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Pornnoppadol G, Zhang B, Desai AA, Berardi A, Remmer HA, Tessier PM, Greineder CF. A hybridoma-derived monoclonal antibody with high homology to the aberrant myeloma light chain. PLoS One 2021; 16:e0252558. [PMID: 34634047 PMCID: PMC8504763 DOI: 10.1371/journal.pone.0252558] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2021] [Accepted: 09/20/2021] [Indexed: 11/23/2022] Open
Abstract
The identification of antibody variable regions in the heavy (VH) and light (VL) chains from hybridomas is necessary for the production of recombinant, sequence-defined monoclonal antibodies (mAbs) and antibody derivatives. This process has received renewed attention in light of recent reports of hybridomas having unintended specificities due to the production of non-antigen specific heavy and/or light chains for the intended antigen. Here we report a surprising finding and potential pitfall in variable domain sequencing of an anti-human CD63 hybridoma. We amplified multiple VL genes from the hybridoma cDNA, including the well-known aberrant Sp2/0 myeloma VK and a unique, full-length VL. After finding that the unique VL failed to yield a functional antibody, we discovered an additional full-length sequence with surprising similarity (~95% sequence identify) to the non-translated myeloma kappa chain but with a correction of its key frameshift mutation. Expression of the recombinant mAb confirmed that this highly homologous sequence is the antigen-specific light chain. Our results highlight the complexity of PCR-based cloning of antibody genes and strategies useful for identification of correct sequences.
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Affiliation(s)
- Ghasidit Pornnoppadol
- Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan, United States of America
- BioInterfaces Institute, University of Michigan, Ann Arbor, Michigan, United States of America
| | - Boya Zhang
- BioInterfaces Institute, University of Michigan, Ann Arbor, Michigan, United States of America
- Department of Pharmacology, University of Michigan, Ann Arbor, Michigan, United States of America
| | - Alec A. Desai
- BioInterfaces Institute, University of Michigan, Ann Arbor, Michigan, United States of America
- Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America
| | - Anthony Berardi
- BioInterfaces Institute, University of Michigan, Ann Arbor, Michigan, United States of America
- Department of Macromolecular Science & Engineering, University of Michigan, Ann Arbor, Michigan, United States of America
| | - Henriette A. Remmer
- Proteomics & Peptide Synthesis Core, University of Michigan, Ann Arbor, Michigan, United States of America
| | - Peter M. Tessier
- Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan, United States of America
- BioInterfaces Institute, University of Michigan, Ann Arbor, Michigan, United States of America
- Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America
| | - Colin F. Greineder
- BioInterfaces Institute, University of Michigan, Ann Arbor, Michigan, United States of America
- Department of Pharmacology, University of Michigan, Ann Arbor, Michigan, United States of America
- Department of Emergency Medicine, University of Michigan, Ann Arbor, Michigan, United States of America
- * E-mail:
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Bradbury ARM, Trinklein ND, Thie H, Wilkinson IC, Tandon AK, Anderson S, Bladen CL, Jones B, Aldred SF, Bestagno M, Burrone O, Maynard J, Ferrara F, Trimmer JS, Görnemann J, Glanville J, Wolf P, Frenzel A, Wong J, Koh XY, Eng HY, Lane D, Lefranc MP, Clark M, Dübel S. When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions. MAbs 2018; 10:539-546. [PMID: 29485921 PMCID: PMC5973764 DOI: 10.1080/19420862.2018.1445456] [Citation(s) in RCA: 72] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.
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Affiliation(s)
| | | | - Holger Thie
- c Miltenyi Biotec GmbH , Friedrich-Ebert-Str. 68, Bergisch Gladbach , Germany
| | - Ian C Wilkinson
- d Absolute Antibody, Wilton Centre , Redcar , Cleveland TS10 4RF , United Kingdom
| | - Atul K Tandon
- e NeoBiotechnologies , 2 Union Square, Union City , CA , USA
| | - Stephen Anderson
- d Absolute Antibody, Wilton Centre , Redcar , Cleveland TS10 4RF , United Kingdom
| | - Catherine L Bladen
- d Absolute Antibody, Wilton Centre , Redcar , Cleveland TS10 4RF , United Kingdom
| | - Brittany Jones
- e NeoBiotechnologies , 2 Union Square, Union City , CA , USA
| | | | - Marco Bestagno
- f International Centre for Genetic Engineering and Biotechnology (ICGEB) , Padriciano 99, Trieste , Italy
| | - Oscar Burrone
- f International Centre for Genetic Engineering and Biotechnology (ICGEB) , Padriciano 99, Trieste , Italy
| | - Jennifer Maynard
- g The University of Texas at Austin, Cockrell School of Engineering , McKetta Department of Chemical Engineering , 200 E Dean Keeton St. Stop C0400, Austin , Texas , USA
| | | | - James S Trimmer
- h Department of Physiology and Membrane Biology , University of California , Davis, One Shields Avenue, Davis , CA , USA
| | - Janina Görnemann
- i Institute for Molecular Genetics , University of Heidelberg , Im Neuenheimer Field 260, Heidelberg , Germany
| | - Jacob Glanville
- j Stanford University, School of Medicine , Stanford , California , USA
| | - Philipp Wolf
- k Department of Urology , Medical Center, University of Freiburg , Breisacher Str. 66, Freiburg , Germany
| | - Andre Frenzel
- l Yumab GmbH , Inhoffenstr. 7, Braunschweig , Germany.,p Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics , Spielmannstr. 7, Braunschweig , Germany
| | - Julin Wong
- m A*Star p53 laboratory , 06-06 Immunos, Singapore , Singapore
| | - Xin Yu Koh
- m A*Star p53 laboratory , 06-06 Immunos, Singapore , Singapore
| | - Hui-Yan Eng
- m A*Star p53 laboratory , 06-06 Immunos, Singapore , Singapore
| | - David Lane
- m A*Star p53 laboratory , 06-06 Immunos, Singapore , Singapore
| | - Marie-Paule Lefranc
- n IMGT®, the international ImMunoGeneTics information system®, Laboratoire d'ImmunoGénétique Moléculaire LIGM, Institut de Génétique Humaine IGH, UPR CNRS 1142, Montpellier University , Montpellier cedex 5 , France
| | - Mike Clark
- o Clark Antibodies Ltd , 10 Wellington Street, Cambridge , CB1 1HW , United Kingdom
| | - Stefan Dübel
- p Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics , Spielmannstr. 7, Braunschweig , Germany
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3
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Identification and verification of hybridoma-derived monoclonal antibody variable region sequences using recombinant DNA technology and mass spectrometry. Mol Immunol 2017; 90:287-294. [PMID: 28865256 DOI: 10.1016/j.molimm.2017.08.014] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2017] [Revised: 08/10/2017] [Accepted: 08/16/2017] [Indexed: 11/24/2022]
Abstract
Antibody engineering requires the identification of antigen binding domains or variable regions (VR) unique to each antibody. It is the VR that define the unique antigen binding properties and proper sequence identification is essential for functional evaluation and performance of recombinant antibodies (rAb). This determination can be achieved by sequence analysis of immunoglobulin (Ig) transcripts obtained from a monoclonal antibody (MAb) producing hybridoma and subsequent expression of a rAb. However the polyploidy nature of a hybridoma cell often results in the added expression of aberrant immunoglobulin-like transcripts or even production of anomalous antibodies which can confound production of rAb. An incorrect VR sequence will result in a non-functional rAb and de novo assembly of Ig primary structure without a sequence map is challenging. To address these problems, we have developed a methodology which combines: 1) selective PCR amplification of VR from both the heavy and light chain IgG from hybridoma, 2) molecular cloning and DNA sequence analysis and 3) tandem mass spectrometry (MS/MS) on enzyme digests obtained from the purified IgG. Peptide analysis proceeds by evaluating coverage of the predicted primary protein sequence provided by the initial DNA maps for the VR. This methodology serves to both identify and verify the primary structure of the MAb VR for production as rAb.
