Abstract 1545: Development of FGFR4-targeted chimeric antigen receptors (CARs) for the treatment of rhabdomyosarcoma

Abstract

Abstract Rhabdomyosarcoma (RMS) is a soft tissue cancer commonly arising in muscle, and is the most common soft tissue cancer in children. Patients with metastatic RMS have a poor prognosis that has not improved in decades, highlighting the need for novel therapies. Pediatric tumors, and especially the fusion-gene driven alveolar RMS (aRMS) subset, have among the lowest mutational burden of any tumor, suggesting that checkpoint therapies alone will be insufficient to control tumors. CAR-T cells are genetically engineered T lymphocytes expressing an extracellular binding domain and intracellular T cell signaling domains. Upon engagement of the binding domain (often antibody-derived) to the antigenic target on a tumor cell, activation and degranulation occurs resulting in cell-mediated toxicity induced death of the tumor cells. The key to CAR specificity is to guide engineered T cells to a molecular target that is tumor specific, expressed on the cell surface, and expressed at high enough levels for CAR-T activation. Previous work identified fibroblast growth factor receptor 4 (FGFR4, CD334) as specifically upregulated in RMS tumors compared to normal tissue, making it a candidate for the selective targeting of CAR T cells. A previous CAR designed to target FGFR4 (m410) showed in vitro efficacy, and some control of tumor in a metastatic (intravenous) RMS model but failed to control orthotopic (intramuscular) RMS tumors in vivo. We have developed a new generation of FGFR4-binding moieties to be tested as new components of CARs. Our new binders were derived from both human F(ab)- and human VH-only (single chain domain antibodies, dAbs) phage display libraries. Binders were cloned into a CAR featuring a CD8 hinge and transmembrane domain, 4-1BB signaling domain, and CD3zeta signaling domain. FGFR4 CARs were screened for surface expression by flow cytometry, cytotoxicity against target and non-target cells, and cytokine production in response to RMS cell lines in vitro. Two F(ab)-based FGFR4-binders engineered into CARs were selected for further development based on high cellular cytotoxicity and tumor specificity. These will be tested for in vivo efficacy against metastatic and intramuscular RMS tumor models in NSG mice. Additionally, we have tested RMS tumor cell lines, with and without exposure to IFN-gamma, to model induced mechanisms of tumor defense against immune cell attack. Pathway analysis identified several potential mechanisms of tumor resistance to CAR T therapy which will be exploited to help armor engineered CAR-T cells to control RMS tumors. This data will determine whether our new FGFR4 CARs are sufficient for targeting and eliminating RMS tumors, provide insight into which armoring approaches are best suited to control RMS tumors, and thus develop a potent new therapeutic approach for rhabdomyosarcoma.​ Citation Format: Peter Sullivan, Rajesh Kumar, Wei Li, Lingyang Wang, Yue Zhang, Adam Cheuk, Nityashree Shivaprasad, Javed Khan, Dimiter S. Dimitrov, Rimas J. Orentas. Development of FGFR4-targeted chimeric antigen receptors (CARs) for the treatment of rhabdomyosarcoma [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 1545.