, Sudhir; Rodland, Karin D.

    2010-10-01

    Monoclonal antibodies (mAbs) have an essential role in biomarker validation and diagnostic assays. A barrier to pursuing these applications is the reliance on immunization and hybridomas to produce mAbs, which is time-consuming and may not yield the desired mAb. We recommend a process flow for affinity reagent production that utilizes combinatorial protein display systems (eg, yeast surface display or phage display) rather than hybridomas. These systems link a selectable phenotype-binding conferred by an antibody fragment-with a means for recovering the encoding gene. Recombinant libraries obtained from immunizations can produce high-affinity antibodies (<10 nM) more quickly than other methods. Non-immune libraries provide an alternate route when immunizations are not possible, or when suitable mAbs are not recovered from an immune library. Directed molecular evolution (DME) is an integral part of optimizing mAbs obtained from combinatorial protein display, but can also be used on hybridoma-derived mAbs. Variants can easily be obtained and screened to increase the affinity of the parent mAb (affinity maturation). We discuss examples where DME has been used to tailor affinity reagents to specific applications. Combinatorial protein display also provides an accessible method for identifying antibody pairs, which are necessary for sandwich-type diagnostic assays.

  13. Ebola Virus Antibodies in Fruit Bats, Bangladesh

    OpenAIRE

    Kevin J Olival; Islam, Ariful; Yu, Meng; Anthony, Simon J.; Jonathan H Epstein; Khan, Shahneaz Ali; Khan, Salah Uddin; Crameri, Gary; Wang, Lin-Fa; Lipkin, W. Ian; Luby, Stephen P.; Daszak, Peter

    2013-01-01

    To determine geographic range for Ebola virus, we tested 276 bats in Bangladesh. Five (3.5%) bats were positive for antibodies against Ebola Zaire and Reston viruses; no virus was detected by PCR. These bats might be a reservoir for Ebola or Ebola-like viruses, and extend the range of filoviruses to mainland Asia.

  14. Neutralizing antibodies in hepatitis C virus infection

    Institute of Scientific and Technical Information of China (English)

    Mirjam B Zeisel; Samira Fafi-Kremer; Isabel Fofana; Heidi Barth; Fran(c)oise Stoll-Keller; Michel Doffo(e)l; Thomas F Baumert

    2007-01-01

    Hepatitis C virus (HCV) is a major cause of hepatitis world-wide. The majority of infected individuals develop chronic hepatitis which can then progress to liver cirrhosis and hepatocellular carcinoma. Spontaneous viral clearance occurs in about 20%-30% of acutely infected individuals and results in resolution of infection without sequaelae. Both viral and host factors appear to play an important role for resolution of acute infection. A large body of evidence suggests that a strong, multispecific and long-lasting cellular immune response appears to be important for control of viral infection in acute hepatitis C. Due too the lack of convenient neutralization assays,the impact of neutralizing responses for control of viral infection had been less defined. In recent years, the development of robust tissue culture model systems for HCV entry and infection has finally allowed study of antibody-mediated neutralization and to gain further insights into viral targets of host neutralizing responses.In addition, detailed analysis of antibody-mediated neutralization in individual patients as well as cohorts with well defined viral isolates has enabled the study of neutralizing responses in the course of HCV infection and characterization of the impact of neutralizing antibodies for control of viral infection. This review will summarize recent progress in the understanding of the molecular mechanisms of antibody-mediated neutralization and its impact for HCV pathogenesis.(C) 2007 The WJG Press. All rights reserved.

  15. Greasing the SCIDs for Universal Flu Antibodies

    Science.gov (United States)

    Yewdell, Jonathan W.; Ince, William L.

    2013-01-01

    Previews In this issue, Nakamura et al. describe a robust SCID mouse-based method for isolating human monoclonal antibodies of desired specificity from adoptively transferred human B cells. As proof-of principle, they isolate human mAbs that could potentially be used to treat or prevent human infection with any influenza A virus strain. PMID:23870308

  16. Burkholderia pseudomallei Antibodies in Children, Cambodia

    OpenAIRE

    Wuthiekanun, Vanaporn; Pheaktra, Ngoun; Putchhat, Hor; Sin, Lina; Sen, Bun; Kumar, Varun; Langla, Sayan; Peacock, Sharon J.; Nicholas P. Day

    2008-01-01

    Antibodies to Burkholderia pseudomallei were detected in 16% of children in Siem Reap, Cambodia. This organism was isolated from 30% of rice paddies in the surrounding vicinity. Despite the lack of reported indigenous cases, melioidosis is likely to occur in Cambodia.

  17. IgA as therapeutic antibody

    NARCIS (Netherlands)

    Leusen, Jeanette H W

    2015-01-01

    This review is focused on the promises of IgA as a new therapeutic antibody. For more than 30 years IgG molecules have been used in the clinic in the fields of oncology, hematology, auto immune diseases and infections. However, IgA might be a good alternative, since it recruits different effector ce

  18. Antiphospholipid Antibody Syndrome Presenting with Hemichorea

    Directory of Open Access Journals (Sweden)

    Yezenash Ayalew

    2012-01-01

    Full Text Available A 25-year-old Bangladeshi lady presented to neurology with a three-month history of involuntary movements of her right arm, associated with loss of power. There was progression to the right leg, and she subsequently developed episodes of slurred speech and blurred vision. At the time of presentation, she was 12 weeks pregnant and the symptoms were reported to have started at conception. Past medical history was unremarkable apart from one first trimester miscarriage and there was no significant family history suggestive of a hereditary neurological condition. MRI of the head revealed no abnormalities but serology showed positive antinuclear antibodies (ANAs at a titre of 1/400. Further investigations revealed strongly positive anticardiolipin antibodies (>120 and positive lupus anticoagulant antibodies. The patient had a second miscarriage at 19 weeks gestation strengthening the possibility that the chorea was related to antiphospholipid antibody syndrome and she was started on a reducing dose of Prednisolone 40 mg daily and aspirin 300 mg daily. Six months later, she had complete resolution of neurological symptoms. There are several reports of chorea as a feature of antiphospholipid syndrome, but no clear consensus on underlying pathophysiology.

  19. PRODUCTION OF MONOCLONAL ANTIBODY AGAINST HUMAN IMMUNOGLOBULIN

    Directory of Open Access Journals (Sweden)

    J. Majidi

    2000-04-01

    Full Text Available Immunoglobulin E is one of the five classes of immonoglobulins that plays an important role in allergic diseases. Production of monoclonal antibodies by a single clonotype against different epitopes of immunoglobulin E has high priority in development of diagnostic kits.In this study, an attempt was made to produce monoclonal antibodies against human immunoglobulin E. Balb/c mice were immunized with semipurified immunoglobulin E and spleen cells fused with SP2.0 mouse myeloma eel! line in the presence of polyethylene glycol. Supernatant of hybridoma cells was screened for detection of antibody by enzyme linked immonosorbent assay method. Cloning of selective high absorbance wells were done with limiting dilution method. The suitable clone (monoclone was selected by enzyme linked immunosorbent assay and confirmed by immunoblot. The subclass of the chosen monoclonal antibodies was determined and the clones freezed and kept in liquid nitrogen.During this study three successful fusions were carried out, which resulted in development of 156 clones with high production of anti-IgE. Fourteen clones with the highest titres were selected for cloning. After limiting dilution more than 100 monoclonal antibodies were produced and the suitable (me (GJ0F7, i.e.; the clone which displayed the high absorbance in reaction with purified immunoglobulin E and the lowest cross-reactivity with immunoglobulin M, immunoglobulin G and immoglobulin A was chosen. In immunoblotting, presence of high density band in reaction with immunoglobulin E was confirmed. The suitable mab was shown to be IgG 1 subclass with kappa light chain. It seems that, this mab could be successfully used in diagnostic kits.

  20. The antiphospholipid antibody syndrome: a case report

    Directory of Open Access Journals (Sweden)

    Luma HN

    2012-10-01

    Full Text Available Henry Namme Luma,1,2 Marie-Solange Doualla,1,2 Elvis Temfack,1 Servais Albert Fiacre Eloumou Bagnaka,1 Emmanuella Wankie Mankaa,3 Dobgima Fofung41Department of Internal Medicine, Douala General Hospital, Douala, Cameroon; 2Faculty of Medicine and Biomedical Sciences, University of Yaoundé I, Yaoundé, Cameroon; 3Department of Radiology, Douala General Hospital Douala, Cameroon; 4Department of Abdominal Surgery, Daniel Muna Memorial Clinic, Douala, CameroonAbstract: Antiphospholipid antibody syndrome is defined by the presence of thromboembolic complications and/or pregnancy morbidity in the presence of persistently increased titers of antiphospholipid antibodies. Its clinical presentation can be diverse and any organ can be involved, with a current impact in most surgical and medical specialties. The authors present the case of a 43-year-old man who, over a 13-year period of follow-up, presented with thrombosis of the mesenteric vein, inferior vena cava, and axillary and subclavian veins in a setting where diagnostic and therapeutic options are limited and costly. Through this case report, the authors aim to describe the evolution of this complex pathology, which to date has not been described in the authors' milieu – probably because of its challenging diagnosis and the limited treatment options available. The authors conclude that clinicians need to have a high index of suspicion of APS in patients who present with a thrombotic episode – clinicians should investigate for the presence of antiphospholipid antibodies, as early diagnosis may influence the course of the disease. Furthermore, resources for the detection of antiphospholipid antibodies should be made readily available in resource-limited settings. Finally, patient education on the importance of drug compliance, periodic monitoring, and prevention of thrombosis is indispensable, especially as mortality could be associated with the effects of vascular thrombosis and/or the effects

  1. B cells contribute to MS pathogenesis through antibody-dependent and antibody-independent mechanisms

    Directory of Open Access Journals (Sweden)

    Wilson HL

    2012-05-01

    Full Text Available Heather L Wilson1,21Vaccine and Infectious Disease Organization-International Vaccine Center, 2Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, CanadaAbstract: For many years, central dogma defined multiple sclerosis (MS as a T cell-driven autoimmune disorder; however, over the past decade there has been a burgeoning recognition that B cells contribute to the pathogenesis of certain MS disease subtypes. B cells may contribute to MS pathogenesis through production of autoantibodies (or antibodies directed at foreign bodies, which unfortunately cross-react with self-antigens, through promotion of T cell activation via antigen presentation, or through production of cytokines. This review highlights evidence for antibody-dependent and antibody-independent B cell involvement in MS pathogenesis.Keywords: autoantibodies, antibody targets, clinically isolated MS, primary progressive MS, secondary progressive MS, relapsing and remitting MS, T cells, T regulatory cells

  2. Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M. (National Inst. for Biological Standards and Control, London (UK))

    1982-10-01

    An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys.

  3. High-Throughput Tools for Characterization of Antibody Epitopes

    DEFF Research Database (Denmark)

    Christiansen, Anders

    Antibodies are molecules of tremendous importance. In their primary role, they protect our bodies against disease. However, in recent decades, scientists have harnessed the binding capabilities of antibodies and have applied them widely in research, diagnostics and therapeutics. Consequently, it ...

  4. Antibody avidity in swine lymphocyte antigen-defined miniature pigs.

    Science.gov (United States)

    Appleyard, G D; Mallard, B A; Kennedy, B W; Wilkie, B N

    1992-01-01

    Antibody avidity to hen egg white lysozyme (HEWL) was measured by thiocyanate ion elution enzyme-linked immunosorbent assay (ELISA) in swine lymphocyte antigen (SLA) defined miniature pigs. Serum antibody avidity was evaluated on day 14 and 30 after primary (day 0) and secondary (day 14) immunizations in eight to ten week old miniature pigs previously typed for swine lymphocyte antigen genotype. The effect of SLA genotype, litter, and gender on anti-HEWL antibody avidity was determined by least squares. Antibody avidity varied amongst individuals. Antibody avidity maturation was observed as a mean rise in antibody avidity from primary response (0.89 +/- 0.64) to secondary response (1.23 +/- 0.54) (p < 0.0005). Overall, SLA genotype did not significantly influence antibody avidity or avidity maturation, but pigs of dd genotype had greater avidity maturation between primary and secondary responses than other genotypes. Litter effects significantly affected antibody avidity and maturation. PMID:1477799

  5. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

    Science.gov (United States)

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-01-01

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  6. Quality of histone modification antibodies undermines chromatin biology research

    OpenAIRE

    Goran Kungulovski; Albert Jeltsch

    2015-01-01

    Histone post-translational modification (PTM) antibodies are essential research reagents in chromatin biology. However, they suffer from variable properties and insufficient documentation of quality. Antibody manufacturers and vendors should provide detailed lot-specific documentation of quality, rendering further quality checks by end-customers unnecessary. A shift from polyclonal antibodies towards sustainable reagents like monoclonal or recombinant antibodies or histone binding domains wou...

  7. Anti-Cardiolipin Antibody in Acute Myocardial Infarction

    OpenAIRE

    Abdolreza S. Jahromi; Mohammad Shojaie; Samira Dana; Abdoulhossain Madani

    2010-01-01

    Problem statement: Myocardial infarction is the combined result of environmental and personal factors. Data concerning the relation between anti-Phospholipid (aPL) antibodies and myocardial infarction in subjects without evidence of overt autoimmune disease are conflicting. Anticardiolipin antibody is detected in various diseases like rheumatoid arthritis, systemic lupus erythematosus and anti-phospholipid antibody syndrome. The study of Anticardiolipin antibody in Acute Myocardial Infarction...

  8. ANTI-PHOSPHATIDYLSERINE ANTIBODIES IN ACUTE MYOCARDIAL INFARCTION

    OpenAIRE

    Abdolreza Sotoodeh Jahromi; Mohammad Shojaei; Mohammad Reza Farjam; Abdolhossien Madani

    2013-01-01

    Acute Myocardial Infarction (AMI) is the combined result of environmental factors and personal predispositions. Many factors play a role in AMI including anti-Phospholipid (aPL) antibodies, that may act in the induction of immunological response leading to the development of AMI. Anti-Phosphatidylserine (PS) antibody is detected in various diseases like rheumatoid arthritis, systemic lupus erythematosus and anti-phospholipid antibody syndrome. The study of anti-PS antibody in AMI might shed l...

  9. Evaluation of Salivary Antibodies to Detect Infection with Helicobacter pylori

    OpenAIRE

    1997-01-01

    Helicobacter pylori infection is an important cause of peptic ulcer disease and chronic gastritis. Infection with this bacterium stimulates the production of immunoglobulin (Ig) G antibody. Salivary IgG antibody tests to detect H pylori infection offer a convenient and noninvasive method of diagnosis. To evaluate an IgG salivary antibody kit, saliva was collected from 157 out-patients with dyspepsia referred for endoscopy to a tertiary centre. A salivary IgG ELISA antibody assay was performed...

  10. Cell-Free Synthesis Meets Antibody Production: A Review

    OpenAIRE

    Marlitt Stech; Stefan Kubick

    2015-01-01

    Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG) molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv) and antigen binding fragments (Fab), have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufactu...

  11. Production and characterization of antibody against aflatoxin Q1.

    OpenAIRE

    Fan, T. S.; Zhang, G S; Chu, F. S.

    1984-01-01

    Antibodies against aflatoxin Q1 (AFQ1) were obtained from rabbits after immunization of either AFQ1-hemisuccinate or AFQ2a conjugated to bovine serum albumin. Both radioimmunoassay and enzyme-linked immunosorbent assaY (ELISA) were used for the determination of antibody titers and specificities. Antibodies obtained from rabbits after immunization with AFQ1-hemisuccinate-bovine serum albumin had the highest affinity to aflatoxin B1, whereas antibodies obtained from rabbits after immunization w...

  12. Development of syngeneic monoclonal anti-idiotype antibodies to mouse monoclonal anti-asialoglycoprotein receptor antibody.

    Directory of Open Access Journals (Sweden)

    Hirai M

    2002-06-01

    Full Text Available Anti-idiotype antibodies (Ab2 play an important role in the homeostasis of immune responses and are related to the development and the disease activity of certain autoimmune diseases. The asialoglycoprotein receptor (ASGPR is considered one of the target antigens in the pathogenesis of autoimmune chronic active hepatitis (AIH. We previously developed a mouse monoclonal antibody (clone 8D7 which recognizes rat and human ASGPR. In this study, to help investigate the anti-ASGPR antibody-anti-idiotype antibody network in patients with AIH, we developed a syngeneic mouse monoclonal Ab2 to the 8D7 anti-ASGPR antibody (Ab1. One clone, designated as 3C8, tested positive for specific reactivity to 8D7-Ab1 and did not bind to other irrelevant immunoglobulins. By competitive inhibition assays, the binding of 8D7-Ab1 to liver membrane extracts, i.e., the crude antigen preparation, was inhibited by 3C8-Ab2 in a dose-dependent manner, and the binding of 8D7-Ab1 to 3C8-Ab2 was inhibited by the liver membrane extracts. In the immunohistochemical analysis, 3C8-Ab2 blocked the specific staining of sinusoidal margins of rat hepatocytes by 8D7-Ab1. These results suggest that 3C8 anti-idiotype antibody recognizes the specific idiotypic determinants within the antigen-binding site of 8D7-Ab1.

  13. Development of monoclonal antibodies suitable for rabies virus antibody and antigen detection.

    Science.gov (United States)

    Chander, Vishal; Singh, R P; Verma, P C

    2012-12-01

    The control of an infectious viral disease as rabies is made easier by rapid and accurate diagnosis. Successful rabies prophylaxis is dependent upon the active immunization with vaccine along with passive administration of rabies virus neutralizing antibodies which together clear the virus before widespread infection of central nervous system occurs. The present study aimed at the development of monoclonal antibodies (MAbs) suitable for rabies virus antibody and antigen detection. For the production of rabies specific MAbs, immunization of Swiss albino mice with a commercially available vaccine was done and Polyethylene glycol mediated fusion of spleenocytes with myeloma cells was performed. The positive clones were selected on the basis of distinct reactivity by cell Enzyme linked immunosorbent assay and fluorescence in Indirect Fluorescent antibody test. The positive clones obtained were subjected to single cell cloning by limiting dilution method. The reactive clones were further titrated and employed for virus titration and virus neutralization. The neutralizing activity was evaluated using Fluorescence Activated Cell Sorter technique. Three MAb clones showed a distinct percent inhibition in the presence of positive serum. One of the MAb clone No. 5C3 was relatively more specific in detecting rabies antibodies and also found suitable for competitive ELISA to assess the antibody level in vaccinated subjects.

  14. Development of monoclonal antibodies suitable for rabies virus antibody and antigen detection.

    Science.gov (United States)

    Chander, Vishal; Singh, R P; Verma, P C

    2012-12-01

    The control of an infectious viral disease as rabies is made easier by rapid and accurate diagnosis. Successful rabies prophylaxis is dependent upon the active immunization with vaccine along with passive administration of rabies virus neutralizing antibodies which together clear the virus before widespread infection of central nervous system occurs. The present study aimed at the development of monoclonal antibodies (MAbs) suitable for rabies virus antibody and antigen detection. For the production of rabies specific MAbs, immunization of Swiss albino mice with a commercially available vaccine was done and Polyethylene glycol mediated fusion of spleenocytes with myeloma cells was performed. The positive clones were selected on the basis of distinct reactivity by cell Enzyme linked immunosorbent assay and fluorescence in Indirect Fluorescent antibody test. The positive clones obtained were subjected to single cell cloning by limiting dilution method. The reactive clones were further titrated and employed for virus titration and virus neutralization. The neutralizing activity was evaluated using Fluorescence Activated Cell Sorter technique. Three MAb clones showed a distinct percent inhibition in the presence of positive serum. One of the MAb clone No. 5C3 was relatively more specific in detecting rabies antibodies and also found suitable for competitive ELISA to assess the antibody level in vaccinated subjects. PMID:24293819

  15. Detection of antitrophoblast antibodies in the sera of patients with anticardiolipin antibodies and fetal loss.

    Science.gov (United States)

    McCrae, K R; DeMichele, A M; Pandhi, P; Balsai, M J; Samuels, P; Graham, C; Lala, P K; Cines, D B

    1993-11-01

    Women with anticardiolipin antibodies (ACLA) are at increased risk for fetal loss. One potential explanation for this outcome is that sera from these individuals contain antibodies reactive with trophoblast cells, which are involved in the establishment of the uteroplacental vasculature and maintenance of placental blood fluidity. To examine this hypothesis, we compared the incidence of trophoblast-reactive antibodies in 27 patients with ACLA and a history of fetal loss with that in 29 normal pregnant women. Sera from 20 patients, but only one control, contained trophoblast-reactive antibodies (P trophoblast cells. In most cases, sera from which ACLA were adsorbed by cardiolipin-containing liposomes maintained reactivity against cells. In addition, patient Ig fractions immunoprecipitated an approximately 62-kD protein from the trophoblast cell surface, stimulated the release of arachidonic acid and thromboxane A2 by trophoblasts, and inhibited the binding of prourokinase to trophoblast urokinase receptors. These observations show that sera from women with ACLA and a history of fetal loss contain antitrophoblast antibodies. These antibodies may be serologically distinct from ACLA, and may contribute to the pathogenesis of fetal demise. PMID:7693045

  16. Behavioral and Psychological Responses to HIV Antibody Testing.

    Science.gov (United States)

    Jacobsen, Paul B.; And Others

    1990-01-01

    Considers effects of informing individuals of their antibody status as determined by human immunodeficiency virus (HIV) antibody testing. Reviews research examining changes in psychological distress and in behaviors associated with HIV infections among individuals who have undergone antibody testing. Identifies methodological issues in studying…

  17. Donor non-specific MICA antibodies in renal transplant recipients.

    Science.gov (United States)

    Sapák, Michal; Chreňová, Silvia; Tirpáková, Jana; Žilinská, Zuzana; Ďurmanová, Vladimíra; Shawkatová, Ivana; Jakuš, Vladimír; Kuba, Daniel; Buc, Milan

    2014-02-01

    Despite recent advances in solid organ transplantations, an antibody mediated rejection caused by donor specific antibodies is still a major problem in kidney graft survival. Besides HLA-induced humoral response, antibodies against MICA antigens have recently attracted attention because of their possible role in graft rejection. The aim of our study was to establish whether renal recipients produce antibodies against MICA molecules due to the transplantation and if they are specific for MICA antigens of the donors. MICA antibody screening was performed in 124 kidney recipient sera. 22 sera, that were found to be MICA antibody positive, were further examined for MICA antibody profiles and compared with donor MICA alleles. The analysis of MICA antibody positive sera showed mostly more complex reactivity patterns. A significant fraction of patient sera (59%) reacted not only with the donor MICA antigens, but also with other MICA patterns. A match between antibody specificities and MICA antigens was observed in 41% of renal recipients only. On the other hand, as much as in 36% of recipient sera were detected antibodies against their own MICA molecules. We did not prove a complete correlation between the recipient MICA antibody specificities and MICA antigens of the donor. We assume that MICA antibody induction occurs not only due to the allogeneic stimulation itself but also due to other factors that need to be elucidated.

