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Service Overviews
Based on “Integrated and Innovative Antibody-drug Conjugates R&D Platform” and “Super Trillion Antibody Library Platform”, we can customize the screening of the best antibody suitable for ADC drug development for partners, and can also provide overall R&D services for ADC drugs from target to Pre-clinical candidates.

Service Overview Based on “Integrated and Innovative Antibody-drug Conjugates R&D Platform” and “Super Trillion Antibody Library Platform”, we can customize the screening of the best antibody suitable for ADC drug development for partners, and can also provide overall R&D services for ADC drugs from target to Pre-clinical candidates. Service Contents Name Content Provided by Clients Deliverables and Criteria Timeline(month) ADC development 1. Raw material preparation 2. Antibody generation 3. Antibody engineering 4. Drug conjugation 5. In vitro efficacy screening 6. In vivo efficacy evaluation 1. Targets 2. Mechanism 3. Linker-drugs 1. Data Reports: phase reports and project data packages 2. Drug Efficacy: candidate ADCs are comparable to or better than positive controls 9-13 ADC Drug Development Phase Raw Material Preparation 1. Human/monkey antigens, ligands and other proteins 2. Positive controls 3. Overexpression cell lines Targets 1. SDS-PAGE: reduced purity>85% 2. ELISA binding: result is in line with literature reports 3. FACS binding: target expression level 10 times higher than the background 1 Antibody Generation 1. Over-trillion antibody phage-display library, mouse/alpaca immunization 2. Affinity maturation (if required) 3. Humanization (if required) Raw material 1. ≥20 unique sequences with cross-reaction between human and monkey 2. Affinity of engineered antibody is 5-20 times higher than before 3. >90% humanization, and the efficacy is similar or better than parental antibodies 1-2 1 2 Drug Conjugation 1. Conjugation between antibody and drugs 2. Quality assessment of ADCs Purified ADCs 1. Qualified ADCs in purity, binding activity 2. In vitro efficacy screening for candidate ADCs by cell-based assay 3. Data reports: ADC conjugation reports 1 In vitro model screening 1. Method development: two ADC screening related methods, e.g. internalization or ADC kill 2. Candidates screening Purified candidates or ADCs 1. Efficacy screening: ≥3 equivalent or superior ADCs 2. Data reports: in vitro screening reports 1-2 In vivo model screening 1Method development involving 2 models of target conditions 2. ADC assays involving 2 models of target conditions 3. PK assay Preferred ADCs 1. Efficacy screening: ≥3 equivalent or superior ADCs 2. Data reports: in vivo drug efficacy screening reports 2-4 Service Highlights 1. Integrated platform for innovative ADC discovery 10 functional modules and 40+ sub-platforms cover the whole antibody discovery process. Professional project management system, seamless integration among all modules, and high efficiency for PCC completion (12 months). 2. Unique multi-pathway antibody generation platform Seven trillion large-capacity antibody library, thousands of lead molecules for conventional targets can be obtained. Diverse antibody sources: natural B-cell antibodies, B-cell rearranged antibodies, synthetic and semi-synthetic antibodies, mouse immunization library, alpaca immunization library. Full coverage of molecular formats: full antibodies, single domain antibodies, bispecific antibodies, mouse monoclonal antibodies, rat monoclonal antibodies. 3. Rapid & high-throughput conjugation platform High-throughput, stable and controlled conjugation platform for efficient screening of lead antibodies. Multiple conjugation methods evaluation: flexible combination of payloads and linkers. 4. Experienced & professional research team 20+ scientists in various fields, systematic and scientific project development. 100+ projects development experience, high rate for successful project deliver. 5. Flexible customization services Customized innovative ADC R&D with diversified service forms. Flexible combination of ADC three core elements to suit different R&D needs. Case Presentations 1. In vitro Efficacy Evaluation based on ADC mechanism 1.1. Binding activity on different Cells and Epitope Analysis Binding activity on different cells method:Flow cytometry assay is applied for the binding ability of different candidates on different cells, include tumor cells and overexpressed cell lines. Binding activity on different cells result: Candidates are ranked by affinity to select the candidates which exhibited superior or similar binding activity to the positive controls. Fig. 1 Binding activity on different Cells Epitope analysis method: Candidates were labeled with biotins, then they are analysed by competitive binding method, and can be grouped by epitopes. Epitope analysis result: All candidates competitively inhibit the binding of target antibody to target antigen, indicating that the candidates recognize the similar epitope. Fig. 2 Competitive binding assay 1.2. Multiple Internalization Assays Testing Method Equipment Application Mechanism Fab-Zap Assay Spectrometer Preliminary antibody screening Coupling a small molecule inhibitor that enters cells through antibody-mediated endocytosis and inhibits cell growth. Dual Fluorescence Permeabilization Flow cytometer ADC screening Antibody triggers endocytosis at 37°C and the amount of endocytosed antibody is detected by dual fluorescence permeabilization staining. pH-Rodo Assay Spectrometer Preliminary antibody screening Antibodies labeled with pH-sensitive dyes are endocytosed by target cells and are acidified to release fluorescent signals. Confocal Microscopy Confocal Microscopy ADC screening Laser scanning confocal microscopy real-time detects transportation and endocytosis rate of ADCs drug in cells. Fluorescence Quenching Assay Flow cytometer Preliminary antibody screening Antibody triggers endocytosis at 37°C, quenching extracellular fluorescence. The amount of endocytosed antibody is detected by permeabilization staining. doi.org/10.1007/s11912-022-01266-4 Fig. 3 Fab-Zap assay 1.3. ADCs Killing Experiment 1.3.1. Conjugating of Different Toxins for Killing Experiment Diverse conjugating methods. Multiple linker-toxin conjugating and screening. Standardized in vitro evaluation methods for ADCs. Fig. 4 Conjugating of toxin DM1 killing experiment Fig. 5 Conjugating of toxin MMAE killing experiment Fig. 6 Conjugating of toxin MMAF killing experiment 1.3.2. Head-to-head Comparison of ADC Killing Activity for Candidates Method: Toxin (MMAE) is coupled to the candidate molecule by cysteamine, and after conjugating, relevant quality control analyses are performed, followed by ADC killing activity analysis. Result: : Through head-to-head comparison of the ADC killing activity after conjugating, the candidates showed superior or similar ADC killing activity to the positive control. Fig. 7 ADC1 Fig. 8 ADC2 ADC ADC1 ADC2 Benchmark DAR Value 3.6 3.6 3.5 Purity(SEC) 99.7% 99.4% 99.9% Conc.(mg/mL) 4.1 4.8 5.7 Free Drug < LOD* < LOD* N.D.* Background: After in vitro screening, control and candidate molecules are usually coupled and evaluated by quality inspection then followed by in vivo screening. Method: : Mice are tumor-loaded, grouped and dosed according to a predetermined protocol. The tumor growth state is observed and recorded. Result: After multiple dosing, mAb2-MMAE and the benchmark-MMAE showed similar tumor suppressive effects; the tumor suppressive effect of the naked antibody was weaker than that of the ADC molecules. Conclusion: The candidate ADC shows superior anti-tumor effect than the benchmark, and the efficacy is dependent on the payload. Fig. 9 ADC in vivo Efficacy Evaluation Additional Services Based on the targeting of antibodies, innovative antibodies will become the core elements of drug design in the future.

