菜单
Service Overviews

Background: Phage display technology is a powerful technique to develop human antibodies. It has many outstanding advantages over hybridoma antibody technology. Phage displayed human antibody libraries in the market generally have the problems of few donors, low capacity, low diversity, low affinity and poor druggability. In order to obtain more quantity and better quality antibody candidates, Sanyou launches the sub-library of super-trillion fully human antibody library screening service.


Methods: This library with over 100-billion diversity was derived from over 10-billion human recombinant monoclonal antibody library through molecular block library construction and multiple rounds of library recombination.


Advantages: Hundreds of high affinity lead antibodies can be obtained, and the affinity, specificity, category richness and other indicators of the obtained antibodies are at the international leading level.


Cases: Sanyou Bio has completed screening and verifying of dozens of projects through fully human antibody library. For different targets, an average of 286 lead antibody molecules with unique sequence and good affinity can be obtained.



Fig. 1 Statistics of the number of leading molecules of different difficulty targets

Service Overview Background: Phage display technology is a powerful technique to develop human antibodies. It has many outstanding advantages over hybridoma antibody technology. Phage displayed human antibody libraries in the market generally have the problems of few donors, low capacity, low diversity, low affinity and poor druggability. In order to obtain more quantity and better quality antibody candidates, Sanyou launches the privileged over 100-billion human recombinant monoclonal antibody customized services. Methods: This library with over 100-billion diversity was derived from over 10-billion human recombinant monoclonal antibody library through molecular block library construction and multiple rounds of library recombination. Advantages: Hundreds of high affinity lead antibodies can be obtained, and the affinity, specificity, category richness and other indicators of the obtained antibodies are at the international leading level. Case:SanyouBio has completed screening and verifying of dozens of projects through human recombinant antibody library. For different targets, an average of 286 lead antibody molecules with unique sequence and good affinity can be obtained. Fig. 1 Statistics of the number of leading molecules of different difficulty targets Service Contents Service Name Service Content Customer Provided Deliverables Lead Time Restructuring of 100 billion people Antibody library screening 1. High throughput screening 2. Eukaryotic expression verification Target antigen (provide overexpressed cell lines on demand) Deliverables:1. > 100 sequence specific lead antibody moleculesDelivery standard:1. Antibody affinity ranking 2. Molecular affinity kinetics or FACS validation is preferred 6-8 weeks Option Service: MIT mouse recombinant mAb customization | Ultimate antibody affinity maturation Service Highlights 1. From clearly defined gene sequences, to directly generate human antibody The service adopts genetic engineering and phage display technology, which can directly screen the human recombinant antibody gene sequence and reduce unnecessary screening work. 2. With an over 100-billion library capacity, to easily obtain high antibody diversity The capacity of sub-trillion human recombinant antibody library is as high as 1.55×1011 CFU. The huge number of hits ensures the diversity of antibodies and lays the foundation for later research and development. 3. By integrated high-throughput panning technology, to significantly improve screening efficiency A one-stop, solid-phase and liquid-phase, high-throughput automatic screening platform is adopted, which greatly shortens the screening time, improves the screening flux and greatly improves the screening efficiency. 4. Based upon numerous antibody binders, to firmly secure hundreds of lead molecules After the verification of dozens of targets, hundreds of lead antibody binders can be obtained for each target by using protein-protein, protein-cell and cell-cell screening methods. 5. Through eukaryotic expression platform, to convincingly perform cell-based function verification Using a mammalian cell expression system, the expressed recombinant protein is close to the natural conformation and has good biological activity. Supplemented by multi-dimensional quality control, the antibody quality is fully verified. Service Features 1. Characteristics of germline genes distribution The gene coverage of the fully human recombinant antibody library is relatively comprehensive. The light chain subtypes of the fully human recombinant antibody library are divided intoκandλ.By analyzing the gene distribution proportion of twosubtypes of light and heavychaingermline,theirdistributioncharacteristicscomplywiththelaw of antibody-drug germline . 