  18. Production and characterization of monoclonal antibodies against mink leukocytes

    DEFF Research Database (Denmark)

    Chen, W.S.; Pedersen, Mikael; Gram-Nielsen, S.;

    1997-01-01

    Three monoclonal antibodies (mAbs) were generated against mink leukocytes. One antibody reacted with all T lymphocytes, one with all monocytes and one had platelet reactivity. Under reducing conditions, the T lymphocyte reactive antibody immunoprecipitated 18 kDa, 23 kDa, 25 kDa and 32-40 kDa pol...

  19. Necrotizing and Crescentic Lupus Nephritis with Antineutrophil Cytoplasmic Antibody Seropositivity

    OpenAIRE

    Nasr, Samih H.; D'Agati, Vivette D; Park, Hye-Ran; Sterman, Paul L.; Goyzueta, Juan D.; Dressler, Robert M.; Hazlett, Shawn M.; Pursell, Robert N.; Caputo, Christopher; Markowitz, Glen S.

    2008-01-01

    Background and objectives: Lupus nephritis is a classic immune complex glomerulonephritis. In contrast, antineutrophil cytoplasmic antibodies are associated with necrotizing and crescentic glomerulonephritis, in the absence of significant immune deposits. Antineutrophil cytoplasmic antibodies are detected by indirect immunofluorescence in 20% of patients with systemic lupus erythematosus. We report 10 cases of necrotizing and crescentic lupus nephritis with antineutrophil cytoplasmic antibody...

  20. Antibody Based Surgical Imaging and Photodynamic Therapy for Cancer

    NARCIS (Netherlands)

    de Boer, Esther

    2016-01-01

    In 1944 Albert Coons was the first to show that a fluorescent molecule could be conjugated directly to an antibody made against a target site of interest. This binding does not affect antibody specificity so that labeled antibodies can be used to visualize the location and distribution of the target

  1. Serum Antibody Response to Clostridium botulinum Toxin in Infant Botulism

    OpenAIRE

    Rubin, Lorry G.; Dezfulian, Manuchehr; Yolken, Robert H.

    1982-01-01

    A serum antibody response has not been previously demonstrated after infection with Clostridium botulinum. We developed an enzyme immunoassay for measuring serum antibody to C. botulinum toxins A, B, and E. This assay system detected a specific immunoglobulin G and immunoglobulin M antibody response to C. botulinum toxin in two patients with infant botulism.

  2. Assay for the specificity of monoclonal antibodies in crossed immunoelectrophoresis

    DEFF Research Database (Denmark)

    Skjødt, K; Schou, C; Koch, C

    1984-01-01

    A method is described based on crossed immunoelectrophoresis of a complex antigen mixture in agarose gel followed by incubation of the gel with the monoclonal antibody. The bound monoclonal antibody is detected by the use of a secondary enzyme-labelled antibody. Using this technique we have been ...

  3. Stability of llama heavy chain antibody fragments under extreme conditions

    NARCIS (Netherlands)

    Dolk, E.

    2004-01-01

    Camelids have next to their normal antibodies, a unique subset of antibodies lacking light chains. The resulting single binding domain, VHH, of these heavy chain antibodies consequently have unique properties. A high stability is one of these properties, which was investigated in this thesis. The a

  4. [Reactivity of antibodies to collagen types I to IV and antibodies to chondroitin sulfate in the spleen].

    Science.gov (United States)

    Galbavý, S; Ruzicková, M; Surmíková, E; Danihel, L; Porubský, J; Papincák, J; Holesa, S; Trnka, J

    1996-02-01

    Antibodies to collagen type I and III reacted negatively, antibodies to collagen type IV positively with reticulin, trabeculae and circumferent reticulum of lymphatic sheaths, poorly positively with capsula, strongly positively with subcapsular zone. Antibodies to collagen type II reacted positively with capsula, poorly with subcapsular zone, strongly with sinus wall and poorly with trabeculae. They did not react with circumferent reticulum of periarterial lymphoid sheaths. Antibodies to collagen type II and IV reacted positively with central arteries. Antibodies to chondroitinsulphate C reacted poorly and antibodies to chondroitinsulphate B strongly positively with sinus walls and oval cells spread in the white and red pulpa. Antibodies to chondroitin sulphate A reacted similarly as antibodies to chondroitinsulphate B. PMID:9560890

  5. A monoclonal antibody toolkit for C. elegans.

    Directory of Open Access Journals (Sweden)

    Gayla Hadwiger

    Full Text Available BACKGROUND: Antibodies are critical tools in many avenues of biological research. Though antibodies can be produced in the research laboratory setting, most research labs working with vertebrates avail themselves of the wide array of commercially available reagents. By contrast, few such reagents are available for work with model organisms. METHODOLOGY/PRINCIPAL FINDINGS: We report the production of monoclonal antibodies directed against a wide range of proteins that label specific subcellular and cellular components, and macromolecular complexes. Antibodies were made to synaptobrevin (SNB-1, a component of synaptic vesicles; to Rim (UNC-10, a protein localized to synaptic active zones; to transforming acidic coiled-coil protein (TAC-1, a component of centrosomes; to CENP-C (HCP-4, which in worms labels the entire length of their holocentric chromosomes; to ORC2 (ORC-2, a subunit of the DNA origin replication complex; to the nucleolar phosphoprotein NOPP140 (DAO-5; to the nuclear envelope protein lamin (LMN-1; to EHD1 (RME-1 a marker for recycling endosomes; to caveolin (CAV-1, a marker for caveolae; to the cytochrome P450 (CYP-33E1, a resident of the endoplasmic reticulum; to beta-1,3-glucuronyltransferase (SQV-8 that labels the Golgi; to a chaperonin (HSP-60 targeted to mitochondria; to LAMP (LMP-1, a resident protein of lysosomes; to the alpha subunit of the 20S subcomplex (PAS-7 of the 26S proteasome; to dynamin (DYN-1 and to the alpha-subunit of the adaptor complex 2 (APA-2 as markers for sites of clathrin-mediated endocytosis; to the MAGUK, protein disks large (DLG-1 and cadherin (HMR-1, both of which label adherens junctions; to a cytoskeletal linker of the ezrin-radixin-moesin family (ERM-1, which localized to apical membranes; to an ERBIN family protein (LET-413 which localizes to the basolateral membrane of epithelial cells and to an adhesion molecule (SAX-7 which localizes to the plasma membrane at cell-cell contacts. In addition to

  6. Comparisons of the effect of naturally acquired maternal pertussis antibodies and antenatal vaccination induced maternal tetanus antibodies on infant's antibody secreting lymphocyte responses and circulating plasma antibody levels.

    Science.gov (United States)

    Ahmad, Shaikh Meshbahuddin; Alam, Jahangir; Afsar, Nure Alam; Huda, Nazmul; Kabir, Yearul; Qadri, Firdausi; Raqib, Rubhana; Stephensen, Charles B

    2016-04-01

    The goal of this study was to explore the effects of trans-placental tetanus toxoid (TT) and pertussis (PT) antibodies on an infant's response to vaccination in the context of antenatal immunization with tetanus but not with pertussis. 38 mothers received a single dose of TT vaccine during pregnancy. Infants received tetanus and pertussis vaccines at 6, 10 and 14 wk of age. TT and PT anti-IgG secretion by infant lymphocytes was measured at 15 wk. Plasma antibodies were measured at 6 wk (pre-vaccination), 15 wk and 1 y of age. Prior to vaccination, TT and PT antibody were detected in 94.6% and 15.2% of infants. At 15 wk anti-TT-IgG and anti-PT-IgG in plasma was increased by 7-9 fold over pre-vaccination levels, while at 1 y plasma anti-TT-IgG was decreased by approximately 5-fold from the peak and had returned to near the pre-vaccination level. At 1 y plasma anti-PT-IgG was decreased by 2-fold 1 yfrom the 15 wk level. However, 89.5% and 82.3% of infants at 1 y had protective levels of anti-TT and anti-PT IgG, respectively. Pre-vaccination plasma IgG levels were associated with lower vaccine-specific IgG secretion by infant lymphocytes at 15 wk (p < 0.10). This apparent inhibition was seen for anti-TT-IgG at both 15 wk (p < 0.05) and t 1 y (p < 0.10) of age. In summary, we report an apparent inhibitory effect of passively derived maternal antibody on an infants' own antibody response to the same vaccine. However, since the cut-off values for protective titers are low, infants had protective antibody levels throughout infancy. PMID:27176823

  7. Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

    OpenAIRE

    Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús

    2013-01-01

    High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel ...

  8. Transfusion management of patients with red blood cell antibodies

    Directory of Open Access Journals (Sweden)

    Bujandrić Nevenka B.

    2013-01-01

    Full Text Available Introduction. Red blood cell antibodies may cause a positive result of pre-transfusion blood compatibility testing (crossmatch test. It can be a problem to provide suitable blood units for patients with clinically significant antibodies to high-frequency antigens as well as for those with multiple alloantibody specificities. This study was aimed at identifying transfused patients in the population of South-Backa who had developed clinically significant red blood cell alloantibodies. Material and methods. We analyzed the records of crossmatch results and antibody screening performed at the Blood Transfusion Institute of Vojvodina during 2012. Results. Antibodies were found in 103 patients: A 63 patients with single antibodies: 1 16 with antibodies of unknown specificity (3 autoantibodies, 13 alloantibodies; 2 39 with clinically significant antibodies (23 from Rh system (2 anti-C, 2 anti-D, 12 anti-E, 7 anti-c, 4 anti-K, 3 anti-Fya, 7 anti-Jka, 2 anti-S; 3 8 with usually not significant antibodies (6 anti-M, 1 anti-A1, 1 anti- Cw; B 40 patients developed multiple antibodies: 1 all patients had at least one clinically significant antibody from various blood group system (44 Rh, 13 Kell, 7 Kidd, 7 MNSs (S, s; 2 3 patients had usually not significant antibodies (1 Lewis, 2 Lutheran; 3 3 patients occasionally had clinically significant antibody (3 anti- Yta; 4 3 patients had antibodies of unknown specificity (2 autoantibodies, 1alloantibody. Antibodies detected in the majority of patients (65-63.1% had a specificity of Rh and/or the Kell system. Conclusions. The main goal of pre-transfusion blood compatibility testing is to detect clinically significant antibodies. The provision of antigen negative blood units for those patients is a special challenge for blood establishments. Database with a sufficient number of typed blood donors can help to resolve this problem.

  9. A MONOCLONAL-ANTIBODY AGAINST HUMAN BETA-GLUCURONIDASE FOR APPLICATION IN ANTIBODY-DIRECTED ENZYME PRODRUG THERAPY

    NARCIS (Netherlands)

    Haisma, Hidde; VANMUIJEN, M; SCHEFFER, G; SCHEPER, RJ; PINEDO, HM; BOVEN, E

    1995-01-01

    The selectivity of anticancer agents may be improved by antibody-directed enzyme prodrug therapy (ADEPT), The immunogenicity of antibody-enzyme conjugates and the low tumor to normal tissue ratio calls for the use of a human enzyme and the development of a monoclonal antibody (MAb) against that enzy

  10. High throughput screening for antibody induced complement-dependent cytotoxicity in early antibody discovery using homogeneous macroconfocal fluorescence imaging

    NARCIS (Netherlands)

    Gerritsen, Arnout F.; Bosch, Martijn; de Weers, Michel; van de Winkel, Jan G. J.; Parren, Paul W. H. I.

    2010-01-01

    Complement-dependent cytotoxicity (CDC) represents an important Fc-mediated effector function of antibodies and is a quality often sought in candidates for therapeutic antibody development in cancer. Antibodies inducing potent CDC are relatively rare as the ability to induce CDC is strongly dependen

  11. Molecular aspects of antibody-antigen interactions : size reduction of a herpes simplex virus neutralizing antibody and its antigen

    NARCIS (Netherlands)

    Schellekens, Gerardus Antonius

    1996-01-01

    Antibody molecules, produced as a response against foreign substances, interact with their antigen in a very specific manner. Antibodies with a predetermined specificity (monoclonal antibodies) can be produced and are widely used in medicine and science as indicator molecules. Genetic engineering of

  12. Monoclonal antibodies against rabbit mammary prolactin receptors. Specific antibodies to the hormone binding domain

    International Nuclear Information System (INIS)

    Three monoclonal antibodies (M110, A82, and A917) were obtained by fusing myeloma cells and spleen cells from mice immunized with partially purified rabbit mammary gland prolactin (PRL) receptors. All 3 antibodies were capable of complete inhibition of 125I-ovine prolactin (oPRL) binding to rabbit mammary PRL receptors in either particulate or soluble form. M110 showed slightly greater potency than oPRL in competing for 125I-oPRL binding. These antibodies also inhibited PRL binding to microsomal fractions from rabbit liver, kidney, adrenal, ovary, and pig mammary gland, although A82 showed poor inhibition in pig mammary gland. There was no cross-reaction of any of the 3 monoclonal antibodies (mAbs) for the other species tested: human (T-47D breast cancer cells) and rat (liver, ovary). In order to confirm that these antibodies are specific to the binding domain, antibodies were purified, iodinated, and binding characteristics were investigated. 125I-M110 and 125I-A82 binding was completely inhibited by lactogenic hormones, whereas nonlactogenic hormones did not cross-react. Competition of 125I-M110 by oPRL was comparable to that of 125I-oPRL by unlabeled oPRL, while 125I-A917 binding was only partially competed (30-60%) by lactogenic hormones. Tissue and species specificity of labeled antibody binding paralleled results of binding inhibition experiments using 125I-oPRL. In addition, A82 and A917 completely inhibited 125I-M110 binding. In contrast, 125I-A82 binding was stimulated by A917 and 125I-A917 binding was stimulated by A82

  13. ON THE NOTION OF SYNERGY OF MONOCLONAL ANTIBODIES AS DRUGS

    Directory of Open Access Journals (Sweden)

    Michael Sela

    2013-08-01

    Full Text Available History of developing synergy between monoclonal antibodies, anti-tumor activity of monoclonal antibodies against tyrosine-kinases receptors EGFR/ErbB-1 and HER2/ErbB-2 as well as growth factor VEGF in various combinations are considered in the article. There were proposed hypotheses about potential molecular mechanisms underlay synergy between monoclonal antibodies (for homo- and hetero combinations of antibodies appropriately specific for antigenic determinants on the same or different receptors. Future trends in researches necessary to deeper understanding causes of this phenomenon and perspectives for practical application of monoclonal antibodies acted synergistically as immunotherapeutic drugs for human tumors treatment are reviewed.

  14. Antissaliva Antibodies of Lutzomyia Longipalpis in area of Visceral Leishmaniasis.

    Science.gov (United States)

    Fraga, Thiago Leite; Fernandes, Magda Freitas; Pontes, Elenir Rose Jardim Cury; Levay, Ana Paula Silva; Almeida da Cunha, Elenice Brandão; França, Adriana de Oliveira; Dorval, Maria Elizabeth Cavalheiros

    2016-07-01

    The aim of the present study was to assess the presence of antissaliva antibodies of Lutzomyia longipalpis in human hosts living in area of visceral leishmaniasis, located in the Center-West region of Brazil. The presence of antissaliva antibodies of L. longipalpis exhibited a strong correlation with the protection and development of antibodies against Leishmania sp. Of the 492 children studied, elevated antissaliva antibodies of L. longipalpis were detected in 38.4% of the participants. There was a higher percentage of positivity (64.7%) among children who exhibited anti-Leishmania sp. antibodies and among those who were positive in the delayed hypersensitivity test (34.8%). PMID:27093167

  15. Antissaliva Antibodies of Lutzomyia Longipalpis in area of Visceral Leishmaniasis.

    Science.gov (United States)

    Fraga, Thiago Leite; Fernandes, Magda Freitas; Pontes, Elenir Rose Jardim Cury; Levay, Ana Paula Silva; Almeida da Cunha, Elenice Brandão; França, Adriana de Oliveira; Dorval, Maria Elizabeth Cavalheiros

    2016-07-01

    The aim of the present study was to assess the presence of antissaliva antibodies of Lutzomyia longipalpis in human hosts living in area of visceral leishmaniasis, located in the Center-West region of Brazil. The presence of antissaliva antibodies of L. longipalpis exhibited a strong correlation with the protection and development of antibodies against Leishmania sp. Of the 492 children studied, elevated antissaliva antibodies of L. longipalpis were detected in 38.4% of the participants. There was a higher percentage of positivity (64.7%) among children who exhibited anti-Leishmania sp. antibodies and among those who were positive in the delayed hypersensitivity test (34.8%).

  16. Antiphospholipid antibody syndrome presenting as transverse myelitis

    Directory of Open Access Journals (Sweden)

    Javvid M Dandroo

    2015-01-01

    Full Text Available The antiphospholipid syndrome (APS is characterized by arterial and/or venous thrombosis and pregnancy morbidity in the presence of anticardiolipin antibodies and/or lupus anticoagulant. APS can occur either as a primary disorder or secondary to a connective tissue disease, most frequently systemic lupus erythematosus. Central nervous system involvement is one of the most prominent clinical manifestations of APS, and includes arterial and venous thrombotic events, psychiatric features, and a variety of other nonthrombotic neurological syndromes. Although the mechanism of neurological involvement in patients with APS is thought to be thrombotic in origin and endothelial dysfunction associated with antiphospholipid antibodies. APS presenting as acute transverse myelitis is very rarely seen with a prevalence rate of 1%. We are describing a foreigner female presenting as acute transverse myelitis which on evaluation proved to be APS induced. So far, very few cases have been reported in literature with APS as etiology.

  17. Monoclonal Antibodies to Plant Growth Regulators

    Science.gov (United States)

    Eberle, Joachim; Arnscheidt, Angelika; Klix, Dieter; Weiler, Elmar W.

    1986-01-01

    Four high affinity monoclonal antibodies, which recognize two plant growth regulators from the cytokinin group, namely trans-zeatin riboside and dihydrozeatin riboside and their derivatives are reported. Six hybridomas were produced from three independent fusions of Balb/c spleen cells with P3-NS1-Ag 4-1 (abbreviated NS1) or X63-Ag 8.653 (X63) myeloma cells. The mice had been hyperimmunized with zeatin riboside-bovine serum albumin conjugate or dihydrozeatin riboside-bovine serum albumin conjugate for 3 months. The hybridomas secrete antibodies of the IgG 1 or IgG 2b subclass and allow the detection of femtomole amounts of the free cytokinins, their ribosides, and ribotides in plant extracts. The use of these monoclonals in radio- and enzyme-linked immunosorbent assay is also discussed. PMID:16664848

  18. Poliarterite nodosa due to anti elastase antibody

    Directory of Open Access Journals (Sweden)

    Caterina Defendenti

    2008-09-01

    Full Text Available The Authors related one case of polyarteritis nodosa occurred to a men forty eight years old.The clinical was characterized by mesenteric and femoral arteries occlusion and chronic cutaneous ulcers to legs. There were bioptical aspects of systemic vasculitis with necrotizing inflammation and a paucity of immune deposit. It was effective oral cyclophosphamide plus steroids. This disease was closely associated with antibodies anti elastase (HLE.The patient had not a history of cocaine abuse or LES disease but the nucleolar pattern ANA was positive >1:640 (anti-nDNA negative. Similar case ANA positive associated with the anti-elastase antibodies, was described by Nassberger (Lancet 1989 for 6/104 patients with LES, anti-nDNA negative. The patient with the highest anti-elastase concentration subsequentely died after very rapid development of severe brain and kidney involvement.

  19. Hepatitis C Virus Antibodies and Vitiligo Disease

    Directory of Open Access Journals (Sweden)

    Z Jadali

    2005-06-01

    Full Text Available Vitiligo is a common skin disorder, characterized by depigmented patches due to selective destruction of melanocytes. The etiology of this disease is unknown. A number of hypotheses including viral theory have been proposed to explain the etiology. To determine the prevalence of antibody to hepatitis C virus infection in vitiligo patients, the present study was performed. Third generation ELISA test was used for detection of antibodies to HCV in human sera. All normal controls were anti-HCV negative whereas only one patient was positive for anti-HCV and there was no significant difference in the prevalence of anti-HCV between patients and controls. These results indicate that hepatitis C virus has not a direct causal role in the pathogenesis of vitiligo, however, this does not rul out a "hit and run" virus induced disease.

  20. Recent developments in monoclonal antibody radiolabeling techniques

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.; Mease, R.C.

    1989-01-01

    Monoclonal antibodies (MAbs) have shown the potential to serve as selective carriers of radionuclides to specific in vivo antigens. Accordingly, there has been an intense surge of research activity in an effort to develop and evaluate MAb-based radiopharmaceuticals for tumor imaging (radioimmunoscintigraphy) and therapy (radioimmunotherapy), as well as for diagnosing nonmalignant diseases. A number of problems have recently been identified, related to the MAbs themselves and to radiolabeling techniques, that comprise both the selectivity and the specificity of the in vivo distribution of radiolabeled MAbs. This paper will address some of these issues and primarily discuss recent developments in the techniques for radiolabeling monoclonal antibodies that may help resolve problems related to the poor in vivo stability of the radiolabel and may thus produce improved biodistribution. Even though many issues are identical with therapeutic radionuclides, the discussion will focus mainly on radioimmunoscintigraphic labels. 78 refs., 6 tabs.

