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Background Chimeric antigen receptor T cells (CAR-Ts) have demonstrated efficacy for patients with hematologic malignancies, but have limited efficiency against solid tumors. Pediatric solid tumors secrete immunosuppressive cytokines, like TGF-beta (TGFB), which has been associated with tumor metastasis and poor patient responses. Therefore, armouring CAR-Ts against TGFB-suppression poses a unique opportunity to enhance CAR-T activity for these patients. Armoured CAR-Ts have been studied in adult cancers, 1–3 but not in pediatric cancers. We developed B7H3 CAR-Ts expressing a dominant-negative-TGFB-ReceptorII (dnTGFBRII), which lacks the intracellular signaling domain. Using either co-transduction or a novel bicistronic construct, we evaluated the activity of dnTGFBRII B7H3 CAR-Ts against pediatric solid tumors known to produce TGFB.Methods Pediatric solid tumor cell lines, including osteosarcoma (143B, SAOS2), rhabdomyosarcoma (RD, Rh30), and Ewing Sarcoma (TC71), were evaluated by ELISA for TGFB cytokine secretion. We expressed dnTGFBRII in B7H3 CAR-Ts either by co-transduction or by a novel B7H3.P2A.dnTGFBRII bicistronic construct. To evaluate the effect of dnTGFBRII, we stimulated CAR-Ts with 10-40 ng/mL TGFB1 protein for 10-120 min and assessed intracellular pSMAD signaling by flow cytometry and western blot. B7H3 CAR-T activity against 143B was evaluated by IL2 and IFNgamma cytokine ELISAs and by cytotoxicity killing Incucyte assays with serial tumor re-challenges. Statistical analyses were performed using paired and unpaired t-tests or one-way ANOVA, as appropriate.Results Pediatric solid tumor cell lines secreted 10.7-126.4ng/mL of TGFB. 143B cell line (56.4ng/mL) was utilized for subsequent experiments. First, T cells transduced with B7H3 CAR alone or co-transduced with B7H3 CAR and dnTGFBRII (B7H3+dnTGFBRII) demonstrated transduction efficiency of 88% and 79% B7H3 CAR, respectively, and 67% dnTGFBRII expression. pSMAD signaling decreased by 1.6-fold with dnTGFBRII incorporation (p=0.08). In serial re-challenge experiments, B7H3+dnTGFBRII CAR T-cells demonstrated equivalent IL2 and IFNgamma production and improved tumor killing compared to the B7H3 CAR-Ts alone (p=0.0026). Second, T cells were transduced with a single vector of either B7H3.P2A.NGFR or B7H3.P2A.dnTGFBRII with transduction efficiencies of 51.8% and 44%, respectively. Intracellular pSMAD signaling decreased by 2.5-fold in B7H3.P2A.dnTGFBRII compared to B7H3.P2A.NGFR (p=0.02). Tumor killing assays are ongoing to evaluate co-transduced versus bicistronic CAR-Ts with a goal to identify the best CAR-T design to translate clinically.Conclusions dnTGFBRII incorporation into B7H3 CAR-Ts resulted in reduced pSMAD immunosuppressive signaling in T cells, maintained cytokine secretion, and improved killing of pediatric solid tumor cell lines. This work will lay the foundation for next-generation CAR-Ts to address immunosuppressive environments of pediatric solid tumors.References Kloss CC, Lee J, Zhang A, Chen F, Melenhorst JJ, Lacey SF, Maus MV, Fraietta JA, Zhao Y, June CH. Dominant-Negative TGF-β Receptor Enhances PSMA-Targeted Human CAR T Cell Proliferation And Augments Prostate Cancer Eradication. Mol Ther. 2018 Jul 5;26(7):1855-1866. doi: 10.1016/j.ymthe.2018.05.003. Epub 2018 May 8. PMID: 29807781; PMCID: PMC6037129Zanvit P, van Dyk D, Fazenbaker C, McGlinchey K, Luo W, Pezold JM, Meekin J, Chang CY, Carrasco RA, Breen S, Cheung CS, Endlich-Frazier A, Clark B, Chu NJ, Vantellini A, Martin PL, Hoover CE, Riley K, Sweet SM, Chain D, Kim YJ, Tu E, Harder N, Phipps S, Damschroder M, Gilbreth RN, Cobbold M, Moody G, Bosco EE. Antitumor activity of AZD0754, a dnTGFβRII-armored, STEAP2-targeted CAR-T cell therapy, in prostate cancer. J Clin Invest. 2023 Nov 15;133(22):e169655. doi: 10.1172/JCI169655. PMID: 37966111; PMCID: PMC10645390Hou AJ, Shih RM, Uy BR, Shafer A, Chang ZL, Comin-Anduix B, Guemes M, Galic Z, Phyu S, Okada H, Grausam KB, Breunig JJ, Brown CE, Nathanson DA, Prins RM, Chen YY. IL-13Rα2/TGF-β bispecific CAR-T cells counter TGF-β-mediated immune suppression and potentiate anti-tumor responses in glioblastoma. Neuro Oncol. 2024 Oct 3;26(10):1850-1866. doi: 10.1093/neuonc/noae126. PMID: 38982561; PMCID: PMC11449012