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Fab arm exchange

Frederick W Jacobsen, Riki Stevenson, Cynthia Li, Hossein Salimi-Moosavi, Ling Liu, Jie Wen, Quanzhou Luo, Kristine Daris, Lynette Buck, Sterling Miller, Shu-Yin Ho, Wei Wang, Qing Chen, Kenneth Walker, Jette Wypych, Linda Narhi, Kannan Gunasekaran
IgG isotypes can differentially bind to Fcγ receptors and complement, making the selection of which isotype to pursue for development of a particular therapeutic antibody important in determining the safety and efficacy of the drug. IgG2 and IgG4 isotypes have significantly lower binding affinity to Fcγ receptors. Recent evidence suggests that the IgG2 isotype is not completely devoid of effector function, whereas the IgG4 isotype can undergo in vivo Fab arm exchange leading to bispecific antibody and off-target effects...
February 3, 2017: Journal of Biological Chemistry
Inga Koneczny, Jo A A Stevens, Anna De Rosa, Saif Huda, Maartje G Huijbers, Abhishek Saxena, Michelangelo Maestri, Konstantinos Lazaridis, Paraskevi Zisimopoulou, Socrates Tzartos, Jan Verschuuren, Silvère M van der Maarel, Philip van Damme, Marc H De Baets, Peter C Molenaar, Angela Vincent, Roberta Ricciardi, Pilar Martinez-Martinez, Mario Losen
Autoimmunity mediated by IgG4 subclass autoantibodies is an expanding field of research. Due to their structural characteristics a key feature of IgG4 antibodies is the ability to exchange Fab-arms with other, unrelated, IgG4 molecules, making the IgG4 molecule potentially monovalent for the specific antigen. However, whether those disease-associated antigen-specific IgG4 are mono- or divalent for their antigens is unknown. Myasthenia gravis (MG) with antibodies to muscle specific kinase (MuSK-MG) is a well-recognized disease in which the predominant pathogenic IgG4 antibody binds to extracellular epitopes on MuSK at the neuromuscular junction; this inhibits a pathway that clusters the acetylcholine (neurotransmitter) receptors and leads to failure of neuromuscular transmission...
February 2017: Journal of Autoimmunity
Shigeyuki Kawa
High serum immunoglobulin (Ig) G4 concentration and abundant IgG4-bearing plasma cell infiltration are characteristic features in autoimmune pancreatitis (AIP). AIP is also complicated with a variety of other organ involvements that commonly share marked IgG4-bearing plasma cell infiltration, suggesting the existence of a systemic disease associated with IgG4 currently recognized as IgG4-related disease (IgG4-RD). However, it is controversial whether IgG4 plays a role in the pathogenesis of AIP or IgG4-RD through such characteristic attributes as Fab-arm exchange and rheumatoid factor (RF)-like activity...
2017: Current Topics in Microbiology and Immunology
Xiaoyu Yang, Fengqiang Wang, Ying Zhang, Larry Wang, Svetlana Antonenko, Shuli Zhang, Yi Wei Zhang, Mohammad Tabrizifard, Grigori Ermakov, Derek Wiswell, Maribel Beaumont, Liming Liu, Daisy Richardson, Mohammed Shameem, Alexandre Ambrogelly
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...
December 2015: Journal of Pharmaceutical Sciences
Stephen W Jarantow, Barbara S Bushey, Jose R Pardinas, Ken Boakye, Eilyn R Lacy, Renouard Sanders, Manuel A Sepulveda, Sheri L Moores, Mark L Chiu
The efficacy of engaging multiple drug targets using bispecific antibodies (BsAbs) is affected by the relative cell-surface protein levels of the respective targets. In this work, the receptor density values were correlated to the in vitro activity of a BsAb (JNJ-61186372) targeting epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-MET). Simultaneous binding of the BsAb to both receptors was confirmed in vitro. By using controlled Fab-arm exchange, a set of BsAbs targeting EGFR and c-MET was generated to establish an accurate receptor quantitation of a panel of lung and gastric cancer cell lines expressing heterogeneous levels of EGFR and c-MET...
October 9, 2015: Journal of Biological Chemistry
Xiaoyu Yang, Ying Zhang, Fengqiang Wang, Larry Jin Wang, Daisy Richardson, Mohammed Shameem, Alexandre Ambrogelly
Therapeutic non-hinge-modified IgG4 molecules form bispecific hybrid antibodies with endogenous human IgG4 molecules via a process known as Fab-arm exchange (or called half molecule exchange). Analysis of the bispecific hybrids is critical for studies of half molecule exchange. A number of analytical methods are available to detect IgG4 hybrids. These methods mostly necessitate labeling or alteration of the model IgG4 molecules, or rely on time-consuming immunoassays and mass spectrometry. In addition, these methods do not allow isolation of hybrid antibodies...