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Tu B, Tieman B, Moore J, Pan Y, Muerhoff AS. Myeloma-Derived Light Chain Paired with a Diagnostic Monoclonal Antibody Hinders Immunoassay Performance. Monoclon Antib Immunodiagn Immunother 2017; 36:113-118. [PMID: 28557609 DOI: 10.1089/mab.2017.0009] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Monoclonal antibodies are widely used as the capture and detection reagents in diagnostic immunoassays. In the past, myeloma fusion partners expressing endogenous heavy and/or light chains were often used to generate hybridoma cell lines. As a result, mixed populations of antibodies were produced that can cause inaccurate test results, poor antibody stability, and significant lot-to-lot variability. We describe one such scenario where the P3U1 (P3X63Ag8U.1) myeloma fusion partner was used in the generation of a hybridoma producing protein induced vitamin K absence/antagonist-II (PIVKA II) antibody. The hybridoma produces three subpopulations of immunoglobulin as determined by ion exchange (IEx) chromatography that exhibit varying degrees of immunoreactivity (0%, 50%, or 100%) to the target antigen as determined by Surface Plasmon Resonance. To produce an antibody with the highest possible sensitivity and specificity, the antigen-specific heavy and light chain variable domains (VH and VL) were cloned from the hybridoma and tethered to murine IgG1 and kappa scaffolds. The resulting recombinant antibody was expressed in Chinese hamster ovary cells and is compatible for use in a diagnostic immunoassay.
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Affiliation(s)
- Bailin Tu
- Abbott Diagnostics Division, Department of Biologics Discovery and Design, Abbott Laboratories , Abbott Park, Illinois
| | - Bryan Tieman
- Abbott Diagnostics Division, Department of Biologics Discovery and Design, Abbott Laboratories , Abbott Park, Illinois
| | - Jeffrey Moore
- Abbott Diagnostics Division, Department of Biologics Discovery and Design, Abbott Laboratories , Abbott Park, Illinois
| | - You Pan
- Abbott Diagnostics Division, Department of Biologics Discovery and Design, Abbott Laboratories , Abbott Park, Illinois
| | - A Scott Muerhoff
- Abbott Diagnostics Division, Department of Biologics Discovery and Design, Abbott Laboratories , Abbott Park, Illinois
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5
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Converting monoclonal antibodies into Fab fragments for transient expression in mammalian cells. Methods Mol Biol 2012; 801:137-59. [PMID: 21987252 DOI: 10.1007/978-1-61779-352-3_10] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/07/2023]
Abstract
In this chapter, protocols are described for converting mouse monoclonal antibodies into recombinant Fabs for transient expression in mammalian cells. Variable region genes are cloned by reverse transcription: PCR using either sequence specific or mixed 5' primers that hybridise to the first framework sequence of the mouse light and heavy chains and 3' primers that bind to the heavy- and light-chain constant regions. The amplified sequences are inserted into mammalian cell expression vectors by In-Fusion™ cloning. This method allows vector and amplified DNA sequences to be seamlessly joined in a ligation-independent reaction. Transient co-expression of light-chain and heavy-chain genes in HEK 293T cells enables production of recombinant Fabs for functional and structural studies.
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6
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Crosnier C, Staudt N, Wright GJ. A rapid and scalable method for selecting recombinant mouse monoclonal antibodies. BMC Biol 2010; 8:76. [PMID: 20525357 PMCID: PMC2898661 DOI: 10.1186/1741-7007-8-76] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2010] [Accepted: 06/04/2010] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Monoclonal antibodies with high affinity and selectivity that work on wholemount fixed tissues are valuable reagents to the cell and developmental biologist, and yet isolating them remains a long and unpredictable process. Here we report a rapid and scalable method to select and express recombinant mouse monoclonal antibodies that are essentially equivalent to those secreted by parental IgG-isotype hybridomas. RESULTS Increased throughput was achieved by immunizing mice with pools of antigens and cloning - from small numbers of hybridoma cells - the functionally rearranged light and heavy chains into a single expression plasmid. By immunizing with the ectodomains of zebrafish cell surface receptor proteins expressed in mammalian cells and screening for formalin-resistant epitopes, we selected antibodies that gave expected staining patterns on wholemount fixed zebrafish embryos. CONCLUSIONS This method can be used to quickly select several high quality monoclonal antibodies from a single immunized mouse and facilitates their distribution using plasmids.
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Affiliation(s)
- Cécile Crosnier
- Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Cambridge CB10 1HH, UK
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7
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Generation and characterization of high affinity humanized fab against hepatitis B surface antigen. Mol Biotechnol 2009; 43:29-40. [PMID: 19326261 DOI: 10.1007/s12033-009-9165-9] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2009] [Accepted: 03/10/2009] [Indexed: 02/06/2023]
Abstract
5S is a mouse monoclonal IgG1 that binds to the 'a' epitope of the Hepatitis B surface antigen (HBsAg) and tested positive in an in vitro test for virus neutralization. We have earlier reported the generation of humanized single chain variable fragment (scFv) from the same. In this article we report the generation of a recombinant Fab molecule by fusing humanized variable domains of 5S with the constant domains of human IgG1. The humanized Fab expressed in E. coli and subsequently purified, retained a high binding affinity (K(D) = 3.63 nmol/L) to HBsAg and bound to the same epitope of HBsAg as the parent molecule. The humanized Fab also maintained antigen binding in the presence of various destabilizing agents like 3 M NaCl, 30% DMSO, 8 M urea, and extreme pH. This high affinity humanized Fab provides a basis for the development of therapeutic molecules that can be safely utilized for the prophylaxis and treatment for Hepatitis B infection.
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Druar C, Yu F, Barnes JL, Okinaka RT, Chantratita N, Beg S, Stratilo CW, Olive AJ, Soltes G, Russell ML, Limmathurotsakul D, Norton RE, Ni SX, Picking WD, Jackson PJ, Stewart DIH, Tsvetnitsky V, Picking WL, Cherwonogrodzky JW, Ketheesan N, Peacock SJ, Wiersma EJ. Evaluating Burkholderia pseudomallei Bip proteins as vaccines and Bip antibodies as detection agents. ACTA ACUST UNITED AC 2007; 52:78-87. [PMID: 17995960 DOI: 10.1111/j.1574-695x.2007.00345.x] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Burkholderia pseudomallei is a biothreat agent and an important natural pathogen, causing melioidosis in humans and animals. A type III secretion system (TTSS-3) has been shown to be critical for virulence. Because TTSS components from other pathogens have been used successfully as diagnostic agents and as experimental vaccines, it was investigated whether this was the case for BipB, BipC and BipD, components of B. pseudomallei's TTSS-3. The sequences of BipB, BipC and BipD were found to be highly conserved among B. pseudomallei and B. mallei isolates. A collection of monoclonal antibodies (mAbs) specific for each Bip protein was obtained. Most recognized both native and denatured Bip protein. Burkholderia pseudomallei or B. mallei did not express detectable BipB or BipD under the growth conditions used. However, anti-BipD mAbs did recognize the TTSS needle structures of a Shigella strain engineered to express BipD. The authors did not find that BipB, BipC or BipD are protective antigens because vaccination of mice with any single protein did not result in protection against experimental melioidosis. Enzyme-linked immunosorbent assay (ELISA) studies showed that human melioidosis patients had antibodies to BipB and BipD. However, these ELISAs had low diagnostic accuracy in endemic regions, possibly due to previous patient exposure to B. pseudomallei.
