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http://dx.doi.org/10.14348/molcells.2017.0106

Generation, Diversity Determination, and Application to Antibody Selection of a Human Naïve Fab Library  

Kim, Sangkyu (Department of Systems Immunology, College of Biomedical Science, Kangwon National University)
Park, Insoo (Therapeutic Antibody Research Center, Korea Research Institute of Bioscience and Biotechnology)
Park, Seung Gu (Department of Medical Biotechnology, College of Biomedical Science, Kangwon National University)
Cho, Seulki (Therapeutic Antibody Research Center, Korea Research Institute of Bioscience and Biotechnology)
Kim, Jin Hong (Therapeutic Antibody Research Center, Korea Research Institute of Bioscience and Biotechnology)
S.Ipper, Nagesh (Institute of Bioscience and Biotechnology, Kangwon National University)
Choi, Sun Shim (Department of Medical Biotechnology, College of Biomedical Science, Kangwon National University)
Lee, Eung Suk (Scripps Korea Antibody Institute)
Hong, Hyo Jeong (Department of Systems Immunology, College of Biomedical Science, Kangwon National University)
Publication Information
Molecules and Cells / v.40, no.9, 2017 , pp. 655-666 More about this Journal
Abstract
We constructed a large $na{\ddot{i}}ve$ human Fab library ($3{\times}10^{10}$ colonies) from the lymphocytes of 809 human donors, assessed available diversities of the heavy-chain variable (VH) and ${\kappa}$ light-chain variable (VK) domain repertoires, and validated the library by selecting Fabs against 10 therapeutically relevant antigens by phage display. We obtained a database of unique 7,373 VH and 41,804 VK sequences by 454 pyrosequencing, and analyzed the repertoires. The distribution of VH and VK subfamilies and germline genes in our antibody repertoires slightly differed from those in earlier published natural antibody libraries. The frequency of somatic hypermutations (SHMs) in heavy-chain complementarity determining region (HCDR)1 and HCDR2 are higher compared with the natural IgM repertoire. Analysis of position-specific SHMs in CDRs indicates that asparagine, threonine, arginine, aspartate and phenylalanine are the most frequent non-germline residues on the antibody-antigen interface and are converted mostly from the germline residues, which are highly represented in germline SHM hotspots. The amino acid composition and length-dependent changes in amino acid frequencies of HCDR3 are similar to those in previous reports, except that frequencies of aspartate and phenylalanine are a little higher in our repertoire. Taken together, the results show that this antibody library shares common features of natural antibody repertoires and also has unique features. The antibody library will be useful in the generation of human antibodies against diverse antigens, and the information about the diversity of natural antibody repertoires will be valuable in the future design of synthetic human antibody libraries with high functional diversity.
Keywords
diversity; human monoclonal antibody; $na{\ddot{i}}ve$ antibody library; phage display; somatic hypermutation;
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