Butyrophilin 1A1 (BTN1A1) is a novel immune checkpoint protein that exhibits an expression pattern mutually exclusive to that of PD-1/PD-L1 and may represent a promising therapeutic target for tumors that are anti-PD-1/PD-L1 antibody refractory. BTN1A1 was identified as a potential stress-inducible candidate immune checkpoint using the STCube in vivo immune target discovery platform. Additional analyses revealed that BTN1A1 was highly expressed in multiple human tumor types including urothelial carcinoma and NSCLC, with no overlap between BTN1A1 and PD-1/PDL-1 expression patterns. Mouse studies demonstrated that an anti-BTN1A1 antibody exhibited antitumor activity both as a single agent and in combination with anti-PD-1/PD-L1 or radiation therapy. A humanized anti-BTN1A1 antibody, hSTC810, was developed and found to suppress tumor growth in A549 CDX humanized murine models. Further studies identified Galectin 9 (Gal9) as a BTN1A1 receptor that can form a three protein complex with BTN1A1 and PD-1, thereby suppressing T cell receptor signaling and T cell activation. 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. Given these findings, a first in human study of hSTC810 is in development to evaluate the safety profile of this therapy and to identify a dose for its further evaluation as an anticancer therapy. PK modeling including allometric scaling was performed to conduct simulations to predict the PK profile of hSTC810 after IV administration in humans and to identify possible doses needed to achieve tumor concentrations corresponding to the minimum anticipated biological effect level (MABEL) of 20% receptor occupancy (RO), 80% RO, or 95% RO. The partition coefficient was used to predict concentrations at the tumor site based on predicted serum concentrations. Simulations were performed for 100 subjects after the administration of multiple Q2W doses. The dose of 0.1 mg/kg was predicted to have <20% RO, 0.3 mg/kg to have ~21-35% RO, 1 mg/kg to have ~47-65% RO and 15 mg/kg to have ~93-96% RO at steady state. We anticipate beginning study enrollment in the first quarter of 2022 at sites in the United States sand South Korea. The study is a standard 3+3 dose-escalation with a dose of 0.3 mg/kg for a single patient followed by a starting dose of 1 mg/kg. Citation Format: Lynn Jackson Howie, Ezra M. Chung, Young-Seung Kim, Yujin Jung, Hyunjin Jung, Stephen Yoo. Humanized STC10 is a monoclonal antibody to BTN1A1, a novel immunotherapeutic target to be evaluated in a planned phase 1 study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5318.

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 protein's 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 KD 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 KD 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.

Cancer immunotherapy is an effective treatment against individuals with late-stage cancer forms. The PD-1/PD-LI axis is a main therapeutic target used in clinical settings, but only 15-20% of cancer patients are responsive. Thus, there is an urgent unmet need to identify other immuno-therapeutic targets to overcome this limitation. Butyrophilin (BTN) belongs to the B7 family, peripheral membrane proteins containing an immunoglobulin domain found in PD-L1 and CTLA4. Among the BTN family, BTN2A2 and BTN3A1 are shown as alternative co-inhibitory or co-stimulatory cancer immune checkpoint(s) in the effector T cell compartment. However, the immune-modulatory roles of BTN1A1 are largely unknown, especially in vivo. Here, we report the immune-modulatory role of BTN1A1 in T cell proliferation and activation in vitro and in vivo. We have found that BTN1A1 inhibits the proliferation of T cells that are activated by anti-CD3 and anti-CD28 antibodies in vitro. The overexpression of BTN1A1 in PC3 cells also inhibits T cell-mediated cancer cell killing. BTN1A1-overexpressing B16-Ova melanoma cancer cell lines also accelerate tumor growth compared to B16-Ova wild type in a syngeneic mouse model. The effect of BTN1A1 was fully attenuated in an immune-compromised SCID mouse. BTN1A1-deficient MC38 colorectal cancer cell lines also grew significantly slower than wild-type MC38 cell lines when they were inoculated subcutaneously into a syngeneic mouse model. This result strongly supports BTN1A1's role in immune evasion. BTN1A1 is expressed on immune cells. These cells include macrophages, B cells or activated CD8 T cells. We found that BTN1A1 is expressed in human tumors, with an expression that was mutually exclusive to PD-L1. We have developed the monoclonal antibody (STC810) against the human BTN1A1. STC810 has anti-tumor activity in a human immune environment in vitro and ex vivo. Since STC810 has no cross-reactivity with mouse BTN1A1 proteins, we generated mouse surrogate antibodies to study the effects of blocking BTN1A1 in vivo in mice tumor models. The anti-BTN1A1 surrogate antibody not only exhibits a single agent but has a synergistic effect in combination with the anti-PD-L1 antibody. Overall, using mouse models, genetic ablation, and antibody tests ex vivo we demonstrate that BTN1A1 is a bona fide immune checkpoint inhibitor. Late-stage preclinical studies of humanized STC810 are underway. The Phase I clinical trial is set to start in late 2021. Citation Format: Ezra M. Chung, Yong-Sik Bong, Young-Seung Kim, Andrew Park, Young-Ok You, Amrish Sharma, Steven H. Lin, Young-Joon Lee, Hyunjin Jung, Stephen S. Yoo. BTN1A1: a novel immune checkpoint for cancer immunotherapy beyond the PD-1/PD-L1 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1643.

