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Service Overviews

Background: In recent years, antibody drugs represented by immune checkpoint inhibitors have become a hot spot in the development of innovative drug. In this process, the in vitro efficacy evaluation model play an irreplaceable role, which can greatly accelerate the process of antibody drugs discovery, screening and optimization. Sanyou Bio focused on the research and development of innovative antibody drugs, and it took 7 years to establish a stable, efficient and reliable in vitro efficacy evaluation platform.


Methods: According to the target mechanism, Sanyou Bio systematically developed stable cell efficacy models of tumor surface antigens, suppressive immune checkpoints, co-stimulated immune checkpoints, and other immunomodulators to ensure the high-quality and efficient promotion of candidate antibody cell efficacy screening.


Advantages: The platform has experience in in vitro efficacy evaluation of 200+ projects, and has established mature cell pharmacodynamic models of 60+ targets, tumor cell lines of 100+ targets, 10+ human primary cells, 40+ SOP guidance. It only takes 6 days to complete all in vitro efficacy evaluations of candidate molecules.


Cases: Sanyou Bio in vitro efficacy evaluation covers primary cell isolation, primary cell induction, flow cytometry screening, luciferase reporting system, cell killing, antibody endocytosis detection, phosphorylation detection, cytokine detection, enzyme bioactivity detection, neutrophil chemotaxis, etc.

