Antibody humanization by molecular dynamics simulations—in‐silico guided selection of critical backmutations

Abstract

Monoclonal antibodies represent the fastest growing class of biotherapeutic proteins. However, as they are often initially derived from rodent organisms, there is a severe risk of immunogenic reactions, hampering their applicability. The humanization of these antibodies remains a challenging task in the context of rational drug design. “Superhumanization” describes the direct transfer of the complementarity determining regions to a human germline framework, but this humanization approach often results in loss of binding affinity. In this study, we present a new approach for predicting promising backmutation sites using molecular dynamics simulations of the model antibody Ab2/3H6. The simulation method was developed in close conjunction with novel specificity experiments. Binding properties of mAb variants were evaluated directly from crude supernatants and confirmed using established binding affinity assays for purified antibodies. Our approach provides access to the dynamical features of the actual binding sites of an antibody, based solely on the antibody sequence. Thus we do not need structural data on the antibody–antigen complex and circumvent cumbersome methods to assess binding affinities.

A Humanized Antibody against LRG1 that Inhibits Angiogenesis and Reduces Retinal Vascular Leakage

“…31 Nevertheless, this may be addressed in future work on Magacizumab with the introduction of appropriate backmutations. 32 For clinical application in ophthalmology there are advantages in using Fab fragments rather than full-length antibodies. Accordingly, as the eye is a site of immune privilege it may be especially sensitive to innate immune responses so Fab fragments, where the Fc portion is removed, will prevent potential activation through Fc receptor engagement.…”

A human monocytic NF-κB fluorescent reporter cell line for detection of microbial contaminants in biological samples

“…Transient expression was performed under serum-free conditions in suspension HEK293-6E as described by Margreitter et al [30]. Briefly, transfection of the pCEP4 vector was performed with polyethylemine (PEI; linear 25 kDA, Polysciences) at a cell density of 1x10 6 /ml.…”

Engineering Stability, Viscosity, and Immunogenicity of Antibodies by Computational Design

“…In another example, Kunert and coworkers have also sought to use MD simulations to predict effects of back-mutations on antibody structures. 215,216 On the assumption that variants with structures and dynamics comparable to those of the original mouse antibody would show significant binding, a similarity score was developed based on the RMSD of all atoms in CDR-H3 to quantify conformational differences between the mouse antibody and humanized variants during the simulations. Starting from a crystal structure of the mouse antibody or the variant models, MD simulations were performed for~100 ns in an explicit solvent.…”