Abstract 1367: Antigen recognition induces chemokine secretion to promote universal CAR-T cell infiltration

The lack of CAR-T cell infiltration and immune-mediated cancer cell destruction is a dominant mechanism of immunotherapy failure. The characteristics and mechanisms of cognate recognition induced CAR-T cell infiltration have yet to be revealed. In this study, we developed an in-vitro 3D collagen droplet co-culture system to simulate CAR-T cell infiltration in the tumor microenvironment and track T cell motility and interactions with tumor cells in real-time under different micro physiological environments. We employed CAR-T cells overexpressing the SNAP tag (SNAP-CAR T cells), where the contacts between CAR-T cells and HER2+ breast tumor cells can be initiated by adding benzyl guanin-conjugated Herceptin (BG-Herceptin). Quantification of infiltrating cell numbers showed that 3D collagen matrices with HER2+ breast tumor cells significantly recruited a higher number of SNAP CAR-T cells compared to cell-free matrices. Upon initiating the contacts between target and effector cells by adding BG-Herceptin, CAR-T cell infiltration to HER2+ breast cancer matrices were significantly increased. To investigate whether chemokine secretion mediates this process, we employed chemokine arrays. Chemokine secretion such as CCL2 were significantly higher with antigen-specific interactions. Further, CAR-T cell infiltration was inhibited by CCL2 neutralization, which indicates CCL2 plays a major role in cognate induced CAR-T cell infiltration. By analyzing CAR-T cell motility patterns, we found that CAR-T cell infiltration and interaction with tumor cells are dependent on cognate recognition. With BG-Herceptin, CAR-T cell interaction with and swarming around HER2+ breast tumor cells were significantly higher. By inhibiting CCL2, CAR-T cell motility was significantly impaired. Our findings suggest that CAR-T cell infiltration towards the solid tumors is dependent on antigen-specific interactions. Moreover, we demonstrated cognate induced chemokine secretion as a crucial factor in CAR-T cell infiltration and motility. Citation Format: Ruxuan Li, Youngbin Cho, Matthew Laird, Walter Kaminski, Elisa Ruffo, Jason Lohmueller, Ioannis Zervantonakis. Antigen recognition induces chemokine secretion to promote universal CAR-T cell infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1367.