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Affiliation(s)
- Chris Druar
- Cangene Corporation, Mississauga, ON, Canada
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9
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Bose B, Khanna N, Acharya SK, Sinha S. High affinity mouse-human chimeric Fab against hepatitis B surface antigen. World J Gastroenterol 2006; 11:7569-78. [PMID: 16437680 PMCID: PMC4727235 DOI: 10.3748/wjg.v11.i48.7569] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM Passive immunotherapy using antibody against hepatitis B surface antigen (HBsAg) has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal (5S) against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody. METHODS We cloned the V(H) and V(L) genes of this mouse antibody, and fused them with CH1 domain of human IgG1 and C(L) domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. coli. RESULTS The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. This chimeric antibody fragment was further expressed in different strains of E. coli to increase the yield. CONCLUSION We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a full-length chimeric antibody for therapeutic uses.
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Affiliation(s)
- Biplab Bose
- Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, PIN-110029, India.
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Qu Z, Griffiths GL, Wegener WA, Chang CH, Govindan SV, Horak ID, Hansen HJ, Goldenberg DM. Development of humanized antibodies as cancer therapeutics. Methods 2005; 36:84-95. [PMID: 15848077 DOI: 10.1016/j.ymeth.2005.01.008] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2004] [Revised: 01/10/2005] [Accepted: 01/17/2005] [Indexed: 11/30/2022] Open
Abstract
Recent success in the development of monoclonal antibody-based anti-cancer drugs has largely benefitted from the advancements made in recombinant technologies and cell culture production. These reagents, derived from the antibodies of mouse origin, while maintaining the exquisite specificity and affinity to the tumor antigens, have low immunogenicity and toxicity in human. High-level expressing cell clones are generated and used to produce large quantities of the recombinant antibodies in bioreactors in order to meet the clinical demand for therapeutic applications. In this report, the systems and general methodologies developed by us to construct and produce humanized antibodies from the parent mouse antibodies are described. Once the humanized antibodies are available, they can be applied in three principal forms for cancer therapy: (1) naked antibodies, (2) drug- or toxin conjugates, and (3) radioconjugates. Using the humanized anti-CD22 (epratuzumab) and anti-carcinoembryonic antigen (ant-CEA; labetuzumab) antibody prototypes, clinical applications of naked and radiolabeled humanized monoclonal antibodies are described.
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Affiliation(s)
- Zhengxing Qu
- Immunomedics, Inc., Morris Plains, NJ 07950, USA.
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11
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Verbeke K, Gils A, Declerck PJ. Inhibition of plasminogen activator inhibitor-1: antibody fragments and their unique sequences as a tool for the development of profibrinolytic drugs. J Thromb Haemost 2004; 2:298-305. [PMID: 14995993 DOI: 10.1111/j.1538-7933.2004.00583.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Physiological inhibition of plasminogen activator inhibitor-1 (PAI-1) might improve the prevention and treatment of various cardiovascular diseases. To date, a variety of monoclonal antibodies that neutralize PAI-1 have been generated. The current study presents the cloning, expression and characterization of four single-chain variable fragments (i.e. scFv-33B8, scFv-33H1F7, scFv-35A5 and scFv-55F4C12) from the corresponding PAI-1 neutralizing monoclonal antibodies. Surprisingly, affinity constants of scFv-33B8, scFv-33H1F7 and scFv-55F4C12 for PAI-1 (KA = 1.4 +/- 0.2 x 1010 m-1, 3.7 +/- 0.1 x 109 m-1, 1.0 +/- 0.2 x 109 m-1, respectively) were only 2- to 4-fold lower compared to those of the respective monoclonal antibodies (MAs). In contrast, scFv-35A5 exhibited a 6250-fold decrease in affinity (KA = 3.2 +/- 0.8 x 106 m-1 vs. 2.0 +/- 0.8 x 1010 m-1 observed for MA-35A5) with a concomitant absence of functional effects on PAI-1 activity. Evaluation of the dose-response curves of the PAI-1 neutralizing effect of the other scFvs revealed a shift towards slightly higher concentrations (in line with the small decrease in affinity) eventually resulting in a similar maximum effect as the corresponding MAs (i.e. 92 +/- 2%, 34 +/- 3% and 66 +/- 5% PAI-1 inhibition for scFv-33B8, scFv-33H1F7 and scFv-55F4C12, respectively). In conclusion, the sequence information of the scFvs allows to humanize MAs with PAI-1 inhibiting properties whereas the scFv constructs serve as an excellent starting point for structure based drug design, both aiming at the reduction of cardiovascular diseases.
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Affiliation(s)
- K Verbeke
- Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium
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12
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Verbeke K, Gils A, Declerck PJ. Cloning and paratope analysis of an antibody fragment, a rational approach for the design of a PAI-1 inhibitor. J Thromb Haemost 2004; 2:289-97. [PMID: 14995992 DOI: 10.1111/j.1538-7933.2004.00582.x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
This study reports the cloning, characterization and paratope analysis of the plasminogen activator inhibitor-1 (PAI-1) neutralizing single-chain variable fragment 56A7C10 (scFv-56A7C10). ScFv-56A7C10-wt exhibits a similar affinity (KA = 1.01 +/- 0.3 x 109 m-1) and PAI-1 inhibitory capacity (90 +/- 6% PAI-1 inhibition at a 16-fold molar excess and IC50 = 44 +/- 14 ng mL-1) as MA-56A7C10 (KA = 1.43 +/- 0.4 x 109 m-1, 90 +/- 2% PAI-1 inhibition at a 16-fold molar excess and IC50 = 122 +/- 26 ng mL-1). Subsequently, alanine scanning of the six complementarity determining regions (CDRs) was performed and the scFv-56A7C10-mutants (n = 26) were analyzed for their PAI-1 binding and PAI-1 inhibitory properties. Mutation of the residues Y32 and V33 in the CDR1 of the heavy chain (HCDR1) and the residues R98, H99, W100 or F100a (HCDR3) resulted in reduced PAI-1 inhibitory capacities (IC50 >/= 418 ng mL-1), confirmed by reduced affinities (14-, 17-, 7-, 9- and 16-fold reduced, respectively, vs. scFv-56A7C10-wt). In the light chain, mutation of the residues W50 (LCDR2), H91, Y92, D93, or W96 (LCDR3) resulted in reduced PAI-1 inhibitory properties (IC50 >/= 160 ng mL-1) and decreased affinities (i.e. 4-, 9-, 3-, 3- and 2-fold reduced affinity, respectively, vs. scFv-56A7C10-wt). Furthermore, an overlapping peptide scan confirmed the importance of the HCDR3 region. These data, combined with a three-dimensional model of scFv-56A7C10, reveal the molecular and structural properties of the paratope and contribute to the rational design of PAI-1 neutralizing compounds.
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Affiliation(s)
- K Verbeke
- Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium
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13
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Kwon MH, Lee MS, Kim KH, Park S, Shin HJ, Jang YJ, Kim HI. Production and characterization of an anti-idiotypic single chain Fv that recognizes an anti-DNA antibody. Immunol Invest 2002; 31:205-18. [PMID: 12472180 DOI: 10.1081/imm-120016241] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
A well-characterized recombinant anti-idiotype to an anti-DNA antibody can be useful for studies of the regulation of anti-DNA-producing B cells. Using a hybridoma technique, a monoclonal anti-idiotypic antibody, designated O2F3, was obtained, and its scFv gene was constructed. O2F3 single chain Fv (scFv) was produced against an idiotope of a monoclonal anti-DNA antibody, 3D8, that was obtained from an autoimmune-prone mouse, MRL-lpr/lpr. Here we describe the production and in vitro characterization of the O2F3 scFv, and compare it with its parent monoclonal antibody, O2F3 IgM. To characterize O2F3 scFv and O2F3 IgM, we generated recombinant 3D8 fragments, including 3D8 scFv, 3D8 VH, and 3D8 VL, that were used as antigens in several assays. ELISA and Western blot analysis showed that both O2F3 scFv and O2F3 IgM recognized a conformational determinant formed by the association of the variable region heavy and light chains of the 3D8 antibody, suggesting that O2F3 scFv retained a similar binding pattern to its parent O2F3 antibody. The idiotope recognized by O2F3 was shown by competitive ELISA to be outside of the DNA binding site of the 3D8 antibody. This characterized O2F3 scFv could be applied for the regulation of anti-DNA antibody production and the manipulation of recombinant antibody-based proteins to which toxins, enzymes, and chemical agents can be connected.
