Selective Recognition of Carbohydrate Antigens by Germline Antibodies Isolated from AID Knockout Mice

Germline
antibodies, the initial set of antibodies produced by
the immune system, are critical for host defense, and information
about their binding properties can be useful for designing vaccines,
understanding the origins of autoantibodies, and developing monoclonal
antibodies. Numerous studies have found that germline antibodies are
polyreactive with malleable, flexible binding pockets. While insightful,
it remains unclear how broadly this model applies, as there are many
families of antibodies that have not yet been studied. In addition,
the methods used to obtain germline antibodies typically rely on assumptions
and do not work well for many antibodies. Herein, we present a distinct
approach for isolating germline antibodies that involves immunizing
activation-induced cytidine deaminase (AID) knockout mice. This strategy
amplifies antigen-specific B cells, but somatic hypermutation does
not occur because AID is absent. Using synthetic haptens, glycoproteins,
and whole cells, we obtained germline antibodies to an assortment
of clinically important tumor-associated carbohydrate antigens, including
Lewis Y, the Tn antigen, sialyl Lewis C, and Lewis X (CD15/SSEA-1).
Through glycan microarray profiling and cell binding, we demonstrate
that all but one of these germline antibodies had high selectivity
for their glycan targets. Using molecular dynamics simulations, we
provide insights into the structural basis of glycan recognition.
The results have important implications for designing carbohydrate-based
vaccines, developing anti-glycan monoclonal antibodies, and understanding
antibody evolution within the immune system.

种系抗体（germline antibodies）是免疫系统产生的初始抗体库，在宿主防御中发挥关键作用；其结合特性相关信息可用于疫苗设计、自身抗体起源解析以及单克隆抗体的开发。多项研究表明，种系抗体具有多反应性，且结合口袋具备易变、灵活的结构特征。尽管该结论颇具洞见，但由于尚有大量抗体家族未被研究，该模型的适用范围仍有待明确。此外，当前获取种系抗体的实验方法通常依赖诸多假设，且对多数抗体而言效果欠佳。本研究提出一种全新的种系抗体分离方法：通过对活化诱导的胞苷脱氨酶（activation-induced cytidine deaminase, AID）基因敲除小鼠进行免疫接种。该策略可扩增抗原特异性B细胞，但由于AID缺失，体细胞高频突变不会发生。研究人员利用合成半抗原、糖蛋白及完整细胞，成功获得了针对一系列临床相关肿瘤相关糖类抗原的种系抗体，包括路易斯Y（Lewis Y）、Tn抗原、唾液酸化路易斯C（sialyl Lewis C）以及路易斯X（CD15/SSEA-1）。通过糖微阵列谱分析与细胞结合实验，本研究证实，除1株外，其余所有种系抗体对其糖类靶标均具有高选择性。借助分子动力学模拟，本研究进一步揭示了糖类识别的结构基础。本研究结果对于糖类疫苗设计、抗糖类单克隆抗体开发以及免疫系统内抗体进化机制的解析均具有重要意义。