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T cell exhaustion resulting from chronic antigen stimulation and immunosuppression in the tumor microenvironment (TME) limits CAR T efficacy in the solid tumor setting. We have previously shown that engineering therapeutic T cell products using CRISPR-based gene insertion of a dual shRNA cassette targeting Fas and PTPN2 significantly increased antitumor efficacy of Integrated Circuit T cells (ICTs) in ovarian cancer models. We sought to build on this finding to induce additional gene perturbations that improve the efficacy of ICTs. Transforming growth factor (TGF)-b is an immunosuppressive cytokine that potently inhibits T cell responses and is present at high levels in numerous solid tumors, including renal cell carcinoma (RCC). In order to render ICT cells less susceptible to TGF-b-mediated suppression, we developed a quadruple shRNA cassette that simultaneously targets Fas, PTPN2, and TGFBR. Candidate TGFBR-targeting shRNAs were selected for their ability to reduce surface TGFBR receptor expression and impair proximal (pSMAD) or distal (CD103, PD-1) signaling through TGFBR. While single shRNAs against TGFBR did not rescue ICT cell activity in the presence of TGF-b, likely due to partial knockdown of TGFBR signaling, a cassette encoding two shRNAs against TGFBR restored ICT function to similar levels observed in the absence of TGF-b. The quadruple shRNA cassettes targeting Fas/PTPN2/TGFBR significantly enhanced antitumor activity of ICT cells in multiple xenograft tumor models relative to Fas/PTPN2 cassettes. These results demonstrate the utility of multiplexed shRNA strategies to render therapeutic T cells resistant to orthogonal suppressive pathways in solid tumors. Citation Format: Thomas J. Gardner, Adam Litterman, Brenal K. Singh, Luisa Silva, Mukund Hari, Stanley Zhou, Colin Tang, Sahil Joshi, John Gagnon, Oliver Takacsi-Nagy, Jason Hall, Hans Pope, James Zhang, Alma Gomez, Jeremy Chen, Suchismita Mohanty, Vince Thomas, Nicholas Quant, Beatriz Millare, Amy-Jo Casbon, Natalie Bezman, Levi Gray-Rupp, Angela C. Boroughs, W. Nicholas Haining. Multiplexed shRNA cassettes targeting orthogonal pathways (FAS/PTPN2/TGFBR) enhance the potency of integrated circuit T cells (ICTs) in multiple solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1768.

Glioblastoma multiforme (GBM) is among the most lethal of cancers. Enhancing anti-tumor killer T cell responses via dendritic cell vaccines has correlated with a positive outcome in selected GBM patients. Nevertheless, the mechanisms by which killer T cell responses to GBM are inhibited or enhanced remain poorly defined. We therefore developed the GL261 “Quad Cassette” glioma cell line that expresses model T cell epitopes in the immunocompetent C57BL/6 mouse. Tumor size and inflammatory profiles observed in these animals was then compared to C57BL/6 mice administered the parent GL261 glioma cell line. Resulting tumors present with a tumor mass surrounded by considerable edema visible by gadolinium enhanced T1 and T2 weighted MRI. Both edema and tumor mass visible by MRI were quantified using Analyze 10.0 software which enables 3D volumetric analysis of MRI images. C57BL/6 mice with GL261 “Quad Cassette”, but not parent GL261 gliomas, presented with smaller tumor mass and significant brain infiltrating tumor specific Kb:ova specific CD8 T cells. We therefore conclude that the GL261 Quag Cassette system is suitable for studying tumor epitope specific CD8 T cell responses using the vast genetic and immunologic resources available for the C57BL/6 mouse background. Furthermore, incorporation of the first true 3D volumetric analysis of GL261 glioma size with small animal MRI will enable investigation of immunotherapeutic treatments in vivo without euthanizing the animal.