Sugar-Binding Proteins from Fish: Selection of High Affinity “Lambodies” That Recognize Biomedically Relevant Glycans

Glycan-binding proteins are important for a wide variety
of basic
research and clinical applications, but proteins with high affinity
and selectivity for carbohydrates are difficult to obtain. Here we
describe a facile and cost-effective strategy to generate monoclonal
lamprey antibodies, called lambodies, that target glycan determinants.
We screened a library of yeast surface-displayed (YSD) lamprey variable
lymphocyte receptors (VLR) for clones that can selectively bind various
biomedically important glycotopes. These glycoconjugates included
tumor-associated carbohydrate antigens (Tn and TFα), Lewis antigens
(LeA and LeX), N-glycolylneuraminic acid, targets
of broadly neutralizing HIV antibodies (poly-Man9 and the HIV gp120),
and the glycoproteins asialo-ovine submaxillary mucin (aOSM) and asialo-human
glycophorin A (aGPA). We isolated clones that bind each of these targets
in a glycan-dependent manner and with very strong binding constants,
for example, 6.2 nM for Man9 and 44.7 nM for gp120, determined by
surface plasmon resonance (SPR). One particular lambody, VLRB.aGPA.23,
was shown by glycan array analysis to be selective for the blood group
H type 3 trisaccharide (BG-H3, Fucα1-2Galβ1-3GalNAcα),
aGPA, and TFα (Galβ1-3GalNAcα), with affinity constants
of 0.2, 1, and 8 nM, respectively. In human tissue microarrays this
lambody selectively detected cancer-associated carbohydrate antigens
in 14 different types of cancers. It stained 27% of non-small cell
lung cancer (NSCLC) samples in a pattern that correlated with poor
patient survival. Lambodies with exquisite affinity and selectivity
for glycans may find myriad uses in glycobiology and biomedical research.

糖结合蛋白（Glycan-binding proteins）在诸多基础研究与临床应用中具有关键价值，但获取对碳水化合物兼具高亲和力与选择性的蛋白质却颇具挑战。本文报道一种简便高效且成本可控的策略，用于制备靶向糖基决定簇的单克隆七鳃鳗抗体——此类抗体被命名为lambodies。我们针对可选择性结合多种具有生物医学重要性糖表位的克隆，筛选了酵母表面展示（Yeast Surface-Displayed, YSD）七鳃鳗可变淋巴细胞受体（Variable Lymphocyte Receptors, VLR）文库。所筛选的糖结合物涵盖肿瘤相关碳水化合物抗原（Tn与TFα）、路易斯抗原（LeA与LeX）、N-羟乙酰神经氨酸（N-glycolylneuraminic acid）、广谱中和HIV抗体靶向靶点（poly-Man9与HIV gp120），以及糖蛋白去唾液酸羊颌下腺黏蛋白（asialo-ovine submaxillary mucin, aOSM）与去唾液酸人类血型糖蛋白A（asialo-human glycophorin A, aGPA）。我们成功分离得到以糖依赖方式结合上述各靶点的克隆，且其结合亲和力极强：经表面等离子体共振（Surface Plasmon Resonance, SPR）测定，Man9的结合常数为6.2 nM，gp120为44.7 nM。其中一款特定的lambody（VLRB.aGPA.23）经糖芯片分析证实，可选择性识别血型H型3三糖（BG-H3, Fucα1-2Galβ1-3GalNAcα）、aGPA与TFα（Galβ1-3GalNAcα），其亲和常数分别为0.2 nM、1 nM与8 nM。在人类组织微阵列实验中，该lambody可选择性检测14种不同癌症中与癌症相关的碳水化合物抗原；其在27%的非小细胞肺癌（Non-Small Cell Lung Cancer, NSCLC）样本中呈阳性染色，且染色模式与患者不良预后显著相关。对糖类具备极致亲和力与选择性的lambodies，有望在糖生物学与生物医学研究中获得广泛应用。