Optimisation of human V_H domain antibodies specific to Mycobacterium tuberculosis heat shock protein (HSP16.3)

Mycobacterium tuberculosis (Mtb) 16.3 kDa heat shock protein 16.3 (HSP16.3) is a latency-associated antigen that can be targeted for latent tuberculosis (TB) diagnostic and therapeutic development. We have previously developed human V_H domain antibodies (dAbs; clone E3 and F1) specific against HSP16.3. In this work, we applied computational methods to optimise and design the antibodies in order to improve the binding affinity with HSP16.3. The V_H domain antibodies were first docked to the dimer form of HSP16.3 and further sampled using molecular dynamics simulation. The calculated binding free energy of the HSP16.3-dAb complexes showed non-polar interactions were responsible for the antigen-antibody association. Per-residue free energy decomposition and computational alanine scanning have identified one hotspot residue for E3 (Y391) and 4 hotspot residues for F1 (M394, Y396, R397 and M398). These hotspot residues were then mutated and evaluated by binding free energy calculations. Phage ELISA assay was carried out on the potential mutants (E3_Y391W, F1_M394E, F1_R397N and F1_M398Y). The experimental assay showed improved binding affinities of E3_Y391W and F1_M394E against HSP16.3 compared with the wild type E3 and F1. This case study has thus showed in silico methods are able to assist in optimisation or improvement of antibody-antigen binding.