2. High antibody diversity The length of CDR-L3 and CDR-H3 of antibodies is an index to evaluate the diversity of antibodies. Hundreds of binders were selected from the constructed library for sequencing and analysis. The length of CDR-L3 ranges 6-13 amino acids, and the length of CDR-H3 ranges 5-28 amino acids, which was consistent with the literature report, with 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 Sanyou sub-trillion human recombinant antibody library. As shown in Fig. 3, through the screening and verification of 10 targets, a total of 1335 antibody clones with unique sequences were obtained, with an average of hundreds of clones for each given target. More than 90% of the projects obtained over 100 clones. Fig. 3 Statistics of Binder amount/project 4. Comprehensive druggability analysis After the full-length construction and expression of candidates obtained from the screening service of sub-trillion human recombinant antibody library, the yield, biochemical and physicochemical properties of the antibody were comprehensively analyzed. As shown in Table 1, Multiple dimensions including purity and concentration determination, primary structure analysis, affinity and affinity kinetics are considered Table 1 Drug developability of antibodies from ST-hRAL category Test contents test methods Purity and concentration determination Purity identification SDS-PAGE/SEC/CE-SDS Purity and concentration determination Concentration identification Protein A-HPLC/UV280 Primary structure analysis Molecular weight analysis LC/MS Primary structure analysis Isoelectric point iCIEF Primary structure analysis Hydrophobicity identification HIC-HPLC Primary structure analysis Charge heterogeneity measurement CEX Primary structure analysis Peptide map analysis LC-UV-MS/MS Primary structure analysis N-sugar spectrum analysis LC/MS Affinity and Affinity Kinetics Affinity detection ELISA Affinity and Affinity Kinetics Affinity kinetic detection BLI/SPR Affinity and Affinity Kinetics Cell binding test (if any) FACS 5. High affinity of lead antibody The molecules from the Sanyou sub-trillion human recombinant antibody library have good affinity activity. The affinity of the obtained antibodies can often reach nM to sub nM. Fig. 4 is the molecular affinity analysis of the candidate antibodies after expression, the results show that most antibody clones have good affinity. Fig. 4 Kinetics determination by ForteBio Case Presentations 1. Development of anti-CD40 fully human antibody Background: up-regulates the expression of costimulatory molecules, and promotes the cross-presentation of antigens. One of the main functions of CD40 is to enhance the presentation of antigen to T cells by activating DC cells, and increase the interaction between DC and T cells through up-regulating membrane proteins such as CD54 and CD86, which promotes the proliferation and activation of T cells. 1.1. Clinical competition pattern Up to now, more than 20 kinds of CD40 antibody drugs are being used in clinical trials against tumors and immune system diseases, most of which are in the early research stage. 1.2. Monoclonal antibody MOA The agonistic anti-CD40 monoclonal antibody can enhance the ability of T cell response by activating DCs. Therefore, the agonistic anti-CD40 antibody can change a "cold" tumor into a "hot" tumor. Anti-CD40 agonist antibodies can overcome the tumor immune tolerance by modifying the immunosuppressive myeloid cell infiltration in tumors. 2. Key results of anti-CD40 antibody 2.1. Function analysis of the binding, blocking and activating activities at the cellular level The constructed full-length antibodies carry out binding, blocking, and activation experiments at the cell level. Fig. 5 shows the cell-based binding activity of antibodies by FACS, and Fig. 6 shows a project case study of the blocking and activating activity of antibodies to receptor-ligand binding based on FACS. It can be seen from the figures that all lead antibody clones showed good cell-level binding activity; Among the 14 antibodies, 8 of which showed good activation activity and 5 of which 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 efficacy in vitro The binding of CD40 and CD40 ligand(CD40L) can activate DC cells, promoting IL-12 secretion and CD83 upregulation of DC cells. The binding of agonistic anti-CD40 mAb and CD40 can also activate DC cells. The results of monocytes induced DC cells(iDC) activation assay by candidate molecules are shown in Fig. 7. The IL-12 secretion and CD83 expression induced by H9 are significantly better than those of the control antibody, showing an excellent activation activity. Fig. 7 iDC activation assay 2.3. validation of efficacy in vivo The in vivo anti-tumor efficacy verification results of the fully human candidate molecule H9 in the CDX model of nude mice are shown in Fig. 8. Mice were divided into the control group and treatment group, which were administered three times a week for two weeks. The results showed that at the dose of 1 mpk, the anti-tumor efficacy of the candidate antibody H9 was better than that of the control antibody; At the dose of 5 mpk, the anti-tumor efficacy of the candidate antibody H9 was comparable to that of the control antibody, and both could completely inhibit tumor growth. Fig. 8 Tumor growth inhibition Represent project R & D Progress

