Allogeneic Polyclonal CD38KO/CD38-CAR γδT Cells For The Treatment Of T Cell Malignancies

Relapsed and refractory T cell malignancies are associated with poor clinical outcomes. Although autologous CAR-αβT cells have been employed in the treatment of several cancers, generating CAR-T cells for T cell malignancies remains challenging. The need to obtain enough healthy αβT cells from patients to generate CAR-αβT cells, combined with the problem of shared antigens such as the pan-cancer target CD38 expressed on both malignant and normal T cells, which can lead to fratricide of healthy CAR-αβT has hindered the application of CAR-T therapy for T-ALL. The use of allogeneic αβT and NK cells has also been explored for treating T-ALL. However deletion of the TCRαβ/CD3 complex to avoid graft-versus-host disease (GvHD) for αβT and multiple infusions of NK cells, can increase the cost of manufacturing and logistics. In this study, we genetically engineered polyclonal gamma delta T (γδT) cells as an alternative allogeneic cell source for cancer immunotherapy. γδT share innate immune properties of NK cells and adaptive immunity of αβT cells. Generating CAR γδT would allow us to minimize the cost of generating allogenic TRAC-KO CAR αβT cells and lower the number of infusions needed when NK cells are used. Utilizing a novel expansion protocol in combination with CRISPR/AAV gene editing, we developed CD38 knockout (CD38KO)/CD38-CAR polyclonal γδT cells that target T-ALL. Our editing strategy enabled site-directed, on-target insertion of the CD38-CAR transgene into the CD38 locus, with no evidence of significant random CAR DNA integration (as commonly seen with lentiviral CAR transduction) or chromatin abnormalities resulting from CRISPR editing. This approach effectively mitigated fratricide through simultaneous CD38 disruption and CAR expression. We evaluated the edited γδT cells in vitro across multiple patient-derived T-ALL samples and demonstrated the efficacy of the CD38KO/CD38-CAR γδT cells against both baseline and relapsed samples. In vivo, a single injection of CD38KO/CD38-CAR γδT cells without cytokine support resulted in potent anti-leukemic efficacy. Fratricide-resistant CD38KO/CD38-CAR polyclonal γδT cells thus represent a promising off-the-shelf therapeutic platform. They may serve as a bridge to allogeneic hematopoietic stem cell transplantation (allo-HSCT), potentially enabling molecular remission in T cell malignancies and other CD38-expressing cancers such as AML.