Chronic inflammation is a critical risk factor in the context of cancer development that promotes the expression of immune checkpoint proteins including PD-1, PD-L1, LAG-3, and TIM-3, which in turn suppress T-cell mediated cytotoxic tumor cell killing. Inflammation induced by treatments including chemotherapy and radiotherapy (RT) can also promote immune checkpoint upregulation and consequent tumor immune escape. However, whether the acute inflammation associated with standard chemotherapy and RT can engage distinct immune checkpoints to further support tumor persistence remains unknown. Using an in vivo screening platform in which tumors were irradiated with high-dose γ-radiation, we identified the B7-related immunoglobulin superfamily protein butyrophilin 1A1 (BTN1A1) as an acute stress-inducible immune checkpoint. Herein, we aimed to investigate how BTN1A1 contributes to post-irradiation intratumoral immunosuppression. To assess BTN1A1 induction in response to γ-radiation, human cell lines were exposed to a range of radiation doses, and cell death and BTN1A1 expression were examined by flow cytometry. Radiation promoted apoptosis and BTN1A1 expression in a dose-dependent manner, and PD-L1 was downregulated by increasing the dosage of γ-radiation. As radiation-induced cell death results in cytosolic DNA accumulation within tumors, which in turn activates the production of type I interferons (IFNs) via the cGAS/STING pathway, we examined the effect of BTN1A1 on cGAS and STING expression. BTN1A1 overexpression in human prostate adenocarcinoma PC3 cells suppressed cGAS and STING expression. IFNs can promote antigen presenting cell activation, thus priming T cell responses. We then examined the impact of BTN1A1 overexpression on type I IFN production as assessed via qRT-PCR, revealing that BTN1A1 overexpression enhanced IFN-β expression in the presence of the STING agonist cGAMP. BTN1A1 was also found to harbor a C-terminal B30.2 domain that was able to specifically interact with xanthine oxidoreductase (XOR), an oxidizing enzyme involved in purine metabolism. BTN1A1 expression induced high levels of intracellular reactive oxygen species (ROS), which have the potential to be more toxic in lymphocytes as compared to tumor cells, and BTN1A1 knockdown decreased these ROS levels. In light of these observations, we assessed the combination therapeutic efficacy of RT and anti-BTN1A1 antibodies in a syngeneic murine Lewis lung carcinoma (LLC) model system, revealing that these two treatments exhibited synergistic anti-tumor activity. FACS and IHC further confirmed that BTN1A1 was upregulated within tumors following irradiation. Together, these data offer new insights regarding the immunomodulatory role of radiation-induced BTN1A1 within tumors, providing a more robust foundation for the development of BTN1A1 as an immunologic target for cancer therapy. Citation Format: Ezra Chung, Young-Seung Kim, Andrew Park, Steven H. Lin, Hyunjin Jung, Stephen S. Yoo. Anti-BTN1A1 exhibits synergistic anti-tumor immunotherapeutic efficacy in combination with radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6321.