Background Prostate-specific membrane antigen (PSMA) is a transmembrane glycosylated homodimer overexpressed in >80% of prostate cancers. PSMA expression is increased in advanced stages of the disease, making it an attractive therapeutic target. Clinically, autologous anti-PSMA αβ CAR T cells have shown initial efficacy with significant CRS-like dose-limiting toxicities. Compared to αβ T cells and other innate cells, γδ T cells are associated with multifunctional innate and adaptive targeting and differentiated biodistribution into tumor-associated tissues. Additionally, γδ CAR T cells have demonstrated enhanced tumoricidal activity and tailored activation-induced cytokine profiles that may decrease toxicities associated with CRS. We characterized γδ T cells modified from a set of novel scFv-based CARs targeting PSMA for prostate cancer.
Methods We used phage panning to identify a library of anti-PSMA scFv sequences, which were reformatted into CARs in VH-VL and VL-VH orientations and screened in Jurkat-Lucia™ NFAT cells to assess CAR expression and activation in the context of target cell-based stimulation. We transduced a set of functional CARs into Vδ1 T cells, a primarily tissue-resident subset, activated and expanded from healthy donor PBMCs. We performed in vitro cell-based cytotoxicity assays and phenotypic assessments of CAR Vδ1 T cells using flow cytometry. Potency was also assessed in NSG mice bearing subcutaneous PSMA-expressing xenografts.
Results Anti-PSMA scFvs ranged in apparent affinities from the low nanomolar to the sub-micromolar range. CAR-expressing Jurkat cells showed target-specific NFAT activation and low tonicity. CAR-engineered Vδ1 T cells demonstrated robust expansion, in vitro cytotoxicity and antigen-specific proliferation against PSMA+ cell lines in a manner comparable to, or greater than, J591 scFv-based Vδ1 CAR. In vitro potency correlated with the release of multiple effector cytokines. Notably, IL-6 and IL-10 production by anti-PSMA Vδ1 CAR T cells was negligible, while TNFα production was low, further supporting the potential of the γδ CAR T platform to demonstrate a favorable cytokine-associated safety profile. Anti-PSMA Vδ1 CAR T cells were also found to be predominantly naïve, with low levels of exhaustion marker expression. Additionally, in vitro (figure 1) and in vivo (figure 2) potency of anti-PSMA Vδ1 CAR T cells was investigated upon co-expression of dominant negative TGFβRII (dnTGFβRII). In xenograft models, anti-PSMA Vδ1 CAR T cells demonstrated potent anti-tumor efficacy.
Conclusions We have engineered, screened and demonstrated preclinical efficacy for “off-the-shelf” PSMA-targeting γδ CAR T cells. Resulting γδ CAR T constructs identified here are candidates for further preclinical and clinical development in the context of armoring technologies.
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