Background: To resolve the heterogenous responses of mouse-derived or alpaca-derived monoclonal antibodies, Sanyou introduces in-depth antibody humanization service. Based on CDR grafting technique, Sanyou utilizes the unique in-house database. AI is highly involved in the sequence design, including 3D structure simulation, surface charge analysis, antigen-antibody binding site prediction, and secondary structure amino acid site distribution. Overall goal is to maximize humanization level without affecting antibody function.
AI prediction with high precision
The 3D structure of antibody molecules is accurately predicted based on the AlphaFold neural network algorithm, furthermore, the antigen-antibody binding sites and spatial conformational can be predicted. Overall, providing scientific and rigorous data support for antibody humanization modification design.
Unique database with scientific references
A unique humanization design database has been developed based on our experienced project design and validation data. The reference sequence is scientifically selected, and the human native antibody with the most similar sequence to the parental antibody is used as the reference template. The over 95% humanization level is achieved by further incorporating the preliminary data, minimizing the heterogeneity of the antibody.
Comprehensive quality control analysis with systemic validation
Sanyou has established a comprehensive and thorough testing system covering a quality control platform of structure validaiton, physicochemical/biochemical analysis, and bioactivity analysis, which complies with the application requirements in China and U.S.
Extensive project experience and high success rate
SanyouBio expert team has rich experience in antibody humanization. We have completed more than 200 cases of humanized antibody design, of which more than 40 designed antibodies are in the clinical application stage, and 7 are in the clinical trial stage.
Fast delivery in 4-5 weeks and with high quality
The one-stop delivery from humanization design, gene synthesis, plasmid construction, protein expression, purification to protein identification only takes 4-5 weeks, which is at the top level in the industry.
1. 3D structure simulation and reference template analysis of mouse-derived antibody sequences
The structure simulation analysis and surface potential analysis of the antibody before and after humanization are shown in the figure below, both of the structure were highly similar.
1.2 Antibody-antigen binding prediction
The binding site is predicted by comparing the mouse-derived antibody sequence with the antigen sequence. Based on avoiding affecting the antigen-antibody interaction interface, the amino acid sites to be mutated are selected and identified by comparative analysis of the human Germline.
1.3 Germline analysis and confirmation
The mouse antibody sequences were aligned with human germline based on the unique database, and a series of germline templates were selected for mouse sequence site confirmation. Humanized templates are usually based on mouse sequences, and four categories of germline (IGHV1/3 and IGKV1/3) with good druggability were selected as design templates.
1.4 Immunogenicity prediction of mouse antibody sequences
Based on the public database, the immunogenicity of the peptide binding to immune cells in mouse antibody sequences was analyzed.
1) VH was predicted to be low immunogenicity
2) VL was predicted to be low immunogenicity
3) The VH was predicted to have an immunogenic peptide sequence of ILYLQMTSLRS
4) The VL was predicted to have an immunogenic peptide sequence of TQSPATLSV
1.5 Prediction of post-translational modification sequence sites
The post-translational modification sites were predicted and analyzed with reference to the unique database.
2. Eukaryotic expression, purification and identification of humanized antibody
Humanization of mouse antibodies is usually designed with 3-5 light and heavy chain sequences, and 9-25 humanized candidates were generated via orthogonal combination, about 10 candidates were selected for eukaryotic expression verification based on the risk assessment eventually. CHO and HEK293 cells were used as host cell and expressed in 10 mL volume for 5-7 days, The samples (milligram level) were obtained by one-step affinity chromatography purification for downstream identification analysis.
3. Quality identification and analysis of humanized antibody
3.1 Analysis of physicochemical properties before and after humanization (SDS-PAGE / SEC / DSF)
The physicochemical properties of the humanized antibody, such as SDS-PAGE, SEC and DSF, are consistent with the parental antibody.
