Rational affinity enhancement of fragmented antibody by ligand-based affinity improvement approach

Antibody engineering is now a noteworthy area in biopharmaceuticals as the next generation of marketed antibodies is engineered antibodies such as affinity- or stability-improved antibodies, fragmented or fused antibodies, antibody drug conjugates (ADCs), and PEGylated antibody fragments. In the current study, affinity enhancement of Nb against PlGF was performed by an in silico affinity maturation and molecular dynamics (MD) simulation. First, 300 single-point mutants were designed by identifying the residues involved in interaction with PlGF and different energy distributions. An energy based screening was performed to select best single-point mutants. Additionally, one variant containing two mutations was designed based on the selected single-point mutants. Finally, mutants-PlGF complexes were analyzed in details by all atom MD simulation. Trajectory analysis revealed that in both single (L112H, S31D, A97K, and R45E) and double (S31D & R45E) mutants, the free binding energies and the stability of complexes were significantly improved. The highest increment in affinity was observed for S31D mutant due to substantial increase in polar and electrostatic interactions. The secondary structure of Nb was intact in all variants and a shrinkage of PlGF over Nb was observed in all mutant-PlGF complexes during simulation. In addition, contact area and hydrogen-bond analysis as well as distance measurement in mutants-PlGF complexes also confirmed the affinity enhancement of variants relative to the native form. Our study showed that ligand-based affinity improvement could be considered as a promising approach for designing high affinity fragmented antibodies.