CAR-T cells co-expressing IL-7 and CCL19 promote epitope spreading to enhance antitumor immunity

Antigen heterogeneity remains a major obstacle to the effective application of chimeric antigen receptor (CAR)-T cell therapy in solid tumors. We investigated the potential of epitope spreading as a strategy to overcome this limitation and examined whether CAR-T cells concomitantly producing IL-7 and CCL19 (7 × 19 CAR-T cells) could act as potent inducers of epitope spreading, as well as the underlying mechanisms. We used a murine model inoculated with a mixture of cancer cell lines-genetically modified to express the CAR target antigen-and their respective parental lines lacking target expression. Following administration of 7 × 19 CAR-T cells, flow cytometry and peptide stimulation assays were performed to evaluate the induction of tumor antigen-specific T cells and dendritic cells within tumor-draining lymph nodes and tumor tissues. We also evaluated the antitumor efficacy of 7 × 19 CAR-T cells derived from an allogeneic donor and their capacity to induce epitope spreading in vivo and ex vivo. In multiple tumor mixture models, 7 × 19 CAR-T cells demonstrated marked antitumor activity and promoted epitope spreading in murine solid tumor models, enabling endogenous T cells to recognize and target tumor-associated antigens. This response was dependent on cross-presentation by defined dendritic cell subsets in both the tumor microenvironment and tumor-draining lymph nodes within 2 weeks of 7 × 19 CAR-T cell administration. Notably, 7 × 19 CAR-T cells derived from allogeneic donors also induced epitope spreading in tumor-bearing hosts, thereby increasing survival. The 7 × 19 CAR system is a promising strategy for overcoming antigen heterogeneity in solid tumors by promoting epitope spreading.