September 1, 2015: Analytical Biochemistry
Aming Zhang, Jing Fang, Robert Y-T Chou, Pavel V Bondarenko, Zhongqi Zhang
Both recombinant and natural human IgG2 antibodies have several different disulfide bond isoforms, which possess different global structures, thermal stabilities, and biological activities. A detailed mapping of the structural difference among IgG2 disulfide isoforms, however, has not been established. In this work, we employed hydrogen/deuterium exchange mass spectrometry to study the conformation of three major IgG2 disulfide isoforms known as IgG2-B, IgG2-A1, and IgG2-A2 in two recombinant human IgG2 monoclonal antibodies...
March 17, 2015: Biochemistry
John-Paul Silva, Olivia Vetterlein, Joby Jose, Shirley Peters, Hishani Kirby
Human immunoglobulin G isotype 4 (IgG4) antibodies (Abs) are potential candidates for immunotherapy when reduced effector functions are desirable. IgG4 Abs are dynamic molecules able to undergo a process known as Fab arm exchange (FAE). This results in functionally monovalent, bispecific antibodies (bsAbs) with unknown specificity and hence, potentially, reduced therapeutic efficacy. IgG4 FAE is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 Abs...
February 27, 2015: Journal of Biological Chemistry
Aran F Labrijn, Joyce I Meesters, Patrick Priem, Rob N de Jong, Ewald T J van den Bremer, Muriel D van Kampen, Arnout F Gerritsen, Janine Schuurman, Paul W H I Parren
The generation of bispecific antibodies (bsAbs) with natural IgG architecture in a practical and efficient manner has been a longstanding challenge. Here we describe controlled Fab-arm exchange (cFAE), which is an easy-to-use method to generate bispecific IgG1 (bsIgG1). The protocol involves the following: (i) separate expression of two parental IgG1s containing single matching point mutations in the CH3 domain; (ii) mixing of parental IgG1s under permissive redox conditions in vitro to enable recombination of half-molecules; (iii) removal of the reductant to allow reoxidation of interchain disulfide bonds; and (iv) analysis of exchange efficiency and final product using chromatography-based or mass spectrometry (MS)-based methods...
October 2014: Nature Protocols
Theo Rispens, Anna M Davies, Pleuni Ooijevaar-de Heer, Samira Absalah, Onno Bende, Brian J Sutton, Gestur Vidarsson, Rob C Aalberse
Interdomain interactions between the CH3 domains of antibody heavy chains are the first step in antibody assembly and are of prime importance for maintaining the native structure of IgG. For human IgG4 it was shown that CH3-CH3 interactions are weak, resulting in the potential for half-molecule exchange ("Fab arm exchange"). Here we systematically investigated non-covalent interchain interactions for CH3 domains in the other human subclasses, including polymorphisms (allotypes), using real-time monitoring of Fab arm exchange with a FRET-based kinetic assay...
February 28, 2014: Journal of Biological Chemistry
Maimuna Ahmad, Vinay S Mahajan, Hamid Mattoo, John H Stone, Shiv Pillai
No abstract text is available yet for this article.
January 2014: Journal of Rheumatology
Yunji Wu, Anthony P West, Helen J Kim, Matthew E Thornton, Andrew B Ward, Pamela J Bjorkman
The human immunoglobulin G (IgG) 2G12 recognizes high-mannose carbohydrates on the HIV type 1 (HIV-1) envelope glycoprotein gp120. Its two antigen-binding fragments (Fabs) are intramolecularly domain exchanged, resulting in a rigid (Fab)2 unit including a third antigen-binding interface not found in antibodies with flexible Fab arms. We determined crystal structures of dimeric 2G12 IgG created by intermolecular domain exchange, which exhibits increased breadth and >50-fold increased neutralization potency compared with monomeric 2G12...
December 12, 2013: Cell Reports
Anna M Davies, Theo Rispens, Pleuni Ooijevaar-de Heer, Hannah J Gould, Roy Jefferis, Rob C Aalberse, Brian J Sutton
Human IgG4, normally the least abundant of the four subclasses of IgG in serum, displays a number of unique biological properties. It can undergo heavy-chain exchange, also known as Fab-arm exchange, leading to the formation of monovalent but bispecific antibodies, and it interacts poorly with FcγRII and FcγRIII, and complement. These properties render IgG4 relatively "non-inflammatory" and have made it a suitable format for therapeutic monoclonal antibody production. However, IgG4 is also known to undergo Fc-mediated aggregation and has been implicated in auto-immune disease pathology...
February 6, 2014: Journal of Molecular Biology
François Debaene, Elsa Wagner-Rousset, Olivier Colas, Daniel Ayoub, Nathalie Corvaïa, Alain Van Dorsselaer, Alain Beck, Sarah Cianférani
Monoclonal antibodies (mAbs) and derivatives such as antibody-drug conjugates (ADC) and bispecific antibodies (bsAb), are the fastest growing class of human therapeutics. Most of the therapeutic antibodies currently on the market and in clinical trials are chimeric, humanized, and human immunoglobulin G1 (IgG1). An increasing number of IgG2s and IgG4s that have distinct structural and functional properties are also investigated to develop products that lack or have diminished antibody effector functions compared to IgG1...