Service Overviews Background: In recent years, antibody drugs represented by immune checkpoint inhibitors have become a hot spot in the development of innovative drug. In this process, the in vitro efficacy evaluation model play an irreplaceable role, which can greatly accelerate the process of antibody drugs discovery, screening and optimization. Sanyou Bio focused on the research and development of innovative antibody drugs, and it took 7 years to establish a stable, efficient and reliable in vitro efficacy evaluation platform. Methods: According to the target mechanism, Sanyou Bio systematically developed stable cell efficacy models of tumor surface antigens, suppressive immune checkpoints, co-stimulated immune checkpoints, and other immunomodulators to ensure the high-quality and efficient promotion of candidate antibody cell efficacy screening. Advantages: The platform has experience in in vitro efficacy evaluation of 200+ projects, and has established mature cell pharmacodynamic models of 60+ targets, tumor cell lines of 100+ targets, 10+ human primary cells, 40+ SOP guidance. It only takes 6 days to complete all in vitro efficacy evaluations of candidate molecules. Cases: Sanyou Bio in vitro efficacy evaluation covers primary cell isolation, primary cell induction, flow cytometry screening, luciferase reporting system, cell killing, antibody endocytosis detection, phosphorylation detection, cytokine detection, enzyme bioactivity detection, neutrophil chemotaxis, etc. Service Contents Service Names Service Details Client material Deliverables and Standards Time In vitro pharmacodynamic method development In vitro pharmacodynamic models for specific targets 1. Reference antibody 2. Mechanism of action A stable system that can evaluate drug efficacy 4-8 weeks In vitro drug efficacy sample detection Detection of In vitro harmacodynamic function of candidate molecules 1. Reference antibody 2. Candidate molecules Candidate Molecular Efficacy Evaluation Report 1-2 weeks Service Highlights 1. Diverse tumor target selection It can evaluate any tumor treatment drug target and different drug mechanisms without fear of difficulty. 2. Well-established in vitro evaluation system in vitro evaluation system. 3. Extensive primary cell models A full range of human/mouse primary cells resource, presenting the in vivo mechanism of action to the greatest extent in vitro . 4. Customized development with high success rate The multi-dimensional pharmacodynamic evaluation system can be customized according to the target pharmacodynamic mechanism. 5. High standard quality system Evaluate the efficacy model from dimensions such as linearity, precision, accuracy, and stability to ensure higher quality. Case Stastics 1. Primary Cell Isolation As shown in Fig. 1, PBMCs were isolated and purified to obtain different types of primary cells such as CD4+ T, CD3+ T, Monocyte, etc. The purity of the sorted cells usually reached 95% or even higher, which provides fully guarantee for the next step of efficacy screening. Fig. 1 Isolation of primary human cells 2. Tumor-specific antigen (TSA) 2.1. Endocytic activity assay Fab-Zap is a small molecule inhibitor that enters cells through antibody-mediated endocytosis and inhibits cell growth. As shown in Fig. 2, after the target cells were treated with the control antibody, it was detected by LDH method and found that the cell growth was significantly inhibited, and the EC50 value was 0.096 μg/mL, indicating that the control antibody had good endocytotic activity. Fig. 2 Endocytosis activity Assay 2.2. ADCC determination SK-BR-3 is a breast cancer cell line that expresses HER2. T drugs targeting HER2 have a strong ADCC effect. In this ADCC experiment, the effector cells were PBMC cells and the target cells were SK-BR-3 cells. As shown in Fig. 3, the cell lysis rate increased significantly under the action of T drug measured by LDH method, and the EC50 value was 0.023 μg/mL, indicating that T drug mediated a strong target cell killing effect. Fig. 3 ADCC activity assay 2.3. CDC assay Antibodies can effectively induce complement-dependent killing effects, causing strong damage to cells. As shown in Fig. 4, the cell lysis rate increased significantly after the target cells were treated with a control antibody, and the EC50 value was 27.90 nM, indicating that the candidate antibody could induce a strong CDC effect, resulting in massive cell death. Fig. 4 CDC activity assay 2.4. Phagocytosis experiment In phagocytosis experiments, blocking the “don't eat me” signal by antibodies can promote the phagocytosis of target cells by macrophages. As shown in Fig. 5, it was found by FACS that the phagocytosis of macrophages on target cells were significantly enhanced by control antibody treatment, and the EC50 value was 0.022 μg/mL, indicating that the candidate antibody had good blocking activity against the “don't eat me” signaling pathway. Fig. 5 macrophage phagocytosis effect 2.5. Proliferation inhibition assay In proliferation inhibition experiments, IL-4 promotes TF-1 cell proliferation, and IL-4 neutralization can inhibit IL-4-mediated cell proliferation. As shown in Fig. 6, cell proliferation was inhibited with an EC50 value of 0.54 μg/mL, indicating