Conserved amino acid networks involved in antibody variable domain interactions

Abstract

Engineered antibodies are a large and growing class of protein therapeutics comprising both marketed products and many molecules in clinical trials in various disease indications. We investigated naturally conserved networks of amino acids that support antibody V(H) and V(L) function, with the goal of generating information to assist in the engineering of robust antibody or antibody-like therapeutics. We generated a large and diverse sequence alignment of V-class Ig-folds, of which V(H) and V(L) domains are family members. To identify conserved amino acid networks, covariations between residues at all possible position pairs were quantified as correlation coefficients (phi-values). We provide rosters of the key conserved amino acid pairs in antibody V(H) and V(L) domains, for reference and use by the antibody research community. The majority of the most strongly conserved amino acid pairs in V(H) and V(L) are at or adjacent to the V(H)-V(L) interface suggesting that the ability to heterodimerize is a constraining feature of antibody evolution. For the V(H) domain, but not the V(L) domain, residue pairs at the variable-constant domain interface (V(H)-C(H)1 interface) are also strongly conserved. The same network of conserved V(H) positions involved in interactions with both the V(L) and C(H)1 domains is found in camelid V(HH) domains, which have evolved to lack interactions with V(L) and C(H)1 domains in their mature structures; however, the amino acids at these positions are different, reflecting their different function. Overall, the data describe naturally occurring amino acid networks in antibody Fv regions that can be referenced when designing antibodies or antibody-like fragments with the goal of improving their biophysical properties.

Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires

“…Conserved V H :V L interactions at FR positions are critical determinants of variable domain stability (47)(48)(49)(50). To explore the relationship between V-gene use and antibody structural similarity within FRs, we calculated the rmsd of antibody models to each other over the FR1-3 backbone within each naive or antigen-experienced computationally predicted antibody model repertoire (V H : 8-25, 36-51, and 57-94 and V L : 10-23, 35-49, and 57-88 backbone residue atoms by the Chothia definition; see SI Appendix, SI Methods).…”

Stable IgG-like Bispecific Antibodies Directed toward the Type I Insulin-like Growth Factor Receptor Demonstrate Enhanced Ligand Blockade and Anti-tumor Activity

“…Stabilizing designs that served as the basis for the scFv libraries were generated using both sequence (28,29) and structure-based methods (30). The expressed libraries were challenged at various temperatures for 1 h prior to assaying scFv activity by DELFIA (27).…”

Rational engineering of antibody therapeutics targeting multiple oncogene pathways

“…This molecule comprised an IgG antibody framework directed at GFR1 and two C-terminally fused scFv modules directed at GFR2, but was not suitable for downstream development, as it contained unstable scFv modules that did not have sufficient intrinsic stability. This phenomenon is well-understood, 72 and engineering of scFv modules for stability by a variety of techniques including linker optimization, 12 disulfide engineering, 73 targeted mutagenesis, 74 co-variation analysis, 75 loop grafting on a stable framework, 76 structure-guided design, 77 focused design, 78 and phage display has been described. 79 To combine optimization of the affinity and stability of the scFv modules within a single campaign, we used a combination of structure-guided design, yeast surface display and micro-scale biophysical characterization.…”