Abstract 6096: Acquired CAR-T therapy resistance in a pre-clinical model of pediatric rhabdomyosarcoma minimal residual disease
Authors: Amanda M. Lulu, Dustin A. Cobb, Nagyeong Park, Lixia Liu, Daniel W. Lee
Journal: Cancer Research (2025)

Abstract Introduction: Current treatments for relapsed/refractory or metastatic rhabdomyosarcoma (RMS) are ineffective. Integrin αvβ3 plays a role in progression, metastasis and neovascularization in several cancers. HER2 is expressed on a majority of RMS and contributes to progression and metastasis in other cancers. Our lab previously demonstrated the efficacy of HER2 and αvβ3 CAR Ts in controlling or eliminating pediatric brain tumors. CAR T therapies for solid tumors face numerous obstacles with limited clinical success. While bispecific CAR Ts can overcome heterogeneous antigen expression and antigen modulation, CAR T suppression and tumor evasion is multifactorial. Success requires understanding the mechanisms driving clinical failure. Here we developed a minimal residual disease (MRD) model of CAR T resistant RMS to evaluate the mechanisms driving acquired resistance after profound initial tumor control. Methods: Control of intramuscular RMS tumors (RH30) by αvβ3 or HER2 CAR Ts was assessed in NSG mice, with CD19 CAR T controls. After 7 weeks, CAR T infiltration, antigen expression and residual disease characteristics were determined by flow cytometry and immunohistochemistry (IHC). In a pilot experiment, mice were treated with a second CAR T dose or rechallenged with a second tumor in the opposite leg. Tumor burden was evaluated by bioluminescence imaging and caliper measurements; MRD and CAR T infiltration were determined by IHC. Results: Both αvβ3 and HER2 CAR Ts nearly eliminate RMS tumors. However, despite robust initial control and CAR T persistence in both treatment groups, MRD remains. αvβ3 expression minimally decreases following αvβ3 CAR T treatment, although expression is 10-fold higher than the parental cell line. Both αvβ3 and HER2 CAR Ts control tumors in a delayed treatment model, despite HER2 down-regulation with tumor progression. By the time of MRD, 60% of tumor-infiltrating αvβ3 CAR Ts have upregulated multiple exhaustion markers (TIM3, LAG3, PD-1 and TIGIT), in contrast to only 10% of circulating αvβ3 CAR Ts and 10% of tumor-infiltrating CD19 CAR Ts. After αvβ3 or HER2 CAR T treatment, surviving tumor cells upregulate cognate exhaustion ligands (HLA-DR, Galectin3, Galectin9 and PD-L1), consistent with the development of a suppressive tumor environment. Despite this, αvβ3 and HER2 CAR Ts, likely from circulation, control a secondary tumor without delay. Neither the original CAR Ts nor a second dose of CAR Ts eliminate MRD at the primary tumor site. Conclusions: We developed a pre-clinical model of CAR T resistant MRD in which to evaluate specific mechanisms of acquired tumor resistance and CAR T dysfunction in RMS. Different mechanisms, distinct from antigen modulation, arise based on the CAR treatment. Continued investigations into these mechanisms will ultimately enable superior CAR T cell design that is resistant to tumor-mediated inhibition. Citation Format: Amanda M. Lulu, Dustin A. Cobb, Nagyeong Park, Lixia Liu, Daniel W. Lee. Acquired CAR-T therapy resistance in a pre-clinical model of pediatric rhabdomyosarcoma minimal residual disease [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 6096.