Molecular Immunology, Vol. 20, No.12, pp. 1369 1377,1983 0161-5890/83 $3.00+0.00 Printed in Great Britain. © 1983 Pergamon Press Ltd IDENTIFICATION AND ISOLATION OF A COMMON TUMOR-ASSOCIATED MOLECULE USING MONOCLONAL ANTIBODY* KOON YAN PAK, MAGDALENA BLASZCZYK, ZENON STEPLEWSKIt and HILARY KOPROWSKI Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, PA 19104, U.S.A. (First received 20 April 1983; accepted in revised form 27 June 1983) Abstraet--A monoclonal antibody, 16B-13, derived from the immunization of BALB/c mice with a lung tumor line, immunoprecipitates a common tumor-associated molecule with an apparent mol. wt of 37, from lactoperoxidase-iodinated lung carcinoma, colon carcinoma, gastric carcinoma, brest carcinoma, melanoma and lymphoma cells, but not from normal fibroblasts. Analysis by two-dimensional gel electrophoresis of the cell surface-labeled 16B-13 antigen from a colorectal and a melanoma cell line reveals four components with similar mol. wts but with different isoelectric points. The antigen purifi from a colorectal carcinoma cell line by immunoatfinity chromatography was shown to be a 37,000 m wt polypeptide similar to that obtained by the lactoperoxidase-labeling procedure. However, the purifi antigen from the melanoma cell line shows the presence of a 65,000 mol. wt polypeptide and the loss the 37,000 tool. wt component as detected by Coomassie blue staining and immunoprecipitation. INTRODUCTION Monoclonal antibodies have proven to be powerful tools for dissecting cell surface antigens [reviewed by Hellstrom et al. (1980), Kennett et al. (1980), Koprowski and Steplewski (1982), Kung and Gold- stein (1981) and Levy et al. (1979)]and receptors (Sutherland et al., 1981; Schneider et al., 1982). Examples of surface molecules purified so far by monoclonal antibody affinity chromatographyin- clude the l l0,000mol, wt plasma membrane gly- coprotein of NIH/3T3 cells (Hughes and August, 1982), p97 from human melanoma (Woodbury et al., 1982), H2 molecules from cells of different haplotypes (Stallcup et al., 1981), Fc receptor from macrophages (Mellman and Unkeless, 1980), the leukocyte- common antigen from rat thymocytes (Sunderland et al., 1979) and the transferrin receptor (Trowbridge and Omary,1981). Monoclonal antibody MBA 9812-16B-13 (16B-13), which binds in vitro to human lung adenocarcinomas, colon and breast carcinomas, but not to normal fibroblasts (Mazauric et al., 1982), was further stud- ied for its biological functions. Antibody 16B-13 (IgG2a isotype) is active in antibody-dependent cell- mediated cytotoxicity (ADCC) in vitro, and mediates tumor destruction in vivo (Herlyn andKoprowski, 1982). This antibody binds equally well to lung, colon, breast and melanoma cells, and the ~3q-labeled F(ab')2 fragments of 16B-13 selectively localize in these tumors in nude mice (Herlyn et al., 1983). However, in nude mice experiments, this antibody *This work was supported by Grants CA-10815, CA-21124, RA-05540 and the W. W. Smith Foundation. tTo whom correspondence should be addressed. does not inhibit the growth of WM9 melanoma cells, to which it binds perfectly well, although it is an effective inhibitor of growth for human lung, colon and breast adenocarcinomas (Herlyn and Ko- prowski, 1982). In our preliminary analysis of antigen immunoprecipitated by 16B-13 monoclonal anti- body, we detected a 37,000 mol. wt polypeptide on both lung and colon carcinomas (Mazauric et al., 1982). It became of interest to determine whether the same molecule(s) is present on WM9 cells in which 16B-13 antibody did not inhibit growth, and on cell lines in which growth in vivo was inhibited by the antibody. We report here the differences in molecules detected by 16B-I 3 antibody and their distribution in different tumor cell lines as determined by immu- noprecipitation. We also report the purification of these molecules by monoclonal antibody affinity chromatography. MATERIALS AND METHODS Monoclonal antibodies 16B-13 monoclonal antibody was the product of a cloned hybridoma generated by fusion of lympho- cytes from BALB/c mice immunized with bron- chogenic carcinoma MBA 9812 cells with a myeloma cell line as describedpreviously (Mazauric et al., 1982). Cell lines Bronchogenic carcinoma MBA 9812 was obtained from the Cell Culture Laboratory, Naval Bioscience Laboratory (Oakland, CA). Colorectal carcinoma SW403, SW948, SW1116 and SW1222 were obtained from Scott and White Clinic (Temple, TX). The other cell lines were established at the Wistar Institute. 1369