  1. [Biophysical Characterization of Biopharmaceuticals, Including Antibody Drugs].

    Science.gov (United States)

    Uchiyama, Susumu

    2016-01-01

    Biopharmaceuticals, including antibody drugs, are now popular because of their high specificity with low adverse effects, especially in the treatment of cancer and autoimmune diseases. However, because the active pharmaceutical ingredients of biopharmaceuticals are proteins, biophysical characterization of these therapeutic proteins should be required. In this manuscript, methods of chemical and physical characterization of therapeutic proteins are described. In terms of chemical characterization, analysis of chemical modifications of the constituent amino acids is explained. Physical characterization includes higher order structural analysis and assessment of protein aggregates. Quantification methods of aggregates with different sizes, recently encouraged by the U.S. Food and Drug Administration (FDA), are introduced. As for the stability of therapeutic proteins, the importance of chemical and physical stability is explained. Finally, the contribution of colloidal and structural stability to the production of an antibody drug less prone to aggregation is introduced.

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

    OpenAIRE

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

    1987-01-01

    The effect of repeated interactions of antibodies with cell surface antigens have been examined in in vitro, but not in in vivo systems. In this study are described the results of multiple antibody-cell surface antigen interactions in vivo. Rabbits were given repeated intravenous injections of goat antibodies to angiotensin converting enzyme (ACE), an antigen expressed on the surface of lung endothelial cells. For prevention of anaphylactic reactions, which would have been induced by multiple...

  3. Technological progresses in monoclonal antibody production systems

    OpenAIRE

    Rodrigues, E.; Costa, A R; Henriques, Mariana; Azeredo, Joana; Oliveira, Rosário

    2009-01-01

    Monoclonal antibodies (mAbs) have become vitally important to modern medicine and are currently one of the major biopharmaceutical products in development. However, the high clinical dose requirements of mAbs demand a greater biomanufacturing capacity, leading to the development of new technologies for their large-scale production, with mammalian cell culture dominating the scenario. Although some companies have tried to meet these demands by creating bioreactors of increased capacity, the op...

  4. Antibody conjugate radioimmunotherapy of superficial bladder cancer

    Directory of Open Access Journals (Sweden)

    Alan Perkins

    2002-09-01

    Full Text Available The administration of antibody conjugates for cancer therapy is now proving to be of clinical value. We are currently undertaking a programme of clinical studies using the monoclonal antibody C595 (IgG3 which reacts with the MUC1 glycoprotein antigen that is aberrantly expressed in a high proportion of bladder tumours. Radioimmunoconjugates of the C595 antibody have been produced with high radiolabelling efficiency and immunoreactivity using Tc-99m and In-111 for diagnostic imaging, and disease staging and the cytotoxic radionuclides Cu-67 and Re-188 for therapy of superficial bladder cancer. A Phase I/II therapeutic trail involving the intravesical administration of antibody directly into the bladder has now begun.A administração de anticorpos conjugados para o tratamento do câncer está agora provando ser de valor clínico. Nós estamos atualmente realizando um programa de estudos clínicos usando o anticorpo monoclonal C595 (IgG3 que reage com a glicoproteína MUC1 que está aberrantemente expressa numa alta proporção de tumores de bexiga. Tem sido produzidos radioimunoconjugados do anticorpo C595, com alta eficiência de radiomarcação e a imunoreatividade, usando-se o Tc-99m e In-111, para o diagnóstico por imagem e estagiamento de doenças. Tem sido produzidos, também, radionuclídeos citotóxicos (Cu-67 e Re-188 para o tratamento de cânceres superficiais de bexiga. A fase terapêutica I/II já se iniciou, envolvendo a administração intravesical do anticorpo diretamente na bexiga.

  5. Feature Selection Approaches In Antibody Display

    OpenAIRE

    Polaka, Inese

    2015-01-01

    Molecular diagnostics tools provide specific data that have high dimensionality due to many factors analyzed in one experiment and few records due to high costs of the experiments. This study addresses the problem of dimensionality in melanoma patient antibody display data by applying data mining feature selection techniques. The article describes feature selection ranking and subset selection approaches and analyzes the performance of various methods evaluating selected feature subsets using...

  6. Antibody induction therapy for lung transplant recipients

    DEFF Research Database (Denmark)

    Penninga, Luit; Møller, Christian H; Penninga, Ida Elisabeth Irene;

    2013-01-01

    Lung transplantation has become a valuable and well-accepted treatment option for most end-stage lung diseases. Lung transplant recipients are at risk of transplanted organ rejection, and life-long immunosuppression is necessary. Clear evidence is essential to identify an optimal, safe...... and effective immunosuppressive treatment strategy for lung transplant recipients. Consensus has not yet been achieved concerning use of immunosuppressive antibodies against T-cells for induction following lung transplantation....

  7. Antibody-Catalyzed Degradation of Cocaine

    Science.gov (United States)

    Landry, Donald W.; Zhao, Kang; Yang, Ginger X.-Q.; Glickman, Michael; Georgiadis, Taxiarchis M.

    1993-03-01

    Immunization with a phosphonate monoester transition-state analog of cocaine provided monoclonal antibodies capable of catalyzing the hydrolysis of the cocaine benzoyl ester group. An assay for the degradation of radiolabeled cocaine identified active enzymes. Benzoyl esterolysis yields ecgonine methyl ester and benzoic acid, fragments devoid of cocaine's stimulant activity. Passive immunization with such an artificial enzyme could provide a treatment for dependence by blunting reinforcement.

  8. Myopathy with anti-HMGCR antibodies

    OpenAIRE

    Alshehri, Ali; Choksi, Rati; Bucelli, Robert; Pestronk, Alan

    2015-01-01

    Objective: To analyze clinical features and myopathology changes in muscle fibers, connective tissue, and vessels in 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) antibody–associated myopathies. Methods: Retrospective review of records and myopathologic features of 49 consecutive patients with myopathies and serum HMGCR antibodies. Results: Clinical features included onset age from 12 to 83 years, female predominance (67%), proximal, symmetric weakness (84%), muscle discomfort (78%)...

  9. Sequencing antibody repertoires: The next generation

    OpenAIRE

    Fischer, Nicolas

    2011-01-01

    Genomic studies have been revolutionized by the use of next generation sequencing (NGS), which delivers huge amounts of sequence information in a short span of time. The number of applications for NGS is rapidly expanding and significantly transforming many areas of life sciences. The field of antibody research and discovery is no exception. Several recent studies have harnessed the power of NGS for analyzing natural or synthetic immunoglobulin repertoires with unprecedented resolution and ex...

  10. Antibody Discovery via Multiplexed Single Cell Characterization

    OpenAIRE

    Harriman, William D.; Collarini, Ellen J.; Sperinde, Gizette V.; Strandh, Magnus; Fatholahi, Marjan M.; Dutta, April; Lee, Yunji; Mettler, Shelley E.; Keyt, Bruce A.; Ellsworth, Stote L.; Kauvar, Lawrence M.

    2008-01-01

    The secreted immunoglobulin footprint of single hybridoma cells, containing ~10 fg of antibody purified in situ, has been probed for 9 properties concurrently by use of detection labels comprising 280 nm combinatorially colored fluorescent latex beads functionalized with proteins. Specificity of each individual hybridoma cell’s product has thereby been assessed in a primary screen. Varying the density of antigen on beads to modulate the avidity of the interaction between bead and secreted ant...

  11. Seropositivity of Dengue Antibodies during Pregnancy

    OpenAIRE

    Nor Azlin Mohamed Ismail; Wan Elly Rushima Wan Abd Rahim; Sharifah Azura Salleh; Hui-Min Neoh; Rahman Jamal; Muhammad Abdul Jamil

    2014-01-01

    Purpose. Malaysia a dengue endemic country with dengue infections in pregnancy on the rise. The present study was aimed at determining dengue seroprevalence (IgG or IgM) during pregnancy and its neonatal transmission in dengue seropositive women. Methods. Maternal with paired cord blood samples were tested for dengue antibodies (IgG and IgM) using an enzyme-linked immunosorbent assay (ELISA). Maternal age, parity, occupation, ethnic group, and gestational age were recorded. Data on neonatal A...

  12. Rare blood donors with irregular antibodies

    Directory of Open Access Journals (Sweden)

    Krga-Milanović Mirjana

    2013-01-01

    Full Text Available Introduction. Blood groups are inherited biological characteristics that do not change throughout life in healthy people. Blood groups represent antigens found on the surface of red blood cells. Kell blood group system consists of 31 antigens. Kell antigen (K is present in 0.2% of the population (the rare blood group. Cellano antigen is present in more than 99% (the high-frequency antigen. These antigens have a distinct ability to cause an immune response in the people after blood transfusion or pregnancy who, otherwise, did not have them before. Case Report. This paper presents a blood donor with a rare blood group, who was found to have an irregular antibody against red blood cells by indirect antiglobulin test. Further testing determined the specificity of antibody to be anti-Cellano. The detected antibody was found in high titers (1024 with erythrocyte phenotype Kell-Cellano+. The blood donor was found to have a rare blood group KellKell. This donor was excluded from further blood donation. It is difficult to find compatible blood for a person who has developed an antibody to the high-frequency antigen. The donor’s family members were tested and Cellano antigen was detected in her husband and child. A potential blood donor was not found among the family members. There was only one blood donor in the Register of blood donors who was compatible in the ABO and Kell blood group system. Conclusion. For the successful management of blood transfusion it is necessary to establish a unified national register of donors of rare blood groups and cooperate with the International Blood Group Reference Laboratory in Bristol with the database that registers donors of rare blood groups from around the world.

  13. Solid-phase fluoroimmunoassay for treponemal antibody.

    OpenAIRE

    Stevens, R W; Schell, R F

    1982-01-01

    An objective, solid-phase fluoroimmunoassay for treponemal antibody was developed with a lysate of virulent Treponema pallidum (Nichols strain) adsorbed on cellulose acetate disks. A probe containing both the antigen and control disks is inserted successively into a serum specimen dilution, a buffer rinse, fluoroscein isothiocyanate-conjugated goat anti-human immunoglobulin G, and a second buffer rinse. Fluorescence signal units are measured with a fluorometer. To establish test calibration c...

  14. DETECTION OF THE BOVINE VIRAL DIARRHEA ANTIBODIES

    Directory of Open Access Journals (Sweden)

    I. V. Goraichuk

    2013-06-01

    Full Text Available Bovine viral diarrhea is a widespread infection of cattle that has a wide range of clinical symptoms in domestic and wild ruminants. It is a major problem in cattle and causes significant economic losses in the cattle industry. The virus infects bovines of all ages and causes both immunosuppression and reproductive, respiratory and digestive disorders. Persistently infected cattle are the main factor in transmission of the disease between and among herds. Comparative results of antibodies presence received by two methods of enzymoimmunoassay and virus neutralization test are given in the paper. During the work, 1010 samples of blood serum of cattle from three farms in the Kharkiv region were selected and analyzed. Bovine viral diarrhea virus concerning antibodies were found by enzymoimmunoassay in 704 samples (69.7% using commercial kit and in 690 samples (68.3% using in house method. After results clarification by virus neutralization test, bovine viral diarrhea antibodies were found in 712 samples (70.5%. Immunoenzyme analysis is recommended for mass screening of cattle for viral diarrhea occurrence. The results confirm that the sensitivity immunoenzyme analysis satisfies the requirements of the diagnostic methods. Using the neutralization reaction of viruses as the «gold standard» of serological methods, it is appropriate to clarify the results of immunoenzyme analysis. Since the results contain a signi ficant number of false positive results, it is necessary to carry out comprehensive studies using both serological and molecular genetics methods.

  15. Polyclonal Antibody Therapies for Clostridium difficile Infection

    Directory of Open Access Journals (Sweden)

    Michael R. Simon

    2014-10-01

    Full Text Available Clostridium difficile infection has emerged as a growing worldwide health problem. The colitis of Clostridium difficile infection results from the synergistic action of C. difficile secreted toxins A and B upon the colon mucosa. A human monoclonal IgG anti-toxin has demonstrated the ability in combination therapy to reduce mortality in C. difficile challenged hamsters. This antibody is currently in a clinical trial for the treatment of human Clostridium difficile infection. More than one group of investigators has considered using polyclonal bovine colostral antibodies to toxins A and B as an oral passive immunization. A significant proportion of the healthy human population possesses polyclonal antibodies to the Clostridium difficile toxins. We have demonstrated that polyclonal IgA derived from the pooled plasma of healthy donors possesses specificity to toxins A and B and can neutralize these toxins in a cell-based assay. This suggests that secretory IgA prepared from such pooled plasma IgA may be able to be used as an oral treatment for Clostridium difficile infection.

  16. Monoclonal antibodies based on hybridoma technology.

    Science.gov (United States)

    Yagami, Hisanori; Kato, Hiroshi; Tsumoto, Kanta; Tomita, Masahiro

    2013-03-01

    Based on the size and scope of the present global market for medicine, monoclonal antibodies (mAbs) have a very promising future, with applications for cancers through autoimmune ailments to infectious disease. Since mAbs recognize only their target antigens and not other unrelated proteins, pinpoint medical treatment is possible. Global demand is dramatically expanding. Hybridoma technology, which allows production of mAbs directed against antigens of interest is therefore privileged. However, there are some pivotal points for further development to generate therapeutic antibodies. One is selective generation of human mAbs. Employment of transgenic mice producing human antibodies would overcome this problem. Another focus is recognition sites and conformational epitopes in antigens may be just as important as linear epitopes, especially when membrane proteins such as receptors are targeted. Recognition of intact structures is of critical importance for medical purposes. In this review, we describe patent related information for therapeutic mAbs based on hybridoma technology and also discuss new advances in hybridoma technology that facilitate selective production of stereospecific mAbs. PMID:24237029

  17. Serum Antibody Repertoire Profiling Using In Silico Antigen Screen.

    Science.gov (United States)

    Liu, Xinyue; Hu, Qiang; Liu, Song; Tallo, Luke J; Sadzewicz, Lisa; Schettine, Cassandra A; Nikiforov, Mikhail; Klyushnenkova, Elena N; Ionov, Yurij

    2013-01-01

    Serum antibodies are valuable source of information on the health state of an organism. The profiles of serum antibody reactivity can be generated by using a high throughput sequencing of peptide-coding DNA from combinatorial random peptide phage display libraries selected for binding to serum antibodies. Here we demonstrate that the targets of immune response, which are recognized by serum antibodies directed against sequential epitopes, can be identified using the serum antibody repertoire profiles generated by high throughput sequencing. We developed an algorithm to filter the results of the protein database BLAST search for selected peptides to distinguish real antigens recognized by serum antibodies from irrelevant proteins retrieved randomly. When we used this algorithm to analyze serum antibodies from mice immunized with human protein, we were able to identify the protein used for immunizations among the top candidate antigens. When we analyzed human serum sample from the metastatic melanoma patient, the recombinant protein, corresponding to the top candidate from the list generated using the algorithm, was recognized by antibodies from metastatic melanoma serum on the western blot, thus confirming that the method can identify autoantigens recognized by serum antibodies. We demonstrated also that our unbiased method of looking at the repertoire of serum antibodies reveals quantitative information on the epitope composition of the targets of immune response. A method for deciphering information contained in the serum antibody repertoire profiles may help to identify autoantibodies that can be used for diagnosing and monitoring autoimmune diseases or malignancies. PMID:23826227

  18. Monoclonal Antibodies Attached to Carbon Nanotube Transistors for Paclitaxel Detection

    Science.gov (United States)

    Lee, Wonbae; Lau, Calvin; Richardson, Mark; Rajapakse, Arith; Weiss, Gregory; Collins, Philip; UCI, Molecular Biology; Biochemistry Collaboration; UCI, Departments of Physics; Astronomy Collaboration

    Paclitaxel is a naturally-occurring pharmaceutical used in numerous cancer treatments, despite its toxic side effects. Partial inhibition of this toxicity has been demonstrated using weakly interacting monoclonal antibodies (3C6 and 8A10), but accurate monitoring of antibody and paclitaxel concentrations remains challenging. Here, single-molecule studies of the kinetics of antibody-paclitaxel interactions have been performed using single-walled carbon nanotube field-effect transistors. The devices were sensitized with single antibody attachments to record the single-molecule binding dynamics of paclitaxel. This label-free technique recorded a range of dynamic interactions between the antibody and paclitaxel, and it provided sensitive paclitaxel detection for pM to nM concentrations. Measurements with two different antibodies suggest ways of extending this working range and uncovering the mechanistic differences among different antibodies.

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

    Science.gov (United States)

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

    2015-02-26

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

  20. Determination of thyroid antimicrosomal antibodies by solid-phase radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Lacka, K.; Kasowicz, J.; Furmaniak-Wehr, J.; Gembicki, M. (Akademia Medyczna, Poznan (Poland))

    1984-01-01

    Thyroid antimicrosomal antibodies have been detected in patient's serum by immunofluorescence and hemagglutination techniques as well as by radioimmunoassay. The aim was to introduce the solid-phase radioimmunoassay for thyroid antimicrosomal antibodies determination. The microsomal fraction was adsorbed on the walls of polystyrene tubes. In the next stage patient's serum was added. The retention of antibodies bound to microsomes on the tube walls was detected by adding /sup 125/I-protein A. The occurrence of antimicrosomal antibodies in the group of patients with Graves' disease was 88%; in case of myxedema autoantibodies were found in 70%. In patients with chronic thyroiditis antimicrosomal antibodies were present in 90% and in the group of non-toxic goiter the incidence was 25%. In the control group antimicrosomal antibodies were found in 8%. The solid-phase radioimmunoassay for thyroid antimicrosomal antibody determination proved to be a useful, sensitive and reproducible method for clinical studies.

  1. Anticardiolipin antibodies in proliferative diabetic retinopathy: An additional risk factor

    International Nuclear Information System (INIS)

    To report the prevalence of anticardiolipin antibodies in patients with proliferative diabetic retinopathy (PDR) having high-risk criteria (HRC). Diabetic patients having PDR with HRC and diabetics free of retinopathy were compared for the presence of anticardiolipin antibodies. Among the 34 patients, 6 (17.7%) of diabetics having PDR with HRC were positive for anticardiolipin antibodies. There was no significant association of aCL antibodies with sex or type of diabetes. Using Pearson's correlation test, no significant associations of aCL antibodies with duration of diabetes or age of patients were found. All patients who were positive for anticardiolipin antibodies had PDR with HRC. The difference was statistically significant. Presence of anticardiolipin antibodies may represent an additional risk factor for PDR. (author)

  2. Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

    Directory of Open Access Journals (Sweden)

    Sindy Liao-Chan

    Full Text Available Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

  3. The INNs and outs of antibody nonproprietary names.

    Science.gov (United States)

    Jones, Tim D; Carter, Paul J; Plückthun, Andreas; Vásquez, Max; Holgate, Robert G E; Hötzel, Isidro; Popplewell, Andrew G; Parren, Paul W H I; Enzelberger, Markus; Rademaker, Hendrik J; Clark, Michael R; Lowe, David C; Dahiyat, Bassil I; Smith, Victoria; Lambert, John M; Wu, Herren; Reilly, Mary; Haurum, John S; Dübel, Stefan; Huston, James S; Schirrmann, Thomas; Janssen, Richard A J; Steegmaier, Martin; Gross, Jane A; Bradbury, Andrew R M; Burton, Dennis R; Dimitrov, Dimiter S; Chester, Kerry A; Glennie, Martin J; Davies, Julian; Walker, Adam; Martin, Steve; McCafferty, John; Baker, Matthew P

    2016-01-01

    An important step in drug development is the assignment of an International Nonproprietary Name (INN) by the World Health Organization (WHO) that provides healthcare professionals with a unique and universally available designated name to identify each pharmaceutical substance. Monoclonal antibody INNs comprise a -mab suffix preceded by a substem indicating the antibody type, e.g., chimeric (-xi-), humanized (-zu-), or human (-u-). The WHO publishes INN definitions that specify how new monoclonal antibody therapeutics are categorized and adapts the definitions to new technologies. However, rapid progress in antibody technologies has blurred the boundaries between existing antibody categories and created a burgeoning array of new antibody formats. Thus, revising the INN system for antibodies is akin to aiming for a rapidly moving target. The WHO recently revised INN definitions for antibodies now to be based on amino acid sequence identity. These new definitions, however, are critically flawed as they are ambiguous and go against decades of scientific literature. A key concern is the imposition of an arbitrary threshold for identity against human germline antibody variable region sequences. This leads to inconsistent classification of somatically mutated human antibodies, humanized antibodies as well as antibodies derived from semi-synthetic/synthetic libraries and transgenic animals. Such sequence-based classification implies clear functional distinction between categories (e.g., immunogenicity). However, there is no scientific evidence to support this. Dialog between the WHO INN Expert Group and key stakeholders is needed to develop a new INN system for antibodies and to avoid confusion and miscommunication between researchers and clinicians prescribing antibodies. PMID:26716992

  4. 一种新型的重组单链三特异抗体的构建与表达%A New Model of Trispecific Antibody Resulting the Cytotoxicity Directed against Tumor Cells

    Institute of Scientific and Technical Information of China (English)

    宋利萍; 程巨龙; 王祥斌; 张众; 方敏; 周志勇; 黄华樑

    2003-01-01

    Bispecific antibodies (BsAb) with specificity to both tumor cells and CD3 molecule were believed to be promising immunological tools for the therapy with minimal residual diseases by activating cytotoxic T cells. However, without costimulatory molecule CD28, the activated T cells tended to apoptosis. In order to kill tumor cells more efficiently, a recombinant multifunctional single-chain trispecific antibody (scTsAb), which contains anti-ovarian carcinoma (OC) scFv, anti-CD3 scFv and VH domain of anti-CD28 antibody, was constructed and expressed in E. coli BL21 Star strain. The scTsAb showed strong binding avidities to membrane antigen of SK-OV-3 cell, CD3 molecule on Jurkat cell, and recombinant CD28 antigen. It was further demonstrated that this scTsAb could activate peripheral blood T cells to elicit strong cytotoxicity against SK-OV-3 cells. This new type of recombinant scFv antibody set up a new technological platform for T cells based immunotherapy against cancer, especially with the failure on MHC antigen presentation or absence of costimulating signal.%双特异抗体由于其缺乏共刺激信号, 激活的T细胞往往会导致凋亡. 为了更有效地杀伤肿瘤细胞, 构建了抗卵巢癌单链×抗CD3单链×抗CD28单域重组三特异抗体的表达载体. 利用大肠杆菌中的分子伴侣FkpA与三特异抗体共表达, 提高了三特异抗体的在BL21 Star菌中的可溶性原核表达. ELISA 结果显示, 可溶性的重组单链三特异抗体(sTRI)与SK-OV-3 细胞的膜抗原, Jurkat细胞上的CD3分子以及重组CD28抗原均有较强的结合活性. 桥连试验证明该抗体能同时与SK-OV-3细胞和Jurkat细胞结合. 体外杀伤试验表明该抗体能激活外周血T细胞来杀伤肿瘤细胞. 形态学观察也进一步说明该抗体具有良好的结合活性以及体外杀伤活性. 这种新型的重组单链三特异抗体为基于T细胞的癌症免疫治疗建立了一个新的技术平台.