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MESH Headings
- Animals
- Antibodies, Anti-Idiotypic/biosynthesis
- Antibodies, Anti-Idiotypic/genetics
- Antibodies, Anti-Idiotypic/immunology
- Antibodies, Anti-Idiotypic/isolation & purification
- Antibodies, Antinuclear/chemistry
- Antibodies, Antinuclear/immunology
- Antibodies, Monoclonal/biosynthesis
- Antibodies, Monoclonal/genetics
- Antibodies, Monoclonal/immunology
- Antibodies, Monoclonal/isolation & purification
- Autoantigens/immunology
- Autoimmune Diseases/immunology
- Binding Sites
- Binding, Competitive
- DNA/immunology
- DNA, Single-Stranded/chemistry
- DNA, Single-Stranded/immunology
- Enzyme-Linked Immunosorbent Assay
- Epitopes/chemistry
- Epitopes/immunology
- Female
- Genes, Immunoglobulin
- Genetic Vectors/genetics
- Hybridomas/immunology
- Immunoglobulin Fragments/biosynthesis
- Immunoglobulin Fragments/genetics
- Immunoglobulin Fragments/immunology
- Immunoglobulin Fragments/isolation & purification
- Immunoglobulin Idiotypes/chemistry
- Immunoglobulin Idiotypes/immunology
- Immunoglobulin M/biosynthesis
- Immunoglobulin M/genetics
- Immunoglobulin M/immunology
- Immunoglobulin M/isolation & purification
- Lupus Erythematosus, Systemic/immunology
- Mice
- Mice, Inbred BALB C
- Mice, Inbred MRL lpr
- Protein Conformation
- Recombinant Fusion Proteins/immunology
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Affiliation(s)
- Myung-Hee Kwon
- Department of Microbiology, Ajou University School of Medicine, Woncheon-dong 5, Suwon 442-749, Korea
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14
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Weston KM, Tangye SG, Dunn RD, Smith A, Morris MB, Raison RL. IgM expressed by leukemic CD5(+) B cells binds mouse immunoglobulin light chain. J Mol Recognit 2001; 14:245-53. [PMID: 11500971 DOI: 10.1002/jmr.539] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Mouse immunoglobulin (Ig) molecules have previously been shown to bind to the surface of CD5(+) B cells from patients with B-cell chronic lymphocytic leukemia (B-CLL). The results indicated that surface IgM was involved in the interaction and suggested the phenomenon was an example of the polyreactive binding capacity of the surface Ig (sIg) expressed by these malignant cells. This article describes the further characterization of the interaction between human IgM and mouse Ig molecules and subunits. Mouse Ig molecules of both kappa and lambda light chain classes bound to the B-CLL cell surface. The dissociation constant for the interaction of mouse IgG1 (K121) with the B-CLL cell surface was 3.6 x 10(-7) M. To confirm the involvement of the human IgM expressed by the B-CLL cells in the interaction, the malignant cells were stimulated in vitro to induce secretion of human IgM. Enzyme immunoassay was used to show that secreted human IgM bound to intact mouse Ig, as occurred with the cell surface analysis. The mouse Ig epitope recognized by the purified secreted human IgM was shown by Western blot analysis to be located on the light chain of the mouse Ig molecule and to be conformationally dependent. K121 light chain was cloned and expressed in E. coli and the recombinant light chain bound to the surface of CLL B cells. The results confirm that human IgM is the reactive ligand in the interaction with mouse Ig and indicate that the interaction of polyreactive IgM with mouse IgG occurs via the light chain component of IgG.
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Affiliation(s)
- K M Weston
- Immunobiology Unit, Department of Cell and Molecular Biology University of Technology, Sydney, Westbourne Street, Gore Hill, 2065, NSW, Australia
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15
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Vidarsson G, van de Winkel JG, van Dijk MA. Multiplex screening for functionally rearranged immunoglobulin variable regions reveals expression of hybridoma-specific aberrant V-genes. J Immunol Methods 2001; 249:245-52. [PMID: 11226481 DOI: 10.1016/s0022-1759(00)00337-9] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
Modification of antibody effector functions is commonly performed by chimerization or humanization. Cloning of antibody variable regions from hybridomas represents a first step that is frequently hampered by the expression of non-functionally rearranged variable regions in hybridoma cells that originate from MOPC21-derived fusion partners. We now present a simple method to clone functionally rearranged V-genes, based on V-gene-specific multiplex PCR screening. Using this method we document the expression of aberrant V-genes that originate from the original B-cell used for the hybridoma generation, not from the fusion partner, and are - thus - hybridoma specific.
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Affiliation(s)
- G Vidarsson
- Immunotherapy Laboratory, Department of Immunology, Rm KC.02-085.2, University Medical Center Utrecht, Lundlaan 6, 3584 EA, Utrecht, The Netherlands
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16
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Park OY, Jin YH, Lee M, Shin HJ, Kim HI, Cho H, Yun CW, Youn JK, Park S. Characterization and gene cloning of monoclonal antibody specific for the hepatitis B virus X protein. Hybridoma (Larchmt) 2000; 19:73-80. [PMID: 10768843 DOI: 10.1089/027245700315815] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
The hepatis B virus X protein (HBx) has been thought to be implicated in the development of hepatocellular carcinoma. Although many functions of HBx have been reported, it is not clear which of HBx functions is important in hepatocellular carcinogenesis. To study HBx function, we produced a monoclonal anti-HBx Ab secreted by hybridoma cell clone H7 and mapped its epitope to a region of HBx between amino acids 29 and 48 by Western blot with truncated forms of HBx and by enzyme-linked immunoadsorbent assay (ELISA) with synthetic HBx peptides. The variable regions of H7 anti-HBx Ab were cloned by polymerase chain reaction using the degenerate-primers and by the 5' rapid amplification-cDNA end method. The sequence analyses revealed that the variable gene segments of the heavy and light chains are the members of mouse heavy chain variable gene 1 family and kappa light chain variable gene 2 family, respectively. In addition, J(H)2 or Jkappa4 gene segment at the end of the heavy-chain or light-chain variable region and DSP2.x gene segment in the CDR 3 of heavy chain were identified.
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Affiliation(s)
- O Y Park
- Department of Microbiology, Ajou University School of Medicine, Suwon, Korea
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17
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Krishnan IS, Hansen HJ, Gold DV, Goldenberg DM, Leung SO. Co-secretion of two distinct kappa light chains by the mu-9 hybridoma. Hybridoma (Larchmt) 1999; 18:325-33. [PMID: 10571262 DOI: 10.1089/hyb.1999.18.325] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Mu-9 is a monoclonal antibody (MAb) specific for the CSAp antigen (Ag) expressed by colorectal cancers. By using variable (V)-region-specific primers, the respective VH and VL sequences of Mu-9 were polymerase chain reaction (PCR)-amplified. However, chimeric Ab (cMu-9-1) constructed from these PCR-amplified V sequences failed to bind the CSAp Ag. Although the light chain of murine Mu-9 was not glycosylated, that of cMu-9-1 was found to be O-glycosylated, as confirmed by reducing SDS-PAGE analyses, glycoprotein blotting and O-linked specific deglycosylation studies. Removal of O-linked oligosaccharides either by enzymatic digestion or by blocking O-glycosylation with a specific inhibitor did not restore the immunoreactivity of cMu-9-1, indicating that light chain O-glycosylation was not the cause for lack of immunoreactivity. We reported earlier that screening of a Mu-9 cDNA library uncovered the presence of an additional light chain sequence that was later proven to be the authentic light chain of Mu-9. Analyses of the cDNA sequence encoding the nonimmunoreactive light chain, however, revealed no defects that would preclude the sequence from being translated and secreted by the murine hybridoma. By adapting the Mu-9 hybridoma culture to serum-free conditions, we confirmed the secretion of low levels of O-glycosylated light chain. The biological significance of the O-glycosylation as well as the cosecretion of both light chains with respect to allelic exclusion are discussed.
