Characterization of H type 1 and type 1 N-acetyllactosamine glycan epitopes on ovarian cancer specifically recognized by the anti-glycan monoclonal antibody mAb-A4.
Characterization of H type 1 and type 1 N-acetyllactosamine glycan epitopes on ovarian cancer specifically recognized by the anti-glycan monoclonal antibody mAb-A4.
复制标题
DOI：
10.1074/jbc.m116.768887
复制
发表时间：
2017-04-14
期刊：
影响因子：
--
通讯作者：
Choo A
中科院分区：
文献类型：
--
作者：
Choo M;Tan HL;Ding V;Castangia R;Belgacem O;Liau B;Hartley-Tassell L;Haslam SM;Dell A;Choo A
卵巢癌特异性聚糖是单克隆抗体（mAb）靶向的有前途的表位。尽管它们具有潜力，但这些聚糖表位的结构表征仍然是mAb临床前开发中的重大挑战。我们的小组产生了抗人胚胎干细胞（hESC）的单克隆抗体mAb-A4，它也特异性结合19个卵巢癌（OC）中的11个和14个乳腺癌细胞系中的8个上存在的N-聚糖。正常细胞系和组织未被mAb-A4染色。为了表征由mAb-A4靶向的OC细胞系上的N-连接聚糖表位，我们使用糖苷酶、聚糖微阵列、siRNA和先进的高灵敏度基质辅助激光解吸/电离质谱（MALDI-MS）。单抗A4的表位为Fucα1-2Galβ1-3GlcNAcβ（H1型）和Galβ1-3GlcNAcβ（LacNAc 1型）。发现这些结构存在于来自hESC和OC的多种蛋白质上。重要的是，内切-β-半乳糖苷酶与MALDI-MS偶联首次允许在SKOV 3、IGROV 1、OV 90和OVCA 433的N-聚糖的聚乳糖胺上直接鉴定这两个表位。此外，SKOV 3中B3 GALT 5表达的siRNA敲低证明mAb-A4结合依赖于B3 GALT 5，提供了表位结构的正交证据。mAb-A4识别OC上的癌胚H 1型和1型LacNAc是一种新颖且有前途的靶向OC的方法，并支持癌症可以获得干细胞样表型的理论。我们建议，在这项工作中使用的正交框架可以是推进抗聚糖mAb表征的基础。
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 to N-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 the N-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 of N-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.