Preclinical development of a chimeric antigen receptor T cell therapy targeting FGFR4 in rhabdomyosarcoma.

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.

FGFR4-Targeted Chimeric Antigen Receptors Combined with Anti-Myeloid Polypharmacy Effectively Treat Orthotopic Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue cancer in children. Treatment outcomes, particularly for relapsed/refractory or metastatic disease, have not improved in decades. The current lack of novel therapies and low tumor mutational burden suggest that chimeric antigen receptor (CAR) T-cell therapy could be a promising approach to treating RMS. Previous work identified FGF receptor 4 (FGFR4, CD334) as being specifically 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. The first barrier we noted was that RMS tumors produce a collagen-rich stroma, replete with immunosuppressive myeloid cells, when T-cell therapy is initiated. This stromal response is not seen in tumor-only xenografts. When scFV-based binders were selected from phage display, CARs targeting FGFR4 were not effective until our screening approach was refined to identify binders to the membrane-proximal domain of FGFR4. Having improved the CAR, we devised a pharmacologic strategy to augment CAR T-cell activity by inhibiting the myeloid component of the T-cell-induced tumor stroma. The combined treatment of mice with anti-myeloid polypharmacy (targeting CSF1R, IDO1, iNOS, TGFbeta, PDL1, MIF, and myeloid misdifferentiation) allowed FGFR4 CAR T cells to successfully clear orthotopic RMS tumors, demonstrating that RMS tumors, even with very low copy-number targets, can be targeted by CAR T cells upon reversal of an immunosuppressive microenvironment.

Solid Tumor Microenvironment Can Harbor and Support Functional Properties of Memory T Cells.

Robust T cell responses are crucial for effective anti-tumor responses and often dictate patient survival. However, in the context of solid tumors, both endogenous T cell responses and current adoptive T cell therapies are impeded by the immunosuppressive tumor microenvironment (TME). A multitude of inhibitory signals, suppressive immune cells, metabolites, hypoxic conditions and limiting nutrients are believed to render the TME non-conducive to sustaining productive T cell responses. In this study we conducted an in-depth phenotypic and functional comparison of tumor-specific T cells and tumor-nonspecific bystander memory T cells within the same TME. Using two distinct TCR transgenic and solid-tumor models, our data demonstrate that despite exposure to the same cell-extrinsic factors of the TME, the tumor-nonspecific bystander CD8 T cells retain the complete panoply of memory markers, and do not share the same exhaustive phenotype as tumor-reactive T cells. Compared to tumor-specific T cells, bystander memory CD8 T cells in the TME also retain functional effector cytokine production capabilities in response to ex vivo cognate antigenic stimulation. Consistent with these results, bystander memory T cells isolated from tumors showed enhanced recall responses to secondary bacterial challenge in a T cell transplant model. Importantly, the tumor-resident bystander memory cells could also efficiently utilize the available resources within the TME to elaborate in situ recall effector functions following intra-tumoral peptide antigen injection. Additionally, CRISPR-Cas9 gene deletion studies showed that CXCR3 was critical for the trafficking of both tumor antigen-specific and bystander memory T cells to solid tumors. Collectively, these findings that T cells can persist and retain their functionality in distinct solid tumor environments in the absence of cognate antigenic stimulation, support the notion that persistent antigenic signaling is the central driver of T cell exhaustion within the TME. These studies bear implications for programming more efficacious TCR- and CAR-T cells with augmented therapeutic efficacy and longevity through regulation of antigen and chemokine receptors.

SMCR8 negatively regulates AKT and MTORC1 signaling to modulate lysosome biogenesis and tissue homeostasis.

The intronic hexanucleotide expansion in the C9orf72 gene is one of the leading causes of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), two devastating neurodegenerative diseases. C9orf72 forms a heterodimer with SMCR8 (Smith-Magenis syndrome chromosome region, candidate 8) protein. However, the physiological function of SMCR8 remains to be characterized. Here we report that ablation of SMCR8 in mice results in splenomegaly with autoimmune phenotypes similar to that of C9orf72 deficiency. Furthermore, SMCR8 loss leads to a drastic decrease of C9orf72 protein levels. Many proteins involved in the macroautophagy-lysosome pathways are downregulated upon SMCR8 loss due to elevated activation of MTORC1 and AKT, which also leads to increased spine density in the Smcr8 knockout neurons. In summary, our studies demonstrate a key role of SMCR8 in regulating MTORC1 and AKT signaling and tissue homeostasis. Abbreviations: ALS: amyotrophic lateral sclerosis; C9orf72: chromosome 9 open reading frame 72; FTLD: frontotemporal lobar degeneration; GEF: guanosine nucleotide exchange factor; GTPase: guanosine tri-phosphatase; KO: knockout; MTOR: mechanistic target of rapamycin kinase; SMCR8: Smith-Magenis chromosome region, candidate 8; WDR41: WD repeat domain 41; WT: wild type.