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In human intestinal malignancy, alterations occur in the expression of mucins defined by the MUC-2 gene. These changes include the unmasking of epitopes in the mucin protein core. In order to probe these modifications associated with mucins of the malignant phenotype, a monoclonal antibody (MAb) was developed against synthetic peptide with a sequence based upon that of the protein core of the MUC-2 mucin. The antibody (designated 996) was shown to recognize a high-molecular-weight glycoprotein from colonic carcinoma tissue. The material reacted uniformly with Concanavalin A but variably with other lectins, indicating heterogeneity in the associated oligosaccharide side chains. The protein core was accessible both to 996 antibody binding and to degradation with proteases. Immunization with the affinity-purified mucin-like material elicited antibodies reactive with both the immunogen and the synthetic peptides, confirming the immunogenic character of protein-core determinants. Epitope mapping studies, using synthetic peptides in solution and synthetic peptides tethered to the heads of plastic pins, indicated that the minimum epitope for the 996 antibody is a tetramer of T G T Q. Antibody interaction with the glutamine (Q) residue was determined to be of major importance in the antigen-antibody reaction. The findings illustrate the characterization of an anti-peptide antibody which may be used to probe alterations in MUC-2 mucin expression associated with human intestinal malignant disease.

Summary Recombinant single-chain fragments (scFv) of the murine anti-MUC1 monoclonal antibody C595 have been produced using the original hybridoma cells as a source of variable heavy (VH)-and variable light (VL)-chain-encoding antibody genes. The use of the polymerase chain reaction (PCR), bacteriophage (phage) display technology and gene expression systems in E. coli has led to the production of soluble C595 scFv. The scFv has been purified from the bacterial supernatant by peptide epitope affinity chromatography, leading to the recovery of immunoreactive C595 scFv, which was similar in activity to the C595 parent antibody. Analysis by DNA sequencing, SDS-PAGE and Western blotting has demonstrated the integrity of the scFv, while ELISA, FACScan analysis, fluorescence quenching, quantitative immunoreactivity experiments and immunohistochemistry confirm that the activity of the scFv compares favourably with that of the parent antibody. The retention of binding activity to MUC1 antigen on human bladder and breast carcinoma tissue specimens illustrates the potential application of this novel product as an immunodiagnostic and immunotherapeutic reagent.

A murine anti-(human gastric carcinoma) monoclonal antibody, GL-013 (IgG1), which reacts with a high-molecular-mass glycoprotein from colorectal tumour tissue [Yang and Price (1989) Anticancer Res 9: 1707], was examined for reactivity against a panel of purified and partially purified antigens associated with tumours of the gastrointestinal tract. These included carcinoembryonic antigen (CEA), normal cross-reacting antigen, Y-hapten glycoproteins, and perchloric acid extracts and glycolipid preparations from colorectal tumours. While the GL-013 antibody failed to bind to these antigens, it was found to react strongly with synthetic peptides with sequences based upon that reported for the protein core of a human gastrointestinal mucin [Barnd et al. (1989) Proc Natl Acad Sci USA 86: 7159; Gum et al. (1989) J Biol Chem 264: 6480]. In control tests, a series of other anti-(colorectal tumour) antibodies (IgG1 and IgG3), with broad reactivity towards gastrointestinal carcinomas, as well as an anti-CEA antibody, (IgG1) failed to react with the synthetic peptides. It is concluded that the anti-(gastric carcinoma) monoclonal antibody GL-013 binds to a threonine-rich peptide epitope expressed within the protein core of gastrointestinal mucins.