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

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.

Rapid liposomal formulation for nucleolin targeting to rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. More effective and less toxic therapies are urgently needed for high-risk patients. Peptide-guided targeted drug delivery can increase the therapeutic index of encapsulated drugs and improve patients' well-being. To apply this strategy to RMS, we identified the peptide F3 in a screening for peptides binding to RMS cells surface. F3 binds to nucleolin, which is present on the surface of RMS cells and is abundantly expressed at the mRNA level in RMS patients' biopsies compared to healthy tissues. We developed a rapid microfluidic formulation of F3-decorated PEGylated liposomes and remote loading of the chemotherapeutic drug vincristine. Size, surface charge, drug loading and retention of targeted and control liposomes were studied. Enhanced cellular binding and uptake were observed in three different nucleolin-positive RMS cell lines. Importantly, F3-functionalized liposomes loaded with vincristine were up to 11 times more cytotoxic than non-targeted liposomes for RMS cell lines. These results demonstrate that F3-functionalized liposomes are promising for targeted drug delivery to RMS and warrant further in vivo investigations.

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

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood, whose prognosis is still poor especially for metastatic, high-grade, and relapsed RMS. New treatments are urgently needed, especially systemic therapies. Chimeric Antigen Receptor T cells (CAR Ts) are very effective against hematological malignancies, but their efficacy against solid tumors needs to be improved. CD276 (B7-H3) is a target upregulated in RMS and detected at low levels in normal tissues. FGFR4 is a very specific target for RMS. Here, we optimized CAR Ts for these two targets, alone or in combination, and tested their anti-tumor activity in vitro and in vivo. METHODS: Four different single-domain antibodies were used to select the most specific FGFR4-CAR construct. RMS cell killing and cytokine production by CD276- and FGFR4-CAR Ts expressing CD8α or CD28 HD/TM domains in combination with 4-1BB and/or CD28 co-stimulatory domains were tested in vitro. The most effective CD276- and FGFR4-CAR Ts were used to generate Dual-CAR Ts. Tumor killing was evaluated in vivo in three orthotopic RMS mouse models. RESULTS: CD276.V-CAR Ts (276.MG.CD28HD/TM.CD28CSD.3ζ) showed the strongest killing of RMS cells, and the highest release of IFN-γ and Granzyme B in vitro. FGFR4.V-CAR Ts (F8-FR4.CD28HD/TM.CD28CSD.3ζ) showed the most specific killing. CD276-CAR Ts successfully eradicated RD- and Rh4-derived RMS tumors in vivo, achieving complete remission in 3/5 and 5/5 mice, respectively. In CD276low JR-tumors, however, they achieved complete remission in only 1/5 mice. FGFR4 CAR Ts instead delayed Rh4 tumor growth. Dual-CAR Ts promoted Rh4-tumors clearance in 5/5 mice. CONCLUSIONS: CD276- and CD276/FGFR4-directed CAR Ts showed effective RMS cell killing in vitro and eradication of CD276high RMS tumors in vivo. CD276low tumors escaped the therapy highlighting a correlation between antigen density and effectiveness. FGFR4-CAR Ts showed specific killing in vitro but could only delay RMS growth in vivo. Our results demonstrate that combined expression of CD276-CAR with other CAR does not reduce its benefit. Introducing immunotherapy with CD276-CAR Ts in RMS seems to be feasible and promising, although CAR constructs design and target combinations have to be further improved to eradicate tumors with low target expression.