Characterization and analysis of scFv-IgG bispecific antibody size variants
Mingyan CaoChunlei WangWai Keen ChungDana MotabarJihong WangElizabeth ChristianShi‐Hua LinAlan K. HunterXiangyang WangDengfeng Liu
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Abstract:
Bispecific antibodies are an emergent class of biologics that is of increasing interest for therapeutic applications. In one bispecific antibody format, single-chain variable fragments (scFv) are linked to or inserted in different locations of an intact immunoglobulin G (IgG) molecule to confer dual epitope binding. To improve biochemical stability, cysteine residues are often engineered on the heavy- and light-chain regions of the scFv to form an intrachain disulfide bond. Although this disulfide bond often improves stability, it can also introduce unexpected challenges to manufacturing or development. We report size variants that were observed for an appended scFv-IgG bispecific antibody. Structural characterization studies showed that the size variants resulted from the engineered disulfide bond on the scFv, whereby the engineered disulfide was found to be either open or unable to form an intrachain disulfide bond due to cysteinylation or glutathionylation of the cysteines. Furthermore, the scFv engineered cysteines also formed intermolecular disulfide bonds, leading to the formation of highly stable dimers and aggregates. Because both the monomer variants and dimers showed lower bioactivity, they were considered to be product-related impurities that must be monitored and controlled. To this end, we developed and optimized a robust, precise, and accurate high-resolution size-exclusion chromatographic method, using a statistical design-of-experiments methodology.Keywords:
Bispecific antibody
Immunoglobulin Fab Fragments
We report the development and application of a method for determining bonding patterns in disulfide-linked peptides containing closely spaced cysteine residues. Through the utility of classic N-terminal sequencing chemistry coupled with facile liquid chromatography and mass spectrometric analysis of the cleavage products, we report the ability to demonstrate unambiguous assignment of paired cysteine residues, using human insulin as a model protein. The conditions of the technique were selected and optimized to maintain disulfide integrity. In a forthcoming article, we will present the results of this method as applied to the complete elucidation of linkages in disulfide variants of a therapeutic monoclonal antibody of the IgG2 subclass.
Disulfide Linkage
Cleavage (geology)
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The Fab'-fragment of a mouse IgA-myeloma (protein-315) was split by pepsin to yield a smaller fragment that retained the anti-2,4-dinitrophenyl activity of the intact protein. This fragment, which we call Fv, has a molecular weight of about 30,000 (half that of Fab'), and is composed of two polypeptide chains (molecular weight 14,000) held together by noncovalent bonds. The N-terminal sequence of Fv suggests that it is composed of the N-terminal half of Fab', and consists of the variable portions of the heavy and light chains. Since Fv has about one binding site with the same association constant as Fab', this experiment provides direct evidence that the antibody site in this protein is contained entirely in the variable portion, and is independent of the constant portion, of the molecule.
Myeloma protein
Immunoglobulin Fab Fragments
Immunoglobulin heavy chain
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Bispecific antibodies that engage immune cells to kill cancer cells are actively pursued in cancer immunotherapy. Different types of bispecific antibodies, including single-chain fragments, Fab fragments, nanobodies, and immunoglobulin Gs (IgGs), have been studied. However, the low molecular weight of bispecific antibodies with single-chain or Fab fragments generally leads to their rapid clearance in vivo, which limits the therapeutic potential of these bispecific antibodies.In this study, we used a site-specific PEGylation strategy to modify the bispecific single-domain antibody-linked Fab (S-Fab), which was designed by linking an anticarcinoembryonic antigen (anti-CEA) nanobody with an anti-CD3 Fab.The half-life (t1/2) of PEGylated S-Fab (polyethylene glycol-S-Fab) was increased 12-fold in vivo with a slightly decreased tumor cell cytotoxicity in vitro as well as more potent tumor growth inhibition in vivo compared to S-Fab.This study demonstrated that PEGylation is an effective approach to enhance the antitumor efficacy of bispecific antibodies.
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Bispecific antibody
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The commercial success of bispecific antibodies generally has been hindered by the complexities associated with generating appropriate molecules for both research scale and large scale manufacturing purposes. Bispecific IgG (BsIgG) based on two antibodies that use an identical common light chain can be combined with a minimal set of Fc mutations to drive heavy chain heterodimerization in order to address these challenges. However, the facile generation of common light chain antibodies with properties similar to traditional monoclonal antibodies has not been demonstrated and they have only been used sparingly. Here, we describe the design of a synthetic human antibody library based on common light chains to generate antibodies with biochemical and biophysical properties that are indistinguishable to traditional therapeutic monoclonal antibodies. We used this library to generate diverse panels of well-behaved, high affinity antibodies toward a variety of epitopes across multiple antigens, including mouse 4-1BB, a therapeutically important T cell costimulatory receptor. Over 200 BsIgG toward 4-1BB were generated using an automated purification method we developed that enables milligram-scale production of BsIgG. This approach allowed us to identify antibodies with a wide range of agonistic activity that are being used to further investigate the therapeutic potential of antibodies targeting one or more epitopes of 4-1BB.