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18
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Chardès T, Villard S, Ferrières G, Piechaczyk M, Cerutti M, Devauchelle G, Pau B. Efficient amplification and direct sequencing of mouse variable regions from any immunoglobulin gene family. FEBS Lett 1999; 452:386-94. [PMID: 10386627 DOI: 10.1016/s0014-5793(99)00649-3] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
We have designed two original sets of oligonucleotide primers hybridizing the relatively conserved motifs within the immunoglobulin signal sequences of each of the 15 heavy chain and 18 kappa light chain gene families. Comparison of these 5' primers with the immunoglobulin signal sequences referenced in the Kabat database suggests that these oligonucleotide primers should hybridize with 89.4% of the 428 mouse heavy chain signal sequences and with 91.8% of the 320 kappa light chain signal sequences with no mismatch. Following PCR amplification using the designed primers and direct sequencing of the amplified products, we obtained full-length variable sequences belonging to major (V(H)1, V(H)2, V(H)3, Vkappa1 and Vkappa21) but also small-sized (V(H)9, V(H)14, Vkappa2, Vkappa9A/9B, Vkappa12/13, Vkappa23 and Vkappa33/34) gene families, from nine murine monoclonal antibodies. This strategy could be a powerful tool for antibody sequence assessment whatever the V gene family before humanization of mouse monoclonal antibody or identification of paratope-derived peptides.
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Affiliation(s)
- T Chardès
- CNRS UMR 9921, Faculté de Pharmacie, Montpellier, France.
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19
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Tsantili P, Tzartos SJ, Mamalaki A. High affinity single-chain Fv antibody fragments protecting the human nicotinic acetylcholine receptor. J Neuroimmunol 1999; 94:15-27. [PMID: 10376932 DOI: 10.1016/s0165-5728(98)00195-7] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
Univalent antibody fragments directed against the main immunogenic region (MIR) of the human acetylcholine receptor (AChR) are capable of protecting the AChR against loss induced by antibodies from myasthenia gravis (MG) patients. Our aim was to construct single-chain Fv (scFv) antibody fragments as a first step towards the production of therapeutic protecting molecules, from two high-affinity anti-MIR monoclonal antibodies (mAb 192 and mAb 195). During the construction of scFv192 fragment, two light chains co-secreted from the hybridoma mAb192 were identified. N-terminal amino acid and cDNA sequence analysis showed that one of the two light chains corresponded to the antigen binding molecule while the other originated from the non-secreting myeloma S194/5.XXO.BU.1 which was used in the production of the hybridoma. Functional scFv 192 and 195 fragments were constructed, expressed in Escherichia coli and affinity purified. The binding affinities of scFv192 and scFv195 (K(D) = 0.6 and 0.8 nM for human AChR) were two orders of magnitude higher than that of the earlier constructed scFv198. The scFv192 almost completely protected human AChR against binding of intact anti-MIR mAbs. Human AChR was also very efficiently protected (74-85%) by the scFv192 against binding of autoantibodies from MG sera with high anti-alpha subunit antibody fractions. These scFvs are good candidates for protection of MG patients after appropriate genetic modifications.
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Affiliation(s)
- P Tsantili
- Department of Biochemistry, Hellenic Pasteur Institute, Athens, Greece
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20
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Abstract
We have previously described the isolation and in vitro binding properties of eight anti-DNA monoclonal antibodies (MAbs) from an MRL-lpr mouse. In light of recent reports that have indicated it is possible to isolate multiple MAbs from a single hybridoma, our pathogenic hybridoma, 11F8, was examined for evidence of similar phenomena. Chromosome counting suggested that 11F8 cells are unusual and might indeed be expressing multiple heavy and/or light chains. PCR, cloning, and sequencing of immunoglobulin heavy and light chains indicate that 11F8 displays expression of both gamma 2a and gamma 3 heavy chains at the DNA level. Flow cytometry and amino acid sequencing reveals that expression of multiple isotypes also occurs at the protein level but only a single heavy- and light-chain sequence is able to bind DNA. Based on these results, we conclude that 11F8 is an unusual hybridoma that secretes two distinct heavy and at least one light chain from a single cell, and may represent a trioma, a stable three-cell fusion.
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Affiliation(s)
- N B Blatt
- University of Michigan, Department of Chemistry, Ann Arbor 48109-1055, USA
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21
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22
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Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard HR, Plückthun A. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods 1997; 201:35-55. [PMID: 9032408 DOI: 10.1016/s0022-1759(96)00208-6] [Citation(s) in RCA: 389] [Impact Index Per Article: 13.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
A prerequisite for the use of recombinant antibody technologies starting from hybridomas or immune repertoires is the reliable cloning of functional immunoglobulin genes. For this purpose, a standard phage display system was optimized for robustness, vector stability, tight control of scFv-delta geneIII expression, primer usage for PCR amplification of variable region genes, scFv assembly strategy and subsequent directional cloning using a single rare cutting restriction enzyme. This integrated cloning, screening and selection system allowed us to rapidly obtain antigen binding scFvs derived from spleen-cell repertoires of mice immunized with ampicillin as well as from all hybridoma cell lines tested to date. As representative examples, cloning of monoclonal antibodies against a his tag, leucine zippers, the tumor marker EGP-2 and the insecticide DDT is presented. Several hybridomas whose genes could not be cloned in previous experimental setups, but were successfully obtained with the present system, expressed high amounts of aberrant heavy and light chain mRNAs, which were amplified by PCR and greatly exceeded the amount of binding antibody sequences. These contaminating variable region genes were successfully eliminated by employing the optimized phage display system, thus avoiding time consuming sequencing of non-binding scFv genes. To maximize soluble expression of functional scFvs subsequent to cloning, a compatible vector series to simplify modification, detection, multimerization and rapid purification of recombinant antibody fragments was constructed.
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Affiliation(s)
- A Krebber
- Biochemisches Institut der Universitat Zurich, Switzerland
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23
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Ostermeier C, Michel H. Improved cloning of antibody variable regions from hybridomas by an antisense-directed RNase H digestion of the P3-X63-Ag8.653 derived pseudogene mRNA. Nucleic Acids Res 1996; 24:1979-80. [PMID: 8657583 PMCID: PMC145887 DOI: 10.1093/nar/24.10.1979] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Affiliation(s)
- C Ostermeier
- Max-Planck-Institut für Biophysik, Frankfurt, Germany
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24
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Wu Y, Duan L, Zhu M, Hu B, Kubota S, Bagasra O, Pomerantz RJ. Binding of intracellular anti-Rev single chain variable fragments to different epitopes of human immunodeficiency virus type 1 rev: variations in viral inhibition. J Virol 1996; 70:3290-7. [PMID: 8627813 PMCID: PMC190196 DOI: 10.1128/jvi.70.5.3290-3297.1996] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Abstract
Intracellular immunization to target the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev has been explored as a genetic therapy for AIDS. Efficient intracellular expression of rearranged immunoglobulin heavy and light chain variable regions of anti-Rev monoclonal antibodies, with various vectors, and subsequent inhibition of HIV-1 replication have been previously reported by our laboratories. To further understand the molecular mechanisms and effects that intracellular anti-Rev single chain variable fragments (SFvs) have against HIV-1, via blocking of Rev function, two anti-Rev SFvs which specifically bind to differing epitopes of the Rev protein have been cloned. One SFv binds to the Rev activation domain, and the second SFv binds to the distal C terminus of Rev in the nonactivation region. Further studies now demonstrate that both anti-Rev SFvs lead to variable resistance to HIV-1 infection. Although binding affinity assays demonstrated that the SFv which specifically recognizes the Rev activation domain (D8) had an extracellular binding affinity significantly lower than that of the SFv specific to the nonactivation region (D1O), the SFv D8 demonstrated more potent activity in inhibiting virus production in human T-cell lines and peripheral blood mononuclear cells than did SFv D10. Thus, extracellular binding affinities of an SFv for a target viral protein cannot be used to directly predict its activity as an intracellular immunization moiety. These data demonstrate potential approaches for intracellular immunization against HIV-1 infection, by efficiently blocking specific motifs of Rev to after the function of this retroviral regulatory protein. These studies extend the understanding of the effects, on a molecular level, of SFvs binding to critical epitopes of Rev and further suggest that rational design of SFvs, with interactions involving specific viral moieties which mediate HIV-1 expression, may hold promise for the clinical application of genetic therapies to combat AIDS.
