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

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.