Surfaceome Profiling of Cell Lines and Patient-Derived Xenografts Confirm FGFR4, NCAM1, CD276, and Highlight AGRL2, JAM3, and L1CAM as Surface Targets for Rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The prognosis for patients with high-grade and metastatic disease is still very poor, and survivors are burdened with long-lasting side effects. Therefore, more effective and less toxic therapies are needed. Surface proteins are ideal targets for antibody-based therapies, like bispecific antibodies, antibody-drug conjugates, or chimeric antigen receptor (CAR) T-cells. Specific surface targets for RMS are scarce. Here, we performed a surfaceome profiling based on differential centrifugation enrichment of surface/membrane proteins and detection by LC-MS on six fusion-positive (FP) RMS cell lines, five fusion-negative (FN) RMS cell lines, and three RMS patient-derived xenografts (PDXs). A total of 699 proteins were detected in the three RMS groups. Ranking based on expression levels and comparison to expression in normal MRC-5 fibroblasts and myoblasts, followed by statistical analysis, highlighted known RMS targets such as FGFR4, NCAM1, and CD276/B7-H3, and revealed AGRL2, JAM3, MEGF10, GPC4, CADM2, as potential targets for immunotherapies of RMS. L1CAM expression was investigated in RMS tissues, and strong L1CAM expression was observed in more than 80% of alveolar RMS tumors, making it a practicable target for antibody-based therapies of alveolar RMS.

CAR T cells recognizing CD276 and Dual-CAR T cells against CD276/FGFR4 promote rhabdomyosarcoma clearance in orthotopic mouse models

“…Antigen loss is a common mechanism of tumor resistance against CAR T cellsthat can be addressed by CAR T cells able to recognize multiple targets on the surface of tumor cells. Therefore, we next focused on FGFR4, another promising target for RMS, as also recently confirmed by our RMS surfaceome profiling (32). Expression levels of FGFR4 on RMS cell lines and PDXs were assessed by Western blotting and Flow Cytometry.…”

“…Therefore, we next focused on FGFR4, another promising target for RMS, as also recently confirmed by our RMS surfaceome profiling (32). Expression levels of FGFR4 on RMS cell lines and PDXs were assessed by Western blotting and Flow Cytometry.…”

The Capacity of Drug-Metabolising Enzymes in Modulating the Therapeutic Efficacy of Drugs to Treat Rhabdomyosarcoma

“…In addition to approaches aimed at regulating the activity of immune effector cells in the TME, these authors are also highlighting the use of neoantigen-displaying MHC receptors alongside differentially overexpressed differentiation antigens, including CD99, CD248, EGFR, GD2, HER2, and IGF1R, for selective target cell killing via, e.g., CAR-T cells or antibody–drug conjugates (ADCs), including recombinant immunotoxins. A recent surfaceome-profiling study based on the differential centrifugation enrichment of surface/membrane proteins and detection via LC-MS of six fusion-positive RMS cell lines, five fusion-negative RMS cell lines, and three RMS patient-derived xenografts (PDXs) indicated a range of potential cell surface targets for RMS immunotherapies, providing opportunities for new drug development strategies [ 131 ]. ADCs represent one of the fastest-growing drug delivery systems, a technology that involves a cancer-cell-targeted monoclonal antibody conjugated with a synthetic cytotoxic payload via a linker.…”

“…This approach could be applied to RMS and, e.g., include the specific delivery of a topoisomerase I inhibitor, as demonstrated for the two recent clinically approved ADCs Enhertu or Trodelvi, which would provide an attractive option for circumventing non-tumour prodrug activation via carboxylesterases [ 132 ]. Although more work is needed to understand how the disease-specific differential expression of such cell surface antigens can benefit RMS patients, they have a great potential to eventually unlock further immunotherapeutic strategies, whether alone or in combination with others [ 131 , 133 ].…”

CD276-CAR T cells and Dual-CAR T cells targeting CD276/FGFR4 promote rhabdomyosarcoma clearance in orthotopic mouse models

“…In our previous work on identifying potential surface antigens by surfaceome profiling of RMS for an immunotherapy approach [ 32 ], we identified two targets that are particularly appealing for a CAR T cell therapy: CD276 and FGFR4.…”