Human gamma delta T lymphocytes (δγT) have many properties of innate lymphocytes including antibody dependent cell mediated cytotoxicity, innate cytotoxicity and capacity for professional antigen presentation. Chimeric antigen receptors (CARs), which combine within a single molecule an scFv targeting a tumor associated antigen with T cell receptor signaling and co-stimulation, are capable of imparting potent effector function but are dependent on a high degree of target antigen specificity to avoid unwanted toxicity. We hypothesized that transduction of δγT with CARs would provide a mechanism to both enhance effector function and increase target antigen specificity.
We have focused on the subset of human peripheral blood γδT harbor the Vγ9Vδ2 TCR, as these cells have innate effector function, mediated by the TCR recognition of stressed cells, against a wide range of human cancers. Moreover, the Vγ9Vδ2 subset can be readily activated and expanded from peripheral blood using zoledronic acid, a clinical agent that leads to engagement of the Vγ9Vδ2 TCR and that can be used within the GMP setting.
Using GD2 as a model antigen, expressed on both cancers and peripheral nerves, we generated anti-GD2 CARs employing a novel scFv that is not prone to tonic signaling. Following stimulation with zoledronate, PBMC were transduced on day 5 with gamma-retroviral vectors expressing CARs, and left to expand for 2 weeks in the absence of antigenic target. Between 10-100 fold expansion was observed and the cell product was typically over 90% pure for Vγ9Vδ2 T cells, with transduction efficiency around 50%. Compared with αβT-CAR cells generated following stimulation of PBMC with CD3/CD28 beads, both CAR-expressing cell populations were a similar mixture of central and effector memory cells and had equivalent dim expression of exhaustion markers.
Using a GD2-CAR with CD3-zeta and CD28 endodomains, γδT-CAR and αβT-CAR cells showed equivalent antigen-specific killing but γδT-CAR cells retained additional innate killing of a range of cancer cells. Both populations were capable of equivalent secondary antigen-specific expansion and interferon gamma secretion. To exploit the innate target recognition of the Vγ9Vδ2 cells through its TCR, we designed CARs for use in γδT cells that provided only antigen-dependent costimulation. Such CARs retain antigen specific target recognition through the TCR but avoid off target toxicity marking a potential new paradigm for enhancing cancer specificity.
Citation Format: Jonathan Fisher, anna capsomidis, Barry Flutter, Gabriel Benthal, Rebcca Wallace, Kenth Gustafsson, Karin Straathof, Martin Pule, John Anderson. Chimeric antigen receptor transduced gamma delta T lymphocytes provide enhanced tumor specificity. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B128.
- ©2016 American Association for Cancer Research.