Selective Recognition of Carbohydrate Antigens by GermlineAntibodies Isolated from AID Knockout Mice br

Germline antibodies, the initial set of antibodiesproduced by the immune system, are critical for host defense, andinformation about their binding properties can be useful fordesigning vaccines, understanding the origins of autoantibodies,and developing monoclonal antibodies. Numerous studies havefound that germline antibodies are polyreactive with malleable,flexible binding pockets. While insightful, it remains unclear howbroadly this model applies, as there are many families of antibodiesthat have not yet been studied. In addition, the methods used toobtain germline antibodies typically rely on assumptions and donot work well for many antibodies. Herein, we present a distinctapproach for isolating germline antibodies that involves immuniz-ing activation-induced cytidine deaminase (AID) knockout mice. This strategy amplifies antigen-specific B cells, but somatichypermutation does not occur because AID is absent. Using synthetic haptens, glycoproteins, and whole cells, we obtained germlineantibodies to an assortment of clinically important tumor-associated carbohydrate antigens, including Lewis Y, the Tn antigen, sialylLewis C, and Lewis X (CD15/SSEA-1). Through glycan microarray profiling and cell binding, we demonstrate that all but one ofthese germline antibodies had high selectivity for their glycan targets. Using molecular dynamics simulations, we provide insightsinto 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.