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      Generation and analysis of the improved human HAL9/10 antibody phage display libraries

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          Abstract

          Background

          Antibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library.

          Results

          The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×10 10 independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations.

          Conclusion

          The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

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          Most cited references54

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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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            Antibody therapeutics in cancer.

            In a relatively short period of time, monoclonal antibodies have entered the mainstream of cancer therapy. Their first use was as antagonists of oncogenic receptor tyrosine kinases, but today monoclonal antibodies have emerged as long-sought vehicles for the targeted delivery of potent chemotherapeutic agents and as powerful tools to manipulate anticancer immune responses. With ever more promising results from the clinic, the future will likely see continued growth in the discovery and development of therapeutic antibodies and their derivatives.
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              A roadmap to generate renewable protein binders to the human proteome.

              Despite the wealth of commercially available antibodies to human proteins, research is often hindered by their inconsistent validation, their poor performance and the inadequate coverage of the proteome. These issues could be addressed by systematic, genome-wide efforts to generate and validate renewable protein binders. We report a multicenter study to assess the potential of hybridoma and phage-display technologies in a coordinated large-scale antibody generation and validation effort. We produced over 1,000 antibodies targeting 20 SH2 domain proteins and evaluated them for potency and specificity by enzyme-linked immunosorbent assay (ELISA), protein microarray and surface plasmon resonance (SPR). We also tested selected antibodies in immunoprecipitation, immunoblotting and immunofluorescence assays. Our results show that high-affinity, high-specificity renewable antibodies generated by different technologies can be produced quickly and efficiently. We believe that this work serves as a foundation and template for future larger-scale studies to create renewable protein binders.
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                Author and article information

                Contributors
                jonas.kuegler@tu-bs.de
                sonja.wilke@mab-factory.com
                d.meier@tu-bs.de
                f.tomszak@tu-bs.de
                andre.frenzel@tu-bs.de
                th.schirrmann@tu-bs.de
                s.duebel@tu-bs.de
                h.garritsen@klinikum-braunschweig.de
                Bjoern.Hock@merckgroup.com
                lars.toleikis@merckgroup.com
                Mark.Schuette@merckgroup.com
                m.hust@tu-bs.de
                Journal
                BMC Biotechnol
                BMC Biotechnol
                BMC Biotechnology
                BioMed Central (London )
                1472-6750
                19 February 2015
                19 February 2015
                2015
                : 15
                Affiliations
                [ ]Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Spielmannstr. 7, 38106 Braunschweig, Germany
                [ ]mAb-factory GmbH, Gelsenkirchenstr. 5, 38108 Braunschweig, Germany
                [ ]YUMAB GmbH, Rebenring 33, 38106 Braunschweig, Germany
                [ ]Klinikum Braunschweig g GmbH, Institut für Klinische Transfusionsmedizin, Celler Str. 38, 38114 Braunschweig, Germany
                [ ]Department Vaccinology, Helmholtz-Zentrum für Infektionsforschung, Inhoffenstraße 7, 38124 Braunschweig, Germany
                [ ]Merck KGaA, Darmstadt, Germany
                Article
                125
                10.1186/s12896-015-0125-0
                4352240
                25888378
                1da37ce1-3aac-4ea8-b479-ebac0b617775
                © Kügler et al.; licensee BioMed Central. 2015

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                Categories
                Research Article
                Custom metadata
                © The Author(s) 2015

                Biotechnology
                scfv,phage display,antibody engineering,library,panning,screening
                Biotechnology
                scfv, phage display, antibody engineering, library, panning, screening

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