  5. Anticardiolipin antibodies in pathogenesis of infertility

    Directory of Open Access Journals (Sweden)

    Lončar Dragan

    2010-01-01

    Full Text Available Background/Aim. Antiphospholipid syndrome (APS is an autoimmune disorder clinically characterized by arterial or venous thrombosis and/or specific obstetric complications and presence of antiphospholipid antibodies (aPL in the serum. It occurs in 0.3% of pregnant women, while 1% of them have two spontaneous abortions. The aim of this study was to analyze the frequency of biphospholipid antibodies in pregnant women with recurrent spontaneous abortions. Methods. We analyzed 60 pregnant women who had two or more recurrent miscarriages. The control group included 60 healthy pregnant women. We analyzed titres of anticardiolipin (aCL IgG and/or IgM with high titres (> 20 U/mL, lupus anticoagulant (LAC antibodies and anti-beta-2 glycoprotein (b2-GP1 IgG as well as parameters of coagulation status of pregnant women. Results. Analyzing Spearman's rank correlation coefficient in a group of affected patients, we noticed a slightly positive correlation of lupus anticoagulants (LAC with aCL antibodies of both classes, while the correlation with b2GP1 IgG was negative. Both classes of aCL antibodies and antib2GP1 IgG were in a discrete positive correlation with the given variables. In the control group, there was a lack of consistency in correlation of the study variables with LAC-aCl IgG, compared to the affected patients, and there was a standard negative coefficient of correlation with anti-b2GP1 IgG. The correlation ratio of anti-b2GP1 IgG was negative for all studied test parameters. Analysis of hemostatic parameters showed a statistically significant difference in the concentration of fibrinogen (p < 0.01 and thrombocyte count (p < 0.05 between the study and the control group of pregnant women. Lower mean values of fibrinogen (2.90 ± 0.45 g/L and lower thrombocyte count [(179.20 ± 6.00 × 109] were found in the study group of pregnant women with secondary infertility compared to the mean values of fibrinogen (3.60 ± 0.55 g/L and thrombocyte count

  6. Antibody induction versus placebo, no induction, or another type of antibody induction for liver transplant recipients

    DEFF Research Database (Denmark)

    Penninga, Luit; Wettergren, André; Wilson, Colin H;

    2014-01-01

    BACKGROUND: Liver transplantation is an established treatment option for end-stage liver failure. To date, no consensus has been reached on the use of immunosuppressive T-cell antibody induction for preventing rejection after liver transplantation. OBJECTIVES: To assess the benefits and harms...... of immunosuppressive T-cell specific antibody induction compared with placebo, no induction, or another type of T-cell specific antibody induction for prevention of acute rejection in liver transplant recipients. SEARCH METHODS: We searched The Cochrane Hepato-Biliary Group Controlled Trials Register, the Cochrane...... Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, Science Citation Index Expanded, and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) until September 2013. SELECTION CRITERIA: Randomised clinical trials assessing immunosuppression with T...

  7. An efficient method for isolating antibody fragments against small peptides by antibody phage display

    DEFF Research Database (Denmark)

    Duan, Zhi; Siegumfeldt, Henrik

    2010-01-01

    not always bind efficiently to passive adsorption surfaces, and we developed a simple method to quantify the binding capacity of surfaces with the peptides. Background binding (the binding of scFvs to the background matrix) is an obstacle for successful selection, and we evaluated two methods......We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of s1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do...

  8. Platelet stimulation by antifibronectin antibodies requires the Fc region of antibody.

    OpenAIRE

    Holderbaum, D; Culp, L. A.; Bensusan, H B; Gershman, H.

    1982-01-01

    Anti-human fibronectin antibodies produced in a goat or in rabbits stimulate the release of serotonin from washed or gelatin/Sepharose-treated human platelets in a dose-dependent manner. This finding led us to propose that fibronectin on the platelet plasma membrane might serve as a collagen receptor on these cells [Bensusan, H. B., Koh, T. L., Henry, K. G., Murray, B. A. & Culp, L. A. (1978) Proc. Natl. Acad. Sci. USA 75, 5864-5868]. To determine whether direct interaction of the antibody wi...

  9. Antigen-Specific Antibody Glycosylation Is Regulated via Vaccination.

    Science.gov (United States)

    Mahan, Alison E; Jennewein, Madeleine F; Suscovich, Todd; Dionne, Kendall; Tedesco, Jacquelynne; Chung, Amy W; Streeck, Hendrik; Pau, Maria; Schuitemaker, Hanneke; Francis, Don; Fast, Patricia; Laufer, Dagna; Walker, Bruce D; Baden, Lindsey; Barouch, Dan H; Alter, Galit

    2016-03-01

    Antibody effector functions, such as antibody-dependent cellular cytotoxicity, complement deposition, and antibody-dependent phagocytosis, play a critical role in immunity against multiple pathogens, particularly in the absence of neutralizing activity. Two modifications to the IgG constant domain (Fc domain) regulate antibody functionality: changes in antibody subclass and changes in a single N-linked glycan located in the CH2 domain of the IgG Fc. Together, these modifications provide a specific set of instructions to the innate immune system to direct the elimination of antibody-bound antigens. While it is clear that subclass selection is actively regulated during the course of natural infection, it is unclear whether antibody glycosylation can be tuned, in a signal-specific or pathogen-specific manner. Here, we show that antibody glycosylation is determined in an antigen- and pathogen-specific manner during HIV infection. Moreover, while dramatic differences exist in bulk IgG glycosylation among individuals in distinct geographical locations, immunization is able to overcome these differences and elicit antigen-specific antibodies with similar antibody glycosylation patterns. Additionally, distinct vaccine regimens induced different antigen-specific IgG glycosylation profiles, suggesting that antibody glycosylation is not only programmable but can be manipulated via the delivery of distinct inflammatory signals during B cell priming. These data strongly suggest that the immune system naturally drives antibody glycosylation in an antigen-specific manner and highlights a promising means by which next-generation therapeutics and vaccines can harness the antiviral activity of the innate immune system via directed alterations in antibody glycosylation in vivo.  .

  10. Antigen-Specific Antibody Glycosylation Is Regulated via Vaccination.

    Directory of Open Access Journals (Sweden)

    Alison E Mahan

    2016-03-01

    Full Text Available Antibody effector functions, such as antibody-dependent cellular cytotoxicity, complement deposition, and antibody-dependent phagocytosis, play a critical role in immunity against multiple pathogens, particularly in the absence of neutralizing activity. Two modifications to the IgG constant domain (Fc domain regulate antibody functionality: changes in antibody subclass and changes in a single N-linked glycan located in the CH2 domain of the IgG Fc. Together, these modifications provide a specific set of instructions to the innate immune system to direct the elimination of antibody-bound antigens. While it is clear that subclass selection is actively regulated during the course of natural infection, it is unclear whether antibody glycosylation can be tuned, in a signal-specific or pathogen-specific manner. Here, we show that antibody glycosylation is determined in an antigen- and pathogen-specific manner during HIV infection. Moreover, while dramatic differences exist in bulk IgG glycosylation among individuals in distinct geographical locations, immunization is able to overcome these differences and elicit antigen-specific antibodies with similar antibody glycosylation patterns. Additionally, distinct vaccine regimens induced different antigen-specific IgG glycosylation profiles, suggesting that antibody glycosylation is not only programmable but can be manipulated via the delivery of distinct inflammatory signals during B cell priming. These data strongly suggest that the immune system naturally drives antibody glycosylation in an antigen-specific manner and highlights a promising means by which next-generation therapeutics and vaccines can harness the antiviral activity of the innate immune system via directed alterations in antibody glycosylation in vivo.  .

  11. Cerebellar Ataxia and Glutamic Acid Decarboxylase Antibodies

    Science.gov (United States)

    Ariño, Helena; Gresa-Arribas, Nuria; Blanco, Yolanda; Martínez-Hernández, Eugenia; Sabater, Lidia; Petit-Pedrol, Mar; Rouco, Idoia; Bataller, Luis; Dalmau, Josep O.; Saiz, Albert; Graus, Francesc

    2016-01-01

    IMPORTANCE Current clinical and immunologic knowledge on cerebellar ataxia (CA) with glutamic acid decarboxylase 65 antibodies (GAD65-Abs) is based on case reports and small series with short-term follow-up data. OBJECTIVE To report the symptoms, additional antibodies, prognostic factors, and long-term outcomes in a cohort of patients with CA and GAD65-Abs. DESIGN, SETTING, AND PARTICIPANTS Retrospective cohort study and laboratory investigations at a center for autoimmune neurologic disorders among 34 patients with CA and GAD65-Abs, including 25 with long-term follow-up data (median, 5.4 years; interquartile range, 3.1-10.3 years). MAIN OUTCOMES AND MEASURES Analysis of clinicoimmunologic features and predictors of response to immunotherapy. Immunochemistry on rat brain, cultured neurons, and human embryonic kidney cells expressing GAD65, GAD67, α1-subunit of the glycine receptor, and a repertoire of known cell surface autoantigens were used to identify additional antibodies. Twenty-eight patients with stiff person syndrome and GAD65-Abs served as controls. RESULTS The median age of patients was 58 years (range, 33-80 years); 28 of 34 patients (82%) were women. Nine patients (26%) reported episodes of brainstem and cerebellar dysfunction or persistent vertigo several months before developing CA. The clinical presentation was subacute during a period of weeks in 13 patients (38%). Nine patients (26%) had coexisting stiff person syndrome symptoms. Systemic organ-specific autoimmunities (type 1 diabetes mellitus and others) were present in 29 patients (85%). Twenty of 25 patients with long-term follow-up data received immunotherapy (intravenous immunoglobulin in 10 and corticosteroids and intravenous immunoglobulin or other immunosuppressors in 10), and 7 of them (35%) improved. Predictors of clinical response included subacute onset of CA (odds ratio [OR], 0.50; 95% CI, 0.25-0.99; P = .047) and prompt immunotherapy (OR, 0.98; 95% CI, 0.96-0.99; P = .01). Similar

  12. A universal strategy for stable intracellular antibodies.

    Science.gov (United States)

    Shaki-Loewenstein, Shelly; Zfania, Rahely; Hyland, Stephen; Wels, Winfried S; Benhar, Itai

    2005-08-01

    The expression of intracellular antibodies (intrabodies) in mammalian cells has provided a powerful tool to manipulate microbial and cellular signalling pathways in a highly precise manner. However, several technical hurdles have thus far restricted their more widespread use. In particular, single-chain antibodies (scFvs) have been reported to fold poorly in the reducing environment of the cytoplasm and as such there has been a reluctance to use scFv-phage libraries as a source of intrabodies unless a preselection step was applied to identify these rare scFvs that could fold properly in the absence of disulfide bonds. Recently, we reported that scFvs can be efficiently expressed within the cytoplasm of bacteria when fused at the C-terminus of the Escherichia coli maltose-binding protein (MBP). Here, we demonstrate that such MBP-scFvs are similarly stabilized when expressed in the mammalian cell cytoplasm as well as other compartments. This was demonstrated by comparing MBP-scFv fusions to the corresponding unfused scFvs that activate a defective beta-galactosidase enzyme, others that neutralize the wild-type beta-galactosidase enzyme, and an antibody that blocks the epidermal growth factor receptor. In all cases, the MBP-scFvs significantly outperformed their unfused counterparts. Our results suggest that fusion of scFvs to MBP, and possibly to other "chaperones in the context of a fusion protein", may provide a universal approach for efficient expression of intrabodies in the mammalian cell cytoplasm. This strategy should allow investigators to bypass much of the in vitro scFv characterization that is often not predictive of in vivo intrabody function and provide a more efficient use of large native and synthetic scFv-phage libraries already in existence to identify intrabodies that will be active in vivo.

  13. Intratypic heterologous vaccination of calves can induce an antibody response in presence of maternal antibodies against foot-and-mouth disease virus

    OpenAIRE

    Dekker, A.; Eble, P.L.; Stockhofe-Zurwieden, N; Chenard, G.

    2014-01-01

    Background - Maternal antibodies can interfere with foot-and-mouth disease vaccination. In this study we determined whether intratypic heterologous vaccination could help to improve herd immunity. Results - In unvaccinated calves, a half-life of maternal antibodies of 21 days was determined. At two weeks of age, calves without maternal antibodies showed a good antibody response against both vaccines used in the trial, while in calves with maternal antibodies no antibody response to homologous...

  14. Presence of Autoimmune Antibody in Chikungunya Infection

    Directory of Open Access Journals (Sweden)

    Wirach Maek-a-nantawat

    2009-01-01

    Full Text Available Chikungunya infection has recently re-emerged as an important arthropod-borne disease in Thailand. Recently, Southern Thailand was identified as a potentially endemic area for the chikungunya virus. Here, we report a case of severe musculoskeletal complication, presenting with muscle weakness and swelling of the limbs. During the investigation to exclude autoimmune muscular inflammation, high titers of antinuclear antibody were detected. This is the report of autoimmunity detection associated with an arbovirus infection. The symptoms can mimic autoimmune polymyositis disease, and the condition requires close monitoring before deciding to embark upon prolonged specific treatment with immunomodulators.

  15. Antipolymer antibody in Italian fibromyalgic patients

    OpenAIRE

    Bazzichi, Laura; Giacomelli, Camillo; De Feo, Francesca; Giuliano, Tiziana; Rossi, Alessandra; Doveri, Marica; Tani, Chiara; Wilson, Russell B.; Bombardieri, Stefano

    2007-01-01

    The objectives of the present study were to evaluate the presence of antipolymer antibody (APA) seropositivity in 285 Italian patients affected by primary fibromyalgia (FM) and to verify whether APA levels correlate with disease severity and with cytokine levels. APA levels were determined on serum samples by an indirect ELISA kit that detects IgG APA. Cytokines (IL-1, IL-6, IL-8, IL-10 and TNFα) were measured by ELISA in plasma. The impact of FM on the quality of life was estimated using the...

  16. Preparation, characterization and radiolabelling of antigranulocytemonoclonal antibody

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    The human granulocytes were isolated. Using hybridoma techniques, a hybridoma cell line(HSN) producing monoclonal antibody (McAb) against human granulocyte was obtained.The antibodybelonged to IgG1 subclass. It was confirmed byimmunohistochemical tests that HSN reacted selectivelynot only with human granulocytes, but also with their bone marrow precursors. Whereas humanlymphocytesand red blood cells retained negative in the tests. No cross-reaction was observedwith theperipheral blood cells in other animals. Its affinity constant was5.7×108L/mol, and the number of epitopesper granulocyte was 4.7×105. Monoclonal antibodydisplayed no loss of immunoreactivity after labelledwith 99mTc.

  17. Development of Anti-Isoproturon Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    LI Fang-shi; SUN Feng; LIU Xian-jin; CUI Heng-hua

    2007-01-01

    A competitive enzyme-linked immunosorbent assay (ELISA) suitable for the determination of the urea herbicide isoproturon,3-(4-isopropylphenyl)-1,1-dimethylurea, in food and environmental samples was developed. Two haptens named 1-(3-carboxypropyl)-3-(4-isopropylphenyl)-1-methylurca (hapten 4C) and 1-(5-carboxypentyl)-3-(4-isopropylphenyl)-1-methylurea (hapten 6C) were synthesized. The haptens were coupled to bovine serum albumin (BSA) and ovalbumin(OVA), respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen,with which a high-titer anti-isoproturon polyclonal antibody (pAb) was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR, and 1H NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36:1 and 46:1, respectively, as calculated by a UV spectrophotometry.The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6×105. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg L-1 and 1.0 × 105, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding (IC50) was 0.07 mg mL-1.The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and

  18. Production of monoclonal antibodies for radioimmunoassays

    International Nuclear Information System (INIS)

    Specific antibodies (Abs) have proven most useful and versatile tools for the identification, quantification, and localization of minute amounts of small and large molecules in biologic materials, e.g., body fluids, specific cells, and other body components. So far the most widely used technique for the production of specific Abs consists in immunization of animals like rabbits, goats, or horses, monitoring of Ab formation in the serum, and selection of animals which produce serum containing Abs sufficiently specific for the use envisaged. Although this approach has yielded many valuable results, it has some deficiencies

  19. Automatic Identification of Antibodies in the Protein Data Bank

    Institute of Scientific and Technical Information of China (English)

    LI Xun; WANG Renxiao

    2009-01-01

    An automatic method has been developed for identifying antibody entries in the protein data bank (PDB). Our method, called KIAb (Keyword-based Identification of Antibodies), parses PDB-format files to search for particular keywords relevant to antibodies, and makes judgment accordingly. Our method identified 780 entries as antibodies on the entire PDB. Among them, 767 entries were confirmed by manual inspection, indicating a high success rate of 98.3%. Our method recovered basically all of the entries compiled in the Summary of Antibody Crystal Structures (SACS) database. It also identified a number of entries missed by SACS. Our method thus provides a more com-plete mining of antibody entries in PDB with a very low false positive rate.

  20. The investigation of relationship between preeclampsia and antiphospholipid antibody syndrome

    Directory of Open Access Journals (Sweden)

    Mehmet Tayyar

    2013-03-01

    Full Text Available Aim. The aim of this study was evaluate the relationship between preeclampsia and antiphospholipid antibodies. Methods. A total of 116 pregnant women between 20th and 40th weeks of gestation admitted to our department were investigated. 63 of them were allocated our preeclampsia group and 53 of them were allocated our control group. Lupus anticoagulant, anti-cardiolipin antibodies (IG G ve M and antiphosphatidylserine antibodies (IG G ve M were measured. Results. There was no statistical significance between preeclampsia and control group for antiphospholipid antibodies but these were two times higher in preeclamptic group compared to control group. (22.2% in preeclampsia, 11.3% in control group p=0.193. Conclusions. In an unselected population we were not able to demonstrate an association between preeclampsia and antiphospholipid antibody syndrome but antiphospholipid antibody ratio elevated in women with preeclampsia. These findings show that, there is a need for large scale studies.

  1. Production of monoclonal antibody with Celline-350 bioreactor

    International Nuclear Information System (INIS)

    Monoclonal antibodies are protein that are highly specific and sensitive in their reaction with specific sites on target molecules that they have become reagents of central importance in the diagnostic and treatment of human diseases. This paper reports the use of CELLine-350 bioreactor to produce continuous supply of serum-free breast cancer monoclonal antibody. Initial volume of 5ml (1.5 x 106 viable cells/ml) is inoculated into the bioreactor and harvesting is done every 5 days to obtain high yield monoclonal antibody. The serum-free supernatant is precipitated with 50% saturated ammonia sulfate and the antibody is purified by protein-G affinity chromatography. The concentration of monoclonal antibody successfully produced by the bioreactor is 0.91mg/ml respectively and it is measured by the Lowry method. This result shows that bioreactor Celline-350 is easy to handle and cost effective for the continuous production of serum free monoclonal antibody. (Author)

  2. Generation and characterization of novel stromal specific antibodies

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Rheumatoid synovial fibroblasts were used as an immunogen to produce monoclonal antibodies selected for their reactivity with stromal cell antigens. Mice were immunised with low passage whole cell preparations and the subsequent hybridomas were screened by immunohistochemistry on rheumatoid synovium and tonsil sections. The aim was to identify those antibodies that recognised antigens that were restricted to stromal cells and were not expressed on CD45 positive leucocytes. A significant number of antibodies detected antigen that identified endothelial cells. These antibodies were further characterised to determine whether the vessels identified by these antibodies were vascular or lymphatic.From five fusions clones were identified with predominant reactivity with: 1) fibroblasts and endothelial cells; or 2)broad stromal elements (fibroblast, endothelium, epithelium, follicular dendritic cells). A fibroblast-specific antibody that did not also identify vessels was not generated. Examples of each reactivity pattern are discussed.

