A novel Vδ1 engager targeting CD19 enhances human Vδ1 γδ T cell responses against CLL and CD19+ hematological malignancies.

γδ T cells are associated with favorable outcomes in many cancers likely through a mechanism of stress-directed cytotoxicity and antitumor cytokine production. Vδ1 γδ T cells are especially promising for immunotherapy due to their broad stress recognition and resistance to activation-induced cell death. Here, we generate a CD19 engager incorporating a novel Vδ1 binding moiety as proof-of-concept for treating CD19+ cancers, including Chronic Lymphocytic Leukemia (CLL). In vitro studies validated Vδ1/CD19 engager-directed cellular binding and Vδ1 activation, where 41BB, CD25 and CD107a were upregulated on Vδ1 T cells following co-culture with CD19+ cancer cell lines and Vδ1/CD19 engager treatment. Ex vivo studies demonstrated engager-mediated Vδ1 T cell proliferation within healthy PBMC cultures that also correlated to basal Vδ1 CD27 expression. Engager-treated PBMCs demonstrated enhanced cytotoxicity against CD19+ cell lines and primary CLL B cells, with minimal killing of healthy B cells and without inducing excessive cytokine release compared with a CD3/CD19 engager. Phenotypic analysis suggested engager treatment encourages maturation of naive Vδ1 T cells, where upregulation of NKG2D, DNAM-1, CD96, CD2, ICOS, and PD-1 were observed, and in culture with cancerous cells, downregulation of CD27 and CD45RA alongside granzyme-B upregulation. Blockade of such co-receptors reduced Vδ1 cytotoxicity following engager treatment whereas co-receptor stimulation modestly enhanced Vδ1 activation. These data suggest that Vδ1/CD19 engager treatment enhances anti-tumoral Vδ1-capabilities while maintaining sufficient dependency on stress ligand availability, supporting selective targeting of malignant over healthy cells. Overall, our data support the rationale for Vδ1-directed immunotherapies and uncovers potential mechanisms that enhance Vδ1 cytotoxic responses against CD19+ cancers.