Functional properties of FC-2.15, a monoclonal antibody that mediates human complement cytotoxicity against breast cancer cells.
FC-2.15 is a murine IgM monoclonal antibody (mAb) that recognizes a cell-surface antigen (Ag2.15) expressed in most tumor-proliferating cells of human breast carcinomas and other neoplasias. In this study the cytotoxic ability of mAb FC-2.15, its cell-surface binding properties and endocytosis in Ag2.15-expressing (Ag2.15+) cells were investigated. A 51Cr-release assay was used to test the FC-2.15-mediated cytotoxicity. When human serum was used as source of complement, FC-2.15 exerted a strong cytotoxic effect against human Ag2.15+ cells such as MCF-7 (breast cancer cell line), primary breast carcinoma cells, polymorphonuclear leukocytes and chronic myeloid leukemia cells. The mAb concentration range was 1-50 micrograms/ml. Cytotoxicity was completely abolished when complement was inactivated. Only 3.8 +/- 2.9% of MCF-7 cells survived the treatment with FC-2.15 in the presence of human serum. A flow-cytometry assay was performed to study the Ag2.15 expression of the surviving cells and they were found to be Ag2.15-. FC-2.15 did not mediate antibody-dependent cell cytotoxicity when different effector cells were used. Scatchard analysis with 125I-FC-2.15 on MCF-7 cells demonstrated an affinity constant of 6.9 x 10(7) M-1 and 2.8 x 10(6) antigenic sites/cell. 125I-FC-2.15 was internalized to cytoplasmic vesicles reaching a maximum of 27% after 6 h incubation, followed by the release of labeled degradation products to the supernatant. FC-2.15 appears to exert its cytotoxic effect mainly in the presence of human complement, it reacts with intermediate affinity with a high-density surface antigen, and it is slowly internalized by Ag2.15+ cells.
Ballaré
C
C
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
Barrio
M
M
Portela
P
P
Mordoh
J
J
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000920:Antibody-Dependent Cell Cytotoxicity; D000936:Antigen-Antibody Complex / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000378:metabolism; D001943:Breast Neoplasms / Q000276:immunology*; D003167:Complement Activation; D003165:Complement System Proteins / Q000276:immunology*; D003602:Cytotoxicity, Immunologic; D004705:Endocytosis; D006801:Humans; D066298:In Vitro Techniques; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000951:Antigens, Neoplasm; D003165:Complement System Proteins