Bispecific gamma delta T cell engagers containing butyrophilin 2A1/3A1 heterodimeric fusion protein efficiently activate Vg9Vd2 T cells and promote tumor cell killing

Abstract A primary mechanism of cancer immunotherapy resistance involves downregulation of specific antigens or major histocompatibility complex (MHC) based antigen presentation, which renders tumor cells invisible to αβ T cells, but not γδ T cells. Recently, a two-step model of γδ T cell activation has emerged, wherein one butyrophilin (BTN, i.e., BTN2A1) directly binds the γδ TCR but is only activated if certain molecular patterns (e.g., phosphoantigens) facilitate recruitment of a second BTN (i.e., BTN3A1) into a complex to form a BTN2A1/3A1 heterodimer. The BTN2A1/3A1 complex specifically activates the Vγ9Vδ2 T cells found in the peripheral blood in response to phosphoantigen build up in tumor cells. The unique mechanism of action and specificity of γδ TCR/BTN interactions suggests that therapeutic proteins comprising specific BTN heterodimers could be used to target specific γδ T cell populations to tumor cells which lack expression of high affinity neoantigens or MHC class I molecules. In this study, we generated bispecific γδ T cell engagers (GADLEN) containing heterodimeric BTN2A1 and BTN3A1 extracellular domains (ECD) fused via inert Fc linkers to scFv domains targeting a tumor-antigen (CD19 or CD33) to test their ability to modulate Vγ9Vδ2 T cells and to promote tumor cell killing in co-culture assays. GADLEN induced proliferation, degranulation and cytokine production including IFNγ and TNFα in Vγ9Vδ2 T cells but requires co-stimulation of a natural cytotoxicity receptor (via anti-NKG2D) or T-cell co-stimulatory receptor (via anti-CD28), highlighting the parallels of signal 1 and signal 2 requirements of TCR activation in αβ- and γδ- T cells. In Vγ9Vδ2 T cells and tumor co-culture assays, the addition of GADLEN alone enhanced killing of CD19+ lymphoma cells such as Daudi and Raji, expressing known ligands for CD28 (e.g., CD80 and CD86). Similarly, GADLEN enhanced Vγ9Vδ2 T cell-mediated killing of K562 cells engineered to express CD19, which express MICA/B (ligands for NKG2D) on the cell surface. In addition, the CD33-targeted GADLEN alone was able to specifically kill acute myeloid leukemia cells expressing CD33 and costimulatory ligands (e.g., CD80 and CD86) in a mechanism similar to the CD19-targeted GADLEN construct. These results highlight the ability of GADLENs in promoting targeted killing of tumor cells by providing the “active” heterodimeric BTN2A1 and BTN3A1 that is critical to enhance cytotoxic killing by Vγ9Vδ2 T cells. Furthermore, this study provides mechanistic insights into BTN-mediated activation of Vγ9Vδ2 T cells and offers proof of concept for using antigen-targeted butyrophilin heterodimeric fusion proteins for the treatment of cancer. Citation Format: Anne Lai, Arpita Patel, Faraha Brewer, Kinsley Evans, Kellsey Johannes, Louis Gonzalez, Kyung Jin Yoo, George Fromm, Keith Wilson, Taylor H. Schreiber, Suresh de Silva. Bispecific gamma delta T cell engagers containing butyrophilin 2A1/3A1 heterodimeric fusion protein efficiently activate Vg9Vd2 T cells and promote tumor cell killing [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 3514.