Novel γδ T cell engager platform for cancer immunotherapy promoted T cell expansion with a persistence-indicating phenotype

Abstract Background: Gamma-delta (γδ) T cells initiate a broad, MHC-independent immune response to cancer, owing to their ability to overcome the challenges of heterogeneity by engaging an array of cytotoxic receptors against multiple stress-associated tumor antigens. An activating T cell engager (TCE) that induces substantial proliferation of γδ T cells would increase the availability of these cytotoxic effector cells for both direct and bystander anti-tumor activity in an environment where γδ T cells are generally suppressed in both number and activity. Here, we present initial findings from a novel activating γδ T cell engager platform (γδ-TCE) targeting CD19+ B cell hematologic malignancies (INB-619). Methods: In vitro cytolytic function and EC50 were determined by addition of incremental concentrations of INB-619 (0 to 10 nM) to co-cultures of equal numbers of the NALM-6 B-ALL cell line and γδ T cells derived from peripheral blood mononuclear cells (PBMCs) in a 1:1 effector to target (E:T) ratio. Following these determinations, peripheral blood anti-leukemia effect was modeled by adding 10% NALM-6 cells to PBMC from two healthy donors followed by increasing concentrations of INB-619 (0 to 5 nM). Controls included PBMC alone, PBMC + 10%NALM-6, and PBMC + 10% NALM-6 + Zoledronate as a control for γδ T cell function. We assessed TCE binding specificity, T cell activation and expansion, TCE-induced target cell cytotoxicity, and cytokine release using flow cytometry. Results: In a 24-hour co-culture assay with γδ T cells and NALM-6 cells at an E:T ratio of 1:1, INB-619 induced dose-dependent, γδ T cell-mediated lysis of NALM-6 cells, with an EC₅₀ in the low picomolar range. In subsequent experiments, PBMCs were co-cultured with 10% NALM-6 cells. INB-619 supplemented PBMC cultures showed initiation of NALM-6 cytolysis beginning on day 2, despite continuing NALM-6 growth in culture, and followed by complete tumor cell elimination by day 6. Concurrently, we observed robust expansion of γδ T cells in culture when compared to untreated PBMC and PBMC + NALM-6 controls. Over a 10-day period, INB-619 treatment of PBMC + NALM-6 co-cultures resulted an increase of γδ T cells from <2% of the culture to >50% showing expansion of both Vδ1+ and Vδ2+ T cell subsets. Vδ1+ T cells predominately expressed a Tscm phenotype (CD45RA+ CD27+) while Vδ2+ T cells expressed a Tcm/Tem phenotype (CD45RA-CD27±), indicating potential for long-term persistence. Degranulation and the release of pro-inflammatory cytokines IFN-γ, TNF-α, granzymes, and perforin were also notably increased over that from PBMC controls, while cytokine release syndrome (CRS) – associated cytokines did not increase or remained below detection. Conclusions: By combining meaningful effector cell expansion, strong anti-tumor activity, and a low CRS-associated cytokine profile, this innovative γδ TCE platform presents a promising new approach to cancer immunotherapy. Additionally, its modular and flexible design suggests potential uses across a wide range of cancer targets by leveraging the innate recognition capabilities of γδ T cells with targeted engagement of specific tumor-associated antigens.