Service Contents

Service

Service Details

Client Providedes

Deliverables and Standards

Time

Sub-library of super-trillion fully human antibody library screening service screening

1. High throughput screening

2. Eukaryotic expression verification

Target antigen (provide overexpressed cell lines on demand)

Deliverables:

1. > 100 sequence specific lead antibody molecules delivery

Standard:

1. Antibody affinity ranking

2. Molecular affinity kinetics or FACS validation is preferred




6-8 weeks


Option Service:
MIT mouse recombinant mAb customization | Ultimate antibody affinity maturation

Service Highlights
  • 1. From clearly defined gene sequences, to directly generate human antibody

    1. The service adopts genetic engineering and phage display technology, which can directly screen the human recombinant antibody gene sequence and reduce unnecessary screening work.
  • 2. With an over 100-billion library capacity, to easily obtain high antibody diversity
    1. The capacity of sub-library of super-trillion fully human antibody library is as high as 1.55×1011 CFU. The huge number of hits ensures the diversity of antibodies and lays the foundation for later research and development.
  • 3. By integrated high-throughput panning technology, to significantly improve screening efficiency
    1. A one-stop, solid-phase and liquid-phase, high-throughput automatic screening platform is adopted, which greatly shortens the screening time, improves the screening flux and greatly improves the screening efficiency.
  • 4. Based upon numerous antibody binders, to firmly secure hundreds of lead molecules
    1. After the verification of dozens of targets, hundreds of lead antibody binders can be obtained for each target by using protein-protein, protein-cell and cell-cell screening methods.
  • 5. Through eukaryotic expression platform, to convincingly perform cell-based function verification
    1. Using a mammalian cell expression system, the expressed recombinant protein is close to the natural conformation and has good biological activity. Supplemented by multi-dimensional quality control, the antibody quality is fully verified.

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 recombinant antibody library are divided intoκandλ.By analyzing the gene distribution proportion of twosubtypes of light and heavychaingermline,theirdistributioncharacteristicscomplywiththelaw of antibody-drug germline .

2. High antibody diversity

The length of CDR-L3 and CDR-H3 of antibodies is an index to evaluate the diversity of antibodies. Hundreds of binders were selected from the constructed library for sequencing and analysis. The length of CDR-L3 ranges 6-13 amino acids, and the length of CDR-H3 ranges 5-28 amino acids, which was consistent with the literature report, with 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 Sanyou sub-library of super-trillion fully human antibody library. As shown in Fig. 3, through the screening and verification of 10 targets, a total of 1335 antibody clones with unique sequences were obtained, with an average of hundreds of clones for each given target. More than 90% of the projects obtained over 100 clones.


