Introduction: Butyrophilins (BTN) are a family of immune regulators in the immunoglobin superfamily, structurally similar to the B7 family. BTN3A, highly expressed in a variety of human cancer cells, suppresses αβ T cell activation by preventing CD45 segregation and immune synapse formation. Human BTN3A has three isoforms with almost identical extracellular domains, but each has unique cytoplasmic tail. BTN3A is highly expressed in a variety of human cancer cells. Therefore, we hypothesize that blocking BTN3A can remove the immunosuppressive effects of cancer cells and therefore enhance the tumor killing effects of immune cells.

Methods: The expression of BTN3A on primary human immune cells was examined by flow cytometry. Human BTN3A2-IgG Fc fusion protein was generated in the Drosophila expression system. BALB/c wild type mice were immunized with the fusion protein and hybridomas were generated. High-affinity monoclonal antibodies (mAb) were screened and purified. NIH 3T3 cells transfected with human BTN3A1, BTN3A2 or BTN3A3 were used to examine if the purified mAbs can recognize each isoform on cell surface. Artificial CHO antigen-presenting cells (APC CHO cells) were transfected with or without hBTN3A2, and then co-cultured with human primary T cells in the presence of mouse IgG isotype or anti-hBTN3A2 mAb. A mixed lymphocyte reaction with different donor dendritic cells and T cells was performed with isotype or anti-hBTN3A mAb. The hBTN3A2 mRNA expression in a variety of tumor cell lines was screened in a gene database, and the protein expression of selected tumor cell lines was examined by flow cytometry with anti-hBTN3A mAb.

Findings: Multiple tumor cell lines, originating from hematologic malignancies, carcinomas, or sarcomas, showed high cell surface expression of hBTN3A by flow cytometry. Two clones of high-affinity, anti-hBTN3A mAb were generated and purified. Both are IgG1 isotypes. Anti-hBTN3A mAb bound to BTN3A1, BTN3A2 or BTN3A3 expressed on NIH 3T3 cells. The majority of human immune cells isolated from peripheral blood from healthy donors expressed hBTN3A, including CD4 and CD8 T cells, B cells, monocytes, natural killer cells and γδ T cells. BTN3A2 expressing APC CHO cells inhibited primary αβ T cell activation, and anti-BTN3A mAb restored T cell proliferation as well as stimulated cytotoxic cytokine production in some healthy donors.

Conclusion: BTN3A is an immunosuppressive receptor that inhibits αβ T cell activation. Anti-hBTN3A mAb restores T cell activation, suggesting the potential for BTN3A to be a target for cancer immunotherapy.

Citation Format: Yixian Li, Xiaoxin Ren, Yao Wei, Mou Peng, Hao Wang, Marc Christopher Pulanco, Christopher David Nishimura, Philip Galbo, Anne Madsen, R. Alejandro Sica, Xingxing Zang. Butyrophilins 3A as an immune checkpoint and a therapeutic target in hematological malignancies [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 6395.