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 breast adenocarcinoma cell line MCF-7 by immunoaffinity using the panreactive anti-class I monoclonal antibodies (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 approximately 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.
Agrawal
B
B
Biomira Inc., Edmonton, Alberta, Canada.
Reddish
M A
MA
Christian
B
B
VanHeele
A
A
Tang
L
L
Koganty
R R
RR
Longenecker
B M
BM
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000276:immunology; D015789:HLA-A2 Antigen / Q000737:chemistry; D015235:HLA-B Antigens / Q000737:chemistry; D059947:HLA-B40 Antigen; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D018396:Mucin-1 / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D015789:HLA-A2 Antigen; D015235:HLA-B Antigens; D059947:HLA-B40 Antigen; D015395:Histocompatibility Antigens Class I; D018396:Mucin-1; D010446:Peptide Fragments