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 K_D < 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.

Figure 1.

Overview of the workflow used to generate the scFv libraries from B cells isolated from Balb/c and SJL mice. B cells are isolated from lymph nodes and encapsulated into droplets with oligo-dT beads and a lysis solution. mRNA-bound beads are purified from the droplets, and then injected into a second emulsion with an OE-RT-PCR amplification mix that generates DNA amplicons that encode scFv with native pairing of heavy and light chain Ig. Libraries of natively paired amplicons are then electroporated into yeast for scFv display. FACS is used to identify high affinity scFv. Finally, deep antibody sequencing is used to identify all clones in the pre- and post-sort scFv libraries.

Figure 2.

(A) Serum ELISA titers for five immunized Balb/c mice. (B) The Balb/c scFv library from mouse 559, subjected to FACS selection for an Fc control or PD-1. Staining for c-Myc (AF488) is used to differentiate yeast cells that express scFv from yeast cells that do not express scFv (x-axis). Staining for biotinylated antigen (PE) is used to identify yeast that express scFv binders. The Fc negative control (left) is used to set gates to capture yeast cells that express scFv and bind antigen (upper right corner of the FACS plot). Gates for yeast selection are indicated by the quadrangle in the upper right corner of each FACS plot. The percentage in each quadrangle (red text) indicates the proportion of c-Myc positive yeast that fell within the gate. The yeast from the first PD-1 sort (middle) are expanded and then subjected to a second PD-1 sort (right).

Figure 3.

(A) Serum ELISA titers for five immunized SJL mice. (B) The SJL scFv libraries from 564, 565, 566, 567, and 568, subjected to FACS selection for an Fc control or PD-1. Staining for c-Myc (AF488) is used to differentiate yeast cells that express scFv from yeast cells that do not express scFv (x-axis). Staining for biotinylated antigen (PE) is used to identify yeast that express scFv binders. The Fc negative control (left) is used to set gates to capture yeast cells that express scFv and bind antigen (upper right corner of the FACS plot). Gates for yeast selection are indicated by the quadrangle in the upper right corner of each FACS plot. The percentage in each quadrangle (red text) indicates the proportion of c-Myc positive yeast that fell within the gate. The yeast from the first PD-1 sort (middle) are expanded and then subjected to a second PD-1 sort (right).

Figure 4.

Amino acid sequence logos for groups of evolutionarily related clones, i.e., putative clonal lineages. CDR3 amino acid sequences from the pre-sort libraries are on the left. Full V(D)J amino acid sequences are shown on the right for post-sort libraries, and an expanded view of the CDR3 amino acid sequences is shown in the middle. Variant amino acids are emphasized with red arrows (the first seven amino acids are omitted due to the high probability of mis-priming during OE-RT-PCR). Though 2–3 such groups were present after each 2nd FACS, we display only a single representative dominant clonal expansion from each mouse: (A) Balb/c 559, (B) SJL 564, (C) SJL 565, (D) SJL 566, (E) SJL 567, and (F) SJL 568.