that the control antibody effectively inhibited the proliferation of TF-1 cells. Fig. 6 Proliferation inhibition experiment 3. Costimulatory immune checkpoint (CIC) The binding of CD40 to CD40L promoted the expression of CD95 and exacerbated apoptosis. As shown in Fig. 7, CD40 candidate antibodies A1-A7 significantly reduced the expression of CD95, indicating that these candidate antibodies blocked the binding of CD40 to CD40L and had good biological activity. Fig. 7 Expression inhibition experiment 4. Inhibitory immune checkpoints (IIC) 4.1. MLR experiment In MLR experiments, CD4+ T cells and DC cells of different donors were co-cultured. Anti-PD-L1 antibodies could effectively break the PD1/PD-L1 inhibitory signal, activate T cells, and promote the secretion of cytokines. As shown in Fig. 8, after treatment with candidate1, the secretion of IL-2 and IFN-γ in cells increased significantly, indicating that it had a highly similar biological activity to the 3 antibody drugs that had been marketed. Fig. 8A IL-2 secretion detection Fig. 8B IFN-γ secretion detection 4.2. Binding and blocking activity analysis at the cellular level Fig. 9A showed a case using FACS to analyze the binding activity of antibodies to cell surface proteins, and Fig. 9B showed a case using FACS to analyze the blocking activity of antibodies binding to receptor ligands. All the lead antibody molecules exhibited stronger binding and blocking activities than the control antibody. Fig. 9A Cell binding activity assay Fig. 9B Cell blocking activity assay 4.3. Antibody epitope analysis As shown in Fig. 10, Candidate 1-5 all competitively inhibited the binding of Target Ab to the target antigen,indicating that these six antibodies recognized the same epitope. Fig. 10 Cell Competitive Binding Assay 4.4. TIGIT luciferase reporter system TIGIT is a surface marker of deeply depletion of T cells, and blocking its negative regulatory signaling pathway with CD155 can effectively activate effector cells. As shown in Fig. 11, under the control antibody treatment, the fluorescence intensity of the cells was significantly enhanced, and the EC50 value was 0.67 μg/mL, indicating that the control antibody could effectively block the interaction between CD155 and TIGIT, thereby reversing the TIGIT negative regulation signal and activating the effector T cell response. Fig. 11 Antibody Neutralizing TGF-β Assay 5. Other Immunomodulation (OIM) 5.1. Neutralization experiment TGF-β can induce tumor cells to express specific cytokines, neutralize TGF-β, and reduce the secretion of corresponding cytokines. As shown in Fig. 12, after treatment with the candidate antibody, the secretion of IL-11 was significantly reduced, indicating that TGF-β was effectively neutralized and the candidate antibody had good biological activity. Fig. 12 Antibody Neutralizing TGF-β Assay 5.2. TGF-β luciferase reporter system Neutralizing TGF-β can effectively block the downstream SMAD phosphorylation signaling pathway. As shown in Fig. 13, the fluorescence intensity of cells was significantly decreased under the control antibody treatment, indicating that the control antibody effectively neutralized TGF-β, thereby reducing the TGF-β-mediated SMAD phosphorylation signal. Fig. 13 TGF-β luciferase reporter system 5.3. Neutrophil chemotaxis assay Interleukin 8, a member of the CXC family of chemokines, is a powerful neutrophil chemotaxis involved in the migration of neutrophils to sites of inflammation. As shown in Fig. 14, Candidate 1-4 blocked the binding of IL-8 to CXCR1 and CXCR2 and inhibited the chemotacticization of neutrophils. Fig. 14 Neutrophil chemotaxis assay Service List Categories Subclasses Categories Subclasses 1. Primary cell isolation 1.1 Human/Mouse CD3+ cells 5. Cell killing 5.1 ADCC 1.2 Human/Mouse CD4+ cells 5.2 CDC 1.3 Human/Mouse CD8+ cells 5.3 ADCP 1.4 Human/Mouse CD14+ cells 5.4 Phagocytosis 1.5 Human CD33+ cells 5.5 Antibodies kill directly 1.6 Human Treg cells 6. Antibody Endocytosis Detection 6.1 Fab-Zap method 2. Primary cell induction 2.1 Macrophages 6.2 double fluorescence permeation method 2.2 dendritic cells 6.3 ADC 3. Streaming Screening 3.1 Affinity 7. Costimulatory immune checkpoint 7.1 T cell costimulation-41BB 3.2 Block 7.2 T cell costimulation -OX40 3.3 Compete 7.3 Cell proliferation/proliferation inhibition 3.4 Epitope grouping 7.4 CD40/CD40L 4. Luciferase reporter system 4.1 PD1/PDL1 7.5 IL-2 factor detection 4.2 PD1/PDL2 7.6 IFN-γ factor detection 4.3 LAG3/MHCII 7.7 TNF-α factor detection 4.4 TIGIT/CD155 8. Suppressive immune checkpoint 8.1 PBMC activation 4.5 PVRIG/PVRL2 8.2 NK cell activation 4.6 CD40/CD40L 8.3 mixed lymphocytereaction 4.7 TGFβ2/TGFβRII 8.4 apoptosis 4.8 VEGF/VEGFR 9. Other immune- related-OIM 9.1 Cytokine neutralization -TGF-β 4.9 IL33/ST2nM 9.2 Cytokine neutralization -IL17 4.10 IL23/IL23R 9.3 neutrophil chemotaxis 4.11 CD3/CD3L 9.4 STAT3 phosphorylation 4.12 4-1BB 9.5 STAT5 phosphorylation 4.13 LILRB1/HLA-G 9.6 STAT6 phosphorylation 4.14 LILRB2/HLA-G 9.7 Tie2 phosphorylation 4.15 TNFα/TNFR2 9.8 Enzyme activity detection 4.16 ADCC 10. Personalization 10.1 Developed under patent 4.17 ADCP 10.2 Developed from the literature 4.18 Phosphorylation 10.3 Developed according

