Chimeric Antigen Receptor T-Cells for the Treatment of Gamma-Delta T-Cell Malignancies

Introduction

Cancers derived from the malignant transformation of gamma delta T-cells are rare but carry very poor prognosis. Hepatosplenic T-cell lymphoma is a highly aggressive condition characterised by hepatosplenic and bone marrow involvement. It has among the worst outcomes of all lymphoma subtypes, with a median survival of only 6-8 months. 95% of cases express the gamma delta T-cell receptor (GDTCR), which is also expressed on a proportion of cases of T-ALL. Treatment for these cancers is based on cytotoxic chemotherapy, with no tumour-specific therapies including immunotherapy available. We have developed a novel chimeric antigen receptor targeting GDTCR and here demonstrate specific in vitro and in vivo efficacy against gamma delta T-cell malignancies.

Results

We cloned anti-GDTCR antibody as a single chain variable fragment (ScFv), and confirmed specific binding to GDTCR-positive T-cell cell lines and primary GD cells. Next, we cloned anti-GDTCR ScFv into a 2^ndgeneration chimeric antigen receptor (CAR) format, including a spacer derived from CD8-stalk, CD28 transmembrane domain and 41BB-zeta endodomain. This construct was stably introduced to primary alpha-beta T-cells by retroviral transduction and surface expression was confirmed by flow cytometry.

We established 48-hour co-cultures of anti-GDTCR CAR T-cells or control anti-CD19 CAR T-cells with T-cell lines positive (Loucy, BE13, MOLT-13) or negative for surface GDTCR (Jurkat, SupT1-CD19). While control anti-CD19 CAR killed only SupT1-CD19 cells, specific cytotoxicity was seen by anti-GDTCR CAR T-cells only against GDTCR-positive cell lines. In addition, anti-GDTCR CAR T-cells demonstrated specific secretion of cytokines including interferon gamma and IL-2, and robust antigen-specific proliferation only in co-culture with GDTCR-positive cells. Expression of exhaustion, activation and differentiation markers in long term co-cultures with target cells was similar to that seen with control anti-CD19 CAR.

To assess the in vivo potency of anti-GDTCR CAR T-cells, we established a murine model of disseminated GDTCR-positive leukaemia. NSG mice were intravenously injected with 4x10^6 Loucy cells, engineered to stably express Firefly luciferase. Tumour engraftment in bone marrow was confirmed at D7 following injection, and mice were treated with 0.8x10^6 anti-GDTCR or control anti-CD19 CAR T-cells. Disease burden was monitored by bioluminescence imaging. Mice receiving anti-GDTCR CAR demonstrated substantial reduction of tumour burden, increased expansion of CAR T-cells and prolonged survival compared to control-CAR treated animals.

Conclusions

We have developed a novel chimeric antigen receptor T-cell treatment for gamma-delta TCR-positive malignancies, including hepatosplenic T-cell lymphoma and some cases of T-ALL. Our approach is, to our knowledge, the first immunotherapeutic strategy proposed for these conditions. Given the restricted expression of GD-TCR on a small subset (0.5-5%) of peripheral T-cells and the absence of a clear human phenotype associated with GD T-cell deficiency, we suggest that this therapy may be well tolerated. Given the very poor prognosis and lack of effective therapies for GD-TCR-positive malignancies, as well as the considerable efficacy of CAR T-cell therapy in analogous B-cell disorders, our approach could bring much needed benefit to patients suffering these conditions.