6 customized solutionsComprehensive upgrade in detection methods
Antibody-Drug Conjugates (ADCs) are biopharmaceutical drugs that combine cytotoxic drugs with monoclonal antibodies. Efficient antibody internalization is critical for developing effective ADCs, which is influenced by antigen density, antibody affinity, and binding site. Varying levels of internalization efficiency can occur among antibodies targeting the same antigen. Screening for high-affinity and high-efficiency internalizing antibodies is crucial for safety and targeting of ADC drugs. ProBio's antibody internalization assay platforms facilitate rapid and high-throughput screening of potential ADC molecules.
GenScript ProBio provides comprehensive solutions for antibody internalization, cell cytotoxicity assays and bystander effect, including 6 different assay formats tailored to meet the specific needs of our clients. Our services cover naked antibodies as well as ADCs.
The Incucyte® live-cell analysis system enables direct detection of antibody internalization in a 96-well plate. By one step of dye addition, a rapid, kinetic and high-throughput analysis of antibody internalization can be achieved.
✓ Single experiment evaluates time-dependent changes and concentration-dependent curves of antibody internalization, suitable for pharmacological dynamic quantitative analysis.
✓ Real-time observation of live cells, with continuous monitoring up to 72 hours with a minimum detection interval of 15 minutes.
✓ Applicable for high-throughput screening.
Fabfluor-pH is a fluorescence dye that is sensitive to changes in pH. It is commonly used in combination with antibodies to create Fab-Ab-Dye complexes. When these complexes are in culture media at a pH of 7.0, they exhibit minimal fluorescence. However, when they are internalized into the nucleus or lysosome, where the pH environment drops to a range of pH 4.7 to 6.3, they emit a vivid red fluorescence. By utilizing the Incucyte® live-cell analysis system, we can track the real-time dynamic changes that occur during antibody internalization within a 96-well plate.
Live-cell analyzer Incucyte
Fig1. Live cell imaging-based assay
Figure 2a depicts the internalization of anti-ROR1 antibody (UC-961) or hIgG1 isotype control labeled with Incucyte® FabFluor, where the internalization level of anti-ROR1 antibody gradually increases with time. Figure 2b shows CHO-K1/DDR1 cells treated with anti-DDR1 antibody (AB3E3) rapidly increasing in the red target area and displaying a dose-response curve.
Figure 2: real-time live cell imaging-based internalization screening assay.
GenScript ProBio employs a recombinant immunotoxin (IT) conjugated to the test antibody. Upon internalization of the mAb-IT complex by target cells, