Rare, high-affinity mouse anti-PD-1 antibodies that function in checkpoint blockade, discovered using microfluidics and molecular genomics

MAbs. 2017 Nov/Dec;9(8):1270-1281. doi: 10.1080/19420862.2017.1371386. Epub 2017 Aug 28.


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.

Keywords: PD-1; checkpoint inhibitors; deep sequencing; microfluidics; mouse repertoire; yeast display.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibodies, Monoclonal / metabolism
  • Antibodies, Monoclonal / pharmacology
  • Antibody Affinity / immunology*
  • Complementarity Determining Regions / genetics
  • Complementarity Determining Regions / immunology
  • Complementarity Determining Regions / metabolism
  • Flow Cytometry
  • Genomics / methods*
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Hybridomas
  • Mice
  • Microfluidics / methods*
  • Peptide Library
  • Programmed Cell Death 1 Receptor / antagonists & inhibitors
  • Programmed Cell Death 1 Receptor / immunology*
  • Programmed Cell Death 1 Receptor / metabolism
  • Protein Binding / immunology
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / immunology
  • Single-Chain Antibodies / metabolism


  • Antibodies, Monoclonal
  • Complementarity Determining Regions
  • Pdcd1 protein, mouse
  • Peptide Library
  • Programmed Cell Death 1 Receptor
  • Single-Chain Antibodies