GPR64, Screened from Ewing Sarcoma Cells, Is a Potential Target for Antibody-Based Therapy for Various Sarcomas

SIMPLE SUMMARY: New strategies for immunotherapy have led to an increased interest in tumor-specific antigens on the cell surface in the field of oncology. Identifying markers in sarcomas is difficult because their tumor mutation burden is less than that of carcinomas. We assumed that a target protein may be acceptable as a therapeutic target, even if it is only expressed in the epididymis along with the tumor, because the epididymis has special barriers, known as the blood–epididymis barrier (BEB). We identified GPR64 as a therapeutic target for Ewing sarcoma via next-generation RNA-sequencing. GPR64 is located on the apical membranes of efferent ductules and separated from antibodies by the BEB. This study revealed, for the first time, that anti-GPR64 antibodies accumulate in various sarcomas and avoid targeting GPR64 in the epididymis in vivo. Furthermore, GPR64 is widely expressed in various sarcomas and is, therefore, a potential antibody-based therapeutic target for sarcomas. ABSTRACT: Ewing sarcoma is an aggressive and the second most common bone tumor in adolescent and young adult patients. The 5-year survival rate is 60–70% for localized disease but 30% for patients with metastases. Here, we aimed to identify a therapeutic target for Ewing sarcoma and evaluate antibody-based therapeutic agents using in vitro and in vivo models. We identified G protein-coupled receptor 64 (GPR64) as a therapeutic target for Ewing sarcoma via next-generation RNA-sequencing. GPR64v205 mRNA was expressed in HTB166, A673, MG63, 143B, HS-Sy II, and HT1080 cell lines as well as in Ewing sarcoma, undifferentiated pleomorphic sarcoma, leiomyosarcoma, dedifferentiated liposarcoma, and synovial sarcoma tissues. GPR64 expression was observed in 62.5% of sarcoma cases and was overexpressed in 33.9% cases. GPR64-specific monoclonal antibodies were tested as near-infrared probes for in vivo imaging using subcutaneous tumor mouse xenografts. Fluorescence intensity was stronger for the AF700-labeled anti-GPR64 antibody than that for the AF700-labeled isotype control antibody. GPR64 was detected in engrafted tumors of A673, 143B, HT1080, and the epididymis but not in other resected tissues. The anti-GPR64 antibody showed excellent binding to GPR64-positive tumors but not to healthy tissues. This antibody has potential for drug delivery in the antibody-based treatment of sarcomas.