The effect of 131I-labeled syngeneic mouse monoclonal anti-melanoma antibodies on tumor growth in vivo were investigated. The injection of unlabeled antibody had little effect on tumor growth, whereas the anti-tumor activity was considerably increased when the antibody was conjugated with 131I and also when a mixture of two different radiolabeled monoclonal antibodies with distinct specificities was used. The anti-tumor effects of the radiolabeled antibody were transient but significant under the present experimental conditions: melanoma growth was completely inhibited for about 10 days or more after the antibody treatment, but resumed thereafter. Possible mechanisms of escape of the tumor from the therapy with radiolabeled monoclonal anti-tumor antibody are discussed.
It has previously been reported that a mouse (C57BL/6) monoclonal antibody, M2590, was established against syngeneic melanoma B16 cells, which was shown to react only with melanoma cells from various species but not with other tumor cells or normal tissues (Taniguchi, M., and Wakabayashi, S. (1984) Gann 75, 418-426). In the present study, the specificity of M2590 antibody was shown to be directed to a saccharide arrangement (NeuAc alpha 2-3Gal beta 1-4Glc (or -GlcNAc)) of gangliosides by three different assay systems including enzyme immunostaining on thin layer plates, sandwich radioimmunoassay, and enzyme-linked immunoadsorbent assays using a variety of glycolipids with known structures. Neither gangliosides having NeuGc terminus, including NeuGc alpha 2-3Gal beta 1-4Glc-ceramide and NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-ceramide, nor ganglio series gangliosides carrying NeuAc reacted with the antibody. An M2590 antibody-reactive antigen was isolated from B16 melanoma cells, and its structure was determined to be NeuAc alpha 2-3Gal beta 1-4Glc-ceramide by fast atom bombardment mass spectrometry, methylation analysis, and exoglycosidase treatment. The ceramide was composed of d18:1 as its long-chain base and C16:0, C24:1, and C24:0 as major fatty acids. The same ganglioside was also detected in the culture supernatant of the melanoma cells as shedding antigen.
The two types of monoclonal anti-melanoma antibodies (M562 and M2590) were obtained by the fusion of P3U1 myeloma cells and spleen cells of C57BL/6 mice hyperimmunized with mitomycin C-treated syngeneic B16 melanoma cells. The M2590 antibody recognized the cross-species melanoma determinant commonly shared among at least mouse, hamster and human melanoma cells, while the M562 antibody reacted with the mouse (B16) specific melanoma antigenic determinant. The M2590 antibody reacted specifically in radioimmunoassay with NP40 tumor cell extracts obtained from melanoma patients but not from other tumor patients. Thus this antibody would provide a powerful tool for immunodiagnostic evaluation of patients with malignant melanoma. Moreover, these antibodies were found to suppress the growth of melanoma cells in vivo.
A sensitive sandwich radioimmunoassay for the detection of minute amounts of melanoma antigens from various mammalian melanomas was established by using a syngeneic mouse monoclonal anti-melanoma antibody (M2590). The assay system detected the soluble mouse and human melanoma antigens equally at a concentration of 10(3)-10(4) cells/ml because the M2590 antibody was found to react specifically with cross-species melanoma antigenic determinants composed of the carbohydrate side chains of the 31,000-dalton glycoprotein uniquely expressed on various mammalian melanoma cells. Furthermore, human melanomas of various types, such as melanotic, amelanotic, primary, metastatic etc., were successfully detected in an antigen-specific manner. Therefore, the present sandwich assay system using a single monoclonal antibody should be quite useful in clinical applications, especially in the diagnosis of human melanomas.
In this article, we summarized syngeneic monoclonal antimelanoma antibodies and their application for chemical characterization of mouse melanoma antigens, cloning of genomic DNA controlling antigen expression, and in vivo/in vitro tumor diagnosis. The melanoma antigen is composed of a protein complex in association with GM3(NeuAc)-like sugar moiety. The GM3 structure expresses the cross-species epitopes shared in various mammalian species, whereas the mouse specific melanoma epitope is present on protein molecules. By using the monoclonal antimelanoma reactive with GM3 epitope, we developed a very sensitive sandwich radioimmunoassay system detecting soluble melanoma antigens equivalent to 10(2)-10(3) cells/ml. The antibody was also useful in imaging tumor in vivo. These results indicate that the antibody with cross-species reactivity has a potential for tumor targeting. The monoclonal antibody M562 recognizing protein molecule with species specific epitope but not other antimelanoma antibodies, however, effectively inhibited experimental lung metastasis of melanoma cells, indicating that the M562 epitope seems to possess important biological functions. Recently, the genomic DNA controlling the antigen expression was successfully isolated by DNA transfection and expression technique with monoclonal anti-melanoma M562 and the fluorescence-activated cell sorter. We also found that genomic DNA possesses transformation-related activity in NIH3T3 cells.
