Unique Antibody Structural Variegation Mechanisms

The cow immune system produces a subset of antibodies with extremely long CDR H3 regions. These CDR H3s can be up to 70 amino acids in length and protrude far from the typical antibody surface, utilizing a -ribbon “stalk” that supports a disulfide bonded “knob” domain that is similar in size and shape to cyclotides and knottins. These antibodies are encoded by specific VH, DH, and JH gene segments, and undergo an atypical VDJ recombination event which inserts a specific A-rich sequence between VH and DH. The molecular mechanism behind this phenomenon is currently unknown. The ultralong CDR H3 antibodies bind to antigen through their knob domain, which we have shown can be produced independently of the antibody. Thus, the cow CDR H3 knob is the smallest known antibody fragment at 1/3rd the size of camelid VHH “nanobodies”. In this proposal we aim to understand the molecular mechanism behind the unusual V(D)J recombination event that produces ultralong CDR H3 genes. The RAG-1/RAG-2 recombinase is known to have transposase activity in vitro, however this activity has not been observed in normal physiology. We will evaluate the source of the unusual A-rich insertional event by bioinformatic, molecular, and genetic analysis. Understanding this molecular mechanism in cows may lead to understanding how rare but important broadly neutralizing anti-viral antibodies with long CDR H3 form in humans, which would be useful information for vaccine approaches attempting to induce these antibodies. Our second major objective is to understand the molecular functions of CDR H3 knobs at a structural and physiological level. We will employ crystallography, electron microscopy, and nuclear magnetic resonance spectroscopy to understand knob structure and antigen binding, and to ascertain whether the free knob structure is identical to its parental antibody. Further, we will investigate the stability, bioavailability and pharmacokinetics of knobs in vivo in mice. Knobs are similar in structure to cyclotides, which, remarkably, are orally bioavailable. If knobs are similarly bioavailable, they could become a novel protein-based orally available class of therapeutic. These studies, therefore, will lay the scientific foundations for knobs as a new modality of research tools, as well as diagnostic and therapeutic agents. Project Number: 1R01AI189981-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Vaughn Smider | Institution: APPLIED BIOMEDICAL SCIENCE INSTITUTE, SAN DIEGO, CA | Award Amount: $976,318 | Activity Code: R01 | Study Section: Macromolecular Structure and Function B Study Section[MSFB] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI18998101A1