Homology Modeling-Based in Silico Affinity Maturation Improves the Affinity of a Nanobody

Abstract

Affinity maturation and rational design have a raised importance in the application of nanobody (VHH), and its unique structure guaranteed these processes quickly done in vitro. An anti-CD47 nanobody, Nb02, was screened via a synthetic phage display library with 278 nM of KD value. In this study, a new strategy based on homology modeling and Rational Mutation Hotspots Design Protocol (RMHDP) was presented for building a fast and efficient platform for nanobody affinity maturation. A three-dimensional analytical structural model of Nb02 was constructed and then docked with the antigen, the CD47 extracellular domain (CD47ext). Mutants with high binding affinity are predicted by the scoring of nanobody-antigen complexes based on molecular dynamics trajectories and simulation. Ultimately, an improved mutant with an 87.4-fold affinity (3.2 nM) and 7.36 °C higher thermal stability was obtained. These findings might contribute to computational affinity maturation of nanobodies via homology modeling using the recent advancements in computational power. The add-in of aromatic residues which formed aromatic-aromatic interaction plays a pivotal role in affinity and thermostability improvement. In a word, the methods used in this study might provide a reference for rapid and efficient in vitro affinity maturation of nanobodies.

A sound strategy for homology modeling-based affinity maturation of a HIF-1α single-domain intrabody

“…Here, we designed a sound strategy for universal, efficient and convenient computer-aided rational design, which was used to significantly increase the binding affinity of a nanobody. Many successful computer-aided affinity maturation cases have been reported in the early stage, which is a highly effective strategy that can often lead from 10 to 100-fold increases in binding affinity (16,18,34). Herein, a three-dimensional structure developed by homology modeling and docking was used instead of crystallographic structures, which broadens application scenarios for our strategy.…”

“…Three-dimensional structures of the parent VHH212 were developed as described in ref. (18). The predicted models were assessed using web server SAVES v5.0 (http://services.mbi.ucla.edu/SAVES/), which contained Ramachandran plots, Verify3D and ERRAT plots.…”

“…In our previous study, the three-dimensional structure of a nanobody was constructed by homology modeling, while complex structures were gained by molecular docking (18). The outcome of such studies also helped us to gain better insight into antibody-antigen interactions and structures.…”

Recombinant expression of nanobodies and nanobody-derived immunoreagents

“…The following examples show some encouraging results of in silico protocols that follow different strategies to anticipate the potential effect of specific (multiple) single mutations. For instance, a successful computational affinity maturation strategy was based on the combination of Rational Mutation Hotspots Design Protocol and Assisted Design of Antibody and Protein Therapeutics [123] applied to the 3D homology model of the target VHH [124]. Molecular dynamics simulation provided single mutation candidates and the successive combinations of four single mutations resulted in a mutant with 87.4-fold affinity improvement.…”

“…More recently, their minimal dimension has been particularly appreciated by scientists looking for binders suitable to optimize the performance of super resolution microscopy [16][17][18] and to create tandem chimeric antigen receptors (CARs) that show higher target specificity due to the possibility of binding simultaneously multiple antigens [19]. Finally, their short sequence makes them the simplest antibody-derived candidate for rational mutagenesis and in silico improvement of biophysical features [20][21][22][23]. In parallel to the interest for their technical advantages, the attention for nanobodies has grown exponentially after the expiration of the patents which restricted their use.…”