Development of a Novel Allogeneic γδ-T Platform for Off-the-Shelf Adoptive Cell Therapy for Oncology and Autoimmune Diseases

Arash Nanbakhsh, Lukasz Wlodarek, Kevin Nguyen, Weijie Lan

Overland Pharmaceuticals, South San Francisco, CA

Introduction: Despite significant clinical benefit for treating hematological malignancies and recent exciting development for autoimmune diseases, autologous cell therapy products face meaningful challenges in terms of expansion to broad patient population, including manufacture scale-up, high cost and certain severe adverse effects. Owing to their innate and MHC-independent cell killing capacities, γδ-T cells have gained increasing interest as a differentiated allogeneic cell type. Establishing robust expansion protocols and elucidating the functional properties of γδ-T cell subsets are fundamental to explore the therapeutic potential of γδ-T cells in both oncology and autoimmune diseases.

Methods: γδ-T cells were enriched from PBMCs using multiple donors. Enriched γδ-T cells were transduced with virus constructs with different promoters and expanded under different culture conditions. The phenotypes of the cells were analyzed using multi-color full spectrum flow cytometry. The innate and CAR-mediated γδ-T cell killing activity were measured using flow cytometry and real-time imaging with Incucyte. In vivo target cell killing activity was demonstrated using NSG mice inoculated with luciferase expressing cancer cell lines.

Results: We found that the abundance and composition of γδ-T varied significantly among different donors. We observed that the cytotoxicity properties of γδ-T cells, both innate- and CAR-dependent, were strongly influenced by donor variability and method of expansion. We developed efficient γδ-T cell expansion protocols that achieved up to >1,000,000-fold cell expansion over a 3-week period of culture without apparent signs of exhaustion or impaired cytotoxicity. Our expansion method generated potent Vδ2 T cells and a unique subset of Vδ1 T cells that highly express CD107, Granzyme B, IFN-γ, and IL-2. We screened viral constructs and identified viral envelops and promoters that support robust viral transduction and CAR expression in γδ-T cells. We found that CAR-γδ-T with mixture of both CAR-Vδ2 and -Vδ1 present maintained complete tumor control during a 30-day serial stimulation assay, whereas CAR-Vδ2 T cells alone failed to supress tumor growth beyond the first 7-days across all tested donors. Sequential rechallenge with target tumor cells ultimately led to selective expansion of CAR- Vδ1 T cells. Vδ1 subtype enrichment could also be achieved in the absence of immune challenge with a small molecule compound. Finally, we demonstrated that CAR- γδ T cells could effectively control tumor growth in a NSG mouse model with disseminated tumor cells.

Conclusions: We have established a unique and scalable allogeneic γδ-T platform with demonstrated CAR mediated in vitro and in vivo efficacies. We are able to generate Vδ1, Vδ2 and mixture of CAR- γδ T cells for potential off-the-shelf adoptive cell therapy in different cancer and autoimmune disease indications.