  3. Rational Design of CXCR4 Specific Antibodies with Elongated CDRs

    Science.gov (United States)

    2015-01-01

    The bovine antibody (BLV1H12) which has an ultralong heavy chain complementarity determining region 3 (CDRH3) provides a novel scaffold for antibody engineering. By substituting the extended CDRH3 of BLV1H12 with modified CXCR4 binding peptides that adopt a β-hairpin conformation, we generated antibodies specifically targeting the ligand binding pocket of CXCR4 receptor. These engineered antibodies selectively bind to CXCR4 expressing cells with binding affinities in the low nanomolar range. In addition, they inhibit SDF-1-dependent signal transduction and cell migration in a transwell assay. Finally, we also demonstrate that a similar strategy can be applied to other CDRs and show that a CDRH2-peptide fusion binds CXCR4 with a Kd of 0.9 nM. This work illustrates the versatility of scaffold-based antibody engineering and could greatly expand the antibody functional repertoire in the future. PMID:25041362

  4. Design and manufacture of monoclonal antibodies for radioimmunotherapy

    International Nuclear Information System (INIS)

    Appropriate design and manufacture of monoclonal antibodies is fundamental to their use for radioimmunotherapy. Besides the right selection of antibody specificity and affinity, recombinant antibodies can be designed to simplify manufacture and minimise unwanted side effects. Although many innovative new technologies have been developed in recent years, antibodies are still most commonly produced from mammalian cells and purified by column chromatography. Purification methods have to be designed and validated to remove potential contaminants, especially retroviruses which in principle might be present in mammalian cell lines. Adherence to relevant Good Manufacturing Practice is mandatory in the production of any medicinal product and there are numerous guidelines regarding the manufacture of antibodies. This article outlines some methods used for fermentation, purification and quality control of antibodies intended for radiolabelling

  5. Engineering broadly neutralizing antibodies for HIV prevention and therapy.

    Science.gov (United States)

    Hua, Casey K; Ackerman, Margaret E

    2016-08-01

    A combination of advances spanning from isolation to delivery of potent HIV-specific antibodies has begun to revolutionize understandings of antibody-mediated antiviral activity. As a result, the set of broadly neutralizing and highly protective antibodies has grown in number, diversity, potency, and breadth of viral recognition and neutralization. These antibodies are now being further enhanced by rational engineering of their anti-HIV activities and coupled to cutting edge gene delivery and strategies to optimize their pharmacokinetics and biodistribution. As a result, the prospects for clinical use of HIV-specific antibodies to treat, clear, and prevent HIV infection are gaining momentum. Here we discuss the diverse methods whereby antibodies are being optimized for neutralization potency and breadth, biodistribution, pharmacokinetics, and effector function with the aim of revolutionizing HIV treatment and prevention options. PMID:26827912

  6. Antibody engineering: facing new challenges in cancer therapy

    Institute of Scientific and Technical Information of China (English)

    Laura SANZ; (A)ngel M CUESTA; Marta COMPTE; Luis (A)LVAREZ-VALLINA

    2005-01-01

    Antibody-based therapeutics are beginning to realize the promise enclosed in their early denomination as "magic bullets". Initial disappointment has turned into clinical and commercial success, and engineered antibodies currently represent over 30% of biopharmaceuticals in clinical trials. Recent structural and functional data have allowed the design of a new generation of therapeutic antibodies, with strategies ranging from complement-mediated and antibody-dependant cellular cytotoxicity enhancement to improved cytotoxic payloads using toxins, drugs,radionucleids and viral delivery. This review considers the structure of different types of recombinant antibodies, their mechanism of action and how their efficacy has been increased using a broad array of approaches. We will also focus on the additional benefits offered by the use of gene therapy methods for the in vivo production of therapeutic antibodies.

  7. Fc-galactosylation modulates antibody-dependent cellular cytotoxicity of therapeutic antibodies.

    Science.gov (United States)

    Thomann, Marco; Reckermann, Katharina; Reusch, Dietmar; Prasser, Jessica; Tejada, Max L

    2016-05-01

    The therapeutic activity of monoclonal antibodies can involve immune cell mediated effector functions including antibody-dependent cellular cytotoxicity (ADCC), an activity that is modulated by the structure of Fc-glycans, and in particular the lack of core fucose. The heterogeneity of these glycostructures and the inherent variability of traditional PBMC-based in vitro ADCC assays, have made it challenging to quantitatively assess the impact of other glycostructures on ADCC activity. We applied a quantitative NK cell based assay to generate a database consisting of Fc-glycostructure and ADCC data from 54 manufacturing batches of a CHO-derived monoclonal antibody. Explorative analysis of the data indicated that, apart from afucosylation, galactosylation levels could influence ADCC activity. We confirmed this hypothesis by demonstrating enhanced ADCC upon enzymatic hypergalactosylation of four different monoclonal antibodies derived using standard CHO manufacturing processes. Furthermore we quantitatively compare the effects of galactosylation and afucosylation in the context of glycan heterogeneity and demonstrate that while galactose can influence ADCC activity, afucosylation remains the primary driver of this activity. PMID:27058641

  8. An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function of a Human Antibody

    Energy Technology Data Exchange (ETDEWEB)

    Krause, Jens C.; Ekiert, Damian C.; Tumpey, Terrence M.; Smith, Patricia B.; Wilson, Ian A.; Crowe, Jr., James E. (Vanderbilt); (Scripps); (CDC)

    2011-09-02

    The structural and functional significance of somatic insertions and deletions in antibody chains is unclear. Here, we demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. The insertion arose through a series of events, including a somatic point mutation in a predicted hot-spot motif, introduction of a new hot-spot motif, a molecular duplication due to polymerase slippage, a deletion due to misalignment, and additional somatic point mutations. Atomic resolution structures of the wild-type antibody and a variant in which the insertion was removed revealed that the three-amino-acid insertion near the base of heavy-chain complementarity-determining region (CDR) H2 resulted in a bulge in that loop. This enlarged CDR H2 loop impinges on adjacent regions, causing distortion of the CDR H1 architecture and its displacement away from the antigen-combining site. Removal of the insertion restores the canonical structure of CDR H1 and CDR H2, but binding, neutralization activity, and in vivo activity were reduced markedly because of steric conflict of CDR H1 with the hemagglutinin antigen.

  9. Antibody structural modeling with prediction of immunoglobulin structure (PIGS)

    DEFF Research Database (Denmark)

    Marcatili, Paolo; Olimpieri, Pier Paolo; Chailyan, Anna;

    2014-01-01

    Antibodies (or immunoglobulins) are crucial for defending organisms from pathogens, but they are also key players in many medical, diagnostic and biotechnological applications. The ability to predict their structure and the specific residues involved in antigen recognition has several useful appl...... on average) to build a structural model of an antibody. It is based on the concept of canonical structures of antibody loops and on our understanding of the way light and heavy chains pack together....

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

    OpenAIRE

    Pierson, Richard N.

    2009-01-01

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

  11. Distinction between antinuclear antibody and P-ANCA.

    OpenAIRE

    Lee, S. S.; Lawton, J W; Chak, W

    1991-01-01

    To differentiate between perinuclear immunofluorescence staining of antinuclear antibody (ANA) and the perinuclear form of anti-neutrophil cytoplasmic antibody (P-ANCA), the pattern after formaldehyde vapour fixation of normal human neutrophils was compared with that of standard ethanol fixation. Fifteen out of 17 myeloperoxidase antibody positive sera showed cytoplasmic staining on formaldehyde vapour fixed cells; 30 of the 32 ANA positive samples became negative or gave weak nuclear stainin...

  12. Critical evaluation of monoclonal antibody staining in breast carcinoma.

    OpenAIRE

    Parham, D M; Coghill, G; Robertson, A.J.

    1989-01-01

    The immunoperoxidase staining of 84 primary invasive breast carcinomas with four monoclonal antibodies (BRST-1, HMFG1, EMA, B72.3) was evaluated by semiquantitative light microscopical examination and quantitative image analysis. Major differences in the staining of the tumours for each of the monoclonal antibodies was observed. Correlation between monoclonal antibody staining and patient age, survival, histological grade, tumour diameter and cellularity was also carried out. This showed a si...

  13. Antibody avidity in swine lymphocyte antigen-defined miniature pigs.

    OpenAIRE

    Appleyard, G D; Mallard, B A; Kennedy, B. W.; Wilkie, B. N.

    1992-01-01

    Antibody avidity to hen egg white lysozyme (HEWL) was measured by thiocyanate ion elution enzyme-linked immunosorbent assay (ELISA) in swine lymphocyte antigen (SLA) defined miniature pigs. Serum antibody avidity was evaluated on day 14 and 30 after primary (day 0) and secondary (day 14) immunizations in eight to ten week old miniature pigs previously typed for swine lymphocyte antigen genotype. The effect of SLA genotype, litter, and gender on anti-HEWL antibody avidity was determined by lea...

  14. Development of antibodies to human embryonic stem cell antigens

    OpenAIRE

    Stanley Marisa; Rao Mahendra S; Olson Judith M; Cai Jingli; Taylor Eva; Ni Hsiao-Tzu

    2005-01-01

    Abstract Background Using antibodies to specific protein antigens is the method of choice to assign and identify cell lineage through simultaneous analysis of surface molecules and intracellular markers. Embryonic stem cell research can be benefited from using antibodies specific to transcriptional factors/markers that contribute to the "stemness" phenotype or critical for cell lineage. Results In this report, we have developed and validated antibodies (either monoclonal or polyclonal) specif...

  15. Anti-collagen antibodies in sera from rheumatoid arthritis patients.

    OpenAIRE

    Beard, H K; Ryvar, R; Skingle, J; Greenbury, C. L.

    1980-01-01

    Anti-cartilage antibodies, demonstrable by immunofluorescence, were found in 3.3% of rheumatoid arthritis patients. In most of these patients antibodies to type II collagen were detected. In specificity studies on these anti-collagen antibodies, they appeared to be type specific, showing no reaction with collagen types I and III. Denatured type II collagen reacted much less well than native type II, but isolated peptides from different regions of the collagen molecule were differentiated by i...

  16. Monoclonal Antibody Expression and Novel Purification in Nicotiana benthamiana

    OpenAIRE

    Fulton, Andrew Dale

    2011-01-01

    Over the past few decades researchers and industrial professionals alike have realized the vast potential of monoclonal antibodies to treat diseases ranging from arthritis, immune and infectious diseases to cancer. There are a number of antibodies on the market that constitute a large portion of the biopharmaceutical niche in the drug industry. Blockbuster drugs (selling greater than $1 billion/year), include antibodies such as Avastin (bevacizumab), Herceptin (trastuzumab), Rituxan (rituxi...

  17. Mechanisms involved in antibody- and complement-mediated allograft rejection

    OpenAIRE

    Wasowska, Barbara A.

    2010-01-01

    Antibody-mediated rejection has become critical clinically because this form of rejection is usually unresponsive to conventional anti-rejection therapy, and therefore, it has been recognized as a major cause of allograft loss. Our group developed experimental animal models of vascularized organ transplantation to study pathogenesis of antibody- and complement-mediated endothelial cell injury leading to graft rejection. In this review, we discuss mechanisms of antibody-mediated graft rejectio...

  18. Monoclonal antibodies against plant proteins recognise animal intermediate filaments.

    Science.gov (United States)

    Parke, J M; Miller, C C; Cowell, I; Dodson, A; Dowding, A; Downes, M; Duckett, J G; Anderton, B J

    1987-01-01

    Four monoclonal antibodies were raised against polypeptides present in a high-salt detergent-insoluble fraction from cells of Chlamydomonas reinhardtii. Indirect immunofluorescence microscopy of fibroblasts and epithelial cells grown in culture using these plant antibodies revealed staining arrays identical to those obtained with well characterised antibodies to animal intermediate filaments. Immunofluorescence microscopy of Chlamydomonas with these monoclonal antibodies and a monoclonal antibody that recognises all animal intermediate filaments (anti-IFA) gave a diffuse, patchy cytoplasmic staining pattern. Both the plant antibodies and anti-IFA stained interphase onion root tip cells in a diffuse perinuclear pattern. In metaphase through to telophase, the labelling patterns colocalised with those of microtubules. Labelling of the phragmoplast was also detected but not staining of the preprophase band. On Western blots of various animal cell lines and tissues, all the antibodies labelled known intermediate filament proteins. On Western blots of whole Chlamydomonas proteins, all the antibodies labelled a broad band in the 57,000 Mr range, and three antibodies labelled bands around 66,000 and 140,000 Mr but with variable intensities. On Western blots of whole onion root tip proteins, all the antibodies labelled 50,000 Mr (two to three bands) polypeptides and a diffuse band around 60,000 Mr and three of the antibodies also labelled several polypeptides in the 90,000-200,000 Mr range. The consistent labelling of these different bands by several different monoclonal antibodies recognising animal intermediate filaments makes these polypeptides putative plant intermediate filament proteins. PMID:2446785

  19. Goat serums for fluorescent antibody conjugates to chlamydial antigens.

    OpenAIRE

    Tessler, J.

    1984-01-01

    Serums from goats hyperimmunized with Chlamydia psittaci consistently produce antichlamydial fluorescent antibody conjugate of high titer. The titer of the fluorescent antibody conjugate prepared from a given serum correlated well with the titer obtained by agar gel precipitin, but not with the complement fixation. The agar gel precipitin test can be used to predict whether a given serum is satisfactory for use in production of a conjugate for direct fluorescent antibody tests. Serums with an...

  20. Purification of Murine Monoclonal IgM Antibody

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    This paper presents the purification of a monoclonal IgM antibody against human tumor associated antigen Lewis-Y by ion exchange chromatography and gel filtration.Enzyme-linked immunosorbent assay (ELISA) and SDS-polyacrylamide gel electrophoresis (PAGE) were used to identify purified IgM antibody.In flow cytometry analysis, the purified IgM antibody recognizes human breast tumor cell line MCF-7 which expresses Lewis-Y antigen.This work presents a new way for the purification of murine monoclonal IgM antibody.

  1. Antibody binding to p-Si using LANL SAM chemistry

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Aaron S [Los Alamos National Laboratory

    2010-12-06

    This NMSBA-sponsored project involves the attachment of antibodies to polymeric silicon (p-Si) surfaces, with the ultimate goal of attaching antibodies to nanowires for Vista Therapeutics, Inc. (Santa Fe, NM). This presentation describes the functionalization of p-Si surfaces. the activation of terminal carboxylates on these surfaces, the conjugation of antibodies, and the analyses undertaken at each step. The results of this work show that antibody conjugation is possible on p-Si coatings using the well-known EDC/NHS activation chemistry.

  2. Antibody-Based Strategies to Prevent and Treat Influenza

    Directory of Open Access Journals (Sweden)

    Ram eSasisekharan

    2015-07-01

    Full Text Available Passive immunization using antibodies has been suggested to offer several benefits in comparison to other antiviral treatment options. The potential for seasonal protection arising from a single injection of antibodies is appealing and has been pursued for a number of infectious agents. However, until recently, antibody-based strategies to combat infectious agents has been hampered due to the fact that typical antibodies have been found to be strain-specific, with the virus evolving resistance in many cases. The discovery of broadly neutralizing antibodies (bNAbs in, for example, influenza, dengue virus, and HIV, which bind to multiple, structurally-diverse strains has provided renewed interest in this area. This review will focus on new technologies that enable the discovery of bNAbs, the challenges and opportunities of immunotherapies as an important addition to existing antiviral therapy, and the role of antibody discovery in informing rational vaccine discovery – with agents targeting influenza specifically addressed. Multiple agents have entered the clinic and raise the possibility that a single antibody or small combination of antibodies can effectively neutralize a wide variety of strains. However, challenges remain - including combating escape variants, pharmacodynamics of antibody distribution, and development of efficacy biomarkers beyond virologic endpoints.

  3. Biophysical characterization of a model antibody drug conjugate.

    Science.gov (United States)

    Arakawa, Tsutomu; Kurosawa, Yasunori; Storms, Michael; Maruyama, Toshiaki; Okumura, C J; Maluf, Nasib Karl

    2016-01-01

    Antibody drug conjugates (ADC) are important next-generation biopharmaceuticals and thus require stringent structure characterization as is the case for monoclonal antibodies. We have tested several biophysical techniques, i.e., circular dichroism, analytical ultracentrifugation, differential scanning calorimetry and fluorescence spectroscopy, to characterize a fluorescein-labeled monoclonal antibody as a model ADC. These techniques indicated possible small structure and stability changes by the conjugation, while largely retaining the tertiary structure of the antibody, consistent with unaltered biological activities. Thus, the above biophysical techniques are effective at detecting changes in the structural properties of ADC. PMID:27534450

  4. Clinical analyses of perinuclear antineutrophil cytoplasmic antibody associated hypertrophic pachymeningitis

    Institute of Scientific and Technical Information of China (English)

    赵鹏

    2013-01-01

    Objective To explore the clinical characteristics,diagnosis,treatment and prognosis of perinuclear antineutrophil cytoplasmic antibody(p-ANCA)associated hypertrophic pachymeningitis.Methods A retrospective study

  5. Antibody Conjugates: From Heterogeneous Populations to Defined Reagents

    Directory of Open Access Journals (Sweden)

    Patrick Dennler

    2015-08-01

    Full Text Available Monoclonal antibodies (mAbs and their derivatives are currently the fastest growing class of therapeutics. Even if naked antibodies have proven their value as successful biopharmaceuticals, they suffer from some limitations. To overcome suboptimal therapeutic efficacy, immunoglobulins are conjugated with toxic payloads to form antibody drug conjugates (ADCs and with chelating systems bearing therapeutic radioisotopes to form radioimmunoconjugates (RICs. Besides their therapeutic applications, antibody conjugates are also extensively used for many in vitro assays. A broad variety of methods to functionalize antibodies with various payloads are currently available. The decision as to which conjugation method to use strongly depends on the final purpose of the antibody conjugate. Classical conjugation via amino acid residues is still the most common method to produce antibody conjugates and is suitable for most in vitro applications. In recent years, however, it has become evident that antibody conjugates, which are generated via site-specific conjugation techniques, possess distinct advantages with regard to in vivo properties. Here, we give a comprehensive overview on existing and emerging strategies for the production of covalent and non-covalent antibody conjugates.

  6. Surface immobilization of antibody on silk fibroin through conformational transition.

    Science.gov (United States)

    Lu, Qiang; Wang, Xiaoqin; Zhu, Hesun; Kaplan, David L

    2011-07-01

    In recent studies silk fibroin has been explored as a new material platform for biosensors. Based on these developments, a procedure for the immobilization of antibodies on silk fibroin substrates was developed as a route to functionalizing these biosensor systems. By controlling the conformational transition of the silk fibroin, a primary antibody was immobilized and enriched at the surface of silk fibroin substrates under mild reaction conditions to maintain antibody function. Compared to chemical crosslinking, the immobilization efficiency in the present approach was increased significantly. This method, achieving high loading of antibody while retaining function, improves the feasibility of silk fibroin as a platform material for biosensor applications.

  7. The Biochemical Properties of Antibodies and Their Fragments.

    Science.gov (United States)

    Hnasko, Robert M

    2015-01-01

    Immunoglobulins (Ig) or antibodies are powerful molecular recognition tools that can be used to identify minute quantities of a given target analyte. Their antigen-binding properties define both the sensitivity and selectivity of an immunoassay. Understanding the biochemical properties of this class of protein will provide users with the knowledge necessary to select the appropriate antibody composition to maximize immunoassay results. Here we define the general biochemical properties of antibodies and their similarities and differences, explain how these properties influence their functional relationship to an antigen target, and describe a method for the enzymatic fragmentation of antibodies into smaller functional parts.

  8. The antibody response in Lyme disease.

    Science.gov (United States)

    Craft, J. E.; Grodzicki, R. L.; Shrestha, M.; Fischer, D. K.; García-Blanco, M.; Steere, A. C.

    1984-01-01

    We determined the antibody response against the Ixodes dammini spirochete in Lyme disease patients by indirect immunofluorescence and an enzyme-linked immunosorbent assay (ELISA). The specific IgM response became maximal three to six weeks after disease onset, and then declined, although titers sometimes remained elevated during later disease. Specific IgM levels correlated directly with total serum IgM. The specific IgG response, often delayed initially, was nearly always present during neuritis and arthritis, and frequently remained elevated after months of remission. Although results obtained by indirect immunofluorescence and the ELISA were similar, the ELISA was more sensitive and specific. Cross-reactive antibodies from patients with other spirochetal infections were blocked by absorption of sera with Borrelia hermsii, but titers of Lyme disease sera were also decreased. To further characterize the specificity of the humoral immune response against the I. dammini spirochete, 35S-methionine-labeled spirochetal antigens were identified by immunoprecipitation with sera from Lyme arthritis patients. These polypeptides had molecular weights of 62, 60, 47, 37, 22, 18, and 15 kDa, and were not recognized by control sera. We conclude that the ELISA, without absorption, is the best method to assay the humoral immune response in Lyme disease, and we have identified methionine-containing spirochetal polypeptides that may be important in Lyme arthritis. PMID:6393607

  9. Drug Development of Therapeutic Monoclonal Antibodies.

    Science.gov (United States)

    Mould, Diane R; Meibohm, Bernd

    2016-08-01

    Monoclonal antibodies (MAbs) have become a substantial part of many pharmaceutical company portfolios. However, the development process of MAbs for clinical use is quite different than for small-molecule drugs. MAb development programs require careful interdisciplinary evaluations to ensure the pharmacology of both the MAb and the target antigen are well-understood. Selection of appropriate preclinical species must be carefully considered and the potential development of anti-drug antibodies (ADA) during these early studies can limit the value and complicate the performance and possible duration of preclinical studies. In human studies, many of the typical pharmacology studies such as renal or hepatic impairment evaluations may not be needed but the pharmacokinetics and pharmacodynamics of these agents is complex, often necessitating more comprehensive evaluation of clinical data and more complex bioanalytical assays than might be used for small molecules. This paper outlines concerns and strategies for development of MAbs from the early in vitro assessments needed through preclinical and clinical development. This review focuses on how to develop, submit, and comply with regulatory requirements for MAb therapeutics. PMID:27342605

  10. Array tomography: immunostaining and antibody elution.

    Science.gov (United States)

    Micheva, Kristina D; O'Rourke, Nancy; Busse, Brad; Smith, Stephen J

    2010-11-01

    Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time-consuming and require some practice to perfect. In this protocol, tissue arrays are prepared for imaging by tagging with primary antibodies against specific cellular targets, followed by labeling with fluorescent secondary antibodies. Alternatively, fluorescent proteins that have been introduced into the tissue before dissection can be used. PMID:21041398

  11. Anti IH: An antibody worth mention.

    Science.gov (United States)

    Mohanan, Nithya; Henry, Nittin; Rafi, Aboobacker Mohamed; Innah, Susheela J

    2016-01-01

    A 72-year-old female with co-morbidities posted for surgical correction of fracture neck of femur without any history of transfusions was noted to have a hemoglobin level of 7 g/dl and packed red blood cells transfusion was ordered. Pretransfusion tests demonstrated A1B group with D positive on forward grouping. Reverse grouping showed a varying grade of agglutination with A, B, and O cells. Agglutination being stronger at 4°C. Antibody screening showed pan-agglutination, direct Coomb's test and auto control were negative. The serum reacted with adult O cells (OIadult) but not with adult Bombay cells (Oh Iadult) or O cord (Oicord) cells. A possibility of a compound cold antibody anti IH was made and A1B compatible cells were transfused to the patient. This case report illustrates anti-IH cold agglutinin with broad thermal amplitude. Uniqueness of this case report was O group incompatibility with A1B group, which was detected earlier and a catastrophic transfusion reaction being subverted.

