Abstract 1372: BTN1A1 regulates T cell-mediated cancer immunotherapy by interacting with Galectin-9, PD-1, and TIM3

Abstract Butyrophilin 1A1 (BTN1A1) is a novel immune checkpoint protein belonging to the butyrophilin family and is expressed on immune cells such as macrophages, B cells, or activated CD8 T cells and in human tumors. Our results indicate that BTN1A1 and PD-L1 expression are mutually exclusive in various human solid tumors. Furthermore, it was observed that BTN1A1 had a significantly higher detection level than PD-L1 in cancer patient samples. We have reported that BTN1A1 plays an immunomodulatory role in T cell activation and proliferation. BTN1A1 inhibited the proliferation of T cells that are activated by anti-CD3 and anti-CD28 antibodies in vitro. Overexpression of BTN1A1 in PC3 cells also inhibits T cell-mediated killing of cancer cells. BTN1A1 is a bona fide immune checkpoint inhibitor and has anti-tumor activity in a human immune setting. BTN1A1 represents a novel cancer immunotherapy target that goes beyond the low expression levels of PD-1/PD-L1 in tumors. BNT1A1 is an immune checkpoint and cell membrane protein that, together with its binding partners, serves as a starting point for signaling pathways. The BNT1A1 complex, which has various abilities by combining with a specific partner, plays a vital role as an immune checkpoint. In this study, we identified binding partners of BTN1A that regulate immune activity in a mutually interaction relationship with BTN1A1 in various ways. We identified binding partners for the extracellular domain of human BTN1A1 using cell microarrays from Retrogenix (Whaley Bridge, UK). 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 protein's unglycosylated (2NQ) form. Of these three targets, immunoprecipitation and Biacore binding assays revealed that Gal9 exhibited the greatest affinity for human BTN1A1. Since Gal9 is a known PD-1 and TIM3 binding protein, we predicted the potential formation of a BTN1A1/Gal9/PD-1 or BTN1A1/Gal9/TIM3 complex. Indeed, it reveals that immunoprecipitation and confocal microscopy assays performed using cells expressing Myc-tagged versions of these four proteins demonstrated the formation of BTN1A1/Gal9, PD-1/Gal9, TIM3/Gal9, BTN1A1/Gal9/TIM3, and BTN1A1/Gal9/PD-1 complexes. In T cell activation assay using these complexes, when present BTN1A1 alongside the complex inhibits activated T cells. As high Gal9 expression levels are correlated with poor prognosis in multiple cancers, our results highlight this BTN1A1-Gal9-PD-1 or BTN1A1-Gal9-TIM3 axis as a novel therapeutic target for immunotherapeutic drug development. Citation Format: Seung Hoon Lee, Young-Seung Kim, Chunai Wu, Andrew H Park, Stephen S. Yoo. BTN1A1 regulates T cell-mediated cancer immunotherapy by interacting with Galectin-9, PD-1, and TIM3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1372.