Abstract
Although chimeric antigen receptor (CAR)-redirected T lymphocytes have achieved unprecedented clinical responses in hematologic neoplasms, their role in solid tumors still needs to be improved. To overcome current limitations, we rationally designed a next-generation CAR-T cell construct incorporating autonomous costimulatory signaling pathways that operate independently of tumor antigen engagement, thus faithfully emulating physiological T cell activation paradigms. We screened seven costimulatory receptors from the TNF receptor superfamily and identified that CD30 is the most effective enhancer of CAR-T cell function. Subsequent structural analysis revealed that the intracellular domain (ICD) of CD30 is primarily responsible for its costimulatory activity. Our data showed that these CAR-T cells co-expressing CD30 ICD have stronger proliferation ability and cytokine secretion function. The results of in vitro experiments showed that CD30 signaling improved the cytotoxicity of CAR-T cells and reduced the expression of exhaustion-related markers. In mouse models of orthotopic renal cancer, lung metastasis, and hematologic malignancies, these CAR-T cells showed better expansion capability and superior antitumor activity. Sequencing results suggest that CD30 signaling may enhance the function of CAR-T cells by increasing the activation of the nuclear factor κB (NF-κB) pathway. Further mechanistic experiments confirmed that the NF-κB pathway is significantly activated in CAIX.CD30(ICD) CAR-T cells compared to CAIX CAR-T cells. Reversal experiments demonstrated that NF-κB pathway inhibitors can reverse the effector function of CAIX.CD30(ICD) CAR-T cells, while CAIX-IKKβ CAR-T cells with self-activated NF-κB pathway also exhibit significant functional advantages. By integrating autonomous costimulatory signaling, we demonstrated improved CAR-T cell persistence, proliferation, and antitumor activity across multiple preclinical models, highlighting the therapeutic potential of this approach for both hematologic malignancies and solid tumors.