Abstract 4819: Pulmonary neutrophil-CAR T cell crosstalk amplifies antigen-specific off tumor toxicity

Abstract Despite the potential for tumor-specific T cells to mediate durable complete responses of advanced disease, no CAR-T cell treatment is currently approved for use against a solid cancer. While nearly all solid cancers express a tumor-associated antigen (TAA) vulnerable to CAR T cell recognition, the same TAAs are also present on healthy tissues. CAR T cells recognize and attack healthy tissues that express their target, even at low levels, provoking lethal and dose-limiting ‘on-target off-tumor’ toxicities, most commonly the lungs, resulting in vanishingly small therapeutic index. Genetically engineered and immunocompetent mouse models continue to inform our understanding of the immunostimulatory and inhibitory roles of myeloid populations in cancer development and antitumor immunity. However, insights into the role of myeloid populations in constraining or promoting CAR T cell function in the tumor and sites of toxicity in cancer-free organs has been limited due to the prominent use of xenograft models when evaluating CAR T cell function. CAR T cell xenograft mouse models (i.e. transfer of human T cells into immunocompromised mice bearing human tumors) fail to capture off-tumor toxicities (due to the lack of host antigen expression) and are compromised in their ability to recapitulate cytokine crosstalk between the transferred and endogenous populations (due to un-even cross-reactivity of heterologous cytokine-receptor interactions).We develop two immunocompetent murine models (C57BL/6) of CAR T cell transfer targeting TAAs abundant in multiple epithelial cancers (EpCAM and CEA). EpCAM and CEA CAR T cells affected moderate control of pancreatic and metastatic colon cancer but also infiltrated and damaged non-cancerous organs that expressed the TAA (lung & colon). The respiratory system was the site of greatest macro and microscopic pathology in both models. Flow cytometry and immunohistochemical analysis of sites of off-tumor toxicity revealed a marked accumulation of macrophages and neutrophils, along with transferred CAR T cells, in the lungs of host mice, along with immune crosstalk mediated activation and proliferation of endogenous CD4 and CD8 T cell populations. To assess their functional significance in mediating off-tumor toxicity, we depleted endogenous macrophage and neutrophil accumulation in the lungs of CEA and EpCAM CAR T cell recipients using clondronate liposomes or anti-Ly6G depleting antibodies (clone 1A8). In both CEA and EpCAM CAR T cell models, we found that depletion of host neutrophils, but not macrophages, prevented CAR T cell accumulation, activation of endogenous T cells, and pathologic tissue destruction in the lungs of host mice. These findings provide strong justification for targeting host myeloid populations for depletion or reprogramming in the setting of CAR T cell transfer to expand their therapeutic index. Citation Format: Robert L. Eil, Alexandra Bartlett, Matthew McWhorter, Chelsea Jenkins, Jessica Briones. Pulmonary neutrophil-CAR T cell crosstalk amplifies antigen-specific off tumor toxicity [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 4819.