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10.1080/19420862.2017.1371386

http://scihub22266oqcxt.onion/10.1080/19420862.2017.1371386
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C5680806!5680806!28846506
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suck abstract from ncbi


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pmid28846506      MAbs 2017 ; 9 (8): 1270-81
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  • Rare, high-affinity mouse anti-PD-1 antibodies that function in checkpoint blockade, discovered using microfluidics and molecular genomics #MMPMID28846506
  • Adler AS; Mizrahi RA; Spindler MJ; Adams MS; Asensio MA; Edgar RC; Leong J; Leong R; Johnson DS
  • MAbs 2017[Nov]; 9 (8): 1270-81 PMID28846506show ga
  • Conventionally, mouse hybridomas or well-plate screening are used to identify therapeutic monoclonal antibody candidates. In this study, we present an alternative to hybridoma-based discovery that combines microfluidics, yeast single-chain variable fragment (scFv) display, and deep sequencing to rapidly interrogate and screen mouse antibody repertoires. We used our approach on six wild-type mice to identify 269 molecules that bind to programmed cell death protein 1 (PD-1), which were present at an average of 1 in 2,000 in the pre-sort scFv libraries. Two rounds of fluorescence-activated cell sorting (FACS) produced populations of PD-1-binding scFv with a mean enrichment of 800-fold, whereas most scFv present in the pre-sort mouse repertoires were de-enriched. Therefore, our work suggests that most of the antibodies present in the repertoires of immunized mice are not strong binders to PD-1. We observed clusters of related antibody sequences in each mouse following FACS, suggesting evolution of clonal lineages. In the pre-sort repertoires, these putative clonal lineages varied in both the complementary-determining region (CDR)3K and CDR3H, while the FACS-selected PD-1-binding subsets varied primarily in the CDR3H. PD-1 binders were generally not highly diverged from germline, showing 98% identity on average with germline V-genes. Some CDR3 sequences were discovered in more than one animal, even across different mouse strains, suggesting convergent evolution. We synthesized 17 of the anti-PD-1 binders as full-length monoclonal antibodies. All 17 full-length antibodies bound recombinant PD-1 with KD < 500 nM (average = 62 nM). Fifteen of the 17 full-length antibodies specifically bound surface-expressed PD-1 in a FACS assay, and nine of the antibodies functioned as checkpoint inhibitors in a cellular assay. We conclude that our method is a viable alternative to hybridomas, with key advantages in comprehensiveness and turnaround time.
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