October 15, 2013: Analytical Chemistry
Michael J Gramer, Ewald T J van den Bremer, Muriel D van Kampen, Amitava Kundu, Peter Kopfmann, Eric Etter, David Stinehelfer, Justin Long, Tom Lannom, Esther H Noordergraaf, Jolanda Gerritsen, Aran F Labrijn, Janine Schuurman, Patrick H C van Berkel, Paul W H I Parren
The manufacturing of bispecific antibodies can be challenging for a variety of reasons. For example, protein expression problems, stability issues, or the use of non-standard approaches for manufacturing can result in poor yield or poor facility fit. In this paper, we demonstrate the use of standard antibody platforms for large-scale manufacturing of bispecific IgG1 by controlled Fab-arm exchange. Two parental antibodies that each contain a single matched point mutation in the CH3 region were separately expressed in Chinese hamster ovary cells and manufactured at 1000 L scale using a platform fed-batch and purification process that was designed for standard antibody production...
November 2013: MAbs
Theo Rispens, Margreet H Hart, Pleuni Ooijevaar-de Heer, Astrid van Leeuwen, Anke Vennegoor, Joep Killestein, Gerrit-Jan Wolbink, Desiree van der Kleij
Direct comparison of immunogenicity data is hampered by differential drug interference in different assay formats. In this paper we identify a drug-related factor that influences the extent of drug interference. We systematically investigated the influence of drug valency of different antibody-derived biologicals on the drug interference, using mono- and bivalent formats of adalimumab as a model system. Our results indicate that compared to regular bivalent antibodies, antibody-derived drugs that are monovalent result in less drug interference...
November 2013: Journal of Pharmaceutical and Biomedical Analysis
Aran F Labrijn, Joyce I Meesters, Bart E C G de Goeij, Ewald T J van den Bremer, Joost Neijssen, Muriel D van Kampen, Kristin Strumane, Sandra Verploegen, Amitava Kundu, Michael J Gramer, Patrick H C van Berkel, Jan G J van de Winkel, Janine Schuurman, Paul W H I Parren
The promise of bispecific antibodies (bsAbs) to yield more effective therapeutics is well recognized; however, the generation of bsAbs in a practical and cost-effective manner has been a formidable challenge. Here we present a technology for the efficient generation of bsAbs with normal IgG structures that is amenable to both antibody drug discovery and development. The process involves separate expression of two parental antibodies, each containing single matched point mutations in the CH3 domains. The parental antibodies are mixed and subjected to controlled reducing conditions in vitro that separate the antibodies into HL half-molecules and allow reassembly and reoxidation to form highly pure bsAbs...
March 26, 2013: Proceedings of the National Academy of Sciences of the United States of America
Anna M Davies, Theo Rispens, Tamara H den Bleker, James M McDonnell, Hannah J Gould, Rob C Aalberse, Brian J Sutton
Antibodies of the human IgG4 subclass uniquely undergo a process of Fab-arm exchange in which the heavy-chains of antibodies of different specificities can dissociate and then recombine. The mechanism by which the resulting functionally monovalent but bi-specific antibodies are formed is not only key to understanding their biological role, but is also important for the design of therapeutic monoclonal antibodies. Both the hinge region and the C(H)3 domain interface are known to be involved, and of the residues that differ between human IgG1 and IgG4 in C(H)3, residue 409, the only difference at the interface itself, has been implicated...
May 2013: Molecular Immunology
Christina Ball, Bernard Fox, Simon Hufton, Giles Sharp, Stephen Poole, Richard Stebbings, David Eastwood, Lucy Findlay, Paul W H I Parren, Robin Thorpe, Adrian Bristow, Susan J Thorpe
The unexpected outcome of the clinical trial of the superagonistic CD28 mAb TGN1412 (IgG4κ) continues to stimulate interest. We show that TGN1412 binds similarly to human and cynomolgus macaque FcγR, eliminating the possibility that differences in Fc-mediated interactions with FcγR contributed to the failure of preclinical testing in macaques to predict toxicity in humans. The influence of the Fc domain and C region structure on the in vitro functional activity of TGN1412 was investigated using F(ab')(2) and Fab fragments derived from TGN1412 recovered from the trial and recombinant TGN1412 subclass variants and mutants...
December 15, 2012: Journal of Immunology: Official Journal of the American Association of Immunologists
Theo Rispens, Joyce Meesters, Tamara H den Bleker, Pleuni Ooijevaar-De Heer, Janine Schuurman, Paul W H I Parren, Aran Labrijn, Rob C Aalberse
Human IgG4 antibodies are remarkable not only because they can dynamically exchange half-molecules (Fab-arm exchange) but also for their ability to interact with the Fc part of IgG4 and other IgG subclasses. This rheumatoid factor-like binding of IgG4 does not appear to take place spontaneously, because it is only observed to solid-phase or antigen-bound IgG. We hypothesized that Fc-Fc interactions might involve (partial) dissociation of heavy chains. We investigated the molecular basis of these Fc-Fc interactions, and found that the structural features important for the exchange reaction also control the Fc binding activity...
January 2013: Molecular Immunology
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