Abstract 6116: Generation of multi-specific CAR T cells using a novel single-marker selection platform to target medulloblastoma heterogeneity

Abstract High-risk Group 3 and Group 4 and recurrent medulloblastoma patients have a dismal prognosis, and emerging targeted therapies have not yielded promising results. While CAR T cell therapy has achieved unprecedented success in treating hematologic malignancies, translating this approach to solid tumors has been challenging due to tumor heterogeneity and the immunosuppressive microenvironment. To overcome these obstacles and address the urgent need for new treatments, we aimed to develop an enhanced multi-specific CAR T cell product targeting the antigens GPC2, GD2, and B7H3, which are described to be highly expressed across cancers, notably in tumors of neuroectodermal origin. We first conducted a comprehensive analysis of bulk and single-cell RNA sequencing data to validate the expression of GPC2, GD2, and B7H3 on medulloblastoma samples. Protein expression of these targets was confirmed on medulloblastoma cell lines and PDX models, and confirmation on patient samples using flow cytometry-based Quantibrite analysis is currently ongoing. Based on these findings, we generated tri-specific CAR T cells using the STASH select platform - a method for enriching cells containing multiple genetic modifications via a single selection marker - and performed phenotypic and functional assays in vitro and in vivo. Our initial results show that the selected tri-specific CAR T cells effectively control pooled populations of tumor cells expressing each of the antigens individually or in combination. However, we observed that the phenotype and function are dominated by the CAR with the highest level of tonic signaling, which decreased the overall efficacy of the tri-specific CAR T cells. By implementing downregulation of MED12, we found that the tri-specific CAR T cells demonstrated increased proliferation upon stimulation and enhanced cytotoxic efficacy in repeated stimulation assays, indicating exhaustion resistance. Further T cell engineering, including overexpression of cJUN and in vivo evaluations of antitumor activity in different mouse models are ongoing. In conclusion, our tri-specific CAR T cell product targeting GPC2, GD2, and B7H3 represents a promising novel therapeutic approach to overcoming T cell-intrinsic (exhaustion) and extrinsic (antigen heterogeneity) limitations in treating high-risk medulloblastoma. Ongoing studies aim to further assess their effectiveness and potential for broader clinical application. Citation Format: Diren Usta, Yushen Feng, Yiyun Chen, Louai Labanieh, Nikoo Jafari, Peng Xu, Sabine Heitzeneder, Elena Sotillo, Crystal Mackall. Generation of multi-specific CAR T cells using a novel single-marker selection platform to target medulloblastoma heterogeneity [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 6116.