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

Abstract

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.

Antibody discovery and engineering by enhanced CRISPR-Cas9 integration of variable gene cassette libraries in mammalian cells

“…43 In recent years, novel single-cell methodologies based on droplet microfluidics have been adapted to screen naturally paired V L and V H genes by yeast display. [44][45][46] A similar approach of cloning natural V H :V L pairs into our synthetic antibody HDR scaffold would offer an attractive way to functionally screen antibody repertoires in mammalian cells.…”

Antibody repertoire analysis of mouse immunization protocols using microfluidics and molecular genomics

“…Using our previously described high-throughput microfluidic antibody discovery platform, 12,18,19 B cells isolated from each tissue (>2 million total cells per immunization method; Supplementary Table S1) were encapsulated into droplets such that each droplet contained a single B cell ( Figure 1b). The heavy and light (kappa only) chain sequences from each cell were amplified within droplets to generate an scFv sequence that retains the proper heavy:light Ig pairing of the original B cell.…”

“…Titer of each mouse was assessed by ELISA on a 1:3 dilution series of antigen, starting at 1000 ng/mL, using a horseradish peroxidase-goat anti-mouse IgG detection antibody (Jackson ImmunoResearch 115-035-071). ELISA was performed prior to the final boosts for ALD/MDP, ISA50 rapid, and Cells/DNA, and prior to injections 2, 3 and 4 for ISA50 12 week.…”

“…Paired heavy and light chain libraries were generated as previously reported. 12,18 During the development of this method, several spike-in experiments with known antibody sequences were performed that indicated proper antibody pairing was maintained; however, there is always the possibility that a minority of sequences could still have improper pairing.…”

“…[6][7][8][9][10][11] To comprehensively characterize binders in diverse repertoires, we previously developed technologies that recreate recombinant, natively paired antibody libraries suitable for high-throughput screening and sequencing. 12,13 The DNA libraries are introduced into a recombinant yeast expression system and multiple rounds of fluorescence-activated cell sorting (FACS) enrich the library for antigen-specific binders.…”