Service Contents

Service Names

Service Details

Client material

Deliverables and

Standards

Time

In vitro pharmacodynamic method development In vitro pharmacodynamic models for specific targets 1. Reference antibody
2. Mechanism of action

A stable system that can evaluate drug efficacy

4-8 weeks

In vitro drug efficacy sample detection Detection of In vitro harmacodynamic function of candidate molecules

1. Reference antibody

2. Candidate molecules

Candidate Molecular Efficacy Evaluation Report

1-2 weeks


Service Highlights
  • 1. Diverse tumor target selection
    1. It can evaluate any tumor treatment drug target and different drug mechanisms without fear of difficulty.

  • 2. Well-established in vitro evaluation system
    1. in vitro evaluation system.
  • 3. Extensive primary cell models
    1. A full range of human/mouse primary cells resource, presenting the in vivo mechanism of action to the greatest extent in vitro.

  • 4. Customized development with high success rate
    1. The multi-dimensional pharmacodynamic evaluation system can be customized according to the target pharmacodynamic mechanism.

  • 5. High standard quality system
    1. Evaluate the efficacy model from dimensions such as linearity, precision, accuracy, and stability to ensure higher quality.


Case Studies
1. Primary Cell Isolation

As shown in Fig. 1, PBMCs were isolated and purified to obtain different types of primary cells such as CD4+ T, CD3+ T, Monocyte, etc. The purity of the sorted cells usually reached 95% or even higher, which provides fully guarantee for the next step of efficacy screening.


Fig. 1 Isolation of primary human cells

2. Tumor-specific antigen (TSA)

2.1. Endocytic activity assay

Fab-Zap is a small molecule inhibitor that enters cells through antibody-mediated endocytosis and inhibits cell growth. As shown in Fig. 2, after the target cells were treated with the control antibody, it was detected by LDH method and found that the cell growth was significantly inhibited, and the EC50 value was 0.096 μg/mL, indicating that the control antibody had good endocytotic activity.


Fig. 2 Endocytosis activity Assay


2.2. ADCC determination

SK-BR-3 is a breast cancer cell line that expresses HER2. T drugs targeting HER2 have a strong ADCC effect. In this ADCC experiment, the effector cells were PBMC cells and the target cells were SK-BR-3 cells. As shown in Fig. 3, the cell lysis rate increased significantly under the action of T drug measured by LDH method, and the EC50 value was 0.023 μg/mL, indicating that T drug mediated a strong target cell killing effect.


Fig. 3 ADCC activity assay


2.3. CDC assay

Antibodies can effectively induce complement-dependent killing effects, causing strong damage to cells. As shown in Fig. 4, the cell lysis rate increased significantly after the target cells were treated with a control antibody, and the EC50 value was 27.90 nM, indicating that the candidate antibody could induce a strong CDC effect, resulting in massive cell death.



Fig. 4 CDC activity assay


2.4. Phagocytosis experiment

In phagocytosis experiments, blocking the “don't eat me” signal by antibodies can promote the phagocytosis of target cells by macrophages. As shown in Fig. 5, it was found by FACS that the phagocytosis of macrophages on target cells were significantly enhanced by control antibody treatment, and the EC50 value was 0.022 μg/mL, indicating that the candidate antibody had good blocking activity against the “don't eat me” signaling pathway.


Fig. 5 macrophage phagocytosis effect


2.5. Proliferation inhibition assay

In proliferation inhibition experiments, IL-4 promotes TF-1 cell proliferation, and IL-4 neutralization can inhibit IL-4-mediated cell proliferation. As shown in Fig. 6, cell proliferation was inhibited with an EC50 value of 0.54 μg/mL, indicating that the control antibody effectively inhibited the proliferation of TF-1 cells.


Fig. 6 Proliferation inhibition experiment

3. Costimulatory immune checkpoint (CIC)

The binding of CD40 to CD40L promoted the expression of CD95 and exacerbated apoptosis. As shown in Fig. 7, CD40 candidate antibodies A1-A7 significantly reduced the expression of CD95, indicating that these candidate antibodies blocked the binding of CD40 to CD40L and had good biological activity.


Fig. 7 Expression inhibition experiment

4. Inhibitory immune checkpoints (IIC)

4.1. MLR experiment

In MLR experiments, CD4+ T cells and DC cells of different donors were co-cultured. Anti-PD-L1 antibodies could effectively break the PD1/PD-L1 inhibitory signal, activate T cells, and promote the secretion of cytokines. As shown in Fig. 8, after treatment with candidate1, the secretion of IL-2 and IFN-γ in cells increased significantly, indicating that it had a highly similar biological activity to the 3 antibody drugs that had been marketed.


Fig. 8A IL-2 secretion detection


Fig. 8B IFN-γ secretion detection


4.2. Binding and blocking activity analysis at the cellular level

Fig. 9A showed a case using FACS to analyze the binding activity of antibodies to cell surface proteins, and Fig. 9B showed a case using FACS to analyze the blocking activity of antibodies binding to receptor ligands. All the lead antibody molecules exhibited stronger binding and blocking activities than the control antibody.


Fig. 9A Cell binding activity assay


Fig. 9B Cell blocking activity assay


4.3. Antibody epitope analysis

As shown in Fig. 10, Candidate 1-5 all competitively inhibited the binding of Target Ab to the target antigen,indicating that these six antibodies recognized the same epitope.


