A Novel High-Throughput Screening Method for a Human Multicentric Osteosarcoma-Specific Antibody and Biomarker Using a Phage Display-Derived Monoclonal Antibody

SIMPLE SUMMARY: Human multicentric osteosarcoma (HMOS) is a variant of human osteosarcoma (HOS) in which multiple bone lesions occur simultaneously or asynchronously before lung metastasis. HOS is associated with a poor prognosis. Using the human antibody phage library, we obtained 95 types of antibody clones against HMOS cell lines and developed a fluorescence probe-based enzyme-linked immunosorbent assay (ELISA) that enables high-throughput screening of the reactivity of these antibodies. The lysate of the HMOS cell line was incubated with the selected antibody, and the antigen–antibody complex was recovered using magnetic beads. Liquid chromatography-mass spectrometry (LC/MS) analysis of the sample bands extracted from gel electrophoresis detected CAVIN1/PTRF specifically in HMOS cells. This novel high-throughput screening assay using antibody–drug conjugate targeting for these specific proteins is promising for clinical applications. ABSTRACT: Osteosarcoma is a malignant tumor that produces neoplastic bone or osteoid osteoma. In human multicentric osteosarcoma (HMOS), a unique variant of human osteosarcoma (HOS), multiple bone lesions occur simultaneously or asynchronously before lung metastasis. HMOS is associated with an extremely poor prognosis, and effective treatment options are lacking. Using the proteins in our previously generated HMOS cell lines as antigens, we generated antibodies using a human antibody phage library. We obtained antibody clones recognizing 95 independent antigens and developed a fluorescence probe-based enzyme-linked immunosorbent assay (ELISA) technique capable of evaluating the reactivity of these antibodies by fluorescence intensity, allowing simple, rapid, and high-throughput selection of antibody clones. These results were highly correlated with those using flow cytometry. Subsequently, the HMOS cell lysate was incubated with the antibody, the antigen–antibody complex was recovered with magnetic beads, and the protein bands from electrophoresis were analyzed using liquid chromatography-mass spectrometry (LC/MS). CAVIN1/polymerase I transcript release factor was specifically detected in the HMOS cells. In conclusion, we found via a novel high-throughput screening method that CAVIN1/PTRF is an HMOS-specific cell membrane biomarker and an antigen capable of producing human antibodies. In the future, antibody–drug conjugate targeting of these specific proteins may be promising for clinical applications.