Preclinical evaluation of intrathecal TRP1-targeted CAR T cells: Effects on durable antitumor immunity in melanoma leptomeningeal disease.

Abstract

2086 Background: Melanoma leptomeningeal disease (LMD) is fatal, with median survival under 3 months and no effective immune-based therapies. Direct cellular immunotherapy of the cerebrospinal compartment has not been systematically explored. Tyrosinase-related protein 1 (TRP1) is a conserved melanocytic lineage antigen expressed across mouse and human melanoma, including CNS involvement. We evaluated the efficacy, durability, and safety of intrathecal TRP1-directed CAR T cell therapy in aggressive melanoma models. Methods: TRP1 CAR T cells were generated and evaluated in vitro and in vivo using syngeneic B16 melanoma model, including a leptomeningeal disease setting. CAR T cells were administered intrathecally. Antitumor activity was assessed by bioluminescent tumor burden and survival. Durability of response was evaluated by secondary tumor rechallenge. Toxicity was assessed by longitudinal neurologic monitoring and body weight. Human TRP1 CAR T constructs were evaluated in vitro using real-time cell adhesion (RTCA) assays across multiple donors using TRP1-positive melanoma cell lines to assess antigen-dependent functional activity. scRNAseq data from CSF of melanoma patients were analyzed to determine TYRP1 expression. Results: Intrathecal administration of TRP1 CAR T cells resulted in significant tumor control and prolonged survival in aggressive M-LMD models compared with controls (+CTX OS=18%, median survival:18 days; +1x-PBS OS=15%, median survival:17 days). Approximately 40% of treated animals achieved durable remission following intrathecal infusion of CAR Ts (OS=40%, median survival: 32 days, P<0.0001). Long-term survivors (eg. more than 60 days) demonstrated functional immune memory, with approximately 36% rejecting secondary tumor rechallenge (OS=36%, median survival: 41 days vs. naïve control OS=17%, median survival: 15 days, P<0.0001). Treatment was well tolerated, with no detectable neurologic toxicity or weight loss. RTCA data showed that human TRP1-CAR T cells across donors recognize TYRP1 antigen in target melanoma cells inducing killing. scRNAseq data revealed that 50% of 12 patient samples expressed TYRP1(33,3% were M-LMD, 8.3% were primary intradural spinal melanoma, and 8.3% from 1 autopsy). Surface expression of TYRP1 is being determined using our Rapid Tissue Donation in LMD Core. Together, our data supports translational relevance. Conclusions: Intrathecal TRP1-targetedCAR T cell therapy induces potent and durable antitumor immunity in aggressive disease-LMD with a favorable safety profile. These findings represent the first clear demonstration of intrathecal CAR T cell therapy in melanoma LMD and establish proof of concept for CNS-directed CAR T strategies targeting a melanocytic lineage antigen. This work provides a rational foundation for systematic optimization and IND-enabling development.