Analysis and Modeling of the Variable Region of Camelid Single-Domain Antibodies

Abstract

Camelids have a special type of antibodies, known as heavy chain antibodies (HCAbs), that are devoid of classical antibody light chains. Relative to classical antibodies, camelid HCAbs (cAbs) have comparable immunogenicity, antigen recognition diversity and binding affinities, higher stability and solubility, and better manufacturability, making them promising candidates for alternate therapeutic scaffolds. Rational engineering of cAbs to improve therapeutic function requires knowledge of the differences of sequence and structural features between cAbs and classical antibodies. Here, amino acid sequences of 27 cAb variable regions (VHH) were aligned with the respective regions of 54 classical antibodies to detect amino acid differences, enabling automatic identification of cAb VHH complementarity determining regions (CDRs). CDR analysis revealed that the H1 often (and sometimes the H2) adopts diverse conformations not classifiable by established canonical rules. Also, while the cAb H3 is much longer than classical H3 loops, it often contains common structural motifs and sometimes a disulfide bond to the H1. Leveraging these observations, we created a Monte Carlo based cAb VHH structural modeling tool, where the CDR H1 and H2 loops exhibited a median root-mean-square-deviation (rmsd) to native of 3.1 and 1.5 Å respectively. The protocol generated 8-12, 14-16 and 16-24 residue H3 loops with a median rmsd to native of 5.7, 4.5 and 6.8 Å respectively. The large deviation of the predicted loops underscores the challenge in modeling such long loops. cAb VHH homology models can provide structural insights into interaction mechanisms to enable development of novel antibodies for therapeutic and biotechnological use.

Global analysis of VHHs framework regions with a structural alphabet

“…This representation is based mainly on the interaction between β-strands. Please note that it had been postulated that VHH have a CDR "4" [71]. The region between residues 71-78 (according to IMGT numbering [69][70]) is close to the other CDRs, leading to a larger paratope [72][73][74].…”

Novel sampling strategies and a coarse-grained score function for docking homomers, flexible heteromers, and oligosaccharides using Rosetta in CAPRI Rounds 37–45

“…Thus, we modeled the constant core of the nanobody and the H1 and H2 loops from available homologs and then we generated 1,000 models with different H3 loop conformations using RosettaAntibody. 36,37 In T123, PorMN-term in complex with nb02, the H3 loop was 12 residues long, whereas in T124, PorMC-term dimer in complex with nb130, the H3 was 21 residues long.…”

Minimalist Antibodies and Mimetics: An Update and Recent Applications

“…However, hcIgG can compensate for this smaller apparent binding surface by expansion of CDR loops. For example, in comparison with IgG, nanobodies typically have longer CDR3 loops (ranging from eight to 24 residues) than those found in mouse or human antibodies (nine and 12 residues, respectively) . The expanded CDR3 can dramatically increase the size of the paratope (the part of the protein that recognizes the epitope).…”

“…The expanded CDR3 can dramatically increase the size of the paratope (the part of the protein that recognizes the epitope). This extended display architecture is generally credited with allowing nanobodies to bind surfaces that challenge or evade IgG, such as deep clefts within enzymes …”