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Monoclonal Antibody to Human Osteosarcoma: A Novel M_r 26,000 Protein Recognized by Murine Hybridoma TMMR-2¹

Departments of Pathology and Orthopedic Surgery, St. Louis University School of Medicine, St. Louis, Missouri 63104

Three murine hybridomas (TMMR-1-3) were developed by repeated immunizations of mice with four different human osteosarcoma cell lines in an alternating sequence of inoculations. The monoclonal antibodies were screened for reactivities to cultured cell lines and tissue sections of osteosarcomas using flow cytometry and immunohistochemical techniques. TMMR-2 is a highly specific antibody (IgG₁) that reacted with all 14 osteosarcoma tumors and eight human osteosarcoma cell lines tested, including the established human osteosarcoma cell lines HOS and Saos-2. Benign neoplastic cells from two osteoblastomas, osteoblasts from regions of reparative osteoid formation and neonatal new bone, are also reactive with TMMR-2. TMMR-1 has mesenchymal specificity while TMMR-3, although reactive with osseous differentiated cells, also reacted with mitotic cells of all cell types. Characterization of antigen structure by Western immunoblotting revealed that TMMR-2 reacted with a 100°C heat labile mercaptoethanol-sensitive M_r 26,000 protein, and TMMR-3 recognized a mercaptoethanol-resistant M_r 97,000 protein whereas TMMR-1 reacted with a series of bands from 65,000 to 85,000 molecular weight, all of which were mercaptoethanol sensitive. TMMR-1 and TMMR-2 monoclonal antibodies showed complement-independent inhibition of [³H]thymidine incorporation into DNA, but did not exhibit cytotoxic activity. The results suggest that TMMR-2 is a specific antibody that recognizes an osteoblast/osteocyte surface antigen present in normal, reactive, and neoplastic disorders of bone. The inhibitory effects on DNA synthesis in cultured osteosarcoma cells by TMMR-2 indicate an important cell growth/proliferation role of this surface antigen. These monoclonal antibodies, in combination with other known antibodies, can be used to characterize mesenchymal cell surface antigenic structure and differentiation.

¹ Supported by a Grant from the Orthopaedic Research and Education Foundation, Chicago, IL 60611 and by a Grant from the American Cancer Society (Institutional Grant IN-124), Atlanta, GA 30329. Presented in part at the Annual Meeting of the International Academy of Pathology, Chicago, IL, 1987; at the 34th Annual Meeting of the Orthopaedic Research Society, February 1–4, 1988, Atlanta, GA; at the 35th Annual Meeting of the Orthopaedic Research Society, February 6–9, 1989, Las Vegas, NV; and at the 73rd Annual Meeting of the Federation of American Societies for Experimental Biology, March 19–23, 1989, New Orleans, LA.

² To whom requests for reprints should be addressed, at Department of Pathology, St. Louis University School of Medicine, St. Louis, MO 63104.

³ Present address: Department of Orthopedic Surgery, Creighton University Medical Center, Omaha, NE 68178.

Received 5/ 4/89. Revised 9/27/89. Accepted 10/ 3/89.