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Phage display technology allows for the production and rapid selection of antigen-specific, Fab antibody fragments. For purposes of immune therapy, though, complete antibodies that retain the Fc domain are often required. In this regard, we designed cassette vectors for converting human Fab fragments selected from combinatorial phage display libraries into full-length IgG1 monoclonal antibodies (MAbs). Two expression vectors, pIEI-Light and pIEI-Heavy, were engineered to contain respective light- and heavy-chain human signal sequences downstream of the baculovirus immediate early gene promoter, IEI. Vector pIEI-Heavy also contains the coding region for each of the human IgG1 constant domains. To generate complete antibody genes, the cassette vectors possess convenient restriction enzyme sites for rapid in-frame cloning of coding regions for full-length light chains in pIEI-Light and for the heavy-chain variable domains in pIEI-Heavy of Fab fragments. Using these constructs and a method that allows for stable transformation of insect cells, complete light- and heavy-chain genes can be inserted into the insect cell genome and subsequently expressed under the control of the baculovirus IEI promoter. This cassette vector system was used to generate stably transformed insect cells that continuously secreted functional full-length, IgG1 MAbs. The expressed antibodies exhibited light and heavy chains of the appropriate molecular sizes and retained the ability to bind antigen. We conclude that our cassette vectors could serve as valuable tools for generating human IgG1 antibodies.
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Immunoglobulin Fab Fragments
Cloning (programming)
Immunoglobulin heavy chain
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Background: Recently a range of alternative novel therapies have been developed to improve treatment options for patients with Hemophilia A. One approach is to generate a Factor VIII (F.VIII) mimetic molecule using a humanised bispecific antibody, as was done for Hemlibra. Taking advantage of Kymab's fully human antibody discovery platform, we describe the selection and optimisation of an FVIII mimetic common light chain (CLC) bispecific antibody which can similarly catalyse the generation of Factor Xa (FXase) and normalise the activated partial thromboplastin time (aPTT). Aims: To generate a functionally active F.VIII mimetic bispecific antibody for Haemophilia A treatment Methods: F.IX and F.X. binding antibodies were generated by immunizing Kymouse™, which contain the full human immunoglobin repertoire, with F.IX or F.X, respectively. Isolated F.IX and F.X specific arms were co‐expressed as 2‐heavy‐2‐light‐chain (2H2L) bispecific antibodies. Purified 2H2L bispecifics were screened using a high‐throughput chromogenic FXase assay. The light chain of a promiscuous F.IX arm was chosen to generate transgenic mice expressing this bespoke common light chain (CLC) in the Kymouse™ background. By immunizing these transgenic mice with F.X, F.X binding antibodies containing the CLC were recovered. The heavy chains of these F.X binding antibodies were co‐expressed with the heavy/light chains of the chosen F.IX arm as CLC bispecific antibodies. One biologically active CLC bispecific antibody was identified by functional assays, and chosen for further optimization. The optimization of the lead bispecific antibody, KY1049, was achieved by data mining of next generation sequencing information using Kymab's IntelliSelect™ bioinformatic platform, coupled with site‐specific mutagenesis. Results: More than 8,000 2H2L bispecifics were screened by a chromogenic FXase assay to select the most active and versatile F.IX arm (Figure 1A). More than 400 F.X heavy chains subsequently isolated from the bespoke CLC Kymouse™ were screened to identify a highly active CLC bispecific (Figure 1B). Further optimisation of the molecule was carried out to iteratively increase FVIII mimetic activity by deep data‐mining of heavy chain NGS sequence data, or site‐specific mutagenesis. The combinatorial optimization process resulted in a highly functional CLC bispecific, KY1049 with comparable F.VIII mimetic activities to a sequence‐identical analogue of Hemlibra (Figure 1C). Summary/Conclusion: Our bispecific antibody discovery platform consisting of four‐chain matrix screening, common light chain transgenic mouse technology, B cell network analysis and site‐specific mutagenesis was applied to develop KY1049, a potent FV.III mimetic bispecific antibody, which shows equivalent activity to Hemlibra in vitro and holds promise as a future therapeutic in Haemophilia A. image
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Factor IX
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Abstract Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies.
Bispecific antibody
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We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This "crossover" retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible "CrossMab" formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMab(CH1-CL) was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.
Heavy chain
Immunoglobulin Fab Fragments
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Thiol
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A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.
Bispecific antibody
Protein Engineering
Immunoglobulin Fab Fragments
Fragment crystallizable region
Protein Stability
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