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Affiliation(s)
- Y Wu
- Dorrance H. Hamilton Laboratories, Center for Human Retrovirology, Division of Infectious Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
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25
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Christian RB, Couto JR, Peterson JA, Ceriani RL. Cloning and expression of cDNAs encoding the variable domains of the antibreast carcinoma antibody Mc5. Hybridoma (Larchmt) 1996; 15:155-8. [PMID: 8743296 DOI: 10.1089/hyb.1996.15.155] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
Mc5, a murine monoclonal antibody that binds to human breast epithelial mucin (BEM), has been shown to be a promising reagent in the diagnosis of breast cancer. We have cloned cDNAs encoding both variable regions of Mc5 (VL and VH) as well as the CL and CH1 constant regions. Mc5 is an IgG1, kappa antibody. We have constructed an IgG1, kappa human/mouse chimeric antibody (by inserting the murine VH and VL-encoding cDNAs into plasmids encoding human constant domains), and expressed it in SP2/0-Ag14 mouse myeloma cells. The affinity of chimeric Mc5 (chMc5) for BEM is 4.4 x 10(8) M-1. Mc5 binds BEM with an affinity constant of 2.8 x 10(8) M-1. Purified chMc5 and purified Mc5 gave similar competition curves when tested against either 125I-labeled Mc5 or 125I-labeled chMc5 for binding to BEM in a competition radioimmunodetection format. Additionally, chMc5 used in breast carcinoma tissue staining stained as well as the original Mc5.
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Affiliation(s)
- R B Christian
- Cancer Research Fund of Contra Costa, Walnut Creek, California 94596, USA
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26
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Bridges A, Birch A, Williams G, Aguet M, Schlatter D, Huber W, Garotta G, Robinson JA. Variable region cDNA sequences and characterization of murine anti-human interferon gamma receptor monoclonal antibodies that inhibit receptor binding by interferon gamma. Mol Immunol 1995; 32:1329-38. [PMID: 8643102 DOI: 10.1016/0161-5890(95)00114-x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
Murine monoclonal antibodies (mAbs) are described that recognize the extracellular human interferon gamma receptor alpha-chain (IFN gamma R) and inhibit the binding to it of interferon gamma. The inhibitory activities (IC50s) of these mAbs, quantified by radioimmunoassay using native receptor on human Raji cells, lie in the range 0.5-24 nM, whereas their relative affinities for the immobilised recombinant extracellular receptor, determined using surface plasmon resonance technology, are in the range 0.6-40.9 nM. Nine mAbs derived from one immunization, were shown by variable region cDNA sequencing to be clonally related, with mAb A6 from this group showing the highest affinity for the receptor. Another two mAbs, gamma R38 and gamma R99, derived from a separate immunization, are clonally unrelated to each other and to those in the A6 family. From the V-region sequences, the L-chains of mAbs A6, gamma R38 and gamma R99 were shown to belong to the V kappa 34C, V kappa 34C and V kappa 1 families, whereas the H-chains belong to the 3069, J606 and J558 families, respectively. The mAbs A6 and gamma R38 recognize overlapping epitopes on the N-terminal Ig-like domain of the IFN gamma R, whereas the gamma R99 epitope is located largely in the membrane proximal Ig-like domain. Sequence comparisons with Ig structures solved by X-ray diffraction allowed deductions concerning likely CDR canonical conformations. These studies provide essential information for crystallographic and mutagenesis experiments aimed at understanding the molecular basis of the interactions of these mAbs with the extracellular IFN gamma R.
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MESH Headings
- Amino Acid Sequence
- Animals
- Antibodies, Monoclonal/chemistry
- Antibodies, Monoclonal/genetics
- Antibodies, Monoclonal/pharmacology
- Antigens, CD/genetics
- Antigens, CD/immunology
- Base Sequence
- Binding Sites, Antibody
- Binding, Competitive/immunology
- DNA, Complementary/isolation & purification
- Humans
- Immunoglobulin Variable Region/chemistry
- Immunoglobulin Variable Region/genetics
- Immunoglobulin Variable Region/pharmacology
- Interferon-gamma/metabolism
- Mice
- Molecular Sequence Data
- Receptors, Interferon/genetics
- Receptors, Interferon/immunology
- Structure-Activity Relationship
- Interferon gamma Receptor
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Affiliation(s)
- A Bridges
- Institute of Organic Chemistry, University of Zurich, Switzerland
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27
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Kleymann G, Ostermeier C, Ludwig B, Skerra A, Michel H. Engineered Fv fragments as a tool for the one-step purification of integral multisubunit membrane protein complexes. BIO/TECHNOLOGY (NATURE PUBLISHING COMPANY) 1995; 13:155-60. [PMID: 9634756 DOI: 10.1038/nbt0295-155] [Citation(s) in RCA: 59] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The preparation of pure and homogeneous membrane proteins or membrane protein complexes is time consuming, and the yields are frequently insufficient for structural studies. To circumvent these problems we established an indirect immunoaffinity chromatography method based on engineered Fv fragments. cDNAs encoding the variable domains of hybridoma-derived antibodies raised against various membrane proteins were cloned and expressed in Escherichia coli. The Fv fragments were engineered to serve as bifunctional adaptor molecules. The Fv fragment binds to the epitope of the membrane protein, while the Strep tag affinity peptide, which was fused to the carboxy-terminus of the VH chain, immobilizes the antigen-Fv complex on a streptavidin sepharose column. The usefulness of this technique is illustrated with membrane protein complexes from Paracoccus denitrificans, namely, the cytochrome c oxidase (EC 1.9.3.1), the ubiquinol:cytochrome c oxidoreductase (EC 1.10.2.2), and subcomplexes or individual subunits thereof. These membrane proteins were purified simply by combining the crude P. denitrificans membrane preparation with the E. coli periplasmic cell fraction containing the corresponding Fv fragment, followed by solubilization and streptavidin affinity chromatography. Pure and highly active membrane protein complexes were eluted in the Fv-bound form using diaminobiotin for mild competitive displacement of the Strep tag. The affinity column could thus be reused under continuous operation for several months. Five to 10 mg of membrane protein complexes could be obtained without any detectable impurities within five hours.
