Advanced Technology in all Key Stages:
1) Antigen Preparation. The methods of preparation for various antigens such as soluble proteins, eukaryotic cells, DNA, and membrane proteins ensure high antigen quality.
2) Antibody Production. A variety of antibody screening methods such as large-capacity phage display, a fully human antibody library, an immunization antibody library, and hybridoma technology, can be used to obtain dozens of preferred antibody candidate molecules with well-defined sequences and high affinity.
3) Antibody engineering. The antibody obtained by 3D deep humanization has a humanization more than 90%, also the pharmacological properties. Based on the affinity maturation technology of the large-capacity phage display antibody library, the obtained antibody candidate molecules are extremely optimized, and the affinity can usually be increased by 5-100 times.
4) Systematic Drug Screening in Vitro. Sanyoubio has established systematic evaluation methods for the biological activity of monoclonal antibody drugs including ADCC, CDC, cell proliferation inhibition, immune cell co-stimulation, macrophage phagocytosis, and other screening methods, which can be used for high efficiency and high throughput screening.
5) Druggability Screening. The combination of the preparation of high expression cell lines, stability studies, physical and chemical analysis, and in vivo pharmacodynamics screening ensures the quality of pre-clinical candidate molecules.
The development of innovative antibody drugs is more efficient and convenient based on the use of
advanced international antibody discovery technologies.
Service Highlight 1: Magnetic Array Immunization Antibody Library (MIT)
The MIT (Magnetic, Immunization, and Tandem Antibody Library) is an antibody preparation method that combines animal immunization, array construction, and magnetic bead high-throughput screening technologies.
Application Case 1: The number of antibodies obtained is extremely large
Through mass selection, primary screening, sequencing analysis and rescreening using MIT immunization antibody library, more than 60 lead antibodies with unique sequences are obtained. The results of the affinity ranking of the obtained antibodies are shown in Figure 1. As can be seen from the figure, the EC50 of the lead antibody binding to the antigen obtained by this project are mostly at the sub-nM level.
Application Case 2: The antibody obtained has higher affinity compared to hybridoma technology
Candidate antibodies tested against a certain target protein were prepared by the MIT immunization library and hybridoma technologies. The results of the affinity comparison test of the antibodies obtained by the two methods are shown in Fig. 2. As seen, the preferred antibody clones B1, C4, and B3 derived from the MIT immunization antibody library have significantly higher affinities than the preferred antibodies derived from hybridomas.
Figure 1. Affinity ranking of antibodies obtained from the MIT immune immunization antibody library
Figure 2. Affinity ratio of antibodies from MIT immunization antibody library to antibodies derived from hybridoma technology
Sanyou bio has accumulated a large number of successful cases in the magnetic array immunization antibody library. By January 2018, Sanyou bio has successfully constructed 298 magnetic array immunization antibody libraries.
Additionally, Sanyou bio has accumulated significant experience in other antibody libraries including 6 human disease antibody libraries, 50 affinity maturation antibody libraries, 28 naive library sub-libraries, and 53 Fab antibody libraries. The accumulated library capacity is more than 1011.
Table 1. Quantity and summary of antibody libraries constructed by Sanyou bio
As of January 2018
Our Advanced integrated antibody library construction platforms guarantee the quality of the MIT antibody library services
Service Highlight 2: Over 1011 Fully Human Antibody Libraries Constructed from Thousands of Samples
At present, the phage display antibody libraries in the world include whole human naive antibody libraries, synthetic antibody libraries, and semi-synthetic antibody libraries. Most antibody libraries are constructed using dozens of donors or synthetic antibody genes and have limitations such as: low library capacity, insufficient diversity, low expression ratio, high mismatch ratio, and insufficient druggability which greatly limit their applications. To solve these problems, Sanyou bio collects four thousand cases of samples to create a world-leading, fully human naive antibody library with a capacity of 1011.
Why Choose a Fully Human Naïve Antibody Library?
Phage display of fully human antibody libraries is a well-proven technique. Currently, 8 of the more than 20 fully human antibodies on the market are screened from phage display of fully human antibody libraries.
Comparison of Sanyou Bio’s Naïve Antibody Library to a Well-Known International Antibody Library
More than 20 lead antibodies with unique sequences were obtained from the fully human antibody library through mass selection, primary screening, sequencing analysis and rescreening.
The results of the affinity ranking of the antibodies obtained are shown in FIG 4. As can be seen from the figure, the EC50 of the lead antibody binding to the antigen obtained by this project are mostly at the sub-nM level
Figure 4. Affinity ranking of antibodies obtained from the fully human antibody library
Service Highlight 3: Extreme Antibody Engineering Assures Druggability
3D antibody humanization refers to the humanized design of the mouse antibody through computer assistance, and the humanized modification of amino acid residues on the surface of the heterologous antibody based on crystal structure analysis. The humanized antibody is humanized to the maximum extent and the degree of humanization is over 90%.