Fig. 10 Cell Competitive Binding Assay


4.4. TIGIT luciferase reporter system

TIGIT is a surface marker of deeply depletion of T cells, and blocking its negative regulatory signaling pathway with CD155 can effectively activate effector cells. As shown in Fig. 11, under the control antibody treatment, the fluorescence intensity of the cells was significantly enhanced, and the EC50 value was 0.67 μg/mL, indicating that the control antibody could effectively block the interaction between CD155 and TIGIT, thereby reversing the TIGIT negative regulation signal and activating the effector T cell response.


Fig. 11 Antibody Neutralizing TGF-β Assay

5. Other Immunomodulation (OIM)

5.1. Neutralization experiment

TGF-β can induce tumor cells to express specific cytokines, neutralize TGF-β, and reduce the secretion of corresponding cytokines. As shown in Fig. 12, after treatment with the candidate antibody, the secretion of IL-11 was significantly reduced, indicating that TGF-β was effectively neutralized and the candidate antibody had good biological activity.


Fig. 12 Antibody Neutralizing TGF-β Assay


5.2. TGF-β luciferase reporter system

Neutralizing TGF-β can effectively block the downstream SMAD phosphorylation signaling pathway. As shown in Fig. 13, the fluorescence intensity of cells was significantly decreased under the control antibody treatment, indicating that the control antibody effectively neutralized TGF-β, thereby reducing the TGF-β-mediated SMAD phosphorylation signal.


Fig. 13 TGF-β luciferase reporter system


5.3. Neutrophil chemotaxis assay

Interleukin 8, a member of the CXC family of chemokines, is a powerful neutrophil chemotaxis involved in the migration of neutrophils to sites of inflammation. As shown in Fig. 14, Candidate 1-4 blocked the binding of IL-8 to CXCR1 and CXCR2 and inhibited the chemotacticization of neutrophils.


Fig. 14 Neutrophil chemotaxis assay


Service List

Categories

Subclasses

Categories

Subclasses

1. Primary cell isolation

1.1 Human/Mouse CD3+ cells

5. Cell killing

5.1 ADCC

1.2 Human/Mouse CD4+ cells

5.2 CDC

1.3 Human/Mouse CD8+ cells

5.3 ADCP

1.4 Human/Mouse CD14+ cells

5.4 Phagocytosis

1.5 Human CD33+ cells

5.5 Antibodies kill directly

1.6 Human Treg cells

6. Antibody Endocytosis Detection

6.1 Fab-Zap method

2. Primary cell induction

2.1 Macrophages

6.2 double fluorescence permeation method

2.2 dendritic cells

6.3 ADC

3. Streaming Screening

3.1 Affinity

7. Costimulatory immun checkpoint

7.1 T cell costimulation-41BB

3.2 Block

7.2 T cell costimulation -OX40

3.3 Compete

7.3 Cell proliferation/proliferation

inhibition

3.4 Epitope grouping

7.4 CD40/CD40L

4. Luciferase reporter system

4.1 PD1/PDL1

7.5 IL-2 factor detection

4.2 PD1/PDL2

7.6 IFN-γ factor detection

4.3 LAG3/MHCII

7.7 TNF-α factor detection

4.4 TIGIT/CD155

8. Suppressive immune checkpoint

8.1 PBMC activation

4.5 PVRIG/PVRL2

8.2 NK cell activation

4.6 CD40/CD40L

8.3 mixed lymphocytereaction

4.7 TGFβ2/TGFβRII

8.4 apoptosis

4.8 VEGF/VEGFR

9. Other immune- related-OIM

9.1 Cytokine neutralization -TGF-β

4.9 IL33/ST2nM

9.2 Cytokine neutralization -IL17

4.10 IL23/IL23R

9.3 neutrophil chemotaxis

4.11 CD3/CD3L

9.4 STAT3 phosphorylation

4.12 4-1BB

9.5 STAT5 phosphorylation

4.13 LILRB1/HLA-G

9.6 STAT6 phosphorylation

4.14 LILRB2/HLA-G

9.7 Tie2 phosphorylation

4.15 TNFα/TNFR2

9.8 Enzyme activity detection

4.16 ADCC

10. Personalization

10.1 Developed under patent

4.17 ADCP

10.2 Developed from the literature

4.18 Phosphorylation

10.3 Developed according