Revitalization of affinity-tuned CAR T cells via antigen-dependent release of interleukin12

Abstract Introduction: The application of chimeric antigen receptor (CAR) T cells in solid tumors has met many challenges, arising from the paucity of effective yet safe targets and T cell dysfunction within the immunosuppressive milieu. Clinical trials over the years have reported severe on-target off-tumor toxicities associated with CAR T cell therapies targeting tumor-associated antigens (TAAs). Here, we propose a strategy to target TAAs effectively and safely: 1) improve tumor-targeting specificity by affinity-tuning; 2) armor T cells with antigen-dependent expression of interleukin-12 (IL-12) to compensate loss of CAR T cell activity caused by affinity-tuning. In this system, IL-12 expression is tightly regulated by the nuclear factor of the activated T cells promoter (NFAT) to limit IL-12 to the environ of activated affinity-tuned CAR T cells within the tumor. We demonstrate here the feasibility of this strategy using epithelial cell adhesion molecule (EpCAM) as a model TAA. Methods: Affinity variants were generated by alanine scanning mutagenesis in the complementary-determining region 3 (CDR3) of an EpCAM antibody (UBS54). The affinity of CAR variants was determined by flow cytometry-based saturation binding assay. On-target off-tumor cytotoxicity of CAR T cells was examined against human primary normal epithelial cells. In vivo anti-tumor efficacy was evaluated in mouse models of gastric cancer with cell line-derived (SNU-638 and MKN-45) and patient-derived xenografts. Results: Substitution of alanine into the UBS54 CDR3 led to identification of a tyrosine (6th residue in heavy chain CDR3) as a hot spot for affinity tuning. Among several more subtle amino acid substitutions for Tyr-6, we found that CAR molecule with valine substituent (Y6V) possesses 10 µM affinity toward EpCAM, rendering CAR T cells to be selective to EpCAM-high tumors while being not reactive to primary normal epithelial cells in vitro. In gastric cancer mouse models, compared to UBS54 CAR T cells that mediated rapid tumor remission, Y6V CAR T cells produced mainly partial responses. To revitalize affinity-tuned CAR T cells, we further engineered CAR T cells to release IL-12 under NFAT. We found that both the activity of the NFAT promoter and the level of IL-12 release were tightly regulated by the antigen density of targets when inducible IL-12 is combined with 10 µM affinity Y6V CAR; however, such dependence is lost when 1 µM affinity UBS54 was examined. In mouse models of gastric cancer, inducible lL-12 armored Y6V CAR T cells produced enhanced anti-tumor responses without elevating systemic exposure to IL-12, evidenced by low levels of IL-12 in mouse sera (below 100 pg/ml). Conclusions: Combination of affinity-tuned CAR with inducible expression of IL-12 is a promising strategy to develop CAR T cell therapy against TAAs with the potential for reduced on-target off-tumor toxicity and tumor resistance. Citation Format: Yanping Yang, Huan Yang, Yago Alcaina, Jaclyn E. McCloskey, Janusz Puc, Alyssa Birt, Yogindra Vedvyas, Juan Gonzalez-Valdivieso, Irene M. Min, Eric von Hofe, Moonsoo M. Jin. Revitalization of affinity-tuned CAR T cells via antigen-dependent release of interleukin-12 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5568.