Chimeric antigen receptor (CAR) T cell therapy has shown remarkable success in hematologic malignancies, prompting efforts to apply this strategy to solid tumors. However, the efficacy of CAR T cells against solid tumors remains limited, mainly due to the immunosuppressive tumor microenvironment (TME). Therefore, enhancing CAR T cell function is crucial for overcoming the barriers, but doing so may increase the risk of on-target, off-tumor toxicity (OTOT), because most solid tumor antigens are also expressed at low levels in normal tissues.

Previous strategies to mitigate OTOT include affinity-tuned CARs and logic-gated CARs. However, affinity-tuned CARs are often ineffective against tumors with low antigen expression. In addition, logic-gated CARs require complex dual gene modifications, which limit their clinical applicability. As an alternative approach, inhibiting the recruitment and retention of CAR T cells in normal tissues may help reduce OTOT. Pre-conditioning regimens have the potential to promote T cell infiltration into inflamed normal tissues, thereby exacerbating OTOT. Thus, CAR T cell therapy without pre-conditioning may represent a promising strategy to minimize OTOT.

In this study, we aimed to enhance the anti-tumor effect of CAR T cells while limiting their migration to normal tissues by improving their function without pre-conditioning. For this purpose, we utilized a murine syngeneic solid tumor model, which allows evaluation of both the efficacy and toxicity of CAR T cells. We previously reported that a CTLA4-CD28 chimera (CTC28) enhances T cell functionality. Additionally, CAR T cells engineered to express interleukin-12 (IL-12) under an inducible promoter have demonstrated improved efficacy in the absence of pre-conditioning. Therefore, we developed murine cMet CAR T cells armored with both CTC28 and inducible IL-12 and sought to validate that these armored CAR T cells can effectively eradicate cMet positive tumors while avoiding OTOT without pre-conditioning.

Our results demonstrate that conventional cMet CAR T cells induced OTOT and lethality in pre-conditioned mice. Furthermore, enhancing CAR T cell function exacerbated OTOT, which was primarily caused by CAR T cell-mediated liver damage under pre-conditioned situations. In contrast, cMet CAR T cells co-modified with CTC28 and inducible IL-12 exhibited potent anti-tumor effects without pre-conditioning and avoided OTOT. This dual-armoring strategy enhanced therapeutic efficacy while preserving safety. Moreover, similar findings were observed in B7H3-targeted CAR T cell models, suggesting broader applicability.

In conclusion, our study provides a novel strategy for safely enhancing CAR T cell function against solid tumors by eliminating the need for lymphodepleting pre-conditioning. This approach could expand the therapeutic window of CAR T cell therapy and enable targeting of solid tumors with improved efficacy and reduced toxicity.