Background. Prostate cancer (PCa) is the most common cancer in men and metastatic disease has no curative options. Here we describe a combinatorial/multi-modular CAR T therapeutic for prostate cancer termed AUTO7. The therapeutic consists of 6 functional modules. The encoded modules are: 1) an anti-PSMA CAR based on a novel humanized binder; 2) the safety switch; 3) dominant negative TGFβRII to induce TGFβ1 resistance (dnTGFβRII); 4) truncated SHP2 (dSHP2) for PD1/PD-L1 pathway inhibition; 5) a constitutively active IL-7 receptor (IL7R_CCR) to induce proliferation and finally 6) an IL-12 module (IL12) for lymphocyte recruitment/activation.

Methods. PSMA-binder was generated by CDR grafting of an anti-PSMA antibody derived from genetically vaccinated rats. CAR T-cells were generated by transduction of T-cells with vectors containing the respective modules. CAR T-cells were evaluated in vitro in co-culture assays with PSMA positive cell lines for redirected lysis, cytokine secretion, T cell proliferation, resistance to immunosuppression and IL-12 expression. Antitumor activity of AUTO7 containing the CAR and all 6 modules was assessed in vivo in an established human xenograft in NSG mice.

Results. AUTO7 CAR T-cells were highly potent in cytotoxicity assays against PSMA positive tumor cell lines showing no difference to a CAR alone control. AUTO7 CAR T cells encoding the dnTGFβRII and the dSHP2 showed resistance to both TGFβ1 and PD1/PDL1 mediated immunosuppression in vitro whilst the IL7R_CCR module conferred cytokine independent viability and proliferation. Finally, a module designed to enable the CAR T-cells to secrete IL-12 showed potent efficacy by its ability to activate fresh PBMC to release IFNγ. We demonstrated AUTO7 efficacy in an in-vivo model in which human PC3-PSMA PCa cells were engrafted into NSG mice for 3 weeks, prior to i.v. infusion of CAR T-cells. Although T cells transduced with CAR alone was unable to control tumor growth, AUTO7 CAR T-cells completely eradicated tumor with no signs of toxicity. Furthermore, examination of the IL12 module in immunocompetent models also showed no signs of cytokine mediated toxicity.

Conclusions. Our results demonstrate the feasibility and efficacy of the multi-modular AUTO7 product. Addition of IL7R_CCR, IL-12, dnTGFβRII and dSHP2 modules to our anti-PSMA CAR product enhanced T cell functions by extending persistence, proliferation, activation and resistance to TGFβ1 and PD1/PDL1 driven immune inhibition.

Citation Format: Marco Della Peruta, Victoria Koullourou, Mohamed El-Kholy, Mathew Robson, Philip Wu, Matteo Righi, Mathieu Ferrari, Giulia Agliardi, James Sillibourne, Simon Thomas, Shimobi Onuoha, Shaun Cordoba, Martin Pule. AUTO7: Anti-PSMA humanized CAR T-cell with improved persistence and resistance to tumor microenvironment for metastatic castration resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1070.