Abstract Antibodies are extremely important as the primary mediators of the immune response to infectious agents and vaccines, but also as recombinant molecules used as therapeutics, diagnostics, and research reagents. Thus, understanding the mechanisms by which antibodies form, bind, and neutralize their targets is very important in multiple biomedical areas. Most vertebrate antibody repertoires form their diversity through V(D)J recombination, where combinatorial rearrangement of V, D, and J genes form a vast repertoire where the complementarity determining regions (CDRs) form a relatively flat binding surface for interaction with antigen. Remarkably, cows appear to have a different mechanism for generating diversity and binding antigen; cow antibodies have particularly long CDR H3 regions, a subset of which can be over 70 amino acids long, which form -ribbon “stalk” and disulfide-bonded “knob” minidomains that protrude far from the typical antibody surface. Remarkably, antibodies from this repertoire can potently and broadly neutralize HIV, whereas normal human or mouse antibody repertoires cannot. Therefore, they have unique abilities to bind and neutralize particularly challenging antigens. Here we propose studies to understand in molecular detail the genetic mechanisms underlying formation of these antibodies, the binding properties of the stalk and knob minidomains, and the unique potential of these antibodes to bind bivalently and bispecifically. These studies will provide insight into mechanisms of viral neutralization generally, and particularly HIV neutralization. Additionally, given the potential of these antibodies to bind recessed epitopes, we will generate additional antibodies against particularly challenging antigens like enzymatic active sites and ion channels. We will employ mutagenesis, binding, and structural methods, as well as cellular assays to evaluate the unique properties of these antibodies. Understanding this novel class of antibodies in detail could provide fundamental insights needed for effective vaccine design as well as discovery and engineering of antibodies against some of the most challenging targets in biomedicine. Similarly, our unique antibodies discovered and characterized in this proposal could eventually become therapeutic candidates themselves.