Abstract 579: Anti-myeloid poly-pharmacy allows FGFR4-targeted chimeric antigen receptors to effectively treat an orthotopic model of rhabdomyosarcoma
Authors: Sullivan Peter M. , Kumar Rajesh , Li Wei , Wang Lingyang , Zhang Yue , Jamet Sophie , Cheuk Adam , Khan Javed , Dimitrov Dimiter S. , Orentas Rimas J. 
Published: June 2022

Abstract
Rhabdomyosarcoma (RMS) is the most common soft tissue cancer in children, yet treatment outcomes, particularly for relapsed/refractory or metastatic disease, have not improved in decades. The lack of novel therapies and limited immune checkpoint blockade efficacy suggests that CAR-T therapy would be a promising therapeutic approach for RMS and other sarcomas. 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 being specifically and consistently upregulated in RMS, making it a candidate target for CAR-T cells. We tested the feasibility of an FGFR4 targeted CAR for treating RMS using an NSG mouse with RH30 orthotopic (intramuscular) tumors. A previous CAR designed to target FGFR4 was active in vitro but failed to control orthotopic tumors in the NSG mouse model. A new generation of FGFR4 binders was produced targeting the membrane proximal domain of FGFR4. When engineered as CARs, these binders exceeded the activity of previous generation binders in vitro with regard to cellular cytotoxicity and cytokine production. Nevertheless, new candidate binders failed to control orthotopic tumors in vivo. We then interrogated the specific tumor defenses employed by RMS to evade immune control. First, we optimized CAR signaling domains to target low density antigen. Quantitative flow analysis determined FGFR4 expression to be a very low 700 molecules per cell on in vivo RH30 tumors, as opposed to 2-3,000 in tissue culture. We also found that RMS tumors produced a collagen-rich stroma, replete with immunosuppressive myeloid cells. Stroma was induced by T cell therapy and absent in untreated mice. This stroma sequesters CAR-T cells, and produces an immune-excluded phenotype, as assessed by immunohistochemical analysis. Immunohistochemistry identified that M2 macrophages, and to a lesser degree MDSC, were the major cellular constituents of the therapy-induced stroma. RNA expression panel analysis (Nanostring) identified the induction of tumor defense-associated transcripts, including MIF, IDO1, and TGFβ, upon T cell therapy. Based on these results, we devised a strategy to augment CAR-T activity while removing the immunosuppressive barriers. The exposure of mice to anti-myeloid poly-pharmacy (targeting CSF1R (PLX3397), IDO1 (epacadostat), iNOS (L-NAME), TGFβ (SD208), PDL1 (αPD1 antibody), MIF (gene knockout), and myeloid misdifferentiation (ATRA)) allowed FGFR4 CAR-T to successfully clear orthotopic RMS tumors. Our results demonstrate that RMS tumors, even with low copy number targets, can be targeted by CAR-T upon reversal of an immunosuppressive microenvironment, modeling an approach to treating pediatric sarcomas with CAR-T therapy.
Citation Format: Peter M. Sullivan, Rajesh Kumar, Wei Li, Lingyang Wang, Yue Zhang, Sophie Jamet, Adam Cheuk, Javed Khan, Dimiter S. Dimitrov, Rimas J. Orentas. Anti-myeloid poly-pharmacy allows FGFR4-targeted chimeric antigen receptors to effectively treat an orthotopic model of rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 579.