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The Anti-MUC1 Monoclonal Antibody BCP8 Can Be Used to Isolate and Identify Putative Major Histocompatibility Complex Class I Associated Amino Acid Sequences

Biomira Inc., Edmonton, Alberta, Canada, T6N 1H1 [B. A., M. A. R., B. C., A. V., R. R. K., B. M. L.], and University of Alberta, Department of Chemistry, Edmonton, Alberta, Canada, T6G 2G2 [L. T.]

MHC class I molecules were isolated from the MUC1-positive human breast adenocarcinoma cell line MCF-7 by immunoaffinity using the panreactive anti-class I monoclonal antibody (MAb) W6/32. Acid-eluted peptides from the class I molecules were separated twice by high-performance liquid chromatography and tested for reactivity with the MAb BCP8, which reacts with the minimal MUC1 core peptide sequence PDTRPA. A peak with strong and specific BCP8 reactivity was found in fractions eluting at 16.5–17.5 min. The protocol used for the MUC1⁺ pancreatic adenocarcinoma cell line CAPAN-1 (HLA.A2) was to perform sequential affinity purifications of class I molecules using MAb W6/32, followed by affinity purification of HLA.A2 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 (HLA.A11,B40), which used A11- and B40-specific MAbs, also resulted in the detection of BCP8-specific peaks at 18–19 min. A preliminary mass spectral analysis of BCP8 affinity-purified class I associated material surprisingly revealed the presence of two 3-mer MUC1 amino acid sequences and one 6-mer sequence. A synthetic 9-mer MUC1 peptide, TSAPDTRPA, containing the isolated fragments was found to cause 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. These studies suggest that MUC1-derived peptides are processed and presented in the context of MHC class I molecules on the surface of tumor cells and support the use of MAb BCP8 to further define MHC class I associated MUC1 motifs.

¹ Present address: ID Vaccine, 19204 North Creek Parkway, #100, Bothell, WA 98011.

² Deceased and to whom this paper is dedicated.

³ To whom requests for reprints should be addressed, at Biomira Inc., 2011-94 Street, Edmonton, Alberta, Canada T6N 1H1. Phone: (403) 450-3761; Fax: (403) 988-5936; E-mail: MLongenecker@Biomira.com.

Received 7/10/98. Accepted 9/16/98.

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