Allo CAR T cells represent an “off-the-shelf” treatment option that addresses the unmet need for immediately available, potentially curative therapies. CRISPR Therapeutics’ first generation allo CAR T cell products, CTX110® (CD19-directed) and CTX130TM (CD70-directed), have shown encouraging efficacy in ongoing clinical trials (NCT04035434, NCT04502446, NCT04438083), including complete and durable responses in B- and T-cell lymphoma and renal cell carcinoma (RCC). However, T cell exhaustion, occurring even prior to CAR T cell clearance by the immune system, can result in diminished efficacy and loss of response, especially in patients with high tumor burden. To address this challenge, we performed a CRISPR screen to discover potency edits to incorporate into next-generation CAR T programs. Herein, we describe two novel allo CAR T cell therapies, CTX112TM and CTX131TM, that incorporate the synergistic potency edits Regnase-1 and TGFBR2. CTX112 (CD19-directed) and CTX131 (CD70-directed) are produced from healthy donor T cells modified ex vivo using CRISPR/Cas9 gene editing. Regnase-1 and TGFBR2 have been added to edits in our first-generation programs: TRAC (T-cell receptor to prevent GvHD), B2M (MHC class I to reduce T-cell mediated rejection), and CD70 (to eliminate fratricide and increase potency), and site-specific CAR insertion at the TRAC locus using an AAV template. These modifications result in next-generation CAR-T therapies that have demonstrated increased potency, prolonged persistence, and sustained antitumor activity. Disruption of Regnase-1 improves functional persistence by increasing cellular expansion potential, correlating with central memory phenotype. TGFBR2 disruption allows CAR T cells to avoid tumor microenvironment suppression. In combination, these edits increase potency at least 10-fold. In addition, long-term maintenance of CM phenotype enables efficient manufacturing. CTX112 and CTX131 demonstrated increased expansion, resistance to exhaustion, and elevated effector cytokine release when tested using in vitro and in vivo models. CTX112 increased survival and tumor regression in CD19+ Nalm6-Luciferase leukemia and Jeko-1 lymphoma models relative to control CAR T cells with only Regnase-1 or TGFBR2 disruption. Similarly, in a “rechallenge model” in which xenografts were serially implanted following a single dose of CAR T cells, CTX131 caused complete regression of NCI-H1975 lung, A498 RCC, and Caki-2 RCC tumors. No evidence of clonal expansion nor cytokine independent growth occurred, suggesting multiplex edited CTX112 and CTX131 cells are safe for clinical use. In summary, CTX112 and CTX131 are the first CAR T programs to incorporate novel Regnase-1 and TGFBR2 edits, with the potential to increase expansion and functional persistence, which may translate clinically to deeper, more durable responses. CTX112 will be developed in patients with B cell malignancies, and CTX131 will be developed in patients with advanced solid tumors, including RCC.

Citation Format: Jonathan A. Terrett, Demetrios Kalaitzidis, Mary-Lee Dequeant, Sushant Karnik, Mohammed Ghonime, Changan Guo, Robert Chain, Heidi Heath, Nivedita Jaishankar, Rachel Yuen, Davis Settipane, Zinkal Padalia, Lauren Zakas, Meghna Kuppuraju, Paul Tetteh, Chandirasegaran Massilamany, Brigid McEwan, Glenn Leary, Henia Dar, Daniel Ferulo, Melanie Allen, Kimberley Tipton, Laura Serwer, Thao Nguyen, Melanie Butler-Gauthier, Parin Sripakdeevong, Shashwat D. Nagar, Yin Tang, Hemangi Chaudhari, Nicole Flanagan, Elaine Huang, Shashwant Phuyal, Elizabeth Koch, Andrew Dunn, Erisa Sula, Jacob Waldman, Cristian Loaiza, Maria Lei Zhang, Erin Thorstensen, Keith Steiger, Katie Schum, Kayla Urbaez, Mark Benton, Anna Ma, Sarah Cohen, Annie Weaver, Christopher Finch, Phuong Khanh Morrow. CTX112 and CTX131: Next-generation CRISPR/Cas9-engineered allogeneic (allo) CAR T cells incorporating novel edits that increase potency and efficacy in the treatment of lymphoid and solid tumors [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 ND02.