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Affiliation(s)
- G Kleymann
- Max-Planck-Institut für Biophysik, Abteilung Molekulare Membranbiologie, Frankfurt, Germany
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28
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Duan L, Zhang H, Oakes JW, Bagasra O, Pomerantz RJ. Molecular and virological effects of intracellular anti-Rev single-chain variable fragments on the expression of various human immunodeficiency virus-1 strains. Hum Gene Ther 1994; 5:1315-24. [PMID: 7893803 DOI: 10.1089/hum.1994.5.11-1315] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
A variety of genetic therapies or intracellular immunization techniques hold promise as modalities to inhibit human immunodeficiency virus type 1 (HIV-1) replication in vivo. We have recently demonstrated that a single-chain variable fragment (SFv) construct, derived from a monoclonal antibody that binds to the HIV-1 regulatory protein Rev, can be expressed intracellularly and potently inhibits HIV-1 replication. This single-chain intracellular antibody, which avidly binds to the effector domain of Rev, is now demonstrated to dramatically inhibit various diverse laboratory and primary clinical strains of HIV-1. Potent suppression of HIV-1 replication by this modality is maintained over several months in long-term cultures. As well, the intracellular expression of anti-Rev SFv is shown to alter HIV-1 replication by specifically affecting Rev function. Importantly, no alterations in HIV-1 internalization, reverse transcription, or initial transcription of multiply spliced viral mRNAs are demonstrated in SFv-immunized cells, as compared to controls. Thus, these studies extend the understanding of the molecular mechanisms involved in the inhibition of lentivirus replication, by these intracellular antibody constructs.
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MESH Headings
- Amino Acid Sequence
- Antibodies, Monoclonal/chemistry
- Antibodies, Monoclonal/genetics
- Antibodies, Monoclonal/immunology
- Antiviral Agents/pharmacology
- Base Sequence
- DNA, Viral/isolation & purification
- Gene Expression Regulation, Viral/drug effects
- Gene Products, rev/antagonists & inhibitors
- Gene Products, rev/immunology
- Gene Products, rev/physiology
- HIV Antibodies/chemistry
- HIV Antibodies/genetics
- HIV Antibodies/immunology
- HIV-1/drug effects
- HIV-1/immunology
- HIV-1/physiology
- HeLa Cells
- Humans
- Immunization, Passive/methods
- Immunoglobulin Fragments/genetics
- Immunoglobulin Fragments/pharmacology
- Immunoglobulin Variable Region/genetics
- Immunoglobulin Variable Region/pharmacology
- Molecular Sequence Data
- Polymerase Chain Reaction
- Proviruses/isolation & purification
- Recombinant Fusion Proteins/biosynthesis
- Recombinant Fusion Proteins/genetics
- Recombinant Fusion Proteins/pharmacology
- Recombinant Proteins
- Single-Chain Antibodies
- Transfection
- Virus Replication/drug effects
- rev Gene Products, Human Immunodeficiency Virus
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Affiliation(s)
- L Duan
- Dorrance H. Hamilton Laboratories, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107
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29
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Mo JA, Bona CA, Holmdahl R. Variable region gene selection of immunoglobulin G-expressing B cells with specificity for a defined epitope on type II collagen. Eur J Immunol 1993; 23:2503-10. [PMID: 7691608 DOI: 10.1002/eji.1830231019] [Citation(s) in RCA: 29] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
Immunization with type II collagen (CII) induces collagen-induced arthritis (CIA) in animals, and B cells reactive with CII are involved in the induction and manifestation of the disease. In this study, B cell hybridomas producing IgG antibodies specific for a major epitope on mouse CII (the "C1" epitope, amino acid 316-333), were isolated 11 days after immunization from draining lymph nodes in DBA/1 mice. Injection into neonatal mice of purified and biotinylated monoclonal antibodies binding the C1 epitope led to a specific binding to joint cartilage, demonstrating that the antibodies interact with native antigen in vivo. cDNA sequencing of the B cell clones revealed that they all expressed the same combination of a variable heavy chain (VH J558 family) and light chain (V kappa 21 family) germ-line gene, apparently lacking somatic mutations. The presence of isotype-switched B cells expressing a certain combination of V genes encoding antibodies that bind epitopes in vivo, indicates that this B cell population has been peripherally selected.
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Affiliation(s)
- J A Mo
- Department of Medical and Physiological Chemistry, Uppsala University, Sweden
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30
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Couto JR, Blank EW, Peterson JA, Ceriani RL. Cloning of cDNAs encoding the variable domains of antibody KC4G3 and construction of a chimeric antibody. Hybridoma (Larchmt) 1993; 12:485-9. [PMID: 8244420 DOI: 10.1089/hyb.1993.12.485] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
We have cloned and sequenced cDNAs encoding the variable regions of the light (VL) and heavy (VH) chains of monoclonal antibody KC4G3. VL belongs to group II and resulted from a V kappa-J kappa 2 recombination. VH belongs to group IIId and arose from a V-D9-JH3 recombination. The VL and VH frameworks are respectively 84% and 83% identical to the corresponding VL and VH human consensus frameworks. The deduced amino acid sequence of VL contains an asparagine-linked glycosylation site in framework 3 (N74 I75 S76). We have determined that a large fraction of the light chains are indeed glycolysated. We constructed an IgG1, kappa human/mouse chimeric antibody (by inserting the murine KC4G3 Fv-encoding cDNAs into plasmids encoding a human IgG1, kappa Fc domain) and expressed it in SP2/0-Ag14 mouse myeloma cells. This chimeric monoclonal antibody is designated ChiKC4. We have determined that the murine monoclonal antibody KC4G3 binds the human breast mucin with an affinity constant of 1.1 x 10(9) M-1. ChiKC4 binds the same antigen with an affinity constant of 1.2 x 10(9) M-1. ChiKC4 binds the carcinoma tissue sections by the ABC immunoperoxidase method in an identical manner as does KC4G3.
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MESH Headings
- Amino Acid Sequence
- Animals
- Antibodies, Monoclonal/genetics
- Antibodies, Monoclonal/immunology
- Antibodies, Monoclonal/isolation & purification
- Antibodies, Neoplasm/genetics
- Antibodies, Neoplasm/immunology
- Antibodies, Neoplasm/isolation & purification
- Antigens, Neoplasm/immunology
- Antigens, Tumor-Associated, Carbohydrate
- Base Sequence
- Breast Neoplasms/immunology
- Cloning, Molecular
- Consensus Sequence
- DNA, Complementary/genetics
- Epithelium/immunology
- Genes, Immunoglobulin/genetics
- Genes, Synthetic
- Glycosylation
- Humans
- Immunoglobulin Constant Regions/genetics
- Immunoglobulin Heavy Chains/genetics
- Immunoglobulin Light Chains/genetics
- Immunoglobulin Variable Region/genetics
- Immunoglobulin kappa-Chains/genetics
- Male
- Mice
- Molecular Sequence Data
- Multiple Myeloma
- Neoplasm Proteins/immunology
- Prostatic Neoplasms/immunology
- Recombinant Fusion Proteins/genetics
- Recombinant Fusion Proteins/immunology
- Recombinant Fusion Proteins/isolation & purification
- Species Specificity
- Tumor Cells, Cultured
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Affiliation(s)
- J R Couto
- Cancer Research Fund of Contra Costa, Walnut Creek, CA 94596
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31
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Kofler H, Schnegg I, Geley S, Helmberg A, Varga JM, Kofler R. Mechanism of allergic cross-reactions--III. cDNA cloning and variable-region sequence analysis of two IgE antibodies specific for trinitrophenyl. Mol Immunol 1992; 29:161-6. [PMID: 1542295 DOI: 10.1016/0161-5890(92)90097-h] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
As a first step toward defining the molecular interactions between ligands and the IgE antigen-combining site, we report here the cDNA cloning and variable (V) region nucleic acid sequences of the heavy (H) and light (L) chains of 2 monoclonal mouse IgE antibodies to trinitrophenyl (ATCC-TIB142 = IGELa2 and ATCC-TIB141 = IGELb4). In all instances, full-length cDNA clones were obtained to facilitate future expression studies. The H chains were encoded by VH genes from the VH3660 and J558 gene families in context with DQ52 and DSP2.2 diversity (D) mini genes, and JH3 and JH4 joining (J) gene segments, respectively. Vk8/Jk2 and Vk1/Jk5 rearrangements encoded the respective L chain V-regions. Both antibodies exhibited considerable conservation of complementarity determining region (CDR) sequences, which will facilitate template-based computer modeling of the three-dimensional structures of complexes formed between various ligands and these antibodies. From sequence comparison between the dinitrophenyl (DNP)-binding myeloma protein MOPC-315 and these IgE antibodies likely candidates for hapten-contact residues within the binding sites of IGELa2 and IGELb4 have been suggested.
