Characterization of H type 1 and type 1N-acetyllactosamine glycan epitopes on ovarian cancer specifically recognized by the anti-glycan monoclonal antibody mAb-A4.
Cancer-specific glycans of ovarian cancer are promising epitopes for targeting with monoclonal antibodies (mAb). Despite their potential, structural characterization of these glycan epitopes remains a significant challenge in mAb preclinical development. Our group generated the monoclonal antibody mAb-A4 against human embryonic stem cells (hESC), which also bound specifically toN-glycans present on 11 of 19 ovarian cancer (OC) and 8 of 14 breast cancer cell lines tested. Normal cell lines and tissue were unstained by mAb-A4. To characterize theN-linked glycan epitopes on OC cell lines targeted by mAb-A4, we used glycosidases, glycan microarray, siRNA, and advanced high sensitivity matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The mAb-A4 epitopes were found to be Fucα1-2Galβ1-3GlcNAcβ (H type 1) and Galβ1-3GlcNAcβ (type 1 LacNAc). These structures were found to be present on multiple proteins from hESC and OC. Importantly, endo-β-galactosidase coupled with MALDI-MS allowed these two epitopes, for the first time, to be directly identified on the polylactosamines ofN-glycans of SKOV3, IGROV1, OV90, and OVCA433. Furthermore, siRNA knockdown of B3GALT5 expression in SKOV3 demonstrated that mAb-A4 binding was dependent on B3GALT5, providing orthogonal evidence of the epitopes' structures. The recognition of oncofetal H type 1 and type 1 LacNAc on OC by mAb-A4 is a novel and promising way to target OC and supports the theory that cancer can acquire stem-like phenotypes. We propose that the orthogonal framework used in this work could be the basis for advancing anti-glycan mAb characterization.
From the Department of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom.|the Bioprocessing Technology Institute, Singapore 138668, Singapore.
Matthew
M
Choo
the Bioprocessing Technology Institute, Singapore 138668, Singapore.
Heng Liang
HL
Tan
the Bioprocessing Technology Institute, Singapore 138668, Singapore.
Vanessa
V
Ding
Shimadzu, Manchester M17 1GP, United Kingdom.
Roberto
R
Castangia
Shimadzu, Manchester M17 1GP, United Kingdom.
Omar
O
Belgacem
the Bioprocessing Technology Institute, Singapore 138668, Singapore.
Brian
B
Liau
the Institute for Glycomics, Griffith University, Southport, Queensland 4215, Australia, and.
Lauren
L
Hartley-Tassell
From the Department of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom.
Stuart M
SM
Haslam
From the Department of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom, a.dell@imperial.ac.uk.
Anne
A
Dell
the Bioprocessing Technology Institute, Singapore 138668, Singapore, andre_choo@bti.a-star.edu.sg.
Andre
A
Choo
D000606:Amino Sugars / Q000276:immunology*; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology*; D000912:Antibodies, Neoplasm / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000276:immunology*; D001943:Breast Neoplasms / Q000276:immunology; D045744:Cell Line, Tumor; D000939:Epitopes / Q000276:immunology*; D005260:Female; D006801:Humans; D014411:Neoplastic Stem Cells / Q000276:immunology*; D010051:Ovarian Neoplasms / Q000276:immunology*
D000606:Amino Sugars; D058846:Antibodies, Monoclonal, Murine-Derived; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D000939:Epitopes; C000458:N-acetyllactosamine
cancer biology; cancer therapy; embryonic stem cell; glycobiology; glycoconjugate; glycomics; mass spectrometry (MS); monoclonal antibody; ovarian cancer; small interfering RNA (siRNA)