Our services are critical for optimizing the design and functionality of Antibody-Drug Conjugates (ADCs). These services provide comprehensive insights into ADC targeting, internalization, cytotoxicity, and stability, ensuring that each ADC is fine-tuned for maximum therapeutic efficacy and safety. By addressing key factors such as binding specificity, payload delivery, and drug stability, we help accelerate the development of highly effective ADCs for targeted cancer therapies.
Target-Specific Binding and Characterization: We ensure precise targeting by evaluating antigen expression, binding affinity, and antibody specificity, minimizing off-target effects.
Internalization and Payload Delivery: We assess ADC internalization efficiency to confirm that the payload is effectively delivered into target cells for maximum therapeutic impact.
Cytotoxicity and Mechanism of Action: Our assays measure the cytotoxic potency of the ADC, providing insights into how effectively it kills target cells while limiting damage to healthy cells.
ADC Design Optimization and Stability: We evaluate the stability and drug-to-antibody ratio (DAR) to ensure the ADC maintains its efficacy and integrity throughout its lifecycle.
We provide comprehensive ADC conjugation and characterization services, including Size-Exclusion Chromatography (SEC) for analyzing ADC size and aggregation state, Hydrophobic Interaction Chromatography (HIC) for characterizing ADC hydrophobicity and conjugation status, and free payload/linker-payload (LP) detection for identifying and quantifying unbound payloads.
DAR Analysis (Drug-to-Antibody Ratio) quantifies the average number of drug molecules conjugated to each antibody molecule. It is crucial for optimizing the therapeutic efficacy and safety profile of the ADC.
Figure 1. DAR characterization of DS-8201.
Our service provides precise measurement of target antigen levels on cell surfaces, crucial for identifying and validating new ADC targets. Utilizing advanced flow cytometry, we quantify antigen density on various cell types, ensuring target suitability for ADC development. This detailed analysis supports the selection of high-value targets, enhancing the specificity and efficacy of next-generation cancer therapies.
Figure 2. Quantitative analysis of HER2 antigen membrane expression in different cancer cell lines with high/medium/low/negative HER2 expression. A. Histogram of Calibration Bead populations. B. Standard curve. C. Assessment of HER2 expression in different cancer cell lines. D. Quantitative analysis of HER2 membrane expression. SABC, Specific Antibody-Binding Capacity.
SPR provides detailed analysis of the binding interactions between therapeutic antibodies and their target antigens, which is essential for designing effective ADCs. The affinity of ADC should be similar as that of naked antibody.
Figure 3. SPR analysis of Trastuzumab (Herceptin) binding affinity to HER2-ECD antigen (left) and Cetuximab (Erbitux) binding affinity to EGFR-ECD antigen (right) using Biacore 8K.
Flow cytometry is a powerful technique for analyzing the binding of antibodies to target antigens, on the surface of different cell lines. This service is essential for evaluating the binding affinity and specificity of antibodies and ADCs across various cell lines. The data is essential for optimizing ADC development, ensuring targeted delivery to specific cancer cells, and minimizing off-target effects.
Figure 4. Cellular affinity comparison of ADCs and corresponding antibodies in SKBR3 (left) and MCF-7 (right) cell lines. Tested samples include T-DM1, DS-8201, RC-48, Trastuzumab, and Disitamab.
Figure 5. Tubulin Polymerization Assay of HCC1954 (HER2+) cell line treated with T-DM1, Trastuzumab, and DM1.
Our Internalization Assay Service utilizes Incucyte live-cell imaging, flow cytometry, and high-content analysis to provide detailed, real-time analysis of ADC internalization into target cancer cells. By combining these advanced technologies, we offer precise, quantitative insights into the uptake and intracellular trafficking of fluorescently labeled ADCs, ensuring optimal design and efficacy of your ADC therapies.
1. Incucyte provides real-time, continuous monitoring of ADC internalization in live cells. This system captures dynamic processes, allowing visualization of ADC internalization and intracellular trafficking over extended periods, which helps in understanding the temporal aspects of ADC behavior.
Figure 6. (A) Internalization activity of ADCs and the corresponding antibodies in SKBR3 cell line with 12 hours incubation, detected by IncuCyte. Tested samples include DS-8201, RC-48, Trastuzumab, and Disitamab. (B) Internalization activity of Disitamab and RC-48 in SKBR3 cell line detected by IncuCyte. Internalization was monitored from 1 to 24 hrs.
2. Temperature shift-based internalization assay is a classical method to evaluate the antibody internalization rate, by calculating MFI of the surface antibody in two groups at 37°C and 4°C respectively.
Figure 7. Internalization rate of Disitamab in SKBR3 cell line was measured at different incubation time points.
3. The internalization efficiency of the antibody can be assessed by measuring the cell killing effect, by using Toxin-based cytotoxicity assay.
Figure 8. Internalization activity of Disitamab in SKBR3 cell line was evaluated by DT3C assay.
4. The pH-indicator based internalization flow cytometry assay provides a specific and accurate method for measuring ADC internalization. By utilizing pH-sensitive dyes, this technique ensures that only ADCs internalized into the acidic compartments of cells are detected, offering precise insights into the efficiency and kinetics of ADC internalization and trafficking.
Figure 9. Internalization activity of Trastuzumab in SKBR3 cell line detected by flow cytometry.
5. High-Content Analysis combines automated microscopy and image analysis to provide detailed, multiparametric data on ADC internalization at the single-cell level. It allows for in-depth analysis of intracellular localization, colocalization with organelles, and morphological changes, offering comprehensive insights into the mechanisms of ADC internalization.
Figure 10. High-Content Analysis of ADC Internalization in cells. The images show cells treated with a fluorescently labeled antibody (red) and the lysosome marker (green). Nuclei are stained with Hoechst dye (blue). The merged image demonstrates the internalization and colocalization of the Antibody within the cells, highlighting its intracellular trafficking and accumulation in specific cellular compartments.
Our live cell imaging-based cytotoxicity assay service provides real-time, kinetic analysis of cell viability and cytotoxic effects of therapeutic agents, including ADCs. Using the Incucyte live-cell imaging system, we offer a detailed and dynamic view of how your ADC affect cancer cell populations over time.
Figure 11. Cytotoxicity of DS-8201 and DXd in the NCI-N87 cell line. The IC50 value was approximately 10 times lower after conjugation to the antibody.
ADC Cytotoxicity Cell Panel is specifically designed for evaluating the efficacy of ADCs for specific targets using cell viability assays. For different ADC targets, we may supply tumor cell lines of different caner types with different expression levels.
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