Service Contents

Service

Service Details

Client Provides

Deliverables and Srandards

Time

ADC development

1. Raw material preparation

2. Antibody generation

3. Antibody engineering

4. Drug conjugation
5. In vitro efficacy screening
6. In vivo efficacy evaluation

1. Targets

2. Mechanism

3. Linker-drugs

1. Data Reports: phase reports and project data packages

2. Drug Efficacy: candidate ADCs are comparable to or better than positive controls

9-13 months

ADC Drug Development Phase

Raw material preparation

1. Human/monkey antigens, ligands and other proteins
2. Positive controls
3. Overexpression cell lines

Targets

1. SDS-PAGE: reduced purity>85%
2. ELISA binding: result is in line with literature reports
3. FACS binding: target expression level 10 times higher than the background

1 months

Antibody generation

1. Over-trillion antibody phage-display library, mouse/alpaca immunization
2. Affinity maturation (if required)
3. Humanization (if required)

Raw material

1. ≥20 unique sequences with cross-reaction between human and monkey

2. Affinity of engineered antibody is 5-20 times higher than before

3. >90% humanization, and the efficacy is similar or better than parental antibodies

1-2 months

1 months

2 months

Drug conjugation

1. Conjugation between antibody and drugs

2. Quality assessment of ADCs

Purified ADCs

1. Qualified ADCs in purity, binding activity
2. In vitro efficacy screening for candidate ADCs by cell-based assay
3. Data reports: ADC conjugation reports

