Service Overview
Background: Phage display technology is a powerful technique to develop human antibodies. It demonstrates advantages over hybridoma antibody technology in many ways. Phage displayed human antibody libraries in the market generally have the problems of few donors, low capacity, low diversity, low affinity and poor pharmacogenicity. In order to obtain large quantities and higher quality antibody candidates, Sanyou launches the screening with sub-library of super-trillion fully human Ab library service.
Service Highlights
Antibody sequences are easily obtained for production of human antibodies
The service adopts genetic engineering and phage display technology, which can directly obtain the fully human antibody gene sequences and reduce unnecessary screening works.
With the over trillion library capacity, antibodies of high diversity are easily obtained
The capacity is as high as 2.16E+12 CFU. The huge number of hits ensures the diversity of antibodies and provides valid candidates for later research and development.
Advanced facilities significantly improve screening efficiency
Using high-throughput and automated batch screening process, matching with the advanced facilities, the whole process takes only 4-6 weeks to deliver, which efficiently meet the clients’ needs for quick antibody discovery.
Using the mammalian cell expressed antibodies, comprehensive drug candidate characterizations are performed
Mammalian cell expression system is used for antibody preparations, and after systematic and comprehensive physicochemical and biochemical analysis and affinity kinetics studies, critical and informative drug developability data are obtained.
Service Content
Service Features
1. Characteristics of germline genes distribution
The gene coverage of the fully human antibody library is relatively comprehensive. The light chain subtypes of the fully human antibody library are κ and λ. After analyzing the gene distribution proportion of two subtypes of light and heavy chain, the distribution characteristics is consistent with the pattern of antibody-drug germline.
2. High antibody diversity
The length of CDR-L3 of antibodies screened by ST-hEAL ranges 6-13 amino acids, and the length of CDR-H3 ranges 5-28 amino acids, which are consistent with the literature, showing high diversity and normal distribution.
Fig. 1 Comparise of CDR-L3 length distrition frequency
Fig. 2 Comparise of CDR-H3 length distrition frequency
3. Numerous lead antibodies
Hundreds of leading molecules can be screened from the sub-library of ST-hRAL. As shown in Fig. 3, through the screening and validation of 16 targets, a total of 5069 antibody clones with unique sequences were obtained, the median number of clones was 345.
Fig. 3 Statistics of Binder amount/project
4. Comprehensive pharmacogenicity analysis
After the full-length construction of candidates obtained from the sub-library of ST-hRAL, the yield, biochemical and physicochemical properties of the antibody are comprehensively analyzed. As shown in Table 1, multiple dimensions including purity, concentration determination, primary structure analysis, affinity and affinity kinetics.
Table 1 Pharmacogenicity analysis of antibodies from ST-hRAL
5. High affinity of lead antibody molecules
The affinity of molecules screened form the sub-library of ST-FHAL can range from nM to sub-nM. As shown in Fig.4, most antibody clones have optimal affinity.
Fig. 3 Statistics of Binder amount/project
Service Process
Case Study
Case 1: Development of Fully Human anti-CD40 Antibody
1. Background
The binding of CD40 to the DC cell surface promotes cytokine and chemokine production, thus inducing the expression of costimulatory molecules and promoting the cross-presentation of antigens. One of the main functions of CD40L is to enhance antigen presentation to T cells by activating DC cells, and to activate T cells by increasing DC interaction with T cells through the upregulation of surface proteins such as CD54 and CD86.
1.1 Clinical Competition Landscape
Currently, more than 20 CD40 antibody drugs are developed in clinical trials for tumors and immune system diseases worldwide, mainly in the early development stage.
1.2 Monoclonal Antibody MOA
Agonistic anti-CD40 monoclonal antibodies can enhance the priming ability of T cell responses by activating dendritic cells (DCs). Anti-CD40 antibodies can turn a "cold tumor" into a "hot tumor" and make the tumor more tolerant to the immune response by modifying immunosuppressive myeloid infiltration in the tumor.
2. Key Results of CD40 Antibody
2.1 Analysis of Binding, Blocking and Activating Activities at Cellular Level
The constructed full-length molecules were tested for binding, blocking, and activation at celluar level. Fig. 5 showed the binding affinity of antibodies to cell surface proteins by FACS. Fig. 6 showed the blocking and activating activity of antibodies on receptor-ligand binding by FACS. The results showed that all lead antibody clones have good binding activity at cellular level; 8 of 14 antibodies showed optimal activation activity, and 5 antibodies showed similar blocking activity to the control antibody.