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Affiliation(s)
- H Kofler
- Department of Dermatology, University of Innsbruck, Austria
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32
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Kütemeier G, Harloff C, Mocikat R. Rapid isolation of immunoglobulin variable genes from cell lysates of rat hybridomas by polymerase chain reaction. Hybridoma (Larchmt) 1992; 11:23-32. [PMID: 1737637 DOI: 10.1089/hyb.1992.11.23] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
The isolation of the rearranged immunoglobulin genes from a hybridoma cell line, which is a prerequisite for the construction of a recombinant antibody, can easily be achieved by polymerase chain reaction. Here we demonstrate that this method, which was originally described for cloning murine immunoglobulin genes from cDNA, is also applicable for rat genes. We show that the procedure also works with crude cell lysates as starting material, thereby greatly reducing the time required for sample preparation. In addition we have sequenced the nonfunctional heavy chain variable gene of the fusion partner X63Ag8.653, which was readily amplified from our hybridoma cells, and whose sequence has been so far unknown.
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Affiliation(s)
- G Kütemeier
- GSF-Forschungszentrum für Umwelt und Gesundheit, Institut für Immunologie, München, Germany
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33
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Abstract
The variable region nucleotide sequences of seven (five IgM and two IgG) anti-histone monoclonal antibodies from a single MRL/Mp-lpr/lpr mouse have been determined. These antibodies are not clonally related and used diverse V, D and J genes. However, six of the seven antibodies have VH segments encoded by genes from the J558 family, two of these (an IgM and an IgG) share an identical VH gene. The isoelectric points of MRA3 and MRA12, the two IgG antibodies of the panel, range from 6.3 to 7.0 and from 6.0 to 6.3, respectively. The second conplementarity-determining region (CDR) of the VH gene of MRA12 (the most acidic and the most strongly histone-reactive antibody) includes only two positively charged but five negatively charged amino acid residues. This feature is unusual since the equivalent CDR in most VHJ558 genes are not comprised predominantly of acidic residues and suggests that such negatively charged residues are important for antibody binding to histones.
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34
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Heavy and light chain variable region sequences and antibody properties of anti-phosphotyrosine antibodies reveal both common and distinct features. J Biol Chem 1991. [DOI: 10.1016/s0021-9258(18)38160-2] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022] Open
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35
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Huston JS, Mudgett-Hunter M, Tai MS, McCartney J, Warren F, Haber E, Oppermann H. Protein engineering of single-chain Fv analogs and fusion proteins. Methods Enzymol 1991; 203:46-88. [PMID: 1762568 DOI: 10.1016/0076-6879(91)03005-2] [Citation(s) in RCA: 164] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
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36
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Lambson BE, Bernstein R, Mendelow BV. Chromosomal evolution in secretory and nonsecretory subline of MOPC 21. SOMATIC CELL AND MOLECULAR GENETICS 1990; 16:91-5. [PMID: 2309143 DOI: 10.1007/bf01650484] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Murine myeloma cell lines are noted for the instability of their immunoglobulin genes and the production of nonsynthesizing variants. In this study, the synthesizing line P3-X63-Ag8 (P3) and its nonsynthesizing subline X63-Ag8-653 (653) were karyotyped and rearrangements of the immunoglobulin carrying chromosomes were investigated. Loss of immunoglobulin synthesis was associated with a reduction in the number of immunoglobulin-carrying chromosomes. When these findings were analyzed in light of published molecular data, it appeared that loss of immunoglobulin synthesis in 653 probably occurred as a result of immunoglobulin gene loss. An unusual finding was the absence of the t(12;15) chromosome in both P3 and 653 cell lines. It was concluded that the t(12;15) chromosome, carried by MOPC 21, has evolved into an unrecognizable form.
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Affiliation(s)
- B E Lambson
- Department of Haematology, School of Pathology of the University of the Witwatersrand, Johannesburg, South Africa
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37
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Strohal R, Helmberg A, Kroemer G, Kofler R. Mouse Vk gene classification by nucleic acid sequence similarity. Immunogenetics 1989; 30:475-93. [PMID: 2574159 PMCID: PMC7087523 DOI: 10.1007/bf02421180] [Citation(s) in RCA: 76] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Analyses of immunoglobulin (Ig) variable (V) region gene usage in the immune response, estimates of V gene germline complexity, and other nucleic acid hybridization-based studies depend on the extent to which such genes are related (i.e., sequence similarity) and their organization in gene families. While mouse Igh heavy chain V region (VH) gene families are relatively well-established, a corresponding systematic classification of Igk light chain V region (Vk) genes has not been reported. The present analysis, in the course of which we reviewed the known extent of the Vk germline gene repertoire and Vk gene usage in a variety of responses to foreign and self antigens, provides a classification of mouse Vk genes in gene families composed of members with greater than 80% overall nucleic acid sequence similarity. This classification differed in several aspects from that of VH genes: only some Vk gene families were as clearly separated (by greater than 25% sequence dissimilarity) as typical VH gene families; most Vk gene families were closely related and, in several instances, members from different families were very similar (greater than 80%) over large sequence portions; frequently, classification by nucleic acid sequence similarity diverged from existing classifications based on amino-terminal protein sequence similarity. Our data have implications for Vk gene analyses by nucleic acid hybridization and describe potentially important differences in sequence organization between VH and Vk genes.
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Affiliation(s)
- R Strohal
- Institute for General and Experimental Pathology, University of Innsbruck Medical School, Austria
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38
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Kofler R, Strohal R, Balderas RS, Johnson ME, Noonan DJ, Duchosal MA, Dixon FJ, Theofilopoulos AN. Immunoglobulin kappa light chain variable region gene complex organization and immunoglobulin genes encoding anti-DNA autoantibodies in lupus mice. J Clin Invest 1988; 82:852-60. [PMID: 3138286 PMCID: PMC303593 DOI: 10.1172/jci113689] [Citation(s) in RCA: 70] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
We have investigated the genetic origin of autoantibody production in several strains of mice that spontaneously develop a systemic lupus erythematosus-like disease. Restriction fragment length polymorphism analyses of gene loci encoding kappa light chain variable regions (Igk-V) demonstrated, as shown previously for the Ig heavy chain locus, that autoantibody production and disease occur in different Igk-V haplotypes. Moreover, autoimmune mice with known genetic derivation inherited their Igk-V loci essentially unaltered from their nonautoimmune ancestors. New Zealand black lupus mice, with unknown genetic derivation, had a possibly recombinant Igk-V haplotype, composed of V kappa loci that were primarily indistinguishable from those of nonautoimmune strains from either of the two potential donor haplotypes. The heavy and light chain gene segments (variable, diversity, joining) encoding anti-DNA antibodies were diverse and often closely related, or even identical, to those found in antibodies to foreign antigens in normal mice. Only 1 of 11 sequenced variable region genes could not be assigned to existing variable region gene families; however, corresponding germline genes were present in the genome of normal mice as well. These data argue against abnormalities in the genes and mechanisms generating antibody diversity in lupus mice and suggest a remarkable genetic and structural diversity in the generation of anti-DNA binding sites.
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Affiliation(s)
- R Kofler
- Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037
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