  12. Anti IH: An antibody worth mention

    Directory of Open Access Journals (Sweden)

    Nithya Mohanan

    2016-01-01

    Full Text Available A 72-year-old female with co-morbidities posted for surgical correction of fracture neck of femur without any history of transfusions was noted to have a hemoglobin level of 7 g/dl and packed red blood cells transfusion was ordered. Pretransfusion tests demonstrated A1B group with D positive on forward grouping. Reverse grouping showed a varying grade of agglutination with A, B, and O cells. Agglutination being stronger at 4°C. Antibody screening showed pan-agglutination, direct Coomb's test and auto control were negative. The serum reacted with adult O cells (OIadult but not with adult Bombay cells (Oh Iadult or O cord (Oicord cells. A possibility of a compound cold antibody anti IH was made and A1B compatible cells were transfused to the patient. This case report illustrates anti-IH cold agglutinin with broad thermal amplitude. Uniqueness of this case report was O group incompatibility with A1B group, which was detected earlier and a catastrophic transfusion reaction being subverted.

  13. Antibody responses in allogeneic radiation chimeras

    International Nuclear Information System (INIS)

    The construction of long-lived allogeneic radiation chimeras, free of graft-versus-host disease, has been achieved using serologic elimination of Thy 1+ cells from donor bone marrow. Humoral immune function was not restored in these animals as evidenced by lack of primary antibody responses to a T cell-dependent antigen, namely, sheep erythrocytes (SRBC) both in vivo and in vitro. No evidence for a suppressor cell-mediated mechanism was found. Using separated chimera spleen cell populations and specific helper cell soluble mediators, the functional capabilities of chimera B cells, T cells, and macrophages were assessed. These findings suggested that the failure of chimeras to produce antibody is not the result of impaired B cell, T cell, or macrophage function, but rather, that it is due to ineffective cellular interactions. Physiologic cellular interactions depend upon the sharing of major histocompatibility complex (MHC) determinants between interacting cells. However, the self-recognition repertoire of developing T cells may be influenced by the environment which these cells differentiate such that they learn to recognize host MHC determinants as self. These findings support the interpretation that the immunologic hyporeactivity of allogeneic bone marrow chimeras reflects the role of the host environment in restricting the interactive capabilities of donor-derived cells

  14. Seropositivity of Dengue Antibodies during Pregnancy

    Directory of Open Access Journals (Sweden)

    Nor Azlin Mohamed Ismail

    2014-01-01

    Full Text Available Purpose. Malaysia a dengue endemic country with dengue infections in pregnancy on the rise. The present study was aimed at determining dengue seroprevalence (IgG or IgM during pregnancy and its neonatal transmission in dengue seropositive women. Methods. Maternal with paired cord blood samples were tested for dengue antibodies (IgG and IgM using an enzyme-linked immunosorbent assay (ELISA. Maternal age, parity, occupation, ethnic group, and gestational age were recorded. Data on neonatal Apgar score and admissions to the Neonatal Intensive Care Unit (NICU were analyzed. Results. Out of 358 women recruited, about 128 (35.8% patients were seropositive. Twelve patients (3.4% had recent infections (IgM positive and another 116 women (32.4% were with past infections (IgG positive. All babies born to seropositive mothers had positive IgG paired cord blood; however, no IgM seropositivity was observed. All neonates had good Apgar scores and did not require NICU admission. Conclusion. In this study, 35.8% pregnant women were found to be dengue seropositive. However, transplacental transfer of IgG antibodies had no detrimental effect on the neonatal outcomes.

  15. Thyroid antibody-negative euthyroid Graves’ ophthalmopathy

    Directory of Open Access Journals (Sweden)

    Arshiya Tabasum

    2016-06-01

    Full Text Available TSH receptor antibodies (TRAbs are the pathological hallmark of Graves’ disease, present in nearly all patients with the disease. Euthyroid Graves’ ophthalmopathy (EGO is a well-recognized clinical entity, but its occurrence in patients with negative TRAbs is a potential source of diagnostic confusion. A 66-year-old female presented to our endocrinology clinic with right eye pain and diplopia in the absence of thyroid dysfunction. TRAbs were negative, as measured with a highly sensitive third-generation thyrotropin-binding inhibitory immunoglobulin (TBII ELISA assay. CT and MRI scans of the orbit showed asymmetrical thickening of the inferior rectus muscles but no other inflammatory or malignant orbital pathology. Graves’ ophthalmopathy (GO was diagnosed on the basis of the clinical and radiological features, and she underwent surgical recession of the inferior rectus muscle with complete resolution of the diplopia and orbital pain. She remained euthyroid over the course of follow-up but ultimately developed overt clinical and biochemical hyperthyroidism, 24 months after the initial presentation. By this time, she had developed positive TRAb as well as thyroid peroxidase antibodies. She responded to treatment with thionamides and remains euthyroid. This case highlights the potential for negative thyroid-specific autoantibodies in the presentation of EGO and underscores the variable temporal relationship between the clinical expression of thyroid dysfunction and orbital disease in the natural evolution of Graves’ disease.

  16. IgE antibodies against snake venoms.

    Science.gov (United States)

    Alonso, A; Scavini, L M; Marino, G A; Rodríguez, S M

    1995-01-01

    A similar event was detected in the clinical records of a small group of atopic patients living in the northern provinces of Argentina, i.e., they were bitten by a snake of the Bothrops species (or yarará) during their rural activities (woodcutters, cattle-drivers and farmers). Those who were bitten twice suffered an acute episode of hives and angioedema within 15 minutes after the snake bite. The presence of specific antibodies against Bothrops alternata (Ba) extract was detected by means of RAST for IgE and Ouchterlony and Boyden for IgG. The Ouchterlony also demonstrated crossreactivity among the venoms of the Bothrops species and the positivity of the six fractions obtained by DEAE-cellulose column fractionation against the horse anti-Ba serum. The Ba antigen induced a definite inhibition of the RAST. We presume that hives and angioedema in atopic patients immediately after a second snake bite could be attributed to the presence of a specific IgE antibody against the venom, and must not be misinterpreted with the toxic effects that appear later. PMID:7551202

  17. Licensed monoclonal antibodies and associated challenges.

    Science.gov (United States)

    Khan, Amjad Hayat; Sadroddiny, Esmaeil

    2015-12-23

    Monoclonal antibodies (mAbs) are the leading class of targeted therapeutics and remarkably effective in addressing autoimmune diseases, inflammations, infections, and various types of cancer. Several mAbs approved by US food and drug administration (FDA), are available on the market and a number are pending for approval. Luckily, FDA approved mAbs have played a pivotal role in the treatment and prevention of lethal diseases. However, claiming that licensed mAbs are 100% safe is still debatable, because infections, malignancies, anaphylactoid, and anaphylactic reactions are the more frequently associated adverse events. To evaluate benefit to risk ratio of mAbs, it is important for the clinical research staff or physicians to monitor and follow-up the patients who are receiving mAbs dozes. It is recommended that patients, physicians, biopharmaceutical companies, and researchers should keep in touch to highlight and resolve antibody-based adverse events. In this review we underscore the associated challenges of mAbs, approved by FDA from 2007-2014. PMID:27472864

  18. Antibody-Mediated Autoimmune Encephalopathies and Immunotherapies.

    Science.gov (United States)

    Gastaldi, Matteo; Thouin, Anaïs; Vincent, Angela

    2016-01-01

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

  19. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity.

    Science.gov (United States)

    Wu, Yuling; Li, Jia J; Kim, Hyun Jun; Liu, Xu; Liu, Weiyi; Akhgar, Ahmad; Bowen, Michael A; Spitz, Susan; Jiang, Xu-Rong; Roskos, Lorin K; White, Wendy I

    2015-11-01

    Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function. PMID:26205082

  20. Role of antiviral antibodies in resistance against coxsackievirus B3 infection: interaction between preexisting antibodies and an interferon inducer.

    OpenAIRE

    Cho, C T; Feng, K. K.; McCarthy, V P; Lenahan, M F

    1982-01-01

    An experimental model of coxsackievirus B3 infection in newborn mice was utilized to examine the protective role of antiviral antibodies and an interferon inducer, polyinosinic acid-polycytidylic acid [poly(I:C)]. Subcutaneous administration to the infected mice of specific antiviral antibodies resulted in significant protection against coxsackievirus B3 infection. Antibody-treated animals had shortened viremia, early clearance of virus from tissues, and a reduced mortality rate. Dose respons...

  1. Free theme; Theme libre

    Energy Technology Data Exchange (ETDEWEB)

    Emptaz, A.; Huglo, D.; Meurisse, H.; Nocaudie, M.; D' Hergomez, M.; Marchandise, X. [Centre Hospitalier Universitaire, Medecine Nucleaire et Imagerie Fonctionnelle, 59 - Lille (France); Kraeber-Bodere, F.; Sai-Maurel, C.; Faivre-Chauvet, A.; Thedrez, P.; Chatal, J.F.; Barbet, J.; Paris, F. [Centre Hospitalier Universitaire, Hotel Dieu, INSERM U 601, 44 - Nantes (France); Thomare, D. [Centre Hospitalier Universitaire, Hotel Dieu, Pharmacie, 44 - Nantes (France); Deleris, G. [INSERM U 577, Universite Victor Segalen Bordeaux 2, 33 - Bordeaux (France); Bikfalvi, A. [Bordeaux-1, INSERM E0113, 33 (France); Py, B.; Karacay, H.; Sharkey, R.M.; Goldenberg, D.M. [CMMI, Garden State Center Belleville NJ (United States); Chang, Ch.; Rossi, E.A. [Garden State Center Belleville, IBC Pharmaceuticals, NJ (United States); McBride, W.J.; Ragland, D.R.; Horak, I.D. [Immunomedics, Inc. Morris Plains NJ (United States)

    2005-11-15

    Efficiency and tolerance are the two important parts of these articles, that treats scintigraphy in tumors having a medullo adrenal origin, radioimmunotherapy in an animal model of thyroid cancer and advantage of pre-targeting with an antibody for the radioimmunotherapy in colon cancers. (N.C.)

  2. DARPins: a true alternative to antibodies.

    Science.gov (United States)

    Stumpp, Michael T; Amstutz, Patrick

    2007-03-01

    Designed ankyrin repeat proteins (DARPins) are a promising class of non-immunoglobulin proteins that can offer advantages over antibodies for target binding in drug discovery and drug development. DARPins have been successfully used, for example, for the inhibition of kinases, proteases and drug-exporting membrane proteins. DARPins specifically targeting the cancer marker HER2 have also been generated and were shown to function in both in vitro diagnostics and in vivo tumor targeting. DARPins are ideally suited for in vivo imaging or delivery of toxins or other therapeutic payloads because of their favorable molecular properties, including small size and high stability. The low-cost production in bacteria and the rapid generation of many target-specific DARPins make the DARPin approach useful for drug discovery. Additionally, DARPins can be easily generated in multispecific formats, offering the potential to target an effector DARPin to a specific organ or to target multiple receptors with one molecule composed of several DARPins.

  3. SPECT assay of radiolabeled monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Jaszczak, R.J.

    1992-02-01

    The long-term goal of this research project is to develop methods to improve the utility of single photon emission computed tomography (SPECI) to quantify the biodistribution of monoclonal antibodies (MoAbs) labeled with clinically relevant radionuclides ({sup 123}I, {sup 131}I, and {sup 111}In) and with another radionuclide,{sup 211}At, recently used in therapy. We describe here our progress in developing quantitative SPECT methodology for {sup 111}In and {sup 123}I. We have focused our recent research thrusts on the following aspects of SPECT: (1) The development of improved SPECT hardware, such as improved acquisition geometries. (2) The development of better reconstruction methods that provide accurate compensation for the physical factors that affect SPECT quantification. (3) The application of carefully designed simulations and experiments to validate our hardware and software approaches.

  4. DARPins: a true alternative to antibodies.

    Science.gov (United States)

    Stumpp, Michael T; Amstutz, Patrick

    2007-03-01

    Designed ankyrin repeat proteins (DARPins) are a promising class of non-immunoglobulin proteins that can offer advantages over antibodies for target binding in drug discovery and drug development. DARPins have been successfully used, for example, for the inhibition of kinases, proteases and drug-exporting membrane proteins. DARPins specifically targeting the cancer marker HER2 have also been generated and were shown to function in both in vitro diagnostics and in vivo tumor targeting. DARPins are ideally suited for in vivo imaging or delivery of toxins or other therapeutic payloads because of their favorable molecular properties, including small size and high stability. The low-cost production in bacteria and the rapid generation of many target-specific DARPins make the DARPin approach useful for drug discovery. Additionally, DARPins can be easily generated in multispecific formats, offering the potential to target an effector DARPin to a specific organ or to target multiple receptors with one molecule composed of several DARPins. PMID:17436550

  5. SPECT assay of radiolabeled monoclonal antibodies

    International Nuclear Information System (INIS)

    The long-term goal of this research project is to develop methods to improve the utility of single photon emission computed tomography (SPECI) to quantify the biodistribution of monoclonal antibodies (MoAbs) labeled with clinically relevant radionuclides (123I, 131I, and 111In) and with another radionuclide,211At, recently used in therapy. We describe here our progress in developing quantitative SPECT methodology for 111In and 123I. We have focused our recent research thrusts on the following aspects of SPECT: (1) The development of improved SPECT hardware, such as improved acquisition geometries. (2) The development of better reconstruction methods that provide accurate compensation for the physical factors that affect SPECT quantification. (3) The application of carefully designed simulations and experiments to validate our hardware and software approaches

  6. Monoclonal antibodies for the control of influenza virus vaccines.

    NARCIS (Netherlands)

    H.J.M. van de Donk; M.F. van Olderen; A.D.M.E. Osterhaus (Albert); J.C. de Jong (Jan)

    1984-01-01

    textabstractHybridomas producing haemagglutination inhibiting monoclonal antibodies against influenza A/Texas/1/77 H3N2 were developed. One hybridoma producing antibodies reacting with Victoria/3/75, Texas/1/77 Bangkok/1/79 and England/496/80 was selected to determine the potency of influenza virusv

  7. Detection of novel diagnostic antibodies in ankylosing spondylitis: An overview.

    Science.gov (United States)

    Quaden, Dana H F; De Winter, Liesbeth M; Somers, Veerle

    2016-08-01

    Ankylosing spondylitis (AS) is a debilitating, chronic, rheumatic disease characterized by inflammation and new bone formation resulting in fusion of the spine and sacroiliac joints. Since early treatment is impeded by a delayed diagnosis, it is highly important to find new biomarkers that improve early diagnosis and may also contribute to a better assessment of disease activity, prognosis and therapy response in AS. Because of the absence of rheumatoid factor, AS was long assumed to have a seronegative character and antibodies are thus not considered a hallmark of the disease. However, emerging evidence suggests plasma cells and autoantibodies to be involved in the disease course. In this review, the role of B cells and antibodies in AS is discussed. Furthermore, an overview is provided of antibodies identified in AS up till now, and their diagnostic potential. Many of these antibody responses were based on small study populations and further validation is lacking. Moreover, most were identified by a hypothesis-driven approach and thus limited to antibodies against targets that are already known to be involved in AS pathogenesis. Hence, we propose an unbiased approach to identify novel diagnostic antibodies. The already successfully applied techniques cDNA phage display and serological antigen selection will be used to identify antibodies against both known and new antigen targets in AS plasma. These newly identified antibodies will enhance early diagnosis of AS and provide more insight into the underlying disease pathology, resulting in a more effective treatment strategy and eventually an improved disease outcome. PMID:27288842

  8. Monoclonal antibodies to Herpes Simplex Virus Type 2

    International Nuclear Information System (INIS)

    In this thesis the production and characterisation of monoclonal antibodies to Herpes Simplex Virus Type 2 is described. The development of a suitable radioimmunoassay for the detection of anti-HSV-2 antibodies, and the selection of an optimal immunisation schedule, is given. Three assay systems are described and their reliability and sensitivity compared. (Auth.)

  9. Human-like antibodies neutralizing Western equine encephalitis virus

    Science.gov (United States)

    Hülseweh, Birgit; Rülker, Torsten; Pelat, Thibaut; Langermann, Claudia; Frenzel, Andrè; Schirrmann, Thomas; Dübel, Stefan; Thullier, Philippe; Hust, Michael

    2014-01-01

    This study describes the development of the first neutralizing antibodies against Western equine encephalitis virus (WEEV), a member of the genus Alphavirus. WEEV is transmitted by mosquitoes and can spread to the human central nervous system, causing symptoms ranging from mild febrile reactions to life-threatening encephalitis. WEEV has been classified as a biological warfare agent by the US Centers for Disease Control and Prevention. No anti-WEEV drugs are currently commercially available. Neutralizing antibodies are useful for the pre- and post-exposure treatment of WEEV infections. In this study, two immune antibody gene libraries were constructed from two macaques immunized with inactivated WEEV. Four antibodies were selected from these libraries and recloned as scFv-Fc, with a human Fc part. These antibodies bound WEEV specifically in ELISA with little or no cross-reaction with other alphaviruses. They were further analyzed by immunohistochemistry. All binders were suitable for the intracellular detection of WEEV particles. Neutralizing activity was determined in vitro. Three of the four antibodies were found to be neutralizing; about 1 ng/mL of the best antibody (ToR69–3A2) neutralized 50% of 5x104 TCID50/mL. Due to its human-like nature with a germinality index of 89% (VH) and 91% (VL), the ToR69–3A2 antibody is a promising candidate for future passive vaccine development. PMID:24518197

  10. Structural Comparison of Different Antibodies Interacting with Parvovirus Capsids

    Energy Technology Data Exchange (ETDEWEB)

    Hafenstein, Susan; Bowman, Valorie D.; Sun, Tao; Nelson, Christian D.S.; Palermo, Laura M.; Chipman, Paul R.; Battisti, Anthony J.; Parrish, Colin R.; Rossmann, Michael G.; Cornell; Purdue

    2009-05-13

    The structures of canine parvovirus (CPV) and feline parvovirus (FPV) complexed with antibody fragments from eight different neutralizing monoclonal antibodies were determined by cryo-electron microscopy (cryoEM) reconstruction to resolutions varying from 8.5 to 18 {angstrom}. The crystal structure of one of the Fab molecules and the sequence of the variable domain for each of the Fab molecules have been determined. The structures of Fab fragments not determined crystallographically were predicted by homology modeling according to the amino acid sequence. Fitting of the Fab and virus structures into the cryoEM densities identified the footprints of each antibody on the viral surface. As anticipated from earlier analyses, the Fab binding sites are directed to two epitopes, A and B. The A site is on an exposed part of the surface near an icosahedral threefold axis, whereas the B site is about equidistant from the surrounding five-, three-, and twofold axes. One antibody directed to the A site binds CPV but not FPV. Two of the antibodies directed to the B site neutralize the virus as Fab fragments. The differences in antibody properties have been linked to the amino acids within the antibody footprints, the position of the binding site relative to the icosahedral symmetry elements, and the orientation of the Fab structure relative to the surface of the virus. Most of the exposed surface area was antigenic, although each of the antibodies had a common area of overlap that coincided with the positions of the previously mapped escape mutations.

  11. Monoclonal Antibodies Specific for Hippurate Hydrolase of Campylobacter jejuni

    OpenAIRE

    Steele, Marina; Gyles, Carlton; Chan, Voon Loong; Odumeru, Joseph

    2002-01-01

    Eleven monoclonal antibodies raised against recombinant Campylobacter jejuni hippurate hydrolase were tested for binding to lysates from 19 C. jejuni strains, 12 other Campylobacter strains, and 21 non-Campylobacter strains. Several monoclonal antibodies bound to C. jejuni but not to other Campylobacter species and may be useful in a species-specific immunoassay.

  12. High throughput production of mouse monoclonal antibodies using antigen microarrays

    DEFF Research Database (Denmark)

    De Masi, Federico; Chiarella, P.; Wilhelm, H.;

    2005-01-01

    Recent advances in proteomics research underscore the increasing need for high-affinity monoclonal antibodies, which are still generated with lengthy, low-throughput antibody production techniques. Here we present a semi-automated, high-throughput method of hybridoma generation and identification...

  13. Antibody function in neutralization and protection against HIV-1

    NARCIS (Netherlands)

    Hessell, A.J.

    2009-01-01

    The ability to induce neutralizing antibodies is generally thought to be of great importance for vaccine efficacy. In HIV-1 research this quality has been elusive as the HIV-1 virus has evolved multiple mechanisms to evade neutralizing antibodies. This thesis traces studies with four broadly neutral

  14. Plasma antibody levels in periodontitis patients and controls

    NARCIS (Netherlands)

    Graswinckel, JEM; van der Velden, U; van Winkelhoff, AJ; Hoek, FJ; Loos, BG

    2004-01-01

    Background: A major aspect of the adaptive host response in periodontitis is the production of antibodies. Several risk and susceptibility factors for periodontitis, including smoking, age and composition of the subgingival microflora, have also been suggested to influence antibody production. Aim:

  15. Anticarbohydrate antibodies in patients with inflammatory bowel disease

    Directory of Open Access Journals (Sweden)

    Mohammed Fakhraldeen

    2010-06-01

    Full Text Available Evaluating the prevalence of antichitobioside carbohydrate antibody (ACCA, antilaminaribioside carbohydrate antibodies (ALCA, antimannobioside carbohydrate antibodies (AMCA and anti-Saccharomyces cerevisiae antibodies (ASCA, in patients with inflammatory bowel disease (IBD. Subjects and methods: 268 serum samples were used; 115 Crohn's disease (CD, 83 ulcerative colitis, and 70 healthy control samples. All samples were evaluated using enzyme-linked immunosorbent assay for the following four anticarbohydrate antibodies: ACCA, ALCA, AMCA, and ASCA. Results: In patients with Crohn's disease the prevalence of the anticarbohydrate antibodies was: ASCA 69%, AMCA 32%, ACCA 28% and ALCA 24% with the highest prevalence being for ASCA (P-value<0.0001 while in patients with ulcerative colitis the prevalence was: ACCA 46%, AMCA 35%, ALCA 23% and ASCA 15% with the highest prevalence being for ACCA (P-value<0.001. Conclusion: Anticarbohydrate antibodies are significantly present in patients with IBD. The use of a panel of anticarbohydrate antibodies may provide additional help in distinguishing IBD from non-IBD disease patterns and narrow the range of differential diagnosis in these patients.