Fig. 5 Binding affinity determination by FACS
Fig. 6 Blocking activity determination by FACS
2.2 Validation of In Vitro Pharmacodynamics
The binding of CD40 to CD40L can activate DC cells and promote the secretion of IL-12 and CD83, and the binding of agonistic anti-CD40 mAb to CD40 can also activate DC cells. The results of iDC activation assay for candidate molecules are shown in Fig. 7. The results showed that H9 promoted IL-12 secretion and CD83-secretion and the promoting activity was significantly higher than the control antibody, demonstrating its excellent DC cell activation activity.
Fig. 7 iDC activation assay
2.3 Validation of In Vivo Pharmacodynamics
The results of in vivo antitumor efficacy validation of the fully human candidate molecule H9 in the Nude mouse CDX model are shown in Fig. 8. Mice were divided into control and treatment groups and dosed three times a week for two weeks. The results showed that at 1 mpk, the tumor inhibition effect of candidate antibody H9 was superior to that of the control antibody, and at 5 mpk, the tumor inhibition effect of the candidate antibody was equivalent to that of the control antibody. All the results above showed that the tumor growth could be completely inhibited.
Fig. 8 Tumor growth inhibition
Case 2: Development of Fully Human anti-IL-23 Antibody
1. Background
IL-23 is a member of the IL-12 cytokine family and plays an important role in inflammatory responses, especially in autoimmune diseases induced by Th17 cells. IL-23 is a heterodimeric cytokine which composed of two subunits, IL-23a p19 and IL 12b p40. IL-23 is produced by activated macrophages or dendritic cells and acts on Th17 cells those expressing the IL-23 receptor to promote their proliferation and stability.
1.1 Clinical Competition Landscape
Currently, there are 7 IL-23 antibody drugs worldwide, 3 of which have been approved and 4 of which are in the preclinical stage and mainly targeting diseases such as psoriasis and Crohn's disease.
1.2 Monoclonal Antibody MOA
Monoclonal antibodies targeting IL-23 can bind IL-23 and block the binding of IL-23 to the IL-23Rα receptor, thereby treating diseases such as psoriasis and Crohn's disease.
2. Key Results of IL-23 Antibody
2.1 Affinity Activity Assay at Protein Level
The affinity of candidate antibodies A1 and A2 to IL-23 was determined by biofilm interferometry (BLI). As shown in Fig. 9, and the results showed that the affinity of candidate antibodies A1 and A2 to IL-23 is comparable to that of the control antibody.
Fig. 9 Affinity testing of antibodies
2.2 Species Cross-activity Assay at Protein Level
Results of the binding affinity of candidate antibodies A1 and A2 to different species of IL-23 were examined by ELISA. As shown in Fig. 10A-C, both candidate antibodies A1 and A2 had cross-activity for human, mouse, and monkey, and the affinity activity is superior to that of the control antibody.The specific binding activity of candidate antibodies A1 and A2 to IL-23 p19 were tested by ELISA. Fig. 10D showed that candidate antibodies A1 and A2 do not bind to the p40 subunit of IL-12 but specifically bind to the p19 subunit of IL-23.
Fig. 10 Binding affinity determination by ELISA
2.3 Blocking Activity Assay at Protein Level
IL-23 plays an important role in many autoimmune diseases by stimulating TyK2 and JAK2 signaling pathways through binding receptor IL-23R, activating the phosphorylation of STAT3, and subsequently producing SEAP-secreted alkaline phosphatase. Candidate antibodies A1 and A2 were tested for their functional activity in blocking IL-23 binding to the receptor IL-23R at the protein level by ELISA. The results in Fig. 11-12 showed candidate antibodies A1 and A2 had comparable blocking activity to the control antibody. As shown in Fig. 12, candidate antibodies A1 and A2 significantly inhibited the phosphorylation of STAT3 compared with control antibodie.
Fig. 11 Blocking assay by ELISA
Fig. 12 STAT3 phosphorylation by ELISA
2.4 In Vitro Efficacy Validation
IL-17A induces the expression of the proinflammatory cytokine IL-6 in mice spleen cells. The neutralization reaction of humanized candidate antibodies A1 and A1 with IL-17A on NHDF cells were validated by testing the expression of IL-6. The experimental results are shown in Fig. 13. Candidate antibodies A1 and A2 neutralize the activity of IL-17A, significantly inhibit the expression of IL-6, and the neutralizing activity is comparable to that of the control antibody.
Fig. 13 Neutralizing assay on NHDF cell
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