208 A novel fully human MSLN CAR armored with dominant negative TGFβ receptor II demonstrates enhanced efficacy in xenograft models of pancreatic cancer

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Background Mesothelin (MSLN) is a tumor-associated antigen highly expressed in multiple solid tumors, including pancreatic cancer. A human second-generation MSLN-targeted CAR T-cell product was developed which demonstrated anti-tumor activity in vitro and in vivo, and was further engineered to express dominant-negative transforming growth factor TGFβ receptor II (TGFβRIIDN) to mitigate TGFb-mediated CAR-T suppression in tumor microenvironment (TME).Methods A second-generation CAR featuring a CD8 hinge and transmembrane domain, 4-1BB and CD3z signaling, an internal EF1-alpha promoter, was generated and featured a scFv targeting the extracellular domain of human MSLN. Human CD4 +/CD8+T cells were purified from leukopaks and transduced with MSLN-CAR lentiviral vector +/- TGFβRIIDN armoring. TGFβRIIDN signaling was demonstrated by flow cytometry for phospho-Smad2/3. CAR-T anti-tumor activity was evaluated using the MSLN+ pancreatic cancer cell line AsPC-1 with direct addition of TGFb1, or AsPC-1 engineered to overexpress TGFb1 (AsPC-1/TGFb1). CAR-T in vitro cytotoxicity was determined using xCELLigence killing assays. MSLN-CAR efficacy was evaluated in vivo using NSG mice xenografted by subcutaneously implantation of AsPC-1 +/- TGFb1 and then treated with MSLN-CAR T cells, with or without armoring. CAR-T and cytokine levels in peripheral blood were determined by flow cytometry and MSD U-plex assay, respectively. Caliper measurements of xenograft tumor size was evaluated to evaluate MSLN-CAR efficacy.Results TGFβRIIDN-armor enhanced CAR-T proliferation, reduced Smad2/3 phosphorylation, and improved cytotoxicity against AsPC-1 by 19% in the presence of TGFb1 or of AsPC-1/TGFb1 by 76%. In AsPC-1 xenograft model expressing low level of TGFb1, armored CAR T cells showed comparable tumor control, CAR-T cell persistence, and overall survival to un-armored CAR. Notably, armored MSLN CAR-T showed reduced T cell exhaustion, number of terminal effector & effector memory phenotypes, and regulatory T cells compared to CAR alone. Importantly, in AsPC-1/TGFβ1 xenograft, TGFβRIIDN-armored CAR T cells exhibited superior proliferation, enhanced antitumor activity (tumor size 750mm 3 vs. 1000mm3 at day 40), and greater persistence compared to non-armored CAR. Armored-CAR T cells also exhibited increased serum IFNγ production (8.5 vs 1.7 ng/mL) at day 5 post-infusion and reduced TGFβ1 levels by day 15.Conclusions TGFβRIIDN-armored MSLN CAR-T cells demonstrated enhanced efficacy and durability in TGFβ1-rich TMEs. Armoring of CAR T cells with TGFβRIIDN provides a promising strategy to overcome a major barrier in solid tumor immunotherapy. Our data support further product development for clinical translation, with the aim of improving patient outcomes with mesothelin-expressing malignancies.

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