Monoclonal antibodies (mAbs) are essential reagents for deciphering gene or protein function and have been a fruitful source of therapeutic and diagnostic agents. However, developing anticarbohydrate antibodies to target glycans for those purposes has been less successful because the molecular basis for glycan-mAb interactions is poorly understood relative to protein- or peptide-binding mAbs. Here, we report our investigation on glycan-mAb interactions by using the unique architectural scaffold of 2G12, an antibody that targets oligomannoses on the HIV-1 glycoprotein gp120, as the template for engineering highly specific mAbs to target glycans. We first analyzed 24 different X-ray structures of antiglycan mAbs from the Protein Data Bank to determine side chain amino acid distributions in of glycan-mAb interactions. We identified Tyr, Arg, Asn, Ser, Asp, and His as the six most prevalent residues in the glycan-mAb contacts. We then utilized this information to construct two phage display libraries (“Lib1” and “Lib2”) in which positions on the heavy chain variable domains of 2G12 were allowed to vary in restricted manner among Tyr, Asp, Ser, His, Asn, Thr, Ala and Pro to interrogate the minimal physicochemical requirements for oligomannose recognition. We analyzed the sequences of 39 variants from Lib1 and 14 variants from Lib2 following selection against gp120, the results showed that there is a high degree of malleability within the 2G12 for glycan recognitions. We further characterized five unique phage clones from both libraries that exhibited a gp120-specific binding profile. Expression of two of these variants as soluble mAbs indicated that, while specificity of gp120-binding was retained, the affinity of these mutants was significantly reduced relative to WT 2G12. Nonetheless, the results indicate these is some malleability in the identity of contact residues and provide a novel insight into the nature of glycan-antibody interactions and how they may differ from protein-antibody binding interactions.

单克隆抗体（mAb）是解密基因或蛋白质功能的重要试剂，并且已成为治疗和诊断试剂的丰硕成果。但是，针对这些目的开发针对目标聚糖的抗碳水化合物抗体的成效较差，因为相对于结合蛋白质或肽的单克隆抗体，人们对聚糖-mAb相互作用的分子基础了解甚少。在这里，我们通过使用独特的2G12结构支架（针对HIV-1糖蛋白gp120上的低聚甘露糖的抗体）作为工程化高特异性mAbs靶向模板的模板，报告了我们对聚糖-mAb相互作用的研究。我们首先分析了来自蛋白质数据库的抗聚糖mAb的24种不同的X射线结构，以确定聚糖-mAb相互作用中的侧链氨基酸分布。我们确定了Tyr，Arg，Asn，Ser，在聚糖-mAb接触中，Asp和His是六个最普遍的残基。然后，我们利用这些信息构建了两个噬菌体展示库（“ Lib1”和“ Lib2”），其中2G12重链可变域上的位置以有限的方式在Tyr，Asp，Ser，His，His，Asn，Thr之间变化。 ，Ala和Pro询问寡甘露糖识别的最低物理化学要求。我们针对gp120进行选择后，分析了来自Lib1的39个变体和来自Lib2的14个变体的序列，结果显示2G12内对于聚糖的识别具有很高的延展性。我们进一步表征了两个库的五个独特的噬菌体克隆，它们均表现出gp120特异性结合谱。这些变体中的两个作为可溶性mAb的表达表明，尽管保留了gp120结合的特异性，但相对于WT 2G12，这些突变体的亲和力显着降低。然而，结果表明这些在接触残基的身份上具有一定的可延展性，并为聚糖-抗体相互作用的性质以及它们与蛋白质-抗体结合相互作用的区别提供了新的见解。