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.

糖结合蛋白在众多基础研究和临床应用中扮演着至关重要的角色，然而，具有高亲和力和选择性结合碳水化合物的蛋白质却难以获得。本研究介绍了一种简便且成本效益高的策略，用于生成针对糖决定簇的单克隆鲟鱼抗体，称之为羊体抗体。我们筛选了一个酵母表面展示（YSD）的鲟鱼可变淋巴受体（VLR）库，以寻找能够选择性结合多种具有生物医学重要性的糖基化表位的克隆。这些糖基共轭物包括肿瘤相关碳水化合物抗原（Tn和TFα）、利氏抗原（LeA和LeX）、N-乙酰神经氨酸酸、广泛中和HIV抗体的靶点（多聚Man9和HIV gp120）以及无唾液酸羊亚唾液酸黏蛋白（aOSM）和无唾液酸人糖基磷脂酰肌醇A（aGPA）。我们分离出能够以糖依赖性方式以及极强的结合常数（例如，Man9的6.2 nM和gp120的44.7 nM，由表面等离子体共振（SPR）测定）结合这些靶点的克隆。其中一种特殊的羊体抗体，VLRB.aGPA.23，通过糖阵列分析显示出对血型H型3三糖（BG-H3，Fucα1-2Galβ1-3GalNAcα）、aGPA和TFα（Galβ1-3GalNAcα）的特异性，其亲和力常数分别为0.2、1和8 nM。在人类组织微阵列中，这种羊体抗体能够选择性地检测出14种不同癌症中的癌症相关碳水化合物抗原。在非小细胞肺癌（NSCLC）样本中，它对27%的样本进行了染色，其模式与患者预后不良相关。具有对糖具有极高亲和力和选择性的羊体抗体，在糖生物学和生物医学研究中可能具有广泛的应用前景。