Highly specific characterization of tyrosine phosphoproteins in tumor cells based on monoclonal antibodies defined by conjugated phosphotyramine.

Phosphotyrosine proteins of four different tumor cell lines were characterized by monoclonal antibodies exhibiting high affinity binding to phosphotyrosine. For the preparation of the antibody-producing mouse hybridoma cell lines we used a novel kind of immunizing antigen with phosphotyramine conjugated directly to carboxylic groups of carrier proteins. Screening for high affinity binding antibodies was based on their selective reactivity in immunoprecipitation, affinity chromatography and immunofluorescence. By means of affinity chromatography we established a one-step purification of phosphotyrosine proteins yielding substantial quantities of highly pure 170kDa EGF receptor from A431 tumor cells, 210kDa bcr-abl gene product from K562 tumor cells and 120 kDa transforming protein of the Abelson murine leukemia virus from TK tumor cells. Cross-reactivity with phosphoproteins containing no phosphotyrosine was not observed.

Monoclonal antibodies produced by in vitro immunization with sera of human-xenotransplant-bearing mice.

In vitro immunization procedures, using sera of athymic mice bearing human WOC ovarian tumors or CM III mammary tumors as immunizing antigen, induced a highly efficient formation of mABs (44% of antibody-producing clones) reacting with human ovarian and/or mammary tumor cells. More than half of these mABs showed cross-reactivity with mouse cell lines. Immunogenicity of normal mouse components in the sera from tumor bearers can be excluded since control immunization with sera of normal athymic mice yielded no mABs reacting with mouse or human cell lines. Furthermore, immunization with sera from tumor bearers did not induce mABs only reacting with mouse cells since 20% of the antibody-producing clones showed an exclusive specificity for the human tumor cells. On the basis of these results we concluded that the human-mouse cross-reacting mABs were induced by circulating human TAA with epitopes shared by mouse cellular components.