Abstract LBA019: The immune checkpoint protein BTN1A1 suppresses T cell activation through interactions with Gal9 and PD-1

Butyrophilin (BTN) proteins are members of the B7 immunoglobulin superfamily and exhibit well-characterized immunomodulatory functions in mammals. We have recently identified BTN1A1 as an immune checkpoint protein prominently upregulated in response to acute inflammatory insults. Further in vitro and in vivo assays have validated BTN1A1 as an immune checkpoint target, particularly for patients refractory to anti-PD-1/PD-L1 antibody treatment. We have also developed a humanized antibody targeting human BTN1A1, hSTC810, which is expected to enter into Phase I clinical trials in the first quarter of 2022. In this study, a cell microarray from Retrogenix (Whaley Bridge, UK) was used to identify binding partner(s) for the extracellular domain of human BTN1A1. Through this screening approach, we found that BTN1A1 binds to galectin-1 (Gal1), galectin-9 (Gal9), and neuropilin 2 (NRP2). These three putative binding partners could specifically bind to wild-type BTN1A1 but not to this protein9s unglycosylated (2NQ) form. Of these three targets, immunoprecipitation and Biacore binding assays revealed that Gal9 exhibited the greatest affinity for human BTN1A1, followed by Gal1, with respective K_D values of 22.7 nM and 1.88 μM - an 83-fold difference. Gal9 binding to human BTN1A1 was dependent on BTN1A1 glycosylation status and required the carbohydrate recognition domain (CRD) of Gal9. As Gal9 is a known PD-1-binding protein, the K_D of Gal9 for PD-1 was additionally assessed and found to be 19.7 nM. These results thus predicted the potential formation of BTN1A1/Gal9/PD-1 complexes. Consistent with these predictions, immunoprecipitation assays performed using cells expressing Myc-tagged versions of these three proteins demonstrated the formation of BTN1A1/Gal9, PD-1/Gal9, and BTN1A1/Gal9/PD-1 complexes. CRISPR-mediated BTN1A1 knockout in Jurkat T cells induced both PD-1 expression and T cell activation. BTN1A1 also suppressed T cell receptor (TCR) signaling in Jurkat cells, and the addition of exogenous recombinant Gal9 protein further blunted such BTN1A1-mediated TCR-signaling downregulation. Such downregulation was not observed in PD-1 knockout Jurkat cells. Together with the observation that BTN1A1 does not bind to PD-1 directly, the data suggest that BTN1A1 suppresses T cell activation by interacting with PD-1 through Gal9. As high Gal9 expression levels are correlated with poor prognosis in multiple cancers, our results highlight this BTN1A1-Gal9-PD-1 axis as a novel therapeutic target for immunotherapeutic drug development. (1) Chung EM, Bong YS, Kim YS, Park A, You YO, Sharma A, Lin SH, Lee YJ, Jung H, Yoo SS. BTN1A1: a novel immune checkpoint for cancer immunotherapy beyond the PD-1/PD-L1 axis. Cancer Res 2021;81(13_Suppl): Abstract nr 1643. Citation Format: Ezra M Chung, Young-Seung Kim, Chunai Wu, Andrew H Park, Hyunjin Jung, Stephen S Yoo. The immune checkpoint protein BTN1A1 suppresses T cell activation through interactions with Gal9 and PD-1 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA019.