  16. Antibody and B cell responses to Plasmodium sporozoites

    Directory of Open Access Journals (Sweden)

    Johanna N Dups

    2014-11-01

    Full Text Available Antibodies are capable of blocking infection of the liver by Plasmodium sporozoites. Accordingly the induction of anti-sporozoite antibodies is a major aim of various vaccine approaches to malaria. In recent years our knowledge of the specificity and quantities of antibodies required for protection has been greatly expanded by clinical trials of various whole sporozoite and subunit vaccines. Moreover, the development of humanized mouse models and transgenic parasites have also aided our ability to assess the specificity of antibodies and their ability to block infection. Nonetheless, considerable gaps remain in our knowledge - in particular in understanding what antigens are recognized by infection blocking antibodies and in knowing how we can induce robust, long-lived antibody responses. Maintaining high levels of circulating antibodies is likely to be of primary importance, as antibodies must block infection in the short time it takes for sporozoites to reach the liver from the skin. It is clear that a better understanding of the development of protective B cell-mediated immunity will aid the development and refinement of malaria vaccines.

  17. Detection of novel diagnostic antibodies in ankylosing spondylitis: An overview.

    Science.gov (United States)

    Quaden, Dana H F; De Winter, Liesbeth M; Somers, Veerle

    2016-08-01

    Ankylosing spondylitis (AS) is a debilitating, chronic, rheumatic disease characterized by inflammation and new bone formation resulting in fusion of the spine and sacroiliac joints. Since early treatment is impeded by a delayed diagnosis, it is highly important to find new biomarkers that improve early diagnosis and may also contribute to a better assessment of disease activity, prognosis and therapy response in AS. Because of the absence of rheumatoid factor, AS was long assumed to have a seronegative character and antibodies are thus not considered a hallmark of the disease. However, emerging evidence suggests plasma cells and autoantibodies to be involved in the disease course. In this review, the role of B cells and antibodies in AS is discussed. Furthermore, an overview is provided of antibodies identified in AS up till now, and their diagnostic potential. Many of these antibody responses were based on small study populations and further validation is lacking. Moreover, most were identified by a hypothesis-driven approach and thus limited to antibodies against targets that are already known to be involved in AS pathogenesis. Hence, we propose an unbiased approach to identify novel diagnostic antibodies. The already successfully applied techniques cDNA phage display and serological antigen selection will be used to identify antibodies against both known and new antigen targets in AS plasma. These newly identified antibodies will enhance early diagnosis of AS and provide more insight into the underlying disease pathology, resulting in a more effective treatment strategy and eventually an improved disease outcome.

  18. MONOCLONAL ANTIBODIES TO IDENTIFY TOMATO MOSAIC TOBAMOVIRUS (TOMV

    Directory of Open Access Journals (Sweden)

    Duarte Keila M.R.

    2001-01-01

    Full Text Available Monoclonal antibodies were obtained against Tomato mosaic tobamovirus (ToMV isolated in Brazil. One antibody (8G7G2 isotyped as IgG2b (kappa light chain showed strong specificity and very low cross reaction with the Tobacco mosaic virus (TMV. It can be used in identification of tomato mosaic virus (ToMV.

  19. Antibodies to Berne virus in horses and other animals

    NARCIS (Netherlands)

    Horzinek, M.C.; Weiss, Marianne; Steck, F.; Kaderli, R.

    1984-01-01

    After inoculation into 2 foals, Berne virus induced neutralizing antibody, but did not cause clinical symptoms. In a horizontal study of seropositive mares and their offspring, a decline of maternal antibodies and a sudden synchronous seroconversion in all foals were observed, again without clinical

  20. Antibody phage display applications for nuclear medicine imaging and therapy

    Energy Technology Data Exchange (ETDEWEB)

    Winthrop, M.D.; Denardo, G.L.; Denardo, S.J. [Sacramento Univ. of California Davis Medical Center, Sacramento, CA (United States). Dept. of Internal Medicine, Div. of Radiodiagnosis and Terapy

    2000-09-01

    Antibody-based constructs genetically engineered from genes of diverse origin provide a remarkable opportunity to develop functional molecular imaging techniques and specific molecular targeted radionuclide therapies. Phage display libraries of antibody fragment genes can be used to select antibody-based constructs that bind any chosen epitope. A large naive human antibody-based library was used to illustrate binding of antibody constructs to a variety of common and unique antigens. Antibody-based libraries from hybridoma cells, lymphocytes from immunized humans or from mice and human antibody repertoires produced in transgenic mice have also been described. Several orders of magnitude of affinity enhancement can be achieved by random or site specific mutations of the selected binding peptide domains of the scFv. Affinities (K{sub d}) as high as 10{sup -}11 M (10 pM) for affinity-matured scFv have been documented. Such gene libraries thus offer an almost limitless variety of antibody-based molecular binding peptide modules that can be used in creative ways for the construction of new targeting agents for functional or molecular imaging and therapy.

  1. Anti-GM-CSF antibodies in paediatric pulmonary alveolar proteinosis

    OpenAIRE

    Latzin, P; Tredano, M.; Wust, Y; J. de Blic; Nicolai, T; Bewig, B; Stanzel, F.; Kohler, D.; Bahuau, M.; Griese, M

    2005-01-01

    Background: Auto-antibodies against granulocyte-macrophage colony stimulating factor (GM-CSF) may be central to the pathogenesis of adult sporadic pulmonary alveolar proteinosis (PAP). The role of anti-GM-CSF auto-antibodies in paediatric forms of PAP is as yet unclear.

  2. Cell-Free Synthesis Meets Antibody Production: A Review

    Directory of Open Access Journals (Sweden)

    Marlitt Stech

    2015-01-01

    Full Text Available Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv and antigen binding fragments (Fab, have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems.

  3. Postbooster Antibodies from Humans as Source of Diphtheria Antitoxin

    OpenAIRE

    Bermejo-Martin, Jesús F; Avila-Alonso, Ana; González-Rivera, Milagros; Tamayo, Eduardo; Eiros, Jose María; Almansa, Raquel

    2016-01-01

    Diphtheria antitoxin for therapeutic use is in limited supply. A potential source might be affinity-purified antibodies originally derived from plasma of adults who received a booster dose of a vaccine containing diphtheria toxoid. These antibodies might be useful for treating even severe cases of diphtheria.

  4. Postbooster Antibodies from Humans as Source of Diphtheria Antitoxin.

    Science.gov (United States)

    Bermejo-Martin, Jesús F; Avila-Alonso, Ana; González-Rivera, Milagros; Tamayo, Eduardo; Eiros, Jose María; Almansa, Raquel

    2016-07-01

    Diphtheria antitoxin for therapeutic use is in limited supply. A potential source might be affinity-purified antibodies originally derived from plasma of adults who received a booster dose of a vaccine containing diphtheria toxoid. These antibodies might be useful for treating even severe cases of diphtheria. PMID:27314309

  5. Development of Biodegradable Nanocarriers Loaded with a Monoclonal Antibody

    Directory of Open Access Journals (Sweden)

    Andrew Gdowski

    2015-02-01

    Full Text Available Treatments utilizing monoclonal antibody therapeutics against intracellular protein-protein interactions in cancer cells have been hampered by several factors, including poor intracellular uptake and rapid lysosomal degradation. Our current work examines the feasibility of encapsulating monoclonal antibodies within poly(lactic-co-glycolic acid (PLGA nanoparticles using a water/oil/water double emulsion solvent evaporation technique. This method can be used to prepare protective polymeric nanoparticles for transporting functional antibodies to the cytoplasmic compartment of cancer cells. Nanoparticles were formulated and then characterized using a number of physical and biological parameters. The average nanoparticle size ranged from 221 to 252 nm with a low polydispersity index. Encapsulation efficiency of 16%–22% and antibody loading of 0.3%–1.12% were observed. The antibody molecules were released from the nanoparticles in a sustained manner and upon release maintained functionality. Our studies achieved successful formulation of antibody loaded polymeric nanoparticles, thus indicating that a PLGA-based antibody nanoformulation is a promising intracellular delivery vehicle for a large number of new intracellular antibody targets in cancer cells.

  6. Functionally fused antibodies--a novel adjuvant fusion system

    DEFF Research Database (Denmark)

    Larsen, Martin; Jensen, Kim Bak; Christensen, Peter Astrup;

    2008-01-01

    Antibodies capable of recognizing key molecular targets isolated e.g. by phage display technology have been used in the pursuit of new and improved therapies for prevalent human diseases. These approaches often take advantage of non-immunogenic antibody fragments to achieve specific toxin-, radio...

  7. Dissecting antibodies with regards to linear and conformational epitopes.

    Science.gov (United States)

    Forsström, Björn; Axnäs, Barbara Bisławska; Rockberg, Johan; Danielsson, Hanna; Bohlin, Anna; Uhlen, Mathias

    2015-01-01

    An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets. PMID:25816293

  8. Dissecting antibodies with regards to linear and conformational epitopes.

    Directory of Open Access Journals (Sweden)

    Björn Forsström

    Full Text Available An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets.

  9. Double-antibody radioimmunoassay for staphylococcal enterotoxin C2

    International Nuclear Information System (INIS)

    A sensitive double-antibody radioimmunoassay for staphylococcal enterotoxin C2 is described. The assay procedure employs anti-rabbit gamma globulin, prepared in goats, to precipitate the antigen-antibody complex of enterotoxin C2 and anti-enterotoxin C2. The test is sensitive to 100 pg of enterotoxin

  10. A Quantitative Method for Comparing the Brightness of Antibody-dye Reagents and Estimating Antibodies Bound per Cell.

    Science.gov (United States)

    Kantor, Aaron B; Moore, Wayne A; Meehan, Stephen; Parks, David R

    2016-01-01

    We present a quantitative method for comparing the brightness of antibody-dye reagents and estimating antibodies bound per cell. The method is based on complementary binding of test and fill reagents to antibody capture microspheres. Several aliquots of antibody capture beads are stained with varying amounts of the test conjugate. The remaining binding sites on the beads are then filled with a second conjugate containing a different fluorophore. Finally, the fluorescence of the test conjugate compared to the fill conjugate is used to measure the relative brightness of the test conjugate. The fundamental assumption of the test-fill method is that if it takes X molecules of one test antibody to lower the fill signal by Y units, it will take the same X molecules of any other test antibody to give the same effect. We apply a quadratic fit to evaluate the test-fill signal relationship across different amounts of test reagent. If the fit is close to linear, we consider the test reagent to be suitable for quantitative evaluation of antibody binding. To calibrate the antibodies bound per bead, a PE conjugate with 1 PE molecule per antibody is used as a test reagent and the fluorescence scale is calibrated with Quantibrite PE beads. When the fluorescence per antibody molecule has been determined for a particular conjugate, that conjugate can be used for measurement of antibodies bound per cell. This provides comparisons of the brightness of different conjugates when conducted on an instrument whose statistical photoelectron (Spe) scales are known. © 2016 by John Wiley & Sons, Inc.

  11. Antibody response and antibody affinity maturation in cats with experimental proliferative immune complex glomerulonephritis.

    Science.gov (United States)

    Bishop, S A; Bailey, M; Lucke, V M; Stokes, C R

    1992-07-01

    An experimental model of proliferative glomerulonephritis (GN) in the cat, which closely resembles human proliferative forms of GN, has been used to study the role of antibody and antibody affinity in the development of immune complex-mediated renal disease. The serum IgG and IgM antibody response to antigen, average antibody affinity (avidity) and affinity heterogeneity of the IgG and IgM populations was assessed at varying times after commencement of chronic immunization with the antigen, human serum albumin (HSA), by enzyme immunoassay. Cats could be classified according to whether they were "low", "intermediate" or "high" IgG responders, by quantification of serum IgG values. Cats with the lowest serum IgG values failed to develop glomerulonephritis. However, there was no relationship between actual IgG values and the severity of the induced disease. In contrast to IgG, there was no division of cats into low or high IgM anti-HSA responders. Again, cats with the lowest IgM values failed to develop GN, but, more interestingly, a late, marked increase in serum IgM anti-HSA occurred only in cats that developed clinical signs of GN (anterior uveitis and nephrotic syndrome). Maturation of average, functional IgG affinity (avidity) for HSA following chronic immunization was clearly demonstrated for all cats. At the end of the experiment, all cats had IgG of high affinity for HSA and the average affinity heterogeneity of the IgG populations was less than in measurements taken earlier. Values of IgG affinity at the end of the experiment were very similar both in cats which developed GN and in those which remained clinically, biochemically and pathologically normal. In contrast to IgG antibody, some cats developed IgM of increased affinity, whilst others produced antibody of reduced affinity, following chronic immunization. There was no correlation between the development of disease and the production of either low or high affinity IgM antibody. Data indicated that an

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

    Science.gov (United States)

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

    2003-12-15

    Parasite drug resistance and difficulties in developing effective vaccines have precipitated the search for alternative therapies for malaria. The success of passive immunization suggests that immunoglobulin (Ig)-based therapies are effective. To further explore the mechanism(s) by which antibody mediates its protective effect, we generated human chimeric IgG1 and IgA1 and a single-chain diabody specific for the C-terminal 19-kDa region of Plasmodium yoelii merozoite surface protein 1 (MSP119), a major target of protective immune responses. These novel human reagents triggered in vitro phagocytosis of merozoites but, unlike their parental mouse IgG2b, failed to protect against parasite challenge in vivo. Therefore, the Fc region appears critical for mediating protection in vivo, at least for this MSP119 epitope. Such antibodies may serve as prototype therapeutic agents, and as useful tools in the development of in vitro neutralization assays with Plasmodium parasites. PMID:12855589

  13. Maturation Pathways of Cross-Reactive HIV-1 Neutralizing Antibodies

    Directory of Open Access Journals (Sweden)

    Dimiter S. Dimitrov

    2009-11-01

    Full Text Available Several human monoclonal antibodies (hmAbs and antibody fragments, including the best characterized in terms of structure-function b12 and Fab X5, exhibit relatively potent and broad HIV-1 neutralizing activity. However, the elicitation of b12 or b12-like antibodies in vivo by vaccine immunogens based on the HIV-1 envelope glycoprotein (Env has not been successful. B12 is highly divergent from the closest corresponding germline antibody while X5 is less divergent. We have hypothesized that the relatively high degree of specific somatic hypermutations may preclude binding of the HIV-1 envelope glycoprotein (Env to closest germline antibodies, and that identifying antibodies that are intermediates in the pathways to maturation could help design novel vaccine immunogens to guide the immune system for their enhanced elicitation. In support of this hypothesis we have previously found that a germline-like b12 (monovalent and bivalent scFv as an Fc fusion protein or IgG lacks measurable binding to an Env as measured by ELISA with a sensitivity in the μM range [1]; here we present evidence confirming and expanding these findings for a panel of Envs. In contrast, a germline-like scFv X5 bound Env with high (nM affinity. To begin to explore the maturation pathways of these antibodies we identified several possible b12 intermediate antibodies and tested their neutralizing activity. These intermediate antibodies neutralized only some HIV-1 isolates and with relatively weak potency. In contrast, germline-like scFv X5 neutralized a subset of the tested HIV-1 isolates with comparable efficiencies to that of the mature X5. These results could help explain the relatively high immunogenicity of the coreceptor binding site on gp120 and the abundance of CD4-induced (CD4i antibodies in HIV-1-infected patients (X5 is a CD4i antibody as well as the maturation pathway of X5. They also can help identify antigens that can bind specifically to b12 germline and

  14. Modification of monoclonal antibodies by polymers possessing chelating properties

    Energy Technology Data Exchange (ETDEWEB)

    Torchilin, V.P.; Khaw, B.A.; Klibanov, A.L.; Slinkin, M.A.; Haber, E.; Smirnov, V.N.

    1986-12-01

    This paper describes a basically new approach to obtaining diagnostic antibodies, consisting of a one-point modification of the antibody, without loss of its activity, by a high-molecular-weight synthetic polymer with the ability of effectively chelating ions of heavy metals. As a result of this approach, preparations of active antibodies containing some tens of atoms of the metal per protein molecule can be obtained. The concentration of radioactive metal (/sup 111/In) was determined with a gamma-counter and the Mn and Cd concentrations by spectroscopy. Gel-filtration of polymer-modified antibodies after binding of /sup 111/InCl/sub 3/ is shown. Also, solid-phase radioimmunoassay of antibodies and Fab fragments, native and modified by chelating polymers, is presented.

  15. Prokaryotic Expression and Polyclonal Antibody Preparation of PRL3

    Institute of Scientific and Technical Information of China (English)

    SHEN Xing-gui; LI Wan-nan; WANG Jing; JIANG Yi-qun; LI Qing-shan; FU Xue-qi

    2007-01-01

    Phosphatase of regenerating liver 3(PRL3), which belongs to the superfamily of protein tyrosine phosphatases (PTPs), represents a group of low molecular weight PTPs that participate in tumorigenesis and metastasis processes.Presented here are the results of cloning, prokaryotic expression, purification, and polyclonal antibody preparation of PRL3. To obtain a specific polyclonal antibody against PRL3, the authors have prepared GST-PRL3 to immunize rabbits and purify an anti-PRL3 polyclonal antibody by negative selection affinity columns. Western blot analysis shows that the anti-PRL3 polyclonal antibody has a specific binding ability with PRL3 protein. The anti-PRL3 polyclonal antibody provides a good tool to further study the function of PRL3.

  16. ELISA Detection of Francisella tularensis using Polyclonaland Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Miroslav Pohanka

    2008-09-01

    Full Text Available The mouse monoclonal and polyclonal antibodies were produced for the detection of intracellular pathogenand potential warfare agent Francisella tularensis. Antibody titers obtained were 1:640 for polyclonal antibodiesand 1:320 for monoclonal antibodies. Both antibodies were used in the indirect enzyme-linked immunosorbentassay (ELISA found to detect F. tularensis whole cells. The limit of detection was 5.4×106 CFU/ml for polyclonalantibodies and 6.9×106 CFU/ml for monoclonal antibodies. The value sample could  be distinguished from anyconcentration of another gram-negative bacterium: Escherichia coli.Defence Science Journal, 2008, 58(5, pp.698-702, DOI:http://dx.doi.org/10.14429/dsj.58.1693

  17. [A spectrum of neurological diseases with anti-VGKC antibody].

    Science.gov (United States)

    Arimura, Kimiyoshi; Watanabe, Osamu; Nagado, Tatsui

    2007-11-01

    Anti-VGKC antibody causing peripheral nerve hyperexcitability is already an established clinical entity. Recently, many patients with non-herpetic limbic encephalitis (NHLE) with anti-VGKC antibody have been reported. The characteristic clinical features are low serum Na+ concentration and good response to immunotherapy. Anti-VGK antibody positive NHLE is relatively frequent among immune-mediated NHLE. It is important to know that this disease is responsive to immunotherapy. Furthermore, anti-VGKC antibody is also positive in some intractable epilepsies. These findings suggest that anti-VGKC is correlated with hyperexcitability in both the peripheral and central nervous system and that the spectrum of anti-VGKC antibody syndrome is now expanding.

  18. Phosphokinase antibody arrays on dendron-coated surface.

    Directory of Open Access Journals (Sweden)

    Ju-Won Kwak

    Full Text Available Monitoring protein phosphorylation at the cellular level is important to understand the intracellular signaling. Among the phosphoproteomics methods, phosphokinase antibody arrays have emerged as preferred tools to measure well-characterized phosphorylation in the intracellular signaling. Here, we present a dendron-coated phosphokinase antibody array (DPA in which the antibodies are immobilized on a dendron-coated glass slide. Self-assembly of conically shaped dendrons well-controlled in size and structure resulted in precisely controlled lateral spacing between the immobilized phosphosite-specific antibodies, leading to minimized steric hindrance and improved antigen-antibody binding kinetics. These features increased sensitivity, selectivity, and reproducibility in measured amounts of protein phosphorylation. To demonstrate the utility of the DPA, we generated the phosphorylation profiles of brain tissue samples obtained from Alzheimer's disease (AD model mice. The analysis of the profiles revealed signaling pathways deregulated during the course of AD progression.

  19. The Role of Complement in Antibody Therapy for Infectious Diseases.

    Science.gov (United States)

    Wibroe, Peter P; Helvig, Shen Y; Moein Moghimi, S

    2014-04-01

    The complement system is part of the innate immune system, eliciting central immunoregulatory functions. Detection of foreign surfaces is either achieved through complement-specific patternrecognition molecules or mediated by antigen recognition of antibodies. Immunoglobulin A (IgA), IgG, and IgM all have the potential to initiate a complement response, with the efficiency and response development closely related to the antibody isotype, multimeric state, and degree of glycosylation. A group of serum proteins constitutes the central effector functions of complement, thus allowing direct cell lysis, opsonization, and inflammation. These effector functions can be used in antibody therapies, especially against infectious diseases, as the target membranes lack complement regulatory proteins. The relative contribution of each function and the interplay with direct antibody-mediated clearance is not fully exploited, thus suggesting an option for further rational optimization of antibody therapies.

  20. Chronic Renal Allograft Dysfunction Antibody-Mediated: An Update

    Directory of Open Access Journals (Sweden)

    Maurizio Salvadori,

    2014-07-01

    Full Text Available This paper reviews the most important studies on chronic antibody-mediated rejection (cABMR, which is an important cause of late graft dysfunction after renal transplantation. Several antibodies seem to be responsible for chronic rejection; new techniques have allowed us to identify these antibodies in circulation. The pathogenetic role of the antibodies generally includes the complement pathway, but may also be complement-independent. This paper also examines the pathogenesis of chronic endothelial lesions, as well as the histopathological aspects. Antibodies responsible for chronic rejection may preexist before transplantation or may develop after transplantation. The possible therapeutic approaches are poor and principally based on early identification and desensitisation techniques. New B cell targeting drugs are aimed at an improved control of the relevant condition.

