Mechanistic principles of an ultra-long bovine CDR reveal strategies for antibody design

Hristo L Svilenov,Ulrike Protzer,Julia Sacherl,Johannes Buchner,Martin Zacharias

doi:10.1038/s41467-021-27103-z

Hristo L Svilenov, Ulrike Protzer + Show 3 more

Open Access

https://doi.org/10.1038/s41467-021-27103-z

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Abstract

Antibodies bind antigens via flexible loops called complementarity-determining regions (CDRs). These are usually 6-20 residues long. However, some bovine antibodies have ultra-long CDRs comprising more than 50 residues organized in a stalk and a disulfide-rich knob. The design features of this structural unit and its influence on antibody stability remained enigmatic. Here, we show that the stalk length is critical for the folding and stability of antibodies with an ultra-long CDR and that the disulfide bonds in the knob do not contribute to stability; they are important for organizing the antigen-binding knob structure. The bovine ultra-long CDR can be integrated into human antibody scaffolds. Furthermore, mini-domains from de novo design can be reformatted as ultra-long CDRs to create unique antibody-based proteins neutralizing SARS-CoV-2 and the Alpha variant of concern with high efficiency. Our findings reveal basic design principles of antibody structure and open new avenues for protein engineering.

Highlights

Antibodies bind antigens via flexible loops called complementarity-determining regions (CDRs)
The ultra-long CDRs are made of two distinct structures – a β-ribbon “stalk” which protrudes from the HC and a “knob” that sits atop of the “stalk”, giving the entire CDR-H3 a characteristic mushroom shape[13]
Our study shows that the stalk provides stability and that the disulfide bonds provide conformational constraints required for antigen binding

Summary

Introduction

Antibodies bind antigens via flexible loops called complementarity-determining regions (CDRs) We show that the stalk length is critical for the folding and stability of antibodies with an ultra-long CDR and that the disulfide bonds in the knob do not contribute to stability; they are important for organizing the antigen-binding knob structure. The antigen-binding site is formed by segments in the variable domains of both the heavy (VH) and the light chains (VL), the so-called complementarity-determining regions (CDRs) Our study shows that the stalk provides stability and that the disulfide bonds provide conformational constraints required for antigen binding This information allowed us to create human antibodies with ultra-long CDR-H3s comprising a naturally occurring bovine knob or a target-binding mini-domain from de novo design

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