Fig. 3 Statistics of Binder amount / project

4. Comprehensive druggability analysis

After the full-length construction and expression of candidates obtained from the screening service of sub-library of super-trillion fully human antibody library, the yield, biochemical and physicochemical properties of the antibody were comprehensively analyzed. As shown in Table 1, Multiple dimensions including purity and concentration determination, primary structure analysis, affinity and affinity kinetics are considered


Table 1 Drug developability of antibodies from ST-hRAL

Category

Test Contents

Test Methods

Purity and concentration determination

Purity identification

SDS-PAGE / SEC / CE-SDS

Purity and concentration determination

Concentration identification

Protein A-HPLC / UV280

Primary structure analysis

Molecular weight analysis

LC / MS

Primary structure analysis

Isoelectric point

iCIEF

Primary structure analysis

Hydrophobicity identification

HIC-HPLC

Primary structure analysis

Charge heterogeneity measurement

CEX

Primary structure analysis

Peptide map analysis

LC-UV-MS / MS

Primary structure analysis

N-sugar spectrum analysis

LC / MS

Affinity and Affinity Kinetics

Affinity detection

ELISA

Affinity and Affinity Kinetics

Affinity kinetic detection

BLI / SPR

Affinity and Affinity Kinetics

Cell binding test (if any)

FACS

5. High affinity of lead antibody

The molecules from the Sanyou sub-library of super-trillion fully human antibody library screening service have good affinity activity. The affinity of the obtained antibodies can often reach nM to sub nM. Fig. 4 is the molecular affinity analysis of the candidate antibodies after expression, the results show that most antibody clones have good affinity.


Fig. 4 Kinetics determination by ForteBio


Case Studies
1. Development of anti-CD40 fully human antibody

Background: up-regulates the expression of costimulatory molecules, and promotes the cross-presentation of antigens. One of the main functions of CD40 is to enhance the presentation of antigen to T cells by activating DC cells, and increase the interaction between DC and T cells through up-regulating membrane proteins such as CD54 and CD86, which promotes the proliferation and activation of T cells.


1.1. Clinical competition pattern

Up to now, more than 20 kinds of CD40 antibody drugs are being used in clinical trials against tumors and immune system diseases, most of which are in the early research stage.


1.2. Monoclonal antibody MOA

The agonistic anti-CD40 monoclonal antibody can enhance the ability of T cell response by activating DCs. Therefore, the agonistic anti-CD40 antibody can change a "cold" tumor into a "hot" tumor. Anti-CD40 agonist antibodies can overcome the tumor immune tolerance by modifying the immunosuppressive myeloid cell infiltration in tumors.

2. Key results of anti-CD40 antibody

2.1. Function analysis of the binding, blocking and activating activities at the cellular level

The constructed full-length antibodies carry out binding, blocking, and activation experiments at the cell level. Fig. 5 shows the cell-based binding activity of antibodies by FACS, and Fig. 6 shows a project case study of the blocking and activating activity of antibodies to receptor-ligand binding based on FACS. It can be seen from the figures that all lead antibody clones showed good cell-level binding activity; Among the 14 antibodies, 8 of which showed good activation activity and 5 of which 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 efficacy in vitro

The binding of CD40 and CD40 ligand (CD40L) can activate DC cells, promoting IL-12 secretion and CD83 upregulation of DC cells. The binding of agonistic anti-CD40 mAb and CD40 can also activate DC cells. The results of monocytes induced DC cells(iDC) activation assay by candidate molecules are shown in Fig. 7. The IL-12 secretion and CD83 expression induced by H9 are significantly better than those of the control antibody, showing an excellent activation activity.



Fig. 7 iDC activation assay


2.3. validation of efficacy in vivo

The in vivo anti-tumor efficacy verification results of the fully human candidate molecule H9 in the CDX model of nude mice are shown in Fig. 8. Mice were divided into the control group and treatment group, which were administered three times a week for two weeks. The results showed that at the dose of 1 mpk, the anti-tumor efficacy of the candidate antibody H9 was better than that of the control antibody; At the dose of 5 mpk, the anti-tumor efficacy of the candidate antibody H9 was comparable to that of the control antibody, and both could completely inhibit tumor growth.


Fig. 8 Tumor growth inhibition


Represent project R&D progress