Stability convergence in natural antibodies with ultra-long hypervariable loops.

Antibodies bind to antigens with hypervariable loops called complementarity-determining regions (CDRs). In contrast to conventional antibodies, a subset of bovine antibodies has an ultra-long CDR (ulCDR) composed of up to 70 residues folded as a stalk and knob. The fundamental principles of how these antibodies maintain their structure and stability remain enigmatic. Here, we investigated how different natural ulCDRs affect antibody structure, stability and function. To this end, we swapped diverse ulCDRs onto the same antibody scaffold. All ulCDR-swap variants exhibit nearly identical secondary structure fingerprints and remarkably similar thermal stability. In addition, specificity and high-affinity binding to the antigens are maintained. Hydrogen-deuterium exchange and molecular dynamics simulations suggest small differences between the variants arising from changed interactions between different stalks and the underlying scaffold. Overall, we reveal principles of grafting natural ulCDRs onto a common Fab scaffold, which have implications for antibody design for biomedical applications.