Summary The anti-breast carcinoma monoclonal antibody (MAb), NCRC-11 defines a polymorphic epithelial mucin (PEM) which is elevated in the circulation of advanced breast carcinoma patients. Here we describe the purification and partial characterisation of this component from patients' sera and its use in the production of a second generation MAb, C568 (IgM). Pooled sera was fractionated by immunoaffinity and size-exclusion chromatography and the purity of preparations assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Serum-derived PEM shows a similar pattern of electrophoretic mobility to PEM isolated from primary breast tumour tissue and migrates as several bands in 4% SDS polyacrylamide gels (Mr > 400,000). The epitope expression of PEMs isolated from either source is also similar, with both bearing topographically distinct determinants for several anti-mucin MAbs. The immunoreactivities of antibodies C568 and NCRC-11 were unaffected by boiling, reduction and alkylation, or by enzyme desialylation of PEM. Periodate oxidation and proteolytic digestion have suggested that the antigenic determinant for C568 is carbohydrate in nature whilst that of NCRC-II is peptidic. In accord with the mucinous nature of the molecule, serum-derived PEM is susceptible to reductive P-elimination, elutes in the void volume of a Sepharose CL-4B column and has a buoyant density of 1.45 g ml-'.
The in vitro anti-proliferative properties of various supernatants from MUC1-expressing cell lines and of purified preparations of MUC1 were evaluated. We have observed that supernatants from the MUC1-and MUC3-positive cell line T47D, but not from the MUC1- and MUC4-positive cell line MCF7, were able to inhibit proliferation of cells from various haematopoietic cell lines. Although the activity of T47D supernatants could be abrogated by immunodepletion of MUC1, immunopurified MUC1 from T47D was unable to inhibit cell proliferation. Significantly, supernatants from mouse 3T3 cells transfected with a secreted form of MUC1 or from BHK-21 cells infected with a recombinant vaccinia virus coding for the secreted form of MUC1, as well as preparations of purified MUC1 from bile or urine, were likewise unable to inhibit T cell proliferation. Surprisingly, a crude mixture of bile mucins had a suppressive effect on T cell growth. Our results suggest that other molecules, such as amino sugars or other mucins, which can associate with MUC1, are likely to be responsible for the observed anti-proliferative effects of T47D cells.
Summary Of 15 anti-CEA monoclonal antibodies, the first 8 were reactive only with CEA, while the remaining 7 antibodies reacted with epitopes commonly expressed on CEA and the normal cross-reacting antigen, NCA. Separate and distinct, conformation-dependent (i.e. susceptible to reduction and alkylation), CEA-associated epitopes were identified using antibodies 1, 2 and 3. Antibodies 4 to 7 defined a series of conformation-independent epitopes which were topographically closely related on the CEA molecule. Antibody number 8 reacted with a separate determinant found on CEA but not NCA, and this also was resistant to reduction and alkylation.Antibody number 9 defined an epitope which was commonly expressed on CEA and NCA. This epitope was conformation-dependent and was the most sensitive to NaIO4. The remaining antibodies, 10 to 15, which also reacted with CEA and NCA, defined an immunodominant region of these molecules since the 6 epitopes were clearly closely related, but not necessarily identical.The findings presented establish a rational basis for the selection of combinations of anti-CEA antibodies for diagnostic and therapeutic purposes.
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