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
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Equipment
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Application
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Mechanism
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Fab-zap assay
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Spectrometer
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Preliminary antibody screening
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Coupling a small molecule inhibitor that enters cells through antibody mediated endocytosis and inhibits cell growth.
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Dual fluorescence permeabilization
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Flow cytometer
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ADC screening
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Antibody triggers endocytosis at 37°C and the amount of endocytosed antibody is detected by dual fluorescence permeabilization staining.
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pH-rodo assay
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Spectrometer
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Preliminary antibody screening
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Antibodies labeled with pH-sensitive dyes are endocytosed by target cells and are acidified to release fluorescent signals.
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Confocal microscopy
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Confocal microscopy
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ADC screening
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Laser scanning confocal microscopy real-time detects transportation and endocytosis rate of ADCs drug in cells.
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Fluorescence quenching assay
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Flow cytometer
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Preliminary antibody screening
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Antibody triggers endocytosis at 37°C, quenching extracellula fluorescence. The amount of endocytosed antibody is detected by permeabilization staining.
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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
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ADC1
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ADC2
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Benchmark
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DAR value
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3.6
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3.6
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3.5
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Purity(SEC)
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99.7%
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99.4%
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99.9%
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Conc.(mg/mL)
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4.1
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4.8
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5.7
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Free drug
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< LOD*
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< LOD*
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N.D.*
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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.