Application Case 1: After humanization, the affinity is consistent with the mouse antibody, and the affinity of some antibodies after humanization is higher than that of the mouse antibody
Through the humanization of the antibody obtained, the results of antibody affinity after humanization are shown in FIG 5. As can be seen from the figure, the affinity of the antibody after humanization remains practically the same as that of the mouse control antibody, and the affinity of some antibodies after humanization is higher than that of the mouse control antibody.
Figure 5. Comparison of the affinity of humanized antibodies to murine antibodies
Figure 6. Crystal structure simulation of a humanized antibody and a murine antibody
Through computer-aid simulation, we can select the CDR combination to be modified which results in 109 or even 1010 of mutant antibody library that can be designed through these methods. Preferred antibodies with higher affinity and higher druggability can be screened through mass selection, affinity screening, sequence analysis, affinity kinetic analysis, and expression characteristics analysis, and finally physicochemical properties analysis.
High-affinity guarantee: Optimize 109 or even 1010 amino acid sites in the original sequence. Through the use of multi-round screening the antibody affinity usually increases by 5-100 times.
Advanced technology collection integrates advanced technologies such as 3D advanced structure simulation, large-capacity antibody library, and physicochemical analysis.
Application Case 2: Extreme affinity maturation modification improves the affinity by 20 times
Here is a study of affinity maturation of the antibodies obtained.
The results of the affinity maturation of the antibodies obtained are shown in Fig. 7. As can be seen from the figure, the project increases the affinity by 5-10 times through one round of mutation, and the affinity increases by more than 20 times through two rounds of mutations.
Application Case 3: multiple candidate antibodies are successfully obtained in PH-sensitive project
This is a study of affinity maturation of the PH-sensitive antibody obtained.
The results of the affinity maturation of the antibodies obtained are shown in Fig. 8. As can be seen from the figure, one round of mutation shows antibodies having a marked difference in binding characteristics between pH 6.0 and 7.2 are selected and compared with the wild type.
Figure 7. Extreme affinity maturation modification
Figure 8. Extremely affinity maturation modification of PH-sensitive antibody
Service Highlight 4: Systematic In Vitro Pharmacodynamic Evaluation for Antibody Drug Screening for Various Targets
Cellular functional testing of antibody drugs is critical for drug screening and quality control. At present, the activity analysis methods of antibody drugs are mainly biochemical and cell-based functional screenings. Sanyou bio has established a systematic evaluation method for the biological activity of monoclonal antibody drugs which can be used for screening drugs against targets in cytotoxicity, cell proliferation inhibition, cell activation and interaction regulation, human autoimmune system regulation, neutralization antigen, and GPCR. These processes allow the development of antibody drugs close to the clinical stage.
Overview of Systematic In Vitro Pharmacodynamic Evaluation
Application 1: Isolating human primary cells with high purity suitable for antibody functional screening
After isolating human primary T cells (negative isolation), CD4+ T cells (negative isolation) and monocytes using the German Miltenyi MACS system, isolated cells were labeled using CD3, CD4 and CD14 flowcytometry antibodies and were tested with flow cytometry. The result is shown in Figure 9. The figure shows the isolated human primary cells are highly purified and are applicable for functional screening of antibodies.
Figure 9. Human primary cell isolation experiment
Application Case 2: FACS Affinity screening of some candidate antibody molecules has the same affinity as the positive control antibody.
The cells tested are sequentially incubated with candidate antibodies of different concentration gradients and a fluorescent secondary antibody. Then the affinity of candidate molecules is detected using a flow cytometer. The result is shown in Figure 10. As seen in the figure, some of the candidate antibodies have the same affinity as the positive control antibody.
Figure 10. FACS affinity screening experiment
Application Case 3: The biological activity of antibody clones obtained by co-stimulation culture experiments is significantly stronger than that of control antibodies.
Human primary T cells are co-stimulated with a control antibody and different concentrations of candidate antibodies. An ELISA assay is used to detect the amount of cytokine secretion in the supernatant of the cultured cells. The result is shown in Figure 11. As seen from the figure, the candidate antibodies can effectively activate the T cell response.
Figure 11. Costimulatory culture experiment
Application Case 4: Flow cytometry detecting the efficiency of macrophages’phagocytic target cells
By differentiating human monocytes to macrophages, the target cells are incubated with macrophages and antibody A or candidate antibodies of different concentration gradients. The efficiency of macrophage phagocytosis of target cells is detected by flow cytometry. The result is shown in Figure 11. As can be seen from the figure, both candidate antibody and antibody A can effectively promote macrophage phagocytosis of target cells, and two of the candidate antibodies are stronger than positive control antibody.
Application Case 5: The screened antibodies effectively enhance immune response
The isolated monocytes are differentiated into macrophages in vitro, and plated with human CD4+ T cells in a certain ratio and different concentrations of the target antibodies that are added to the plate. The result is shown in FIG 13. As can be seen from the figure, antibodies can effectively enhance the immune effect.
Figure 12. Flow cytometry detection of macrophage phagocytosis of target cells
Figure 13. Mixed lymphocyte reaction experiment
Candidate molecules with good druggability can be obtained through four milestones:
Sanyou bio has adopted a variety of flexible service models such as outsourcing, milestone cooperation development, and personalized services, which halve the risk for the customer and double the success rate.