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Pediatric patients with relapsed or refractory rhabdomyosarcoma (RMS) have dismal cure rates, and effective therapy is urgently needed. The oncogenic receptor tyrosine kinase fibroblast growth factor receptor 4 (FGFR4) is highly expressed in RMS and lowly expressed in healthy tissues. Here, we describe a second-generation FGFR4-targeting chimeric antigen receptor (CAR), based on an anti-human FGFR4-specific murine monoclonal antibody 3A11, as an adoptive T cell treatment for RMS. The 3A11 CAR T cells induced robust cytokine production and cytotoxicity against RMS cell lines in vitro. In contrast, a panel of healthy human primary cells failed to activate 3A11 CAR T cells, confirming the selectivity of 3A11 CAR T cells against tumors with high FGFR4 expression. Finally, we demonstrate that 3A11 CAR T cells are persistent in vivo and can effectively eliminate RMS tumors in two metastatic and two orthotopic models. Therefore, our study credentials CAR T cell therapy targeting FGFR4 to treat patients with RMS., Competing Interests: Declaration of interests J. Khan, R.J.O., D.S.D., and A.T.C. are inventors on international patent application no. PCT/US2016/052496. The 3A11 CAR sequence is in this patent application (see https://patents.justia.com/patent/11078286) filed on September 19, 2016, titled “Monoclonal antibodies specific for fibroblast growth factor receptor 4 (FGFR4) and methods of their use.”, (Published by Elsevier Inc.)

Chimeric antigen receptor (CAR) T cell therapies targeting single antigens have performed poorly in clinical trials for solid tumors due to heterogenous expression of tumor-associated antigens (TAAs), limited T cell persistence, and T cell exhaustion. Here, we aimed to identify optimal CARs against glypican 2 (GPC2) or CD276 (B7-H3), which were highly but heterogeneously expressed in neuroblastoma (NB), a lethal extracranial solid tumor of childhood. First, we examined CAR T cell expansion in the presence of targets by digital droplet PCR. Next, using pooled competitive optimization of CAR by cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), termed P-COCC, we simultaneously analyzed protein and transcriptome expression of CAR T cells to identify high-activity CARs. Finally, we performed cytotoxicity assays to identify the most effective CAR against each target and combined the CARs into a bicistronic "OR" CAR (BiCisCAR). BiCisCAR T cells effectively eliminated tumor cells expressing GPC2 or CD276. Furthermore, the BiCisCAR T cells demonstrated prolonged persistence and resistance to exhaustion when compared with CARs targeting a single antigen. This study illustrated that targeting multiple TAAs with BiCisCAR may overcome heterogenous expression of target antigens in solid tumors and identified a potent, clinically relevant CAR against NB. Moreover, our multimodal approach integrating competitive expansion, P-COCC, and cytotoxicity assays is an effective strategy to identify potent CARs among a pool of candidates.