THE ANTI-MUC1 MONOCLONAL-ANTIBODY BCP8 CAN BE USED TO ISOLATE AND IDENTIFY PUTATIVE MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-I ASSOCIATED AMINO-ACID-SEQUENCES

MHC class I molecules were isolated from the MUC1-positive human breas t adenocarcinoma cell line MCF-7 by immunoaffinity using the panreacti ve anti-class I monoclonal antibody (MAb) W6/32, Acid-eluted peptides from the class I molecules were separated twice by high-performance li quid chromatography and tested for reactivity with the MAb BCP8, which reacts with the minimal MUC1 core peptide sequence PDTRPA, A peak wit h strong and specific BCP8 reactivity was found in fractions eluting a t 16.5-17.5 min. The protocol used for the MUC1(+) pancreatic adenocar cinoma cell line CAPAN-1 (HLA.A2) was to perform sequential affinity p urifications of class I molecules using MAb W6/32, followed by affinit y purification of HLA,AZ molecules by the HLA.A2.1-specific MAb, MA2.1 , and high-performance liquid chromatography fractionation of the acid -eluted material. A single peak with MAb BCP8 reactivity was noted at 18-19 min. The protocol for the MUC1+ breast adenocarcinoma cell line SKBr-3 (HA.A11,B40), which used A11- and B10-specific MAbs, also resul ted in the detection of BCP8-specific peaks at similar to 18-19 min. A preliminary mass spectral analysis of BCP8 affinity-purified class I associated material surprisingly revealed the presence of two 3-mer MU C1 amino acid sequences and one 6-mer sequence. A synthetic 9-mer MUC1 peptide, TSAPDTRPA, containing the isolated fragments was found to ca use strong class I up-regulation in T2 cells as well as to serve as an epitope for CTL generated in a primary in vitro immune response. Thes e studies suggest that MUC1-derived peptides are processed and present ed in the context of MHC class I molecules on the surface of tumor cel ls and support the use of MAb BCP8 to further define MHC class I assoc iated MUC1 motifs.