SDS-PAGE Purity of antibody protein> 90%
SEC Main peak of antibody protein> 95%
3.2 Binding activity of humanized antibody (Gator / Biacore)
The affinity kinetics are indispensable for the evaluation of antibody affinity and druggability. While increasing the proportion of humanization, SanyouBio can also ensure that the antibody affinity activity is basically the same with the parent one.
Fig. 1A Affinity kinetic analysis of parental antibody (Gator)
Fig. 2A Affinity kinetic analysis of parental antibody (Biacore)
Fig. 1B Affinity kinetic analysis of humanized antibody (Gator)
Fig. 2B Affinity kinetic analysis of humanized antibody (Biacore)
3.3 Binding activity of humanized antibody (Gator / Biacore)
3.4 Analysis of antigen-antibody binding/in vitro functional activity of humanized antibody
The binding and blocking activity before and after humanization of mouse antibody are shown in Fig. 1 and Fig. 2. The EC50 and the binding curves of humanized antibody (HuAb1-8) are highly similar to the parental antibody, which indicates that the cell binding and blocking activity are highly similar to the parent control before humanization.
Fig. 1 Comparison of the affinity of humanized antibodies to parental antibody
Fig. 2 Comparison of the blocking activity of humanized antibodies to parental antibody
The results of binding and blocking activities of certain mouse-derived antibody before and after humanization are shown in Fig. 3 and Fig. 4. The figures show that the binding and blocking curves of the humanized antibody (huAb1-5) fully overlap with those of the pre-humanized parental antibody (Parental Ab) at the cellular level, with EC50 and IC50 values at around 0.06 μg/mL, indicating that the affinity after humanization modification is well maintained when compared with that of the parental antibody.
Fig. 3 Comparison of the affinity of humanized antibodies to parental antibody
Fig. 4 Comparison of the blocking activity of humanized antibodies to parental antibody
3.5 The binding activity of humanized antibody (FACS / ELISA)
The results of antigen binding and blocking activities of certain mouse-derived antibody before and after humanization are shown in Fig. 5 and Fig. 6. The figures show that the binding and cell blocking activities of the humanized antibody (huAb1-2) are relatively consistent with those of the pre-humanized parental antibody (Parental Ab) at the protein level, with EC50 and IC50 values maintained at 0.03 μg/mL and 0.5 nM, respectively, indicating that the antibody affinity activity after humanization modification is comparable to those of the parental antibody.
Fig. 5 Comparison of the affinity of humanized antibodies to parental antibody
Fig. 6 Comparison of the blocking activity of humanized antibodies to parental antibody
The results of antigen binding and in vitro functional activities of certain mouse-derived antibody before and after humanization are shown in Fig. 7 and Fig. 8. The figures show that the humanized antibody (huAb1-3) significantly enhance their antigen binding and reporter gene activation when compared to the parental antibody (Parental Ab), indicating that the affinity and functional activity after humanization modification are not only unaffected, but also further improved.
Fig. 7 Comparison of the affinity of humanized antibodies to parental antibody
Fig. 8 Comparison of the luciferase activity of humanized antibodies to parental antibody
3.6 The binding activity of humanized antibody (FACS / ELISA)
The results of blocking and in vitro functional activity of ceratin mouse-derived antibody at the cellular level before and after humanization are shown in Fig. 9 and Fig. 10. The figures show that when comparing between humanized antibody (huAb1-5) and Parental Ab, the blocking activity is comparable and the reporter gene activation function is slightly enhanced, indicating that the blocking and functional activity are not affected after humanization modification.
Fig. 9 Comparison of the blocking activity of humanized antibodies to parental antibody
Fig. 10 Comparison of the luciferase activity of humanized antibodies to parental antibody
4. Selection principle of humanized antibody
The selection principles of humanized candidate antibodies are arranged according to the priorities:
1) The binding or blocking activity (Affinity kinetics are generally three times higher than the parent
2) Degree of antibody humanization (number of back mutations < 8)
3) Expression titer and physicochemical properties (the expression titer and physicochemical properties were consistent with the parental antibody)