Mining Cancer-Specific Isoforms As Car T-Cell Therapy Targets For Pediatric Solid And Brain Tumors.

Abstract Immunotherapy with T-cells expressing chimeric antigen receptors (CARs) holds the promise to improve outcomes for children with solid and brain tumors. However, at present, there is a limited array of targetable antigens. We posit here that targets generated by alternative splicing events (ASEs) in cancer cells present an untapped class of CAR targets since many of these are tumor-specific and overexpressed in malignant cells. Thus, we developed Cancer-Specific Isoform Miner (CSI-Miner), a pipeline to perform a comprehensive ASE analysis of pediatric cancers to discover CSIs as CAR T-cell therapy targets. We analyzed 1,938 solid and brain tumor samples collected from PCGP, TARGET, and St. Jude's real-time clinical sequencing data sets. In addition, 7,527 GTEx RNAseq samples across 31 human tissues were harmonized to prioritize exon candidates with minimal expression in normal tissue. Through CSI-Miner, we identified 40,748 highly expressed exons from the tumor's surfaceome and secretome, of which 185 were unannotated novel exons. Taking advantage of available mass-spectrometry proteome data from the Clinical Proteomic Tumor Analysis Consortium and St. Jude's proteomics facility, we validated the presence of peptides encoded by these novel exons for rhabdomyosarcoma, medulloblastoma, high-grade glioma, low-grade glioma, and ependymoma. Moreover, non-malignant mass-spectrometry data from muscle and brain samples were incorporated as negative controls. Several secretome targets were identified that adhered to the surface of cancer cells, including EDB, an oncofetal splice variant of fibronectin. EDB was expressed in a broad range of pediatric solid and brain tumors, including Ewing sarcoma and high-grade glioma. We generated T-cells expressing a 2nd generation EDB-CAR (EDB-CAR T-cells) by standard retroviral transduction. EDB-CAR T-cells recognized and killed EDB+ cancer cells in vitro and had potent antitumor activity in xenograft models, resulting in improved overall survival compared to control mice. Importantly, EDB-CAR T-cells did not induce ‘on target/off cancer' toxicities in mice, which is encouraging since EDB is identical in mice and humans. c-MET was also identified as a candidate surfaceome CAR target for rhabdomyosarcoma and melanoma. In summary, we developed CSI-Miner, an integrative analysis pipeline to discover CSIs as CAR T-cell therapy targets, comprised of 248 surfaceome and 51 secretome candidates, for pediatric solid and brain tumors. At present we have validated these CSIs by mass-spectrometry and successfully generated CAR T-cells for one of the candidates. Collectively, CSIs present an untapped class of immunotherapy targets that hold the promise to improve current CAR T-cell therapy approaches for a wide range of pediatric solid and brain tumors. A web portal has been developed supporting access and visualization of this data set to the research community. Citation Format: Timothy I. Shaw, Jessica Wagner, Elizabeth Wickman, Liqing Tian, Dong Li, Suresh Poudel, Elizabeth Stewart, Yuxin Li, Hong Wang, Mingming Niu, Robin Paul, Colleen Reilly, Xin Zhou, Michael Dyer, Suzanne Baker, Junmin Peng, Jiyang Yu, Paulina Velasquez, Chris DeRenzo, Giedre Krenciute, Jinghui Zhang, Stephen Gottschalk. Mining cancer-specific isoforms as CAR T-cell therapy targets for pediatric solid and brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1543.