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      Productive common light chain libraries yield diverse panels of high affinity bispecific antibodies

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          ABSTRACT

          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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          Most cited references38

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          Diversity in the CDR3 region of V(H) is sufficient for most antibody specificities.

          J. Xu, M Davis (2000)
          All rearranging antigen receptor genes have one or two highly diverse complementarity determining regions (CDRs) among the six that typically form the ligand binding surface. We report here that, in the case of antibodies, diversity at one of these regions, CDR3 of the V(H) domain, is sufficient to permit otherwise identical IgM molecules to distinguish between a variety of hapten and protein antigens. Furthermore, we find that somatic mutation can allow such antibodies to achieve surprisingly high affinities. These results are consistent with a model in which the highly diverse CDR3 loops are the key determinant of specificity in antigen recognition in both T cell receptors (TCR) and antibodies, whereas the germline-encoded CDR1 and CDR2 sequences are much more cross-reactive.
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            Biophysical properties of the clinical-stage antibody landscape.

            Antibodies are a highly successful class of biological drugs, with over 50 such molecules approved for therapeutic use and hundreds more currently in clinical development. Improvements in technology for the discovery and optimization of high-potency antibodies have greatly increased the chances for finding binding molecules with desired biological properties; however, achieving drug-like properties at the same time is an additional requirement that is receiving increased attention. In this work, we attempt to quantify the historical limits of acceptability for multiple biophysical metrics of "developability." Amino acid sequences from 137 antibodies in advanced clinical stages, including 48 approved for therapeutic use, were collected and used to construct isotype-matched IgG1 antibodies, which were then expressed in mammalian cells. The resulting material for each source antibody was evaluated in a dozen biophysical property assays. The distributions of the observed metrics are used to empirically define boundaries of drug-like behavior that can represent practical guidelines for future antibody drug candidates.
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              Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire.

              Antibody repertoire diversity, potentially as high as 10(11) unique molecules in a single individual, confounds characterization by conventional sequence analyses. In this study, we present a general method for assessing human antibody sequence diversity displayed on phage using massively parallel pyrosequencing, a novel application of Kabat column-labeled profile Hidden Markov Models, and translated complementarity determining region (CDR) capture-recapture analysis. Pyrosequencing of domain amplicon and RCA PCR products generated 1.5 x 10(6) reads, including more than 1.9 x 10(5) high quality, full-length sequences of antibody variable fragment (Fv) variable domains. Novel methods for germline and CDR classification and fine characterization of sequence diversity in the 6 CDRs are presented. Diverse germline contributions to the repertoire with random heavy and light chain pairing are observed. All germline families were found to be represented in 1.7 x 10(4) sequences obtained from repeated panning of the library. While the most variable CDR (CDR-H3) presents significant length and sequence variability, we find a substantial contribution to total diversity from somatically mutated germline encoded CDRs 1 and 2. Using a capture-recapture method, the total diversity of the antibody library obtained from a human donor Immunoglobulin M (IgM) pool was determined to be at least 3.5 x 10(10). The results provide insights into the role of IgM diversification, display library construction, and productive germline usages in antibody libraries and the humoral repertoire.
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                Author and article information

                Journal
                MAbs
                MAbs
                KMAB
                kmab20
                mAbs
                Taylor & Francis
                1942-0862
                1942-0870
                Feb-Mar 2018
                14 December 2017
                14 December 2017
                : 10
                : 2
                : 256-268
                Affiliations
                Oncology Research and Development, Pfizer Inc. , South San Francisco, CA, USA
                Author notes
                CONTACT Thomas Van Blarcom thomas.vanblarcom@ 123456pfizer.com
                [*]

                These two authors contributed equally to this work.

                Supplemental data for this article can be accessed on the publisher's website.

                Author information
                https://orcid.org/0000-0001-6059-7425
                https://orcid.org/0000-0002-9673-7394
                https://orcid.org/0000-0002-5034-1722
                Article
                1406570
                10.1080/19420862.2017.1406570
                5825193
                29227213
                3fb1de82-347c-46f3-aeff-11afc06a2bbf
                © 2018 The Author(s). Published with license by Taylor & Francis Group, LLC

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License ( http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

                History
                : 12 October 2017
                : 6 November 2017
                : 11 November 2017
                Page count
                Figures: 5, Tables: 1, Equations: 0, References: 64, Pages: 13
                Categories
                Report

                Immunology
                bispecific antibody,common light chain,synthetic antibody library,high affinity,manufacturing,4-1bb

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