  1. Triiodothyronine uptake test using specific antibody as the secondary binder

    International Nuclear Information System (INIS)

    We describe a specific and reliable triiodithyronine uptake (T3-U) method for the estimation of free thyroxine index(FT4I) using precipitated anti-T3-antibody and second-antibody (anti-rabbit-IgG raised in goat) complex. Since the method measures the partitioning of T3-125I between the binding proteins and antibody, separation of the portion of T3-125I taken up by the antibody from that bound to the binding proteins is achieved by the use of pre-incubated primary and second antibody complex in the form of suspension, and separation of the bound complex was carried out by means of centrifugation. The T3-U method developed is clinically evaluated and compared with the reputed commercial kit and a correlation of 0.96 was obtained. (author)

  2. Development of radioactivity labelling method of new antibody by using the antibody engineering

    International Nuclear Information System (INIS)

    With an aim to develop a method to produce labelled antibodies with low immunogenicity, two recombinant fusion proteins; scFv-His and scFv-MTβ were produced using gene engineering techniques. The former was constructed with scFv-antibody and histidine hexamer, a metal-chelated protein (or peptide). The latter was done with scFv-antibody and β-domain of metallothionein. Then, antigen-binding activity and metal-binding activity of these fusion proteins were determined using gel-filtration chromatography and ELISA. The main antigen-binding activity of scFv-His preparation was detected in a domain of about 25-30 kDa, which agreed with the peak of 29 kDa corresponding to the presumed molecular weight for the protein. Whereas the antigen-binding activity of scFv-MTβ was found in a domain of 30-35 kDa, which agreed with 32 kDa, the presumed molecular weight of scFv-MTβ. Gel-filtration chromatography of scFv-His preparation after the addition of Cu2+ ion revealed an optical absorption at 280 nm and a Cu-peak near at 14 kDa. These results suggested that the metal affinity of the histidine-hexamer was too weak to chelate Cu2+ in a solution. The chromatography of scFv-MTβ preparation added with Cd2+ showed a peak of Cd appeared around a position of about 20 kDa but the peak was not coincident with that of the antigen-binding activity (ca. 30 kDa), suggesting that the present preparation of scFv-MTβ had no Cd-binding activity due to metal-exchange reaction. Based on these results, problems on the production of recombinant scFv-antibody fused with metal-binding domain of cystein-binding type or histidine-binding one were discussed. (M.N.)

  3. Clearance of injected radioactively labelled antibodies to tumour products by liposome-bound second antibodies

    International Nuclear Information System (INIS)

    Liposomes containing anti-goat immunoglobulin were injected 24 h after administration of 125I-labelled goat antibody against the carcinoembryonic antigen (anti-CEA) to groups of nude mice bearing human tumour xenografts, and normal mice. Controls in each group received radioactively labelled anti-CEA only. In liposome-treated mice, blood 125I levels were lower than those of controls 30 min to 24 h after liposome administration, with corresponding accumulation of 125I activity in the liver and spleen for the first 2 h after liposome injection. [14C]Cholesterol or sup(99m)Tc labels in the bilayer were eliminated rapidly from the blood, with uptake in the liver and spleen. In xenograft-bearing mice, 125I activity detected in the tumours up to 6 h after liposome injection was identical to that detected in the tumours of controls. However, 24 h after liposome injection a reduction in the tumour concentration of 125I-labelled anti-CEA was obtained, but the tumour/blood radioactivity was still increased. In two mice given 27 μmol lipid, the blood radioactivity count after 24 h was only 5% of that in the controls. In rabbits, 2 h after administration of liposomes containing anti-goat second antibody, the circulating 125I activity had dropped by 28-40%. The results suggest that administration of liposome-entrapped second antibody approximately 2 h prior to external scintigraphy may clear circulating radioactively labelled primary antibody by up to 50%. (Auth.)

  4. Polyclonal anti-idiotypic antibodies mimicking the small cell lung carcinoma antigen cluster-5A interact with a panel of antibodies and induce specific immune response in animals.

    OpenAIRE

    Zwicky, C.; Stahel, R A; Jaksche, H.; Waibel, R.; Lehmann, H. P.; Loibner, H

    1991-01-01

    Polyclonal anti-idiotypic antibodies (ab2) were generated by immunising goats with the murine IgG2a monoclonal antibody SWA20 which recognises the SCLC antigen cluster-5A, a tumour-associated sialoglycoprotein. Ab2 was shown to bind specifically to antibody SWA20, but not to isotype matched control antibodies. Pre-incubation with ab2 completely inhibited target cell binding of antibody SWA20 and of four other antibodies to cluster-5A antigen, while no effect was seen with antibodies to cluste...

  5. Antiphospholipid antibodies in Brazilian hepatitis C virus carriers

    Directory of Open Access Journals (Sweden)

    A.M. Atta

    2008-06-01

    Full Text Available Hepatitis C, a worldwide viral infection, is an important health problem in Brazil. The virus causes chronic infection, provoking B lymphocyte dysfunction, as represented by cryoglobulinemia, non-organ-specific autoantibody production, and non-Hodgkin's lymphoma. The aim of this research was to screen for the presence of antiphospholipid autoantibodies in 109 Brazilian hepatitis C virus carriers without clinical history of antiphospholipid syndrome. Forty healthy individuals were used as the control group. IgA, IgG, and IgM antibodies against cardiolipin and β2-glycoprotein I were measured with an enzyme-linked immunosorbent assay, using a cut-off point of either 20 UPL or 20 SBU. While 24 (22.0% hepatitis C carriers had moderate titers of IgM anticardiolipin antibodies (median, 22.5 MPL; 95%CI: 21.5-25.4 MPL, only three carriers (<3% had IgG anticardiolipin antibodies (median, 23 GPL; 95%CI: 20.5-25.5 GPL. Furthermore, IgA anticardiolipin antibodies were not detected in these individuals. Male gender and IgM anticardiolipin seropositivity were associated in the hepatitis C group (P = 0.0004. IgA anti-β2-glycoprotein-I antibodies were detected in 29 of 109 (27.0% hepatitis C carriers (median, 41 SAU; 95%CI: 52.7-103.9 SAU. Twenty patients (18.0% had IgM anti-β2-glycoprotein I antibodies (median, 27.6 SMU; 95%CI: 23.3-70.3 SMU, while two patients had IgG antibodies against this protein (titers, 33 and 78 SGU. Antiphospholipid antibodies were detected in only one healthy individual, who was seropositive for IgM anticardiolipin. We concluded that Brazilian individuals chronically infected with hepatitis C virus present a significant production of antiphospholipid antibodies, mainly IgA anti-β2-glycoprotein I antibodies, which are not associated with clinical manifestations of antiphospholipid syndrome.

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

    Science.gov (United States)

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

    2016-01-01

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

  7. Rifampicin-dependent antibodies bind a similar or identical epitope to glycoprotein IX-specific quinine-dependent antibodies

    NARCIS (Netherlands)

    Burgess, J K; Lopez, J A; Gaudry, L E; Chong, B H

    2000-01-01

    The drug-dependent antibody of a patient with rifampicin-induced thrombocytopenia was characterized using the antigen-capture enzyme-linked immunosorbent assay (MAIPA assay), flow cytometry, and immunoprecipitation. The antibody was found to bind glycoprotein (GP) Ib-IX but not GPIIb-IIIa because (1

  8. ANTIBODIES DEFINING RAT ENDOTHELIAL-CELLS - RECA-1, A PAN-ENDOTHELIAL CELL-SPECIFIC MONOCLONAL-ANTIBODY

    NARCIS (Netherlands)

    DUIJVESTIJN, AM; VANGOOR, H; KLATTER, F; MAJOOR, GD; VANBUSSEL, E; VRIESMAN, PJCV

    1992-01-01

    We have been searching for antibodies reactive with rat endothelial cells. Two monoclonal antibodies (mAb), named RECA-1 and RECA-2 were produced and tested in immunoperoxidase staining on frozen sections of various rat tissues. Staining patterns were compared to those obtained with the mAbs OX-2, O

  9. Differentiation of Behcet's disease from inflammatory bowel diseases: Anti-saccharomyces cerevisiae antibody and anti-neutrophilic cytoplasmic antibody

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The differential diagnosis of Behcet's disease (BD) from inflammatory bowel disease (IBD) is sometimes difficult and challenging. Hereby, we suggested the utility of anti-saccharomyces cerevisiae antibody (ASCA) and anti-neutrophilic cytoplasmic antibody (p-ANCA) in the differential diagnosis of BD from IBD.

  10. Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality.

    Science.gov (United States)

    Kibat, Janek; Schirrmann, Thomas; Knape, Matthias J; Helmsing, Saskia; Meier, Doris; Hust, Michael; Schröder, Christoph; Bertinetti, Daniela; Winter, Gerhard; Pardes, Khalid; Funk, Mia; Vala, Andrea; Giese, Nathalia; Herberg, Friedrich W; Dübel, Stefan; Hoheisel, Jörg D

    2016-09-25

    Many diagnostic and therapeutic concepts require antibodies of high specificity. Recombinant binder libraries and related selection approaches allow the efficient isolation of antibodies against almost every target of interest. Nevertheless, it cannot be guaranteed that selected antibodies perform well and interact specifically enough with analytes unless an elaborate characterisation is performed. Here, we present an approach to shorten this process by combining the selection of suitable antibodies with the identification of informative target molecules by means of antibody microarrays, thereby reducing the effort of antibody characterisation by concentrating on relevant molecules. In a pilot scheme, a library of 456 single-chain variable fragment (scFv) binders to 134 antigens was used. They were arranged in a microarray format and incubated with the protein content of clinical tissue samples isolated from pancreatic ductal adenocarcinoma and healthy pancreas, as well as recurrent and non-recurrent bladder tumours. We observed significant variation in the expression of the E3 ubiquitin-protein ligase (CHFR) as well as the glutamate receptor interacting protein 2 (GRIP2), for example, always with more than one of the scFvs binding to these targets. Only the relevant antibodies were then characterised further on antigen microarrays and by surface plasmon resonance experiments so as to select the most specific and highest affinity antibodies. These binders were in turn used to confirm a microarray result by immunohistochemistry analysis. PMID:26709003

  11. Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

    Science.gov (United States)

    Bidlingmaier, Scott; Su, Yang; Liu, Bin

    2015-01-01

    Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties. PMID:26060069

  12. A simple vector system to improve performance and utilisation of recombinant antibodies

    OpenAIRE

    Vincent Karen J; Mitchell Joanne N; Rojas Gertrudis; Martin Cecile D; Wu Jiahua; McCafferty John; Schofield Darren J

    2006-01-01

    Abstract Background Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression. Results We ha...

  13. Discovery and characterization of antibody variants using mass spectrometry-based comparative analysis for biosimilar candidates of monoclonal antibody drugs.

    Science.gov (United States)

    Li, Wenhua; Yang, Bin; Zhou, Dongmei; Xu, Jun; Ke, Zhi; Suen, Wen-Chen

    2016-07-01

    Liquid chromatography mass spectrometry (LC-MS) is the most commonly used technique for the characterization of antibody variants. MAb-X and mAb-Y are two approved IgG1 subtype monoclonal antibody drugs recombinantly produced in Chinese hamster ovary (CHO) cells. We report here that two unexpected and rare antibody variants have been discovered during cell culture process development of biosimilars for these two approved drugs through intact mass analysis. We then used comprehensive mass spectrometry-based comparative analysis including reduced light, heavy chains, and domain-specific mass as well as peptide mapping analysis to fully characterize the observed antibody variants. The "middle-up" mass comparative analysis demonstrated that the antibody variant from mAb-X biosimilar candidate was caused by mass variation of antibody crystalline fragment (Fc), whereas a different variant with mass variation in antibody antigen-binding fragment (Fab) from mAb-Y biosimilar candidate was identified. Endoproteinase Lys-C digested peptide mapping and tandem mass spectrometry analysis further revealed that a leucine to glutamine change in N-terminal 402 site of heavy chain was responsible for the generation of mAb-X antibody variant. Lys-C and trypsin coupled non-reduced and reduced peptide mapping comparative analysis showed that the formation of the light-heavy interchain trisulfide bond resulted in the mAb-Y antibody variant. These two cases confirmed that mass spectrometry-based comparative analysis plays a critical role for the characterization of monoclonal antibody variants, and biosimilar developers should start with a comprehensive structural assessment and comparative analysis to decrease the risk of the process development for biosimilars. PMID:27214604

  14. PK/PD analysis of a novel pH-dependent antigen-binding antibody using a dynamic antibody-antigen binding model.

    Science.gov (United States)

    Haraya, Kenta; Tachibana, Tatsuhiko; Iwayanagi, Yuki; Maeda, Atsuhiko; Ozeki, Kazuhisa; Nezu, Junichi; Ishigai, Masaki; Igawa, Tomoyuki

    2016-04-01

    Previously, we have reported novel engineered antibody with pH-dependent antigen-binding (recycling antibody), and with both pH-dependent antigen-binding and increased FcRn-binding at neutral pH (sweeping antibody). The purpose of this study is to perform PK/PD predictions to better understand the potential applications of the antibodies as therapeutics. To demonstrate the applicability of recycling and sweeping antibodies over conventional antibodies, PK/PD analyses were performed. PK/PD parameters for antibody and antigen dynamics were estimated from the results of a pharmacokinetic study in human FcRn transgenic mice. A simulation study was performed using the estimated PK/PD parameters with various target antigen profiles. In comparison to conventional antibody, recycling antibody enhanced antibody-antigen complex clearance by 3 folds, while sweeping antibody accelerated antigen clearance by 10 folds in a pharmacokinetic study. Simulation results showed that recycling and sweeping antibodies can improve dosage frequency and reduce the required dose for target antigens with various clearances, plasma concentrations or binding kinetics. Moreover, importance of the association rate constant to enhance the beneficial effect of antibodies was shown. These results support the conclusion that recycling and sweeping antibodies can be applied to various target antigens with different profiles, and expand the number of antigens that antibodies can target. PMID:26944099

  15. Tumor targeting of radiolabeled antibodies using HYNIC chelate

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Tae Sup; Chung, Wee Sup; Woo, Kwang Sun; Choi, Tae Hyun; Chung, Hye Kyung; Lee, Myung Jin; Kim, So Yeon; Jung, Jae Ho; Choi, Chang Woon; Lim, Sang Moo [KIRAMS, Seoul (Korea, Republic of); Darwati, Siti [National Nuclear Energy Agency, Tangerang (Indonesia)

    2004-07-01

    There is an increasing interest in the use of labeled antibodies for diagnosis of cancers as well as for therapy. Various radiolabeling methods have been used in order to obtain better tumor specific targeting for detection and therapy. It was generally used to tumor targeted immunotherapy and immunodetection that lym-1, mouse monoclonal antibody, was specific binding to surface antigen of Raji. The 3E8 antibody was produced from humanized anti-TAG-72 monoclonal antibody (AKA) by amino acid change in 95-99 residues of heavy chain complementary determinant regions (HCDRs) 3 using phage displayed library technology. In this study, we are investigating the usefulness of HYNIC chelate as a bifunctional chelating agent in radioimmunodetecton of tumor. Two types of antibodies, Lym-1 and 3E8, were used for the conjugation with HYNIC chelate. Lym-1 and 3E8 are specific antibodies to surface antigen of Non-Hogkin's lymphoma and TAG-72 antigen of colorectal carcinoma, respectively. We prepare HYNIC-antibody conjugates, determine radiolabeling yield with {sup 99m}Tc and evaluate tumor targeting in tumor bearing nude mice model.

  16. Antibody dependent enhancement of frog virus 3 infection

    Directory of Open Access Journals (Sweden)

    Penny Emily

    2010-02-01

    Full Text Available Abstract Background Viruses included in the family Iridoviridae are large, icosahedral, dsDNA viruses that are subdivided into 5 genera. Frog virus 3 (FV3 is the type species of the genus Ranavirus and the best studied iridovirus at the molecular level. Typically, antibodies directed against a virus act to neutralize the virus and limit infection. Antibody dependent enhancement occurs when viral antibodies enhance infectivity of the virus rather than neutralize it. Results Here we show that anti-FV3 serum present at the time of FV3 infection enhances infectivity of the virus in two non-immune teleost cell lines. We found that antibody dependent enhancement of FV3 was dependent on the Fc portion of anti-FV3 antibodies but not related to complement. Furthermore, the presence of anti-FV3 serum during an FV3 infection in a non-immune mammalian cell line resulted in neutralization of the virus. Our results suggest that a cell surface receptor specific to teleost cell lines is responsible for the enhancement. Conclusions This report represents the first evidence of antibody dependent enhancement in iridoviruses. The data suggests that anti-FV3 serum can either neutralize or enhance viral infection and that enhancement is related to a novel antibody dependent enhancement pathway found in teleosts that is Fc dependent.

  17. Quantifying serum antibody in bird fanciers' hypersensitivity pneumonitis

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    Anderson Kenneth

    2006-06-01

    Full Text Available Abstract Background Detecting serum antibody against inhaled antigens is an important diagnostic adjunct for hypersensitivity pneumonitis (HP. We sought to validate a quantitative fluorimetric assay testing serum from bird fanciers. Methods Antibody activity was assessed in bird fanciers and control subjects using various avian antigens and serological methods, and the titer was compared with symptoms of HP. Results IgG antibody against pigeon serum antigens, quantified by fluorimetry, provided a good discriminator of disease. Levels below 10 mg/L were insignificant, and increasing titers were associated with disease. The assay was unaffected by total IgG, autoantibodies and antibody to dietary hen's egg antigens. Antigens from pigeon serum seem sufficient to recognize immune sensitivity to most common pet avian species. Decreasing antibody titers confirmed antigen avoidance. Conclusion Increasing antibody titer reflected the likelihood of HP, and decreasing titers confirmed antigen avoidance. Quantifying antibody was rapid and the increased sensitivity will improve the rate of false-negative reporting and obviate the need for invasive diagnostic procedures. Automated fluorimetry provides a method for the international standardization of HP serology thereby improving quality control and improving its suitability as a diagnostic adjunct.

  18. Phospho-Specific Antibody Probes of Intermediate Filament Proteins.

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    Goto, Hidemasa; Tanaka, Hiroki; Kasahara, Kousuke; Inagaki, Masaki

    2016-01-01

    Intermediate filaments (IFs) form one of the major cytoskeletal systems in the cytoplasm or beneath the nuclear membrane. Accumulating data have suggested that IF protein phosphorylation dramatically changes IF structure/dynamics in cells. For the production of an antibody recognizing site-specific protein phosphorylation (a site- and phosphorylation state-specific antibody), we first employed a strategy to immunize animals with an in vitro-phosphorylated polypeptide or a phosphopeptide (corresponding to a phosphorylated residue and its surrounding sequence of amino acids), instead of a phosphorylated protein. Our established methodology not only improves the chance of obtaining a phospho-specific antibody but also has the advantage that one can predesign a targeted phosphorylation site. It is now applied to the production of an antibody recognizing other types of site-specific posttranslational modification, such as acetylation or methylation. The use of such an antibody in immunocytochemistry enables us to analyze spatiotemporal distribution of site-specific IF protein phosphorylation. The antibody is of great use to identify a protein kinase responsible for in vivo IF protein phosphorylation and to monitor intracellular kinase activities through IF protein phosphorylation. Here, we present an overview of our methodology and describe stepwise approaches for the antibody characterization. We also provide some examples of analyses for IF protein phosphorylation involved in mitosis and signal transduction.

  19. Pathogenic and Epiphenomenal Anti-DNA Antibodies in SLE

    Directory of Open Access Journals (Sweden)

    Mirjana Pavlovic

    2010-01-01

    Full Text Available The discoveries of natural and the development of manufactured highly efficient catalytic antibodies (abzymes opens the door to many practical applications. One of the most fascinating is the use of such antibodies in human therapy and prevention (vaccination, of cancer, AIDS, autoimmune diseases. A special entity of naturally occurring DNA hydrolytic anti-DNA antibodies is emerging within past decades linked to autoimmune and lymphoproliferative disorders, such as systemic lupus erythematosus (SLE, multiple sclerosis (MS, Sjogren Syndrome (SS, B - Chronic lymphocytic leucosis (B-CLL, and Multiple Myeloma (MM. The origin of the antibodies is unknown. The underlying mechanisms of these activities are suggested to be penetration into the living cells and translocation in the nucleus, with recognition of the specific binding sites at particular (ss or ds DNA. There are controversies in the literature whether hydrolysis is a sequence-specific event. The interplay between anti-DNA antibodies and DNA is not yet elucidated. This molecular “twist” also suggests that anti-DNA antibodies with DNA hydrolytic capacity could be the organism's immune response to a microbial attack, with microbial DNA, or specific genes within microbial DNA sequence, as a target for neutralization. The catalytic antibody-based approach can become a key tool in selective chemotherapeutic strategies.

  20. Seroprevalence of dengue virus antibodies in healthy Jamaicans.

    Science.gov (United States)

    Brown, Michelle G; Vickers, Ivan E; Salas, Rose Alba; Smikle, Monica F

    2009-01-01

    Dengue fever, a mosquito borne viral infection, is endemic to Jamaica. The seroprevalence of dengue IgG and IgM antibodies were determined in 277 healthy Jamaicans by enzyme linked immunosorbent assay (ELISA). The seroprevalence of dengue IgG antibodies was 100% (277/277) while dengue IgM antibodies were found in 3.6% (10/277). A statistically significant association was found between the presence of dengue IgM antibodies and gender (males 10/105, 9.5% vs females 0/172, 0.0%); chi(2) = 17.0, p=0.000.The high seroprevalence rate of dengue IgG antibodies and the presence of dengue IgM in the healthy population are in keeping with the endemicity of the virus in Jamaica. Therefore tests for dengue IgG antibodies are of limited usefulness in Jamaica and can be safely excluded from diagnostic testing as a cost saving measure. Serological diagnosis of current dengue infection should be centred around the dengue IgM tests although the limitations in the predictive values of such tests should also be considered. The results also suggest that the risk of emergence of the more severe forms of dengue, dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) in the Jamaican population, due to the presence of enhancing antibodies, is high. PMID:19996526