Abstract
BackgroundHumanization of mouse monoclonal antibodies (mAbs) is crucial for reducing their immunogenicity in humans. However, humanized mAbs often lose their binding affinities. Therefore, an in silico humanization method that can prevent the loss of the binding affinity of mAbs is needed.MethodsWe developed an in silico V(D)J recombination platform in which we used V(D)J human germline gene sequences to design five humanized candidates of anti-tumor necrosis factor (TNF)-α mAbs (C1–C5) by using different human germline templates. The candidates were subjected to molecular dynamics simulation. In addition, the structural similarities of their complementarity-determining regions (CDRs) to those of original mouse mAbs were estimated to derive the weighted interatomic root mean squared deviation (wRMSDi) value. Subsequently, the correlation of the derived wRMSDi value with the half maximal effective concentration (EC50) and the binding affinity (KD) of the humanized anti-TNF-α candidates was examined. To confirm whether our in silico estimation method can be used for other humanized mAbs, we tested our method using the anti-epidermal growth factor receptor (EGFR) a4.6.1, anti-glypican-3 (GPC3) YP9.1 and anti-α4β1 integrin HP1/2L mAbs.ResultsThe R2 value for the correlation between the wRMSDi and log(EC50) of the recombinant Remicade and those of the humanized anti-TNF-α candidates was 0.901, and the R2 value for the correlation between wRMSDi and log(KD) was 0.9921. The results indicated that our in silico V(D)J recombination platform could predict the binding affinity of humanized candidates and successfully identify the high-affinity humanized anti-TNF-α antibody (Ab) C1 with a binding affinity similar to that of the parental chimeric mAb (5.13 × 10−10). For the anti-EGFR a4.6.1, anti-GPC3 YP9.1, and anti-α4β1 integrin HP1/2L mAbs, the wRMSDi and log(EC50) exhibited strong correlations (R2 = 0.9908, 0.9999, and 0.8907, respectively).ConclusionsOur in silico V(D)J recombination platform can facilitate the development of humanized mAbs with low immunogenicity and high binding affinities. This platform can directly transform numerous mAbs with therapeutic potential to humanized or even human therapeutic Abs for clinical use.Graphical
Highlights
Therapeutic monoclonal antibodies have been approved for the treatment of various human diseases including cancer, infections, and immune disorders [1]
IGKV6-21*01-JK2 and IGHV3-15*07-JH5 were selected as the human immunoglobulin frameworks for complementarity-determining regions (CDRs) grafting for the VL and VH, respectively
All the nine humanized antiTNF-α monoclonal antibodies (mAbs) candidates were subjected to Molecular dynamics (MD) simulation, and the resulting trajectories were compared with those of Remicade
Summary
Therapeutic monoclonal antibodies (mAbs) have been approved for the treatment of various human diseases including cancer, infections, and immune disorders [1]. The conventional method used to develop effective mAbs involves the immunization of mice with the target antigen (Ag) and the generation of a hybridoma to acquire the Ag-specific antibody (Ab) [3]. To reduce immunogenicity, humanized mAbs integrating human frameworks were developed. The humanization of murine mAbs can reduce their immunogenicity in humans. A reliable approach for humanizing potential mouse mAbs is necessary for developing therapeutic mAbs. Humanization of mouse monoclonal antibodies (mAbs) is crucial for reducing their immunogenicity in humans. Humanized mAbs often lose their binding affinities. An in silico humanization method that can prevent the loss of the binding affinity of mAbs is needed
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