H. Durbin

Abstract The monoclonal antibodies HMFG-1 and HMFG-21 are produced by hybridomas derived by fusing spleen cells of mice immunized with delipidated preparations of the human milk fat globule. Enzyme-linked immunosorbent (ELISA) assays of Western blots prepared from gels separating components of the milk fat globule showed that both antibodies recognize determinants fouinn dh igh molecular weight components (>400K). Lectin-blocking experiments indicated that both antibodies recognizeo ligosaccharide sequences containing galactose, N-acetyl glucosamine, and, possibly, N-acetyl galactosamine. However, the determinant recognized by HMFG-1 is different from the determinant recognbizye dH MFG-2,w hich may contain or be adjacent to a nonterminal sialic acid residue. The expression of these antigenic determinants by normal mammary epithelial cells cultured from milk (HumE) and by a breast cancer cell line (T47D), was studied. In a live cell, radioimmune assay lower levels of HMFG-1 than HMFG-2 were required for effective binding to HumE cells, whereas HMFG-2 bound to T47D cells at lower concentrations than HMFG-1. Kinetic experiments using 125I-Iabeled antibodies showed that high affinity binding sites for both antibodies are present on HumE cells, but the number of sites binding HMFG-1 is greater than the number of sites binding HMFG-2. Thkei netics of dissociation of labeled antibodies from T47D cells sug gested the presenceo f several sites with different affinities for each antibody. Only in the case of HMFG-2 did some of these sites show a high affinity for the antibody. Western blots of gel-separated components of cell lysates when reacted in an ELISA assay showed that HMFG- 1r eacted with high molecular weight compoinn eHnutms E cells similar to those seen in the HMFG preparation. In contrast, high affinity sites for HMFG-2 were expressed by HumE and T47D on several components of lower molecular weight. All other breast carcinoma lines examined and metastatic cells from two breast cancer patients expressed high affinity binding sites for HMFG-2 on components of varying size (80K to >400K). Only two of five cell lines and cancer cells from one of the two patients expressed high affinity sites for HMFG-1 and these were found on high molecular weight glycoprotein(s3 00-400K). The results are consistent with the interpretation that the HMFG-1 determinant is present in large glycoprotein molecules with complex carbohydrate side chains, and the HMFG-2 determinant is more commonly found in smaller molecules where glycosylation may be incomplete and sialic acid residues more exposed. The preferential binding of HMFG-2 to some breast cancers may therefore reflect a changed glycosylation patteirnn these cells.

Tumor-associated monoclonal antibodies were radiolabeled with 125I and 131I and given i.v. in pairs to 19 patients 1-26 days prior to surgical excision of primary and metastatic breast, ovarian, and gastrointestinal tumors. For individual patients each monoclonal antibody was designated as specific or nonspecific according to prior immunoperoxidase staining results on the appropriate target neoplastic tissues. Quantitation of antibody uptake was performed on resected normal and neoplastic tissues. Although good tumor:non-tumor ratios were obtained with the specific antibodies (maximal tumor:blood ratio, 35.8:1 at 12 days postadministration), the absolute amount of radiolabel detected in tumors was small (mean value of 0.015% of total injected amount per g of tumor occurring 1 day postadministration). Furthermore, both specific and nonspecific antibodies accumulated in normal lymph nodes to a significant extent (mean value of 0.0026% of total injected amount per g of tissue occurring 1 day postadministration). Knowledge of such data is essential prior to considering therapeutic uses of radiolabeled monoclonal antibodies.