Characterization of a new MUC1 monoclonal antibody (VU-2-G7) directed to the glycosylated PDTR sequence of MUC1.
A monoclonal antibody (MAb), VU-2-G7, was generated against a synthetic 60-mer MUC1 triple tandem repeat peptide with N-acetyl-galactosamine (GalNAc) O-linked to the threonine in the PDTR region of each repeat (3M GalNAc). VU-2-G7 and 8 MUC1 MAbs (VU-3-C6, VU-4-H5, 139H2, A76-A/C7, VU-12-E1, BCP9, MF11 and BW835) were tested against various glycosylated and nonglycosylated MUC1 tandem repeat peptides. VU-2-G7 showed strong reactivity with its immunogen, 3M GalNAc, and much lower reactivity with the nonglycosylated 60-mer MUC1 triple tandem repeat peptide. VU-2-G7 showed no reactivity with a 60-mer MUC1 triple tandem repeat peptide modified at the PDTR region or with a 60-mer MUC1 triple tandem repeat peptide with 3 GalNAc per repeat outside the PDTR region (9M GalNAc). In ELISA and flow cytometry, VU-2-G7 ubiquitously reacted with 4 MUC1-expressing breast cancer and 2 ovarian cancer cell lines and with a MUC1-gene-transfected Chinese hamster ovary cell line. The reactivity of VU-2-G7 was always higher than that of VU-4-H5, raised against a nonglycosylated 60-mer MUC1 triple tandem repeat peptide. Immunohistochemical staining of paraffin sections of breast and ovarian tumor tissues showed strong binding of VU-2-G7 predominantly at the cell membrane. The dominant epitope of VU-2-G7 is in the glycosylated PDTR motif of the MUC1 tandem repeat, and this epitope is abundantly present on the surface of tumor cell lines and breast and ovarian tumor tissues. Given the ubiquitously aberrant glycosylation of MUC1 in malignant cells, the production of MAbs against highly purified glycosylated MUC1 tandem repeat peptides may yield MAbs better suited for the immunotherapy of carcinomas than those available at the moment.
Ryuko
K
K
Department of Obstetrics and Gynecology, Shimane Medical University, Izumo, Japan.
Schol
D J
DJ
Snijdewint
F G
FG
von Mensdorff-Pouilly
S
S
Poort-Keesom
R J
RJ
Karuntu-Wanamarta
Y A
YA
Verstraeten
R A
RA
Miyazaki
K
K
Kenemans
P
P
Hilgers
J
J
D000116:Acetylgalactosamine / Q000276:immunology / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology* / Q000378:metabolism; D001943:Breast Neoplasms / Q000276:immunology; D016466:CHO Cells; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D005260:Female; D006031:Glycosylation; D006801:Humans; D007132:Immunoglobulin Isotypes / Q000276:immunology; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000276:immunology* / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology* / Q000378:metabolism; D020080:Tandem Repeat Sequences; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007132:Immunoglobulin Isotypes; C116055:MUC1 tandem repeat peptide; D018396:Mucin-1; D010446:Peptide Fragments; D000116:Acetylgalactosamine