Human single-chain Fv antibodies to MUC1 core peptide selected from phage display libraries recognize unique epitopes and predominantly bind adenocarcinoma.
The tumor-associated antigen MUC1 is overexpressed and underglycosylated in human adenocarcinomas of diverse origins, such as breast, ovary, and colon. We isolated and describe five human single-chain (sc) Fv antibodies specific for the MUC1 variable number of tandem repeats region isolated by in vitro selection from a large naive phage antibody library containing over 6 x 10(9) different scFv antibodies. A synthetic biotinylated 100-mer peptide corresponding to five tandem repeats of the MUC1 peptide core was used for selection. Two of the antibodies were highly specific for MUC1 as judged by ELISA and flow cytometry. In immunohistochemistry, antibody clone 10A stained MUC1 in the cytoplasm and membrane of adenocarcinoma cells of breast and ovary, whereas in normal epithelium, only cytoplasmic or no staining was observed. With antibody clone 10B, staining was less pronounced and was not always membrane associated in adenocarcinoma. Determination of the fine specificity of 10A and 10B using a novel "indirect epitope fingerprinting" ELISA showed that both antibodies recognize unique epitopes that have not been described for hybridoma-derived anti-mucin antibodies of mouse origin. The selected human antibodies, like many of the murine MUC1 antibodies, recognize epitopes on the protein core of MUC1 that are abundantly present in the underglycosylated form of cell surface mucin on adenocarcinoma. The best human scFv, clone 10A, appears to distinguish normal cells from adenocarcinoma cells, which makes it an attractive candidate for use in antibody-based tumor targeting.
University Hospital Maastricht and Maastricht University, Centre for Engineering and Selection of Antibody Molecules Euregio at the Department of Pathology, The Netherlands.
P
P
Henderikx
M
M
Kandilogiannaki
C
C
Petrarca
S
S
von Mensdorff-Pouilly
J H
JH
Hilgers
E
E
Krambovitis
J W
JW
Arends
H R
HR
Hoogenboom
D000230:Adenocarcinoma / Q000737:chemistry* / Q000473:pathology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001666:Binding Sites, Antibody; D001943:Breast Neoplasms / Q000737:chemistry / Q000473:pathology; D003110:Colonic Neoplasms / Q000737:chemistry / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000737:chemistry / Q000166:cytology; D000939:Epitopes / Q000032:analysis*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D007150:Immunohistochemistry; D051379:Mice; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D010051:Ovarian Neoplasms / Q000737:chemistry / Q000473:pathology; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D019151:Peptide Library; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D018396:Mucin-1; D010446:Peptide Fragments; D019151:Peptide Library