Context‐dependent mutations predominate in an engineered high‐affinity single chain antibody fragment

Abstract

A mutational analysis of the femtomolar-affinity anti-fluorescein antibody 4M5.3, compared to its wild-type progenitor, 4-4-20, indicates both context-dependent and -independent mutations are responsible for the 1800-fold affinity improvement. 4M5.3 was engineered from 4-4-20 by directed evolution and contains 14 mutations. The seven mutations identified as present in each of 10 final round affinity maturation clones were studied here. Affinities of the 4-4-20 single mutant addition and 4M5.3 single site reversion mutants were compared. These experiments identified four mutations, of these seven, that were context-dependent in their contribution to higher affinity. A simplified mutant containing only these seven mutations was created to analyze complete double mutant cycles of selected sets of mutations. Specific mutational sets studied included the ligand contact mutations, the heavy chain CDR3 mutations, the heavy chain CDR3 mutations plus the neighboring residue at site H108, and the early and late acquired mutations on the directed evolution pathway. The heavy chain CDR3 mutational set and the ligand-contacting mutations were shown to provide -1.4 and -2.0 kcal/mol, respectively, of the total -3.5 kcal/mol change in free energy of binding of the seven-site consensus mutant. The mutations acquired late in the directed evolution rounds provided much of the change in free energy without the earlier acquired mutations (-3.1 kcal/mol of the total -3.5 kcal/mol). Prior structural data and electrostatic calculations presented several hypotheses for the higher affinity contributions, some of which are supported by these mutational data.

Evolution of an Interloop Disulfide Bond in High-Affinity Antibody Mimics Based on Fibronectin Type III Domain and Selected by Yeast Surface Display: Molecular Convergence with Single-Domain Camelid and Shark Antibodies

“…Library BC7 was constructed by randomizing the 7 residues (23)(24)(25)(26)(27)(28)(29) in loop BC; library FG7 was constructed by randomizing 7 residues (77-83) in loop FG; and libraries 2L14 and BF14 were constructed by simultaneously randomizing the 14 residues in loops BC and FG (23-29 and 77-83). The only difference between libraries 2L14 and BF14 is that the complexity of library BF14 is 10 times higher than the complexity of library 2L14.…”

Computational design of antibody-affinity improvement beyond in vivo maturation

“…Our designed single mutations in 4-4-20 revealed opportunities for improvement based on computed electrostatics at nine positions. Two of the predicted mutations, H31 Asp-to-His and H101 Ser-to-Ala, have already been shown to improve binding affinity as single mutations in 4M5.3 22 . Interestingly, the design missed the H102 Tyr-to-Ser mutation in 4M5.3 due to the rigid backbone constraint.…”

Physicochemical Evaluation of Biofield Treated Peptone and Malmgren Modified Terrestrial Orchid Medium

“…It was previously reported that proteins have a stronger tendency of forming aggregate. They can form self-aggregates in a number of ways such as the formation of structural complexes, and multimeric native states with metal complexation [30][31][32]. These proteins have sufficiently strong inter-protein interactions which induce formation of bigger aggregates [33].…”