PROJECT SUMMARY/ABSTRACT Rahbdomyosarcoma (RMS) is a devastating pediatric malignancy that has not shown improvement in decades for patients with high-risk or recurrent disease. One subset of this disease, ARMS, is usually more severe, is driven by a PAX3/7-FOXO1 fusion gene product, has remarkably few other mutations, and expresses the protein FGFR4 on its surface. In previous work we used extensive genomic analysis to define the relevance of FGFR4 as a CAR-T target and created a set of CARs that target FGFR4 using an scFv expression library. Two of the binders isolated function in vitro, and one was partially effective in vivo using an i.v. model in NSG mice (injection of the Rh30 cell line we engineered to also express CD19). Mice that succumbed to i.v. tumor also had liver metastases with a rich stroma and induced expression of PD-L1, which was not seen in tumor- bearing mice that did not receive either CAR-T or activated non-transduced T cells. Importantly, when Rh30_19 was injected i.m., no tumor control was seen. Upon histological analysis, both liver mets and i.m. tumor had a classic “immune excluded” morphology, in that CD4 and CD8 lymphocytes were present, but primarily were restricted to the tumor periphery. This led us to propose new approaches in this application to improve CAR-T therapy for ARMS. When we mapped the binding site of our current scFv-based CAR, it was quite distal to the target cell membrane. Our earlier work with CD22 demonstrated that a more active CAR can be created by targeting membrane proximal domains. Here we will use engineered domain antibodies (dAb) instead of scFv to target new epitopes on FGFR4. Unique to the Dimitrov lab, dAb are based on a stable, soluble, VH-only domain, and are displayed in an engineered phage library format with incredible large diversity. dAb are smaller in size than scFv and are able to uniquely identify new binding sites on target proteins. We outline an approach that will target a more membrane proximal domain of FGFR4. Secondly, we will armor CARs to subvert the negative signals induced by PD-1 binding, by TGF-beta, and by Fas. Third, we will disrupt the physical structure and the biological formation of tumor stroma through the use of collagenase and all-trans retinoic acid (ATRA). Collagenase breaks down the physical barrier collagen presents to immune cells, and releases chemokines sequestered in the stromal matrix. ATRA directly impacts myeloid-derived suppressor cells activity in the tumor micorenvironment and renders MDSC less immunosuppressive. Through the targeting of a membrane proximal FGFR4 domain, armoring of the CAR, and disruption of the tumor tissue microenvironment we propose to create a new generation of FGFR4-specific CARs that will impact this dire pediatric cancer.