Jacob Bridge

Introduction: γδ T cells are a subset of lymphocytes that recognize non-peptide antigens via canonical γδ T cell receptors (TCRs). Due to their diverse functionality and MHC-independent cytolytic activity, γδ T cells have garnered enthusiasm as a potential allogeneic immunotherapy. However, their heterogeneity and infrequency within peripheral blood, combined with inefficient genetic engineering and expansion of populations bearing polyclonal γδ TCR repertoires, has limited their clinical application. Here we implement a novel procedure for the large-scale production of polyclonal, non-virally engineered CAR-γδ T cells possessing potent anti-cancer activity. In vivo tracking of CAR-γδ T cells reveals selective persistence of specific γδ T cell subsets correlating with therapeutic efficacy, providing opportunities to further refine the composition of γδ T cell products.

Methods: Primary human γδ T cells were isolated from peripheral blood via immunomagnetic separation, then stimulated using plate-bound pan-γδ TCR and soluble CD28 antibodies. Non-viral transposon-based integration of a CD19 chimeric antigen receptor (CAR) construct was employed to direct intrinsic cytolytic activity against target cancer cells in vitro and in vivo. Candidate genes linked to γδ T cell inhibition, including CISH, PD1, and Fas, were inactivated at the genetic level using highly efficient Cas9 adenosine base editor (ABE). Cytotoxicity against tumor lines was first confirmed through in vitro serial killing assays, then evaluated in vivo using NSG mice challenged with Burkitt’s Lymphoma.

Results: Antibody-stimulated γδ T cell populations yielded >10,000 fold expansion by day 22, and maintained greater TCR diversity than populations derived from zoledronate-based expansion methods. γδ T cells were also amenable to highly efficient ABE-mediated triple gene knockout (>90%) without negatively impacting concurrent transposon-mediated CAR integration (>45%). CAR-γδ T cells exhibited potent in vitro anti-cancer activity in serial killing assays, and significantly extended mouse survival relative to PBS controls in vivo (22±1 vs 47±11 days; p < 0.0001). Peripheral Vδ1+ γδ T cells were enriched over time in treated mice, and exhibited increased persistence when engineered to secrete IL15. Vδ1+ γδ T cell persistence also significantly correlated with survival in IL15-expressing groups. Strikingly, a distinct population of αβ TCR-Vδ1-Vδ2- cells representing <7% of injected γδ T cells predominated at endpoint in non-IL15-expressing groups.

Conclusions: High cytolytic activity, potent effector function, and lack of alloreactivity make γδ T cells a prime candidate for use as an off-the-shelf cancer therapeutic. We developed a scalable, clinically-relevant method to expand non-virally engineered human γδ T cells with enhanced cytolytic activity. γδ T cell populations generated using this method achieved >10,000-fold expansion across a 22 day manufacturing process, and, when engineered with a CD19 CAR, displayed highly-efficient killing against tumor lines in vitro and in vivo. Our polyclonal approach supports improved characterization of discrete γδ T cell populations in vivo, informing the development of targeted immunotherapies against diverse subsets of cancer. Ongoing experiments are being conducted to screen for γδ TCRs with the highest reactivity in vivo, and to investigate the αβ TCR-Vδ1-Vδ2- cells identified at endpoint. Updated results from these studies will be presented.