Surface-epitope masking: a strategy for the development of monoclonal antibodies specific for molecules expressed on the cell surface.
BACKGROUND
Producing monoclonal antibodies against specific targets, including tumor-specific antigens, is a tedious and extremely inefficient process.


OBJECTIVE
Our purpose was to determine whether DNA transfection combined with an immunologic masking tactic could be used to efficiently generate hybridomas that secrete monoclonal antibodies. The quest was for monoclonal antibodies that would react with molecules existing on the surface of genetically altered cells.


METHODS
We developed a masking technique called surface-epitope masking (SEM). The SEM procedure involves the selective blocking of surface antigens present in a genetically engineered cell (referred to as a "tester") with high-titer polyclonal antibodies that have been produced against the untransfected parental cell (referred to as a "driver"). Surface-epitope-masked tester cells were injected into BALB/c mice; immune spleen cells then taken from these mice were fused with myeloma cells.


RESULTS
This process resulted in the efficient generation of hybridomas that secreted monoclonal antibodies that reacted with cell-surface antigens on transfected tester cells and with additional cell types that expressed the same surface molecules. In one case, CREF-Trans 6 cells were engineered to express a typical multidrug-resistant (MDR) phenotype. Using CREF-Trans 6:MDR cells as a tester cell line, we utilized the SEM procedure to produce monoclonal antibodies that displayed surface reactivity to both CREF-Trans 6:MDR cells and MDR human breast carcinoma (MCF7) cells. In a second case, human prostatic carcinoma CREF-Trans 6 cells, which were DNA transfected and derived from nude mouse tumors, were used as the tester cell line. The SEM procedure was again used to produce monoclonal antibodies. These antibodies were designed to and did react with: (a) tumor-associated antigens on the surface of the original LNCaP cell line used to obtain human prostatic carcinoma DNA, (b) primary and secondary nude mouse transfectants derived from tumors, and (c) two additional human prostatic carcinoma cell lines, DU-145 and PC-3.


CONCLUSIONS
The SEM approach was used for the efficient and selective development of monoclonal antibodies that react with cell-surface molecules with both known and unknown functions.


CONCLUSIONS
The SEM procedure should be useful in producing monoclonal antibodies and identifying genes associated with important cellular processes, including immunologic recognition, tumorigenesis, metastasis, atypical multidrug resistance, and autoimmune diseases.
Shen
R
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Department of Urology, Columbia University, College of Physicians and Surgeons, New York, NY 10032.
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Olsson
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Goldstein
N I
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Fisher
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology*; D000954:Antigens, Surface / Q000276:immunology*; D002277:Carcinoma / Q000235:genetics / Q000276:immunology; D004351:Drug Resistance / Q000235:genetics / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D005260:Female; D006825:Hybridomas / Q000276:immunology*; D007158:Immunologic Techniques; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011471:Prostatic Neoplasms / Q000235:genetics / Q000276:immunology; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D000939:Epitopes