1 months

In vitro model screening

1. Method development: two ADC screening related methods, etc. internalization or ADC kill
2. Candidates screening

Purified candidates or ADCs

1. Efficacy screening: ≥3 equivalent or superior ADCs
2. Data reports: in vitro screening reports

1-2 months

In vivo model screening

1. Method development involving 2 models of target conditions

2. ADC assays involving 2 models of target conditions

3. PK assay

Preferred ADCs

1. Efficacy screening: ≥3 equivalent or superior ADCs
2. Data reports: in vivo drug efficacy screening reports

2-4 months


Service Highlights
  • 1. Integrated platform for innovative ADC discovery
    1. 10 functional modules and 40+ sub-platforms cover the whole antibody discovery process.
    2. Professional project management system, seamless integration among all modules, and high efficiency for PCC completion (12 months).
  • 2. Unique multi-pathway antibody generation platform
    1. Seven trillion large-capacity antibody library, thousands of lead molecules for conventional targets can be obtained.
    2. Diverse antibody sources: natural B-cell antibodies, B-cell rearranged antibodies, synthetic and semi-synthetic antibodies, mouse immunization library, alpaca immunization library.
    3. Full coverage of molecular formats: full antibodies, single domain antibodies, bispecific antibodies, mouse monoclonal antibodies, rat monoclonal antibodies.
  • 3. Rapid & high-throughput conjugation platform
    1. High-throughput, stable and controlled conjugation platform for efficient screening of lead antibodies.
    2. Multiple conjugation methods evaluation: flexible combination of payloads and linkers.
  • 4. Experienced & professional research team
    1. 20+ scientists in various fields, systematic and scientific project development.
    2. 100+ projects development experience, high rate for successful project deliver.
  • 5. Flexible customization services
    1. Customized innovative ADC R&D with diversified service forms.
    2. Flexible combination of ADC three core elements to suit different R&D needs.

Case Studies
1. In vitro Efficacy Evaluation based on ADC mechanism

1.1. Binding activity on different Cells and Epitope Analysis

Binding activity on different cells method:Flow cytometry assay is applied for the binding ability of different candidates on different cells, include tumor cells and overexpressed cell lines.

Binding activity on different cells result: Candidates are ranked by affinity to select the candidates which exhibited superior or similar binding activity to the positive controls.


Fig. 1 Binding activity on different Cells


Epitope analysis method: Candidates were labeled with biotins, then they are analysed by competitive binding method, and can be grouped by epitopes.

Epitope analysis result: All candidates competitively inhibit the binding of target antibody to target antigen, indicating that the candidates recognize the similar epitope.


Fig. 2 Competitive binding assay


1.2. Multiple Internalization Assays


Testing Method

Equipment

Application

Mechanism

Fab-zap assay

Spectrometer

Preliminary antibody screening

Coupling a small molecule inhibitor that enters cells through antibody mediated endocytosis and inhibits cell growth.

Dual fluorescence permeabilization

Flow cytometer 

ADC screening

Antibody triggers endocytosis at 37°C and the amount of endocytosed antibody is detected by dual fluorescence permeabilization staining.

pH-rodo assay

Spectrometer

Preliminary antibody screening

Antibodies labeled with pH-sensitive dyes are endocytosed by target cells and are acidified to release fluorescent signals.

Confocal microscopy

Confocal microscopy

ADC screening

Laser scanning confocal microscopy real-time detects transportation and endocytosis rate of ADCs drug in cells.

Fluorescence quenching assay

Flow cytometer

Preliminary antibody screening

Antibody triggers endocytosis at 37°C, quenching extracellula fluorescence. The amount of endocytosed antibody is detected by permeabilization staining.


doi.org/10.1007/s11912-022-01266-4


Fig. 3 Fab-Zap assay


1.3. ADCs Killing Experiment

1.3.1. Conjugating of Different Toxins for Killing Experiment

• Diverse conjugating methods.

• Multiple linker-toxin conjugating and screening.

• Standardized in vitro evaluation methods for ADCs.


Fig. 4 Conjugating of toxin DM1 killing experiment


Fig. 5 Conjugating of toxin MMAE killing experiment


Fig. 6 Conjugating of toxin MMAF killing experiment


1.3.2. Head-to-head Comparison of ADC Killing Activity for Candidates

Methods: Toxin (MMAE) is coupled to the candidate molecule by cysteamine, and after conjugating, relevant quality control analyses are performed, followed by ADC killing activity analysis.

Results: : Through head-to-head comparison of the ADC killing activity after conjugating, the candidates showed superior or similar ADC killing activity to the positive control.


Fig. 7 ADC1


Fig. 8 ADC2


ADC

ADC1

ADC2

Benchmark

DAR value

3.6

3.6

3.5

Purity(SEC)

99.7%

99.4%

99.9%

Conc.(mg/mL)

4.1

4.8

5.7

Free drug

< LOD*

< LOD*

N.D.*


Background: After in vitro screening, control and candidate molecules are usually coupled and evaluated by quality inspection then followed by in vivo screening.

Methods: Mice are tumor-loaded, grouped and dosed according to a predetermined protocol. The tumor growth state is observed and recorded.

Results: After multiple dosing, mAb2-MMAE and the benchmark-MMAE showed similar tumor suppressive effects; the tumor suppressive effect of the naked antibody was weaker than that of the ADC molecules.

Conclusions: The candidate ADC shows superior anti-tumor effect than the benchmark, and the efficacy is dependent on the payload.


Fig. 9 ADC in vivo Efficacy Evaluation


Additional Services

Based on the targeting of antibodies, innovative antibodies will become the core elements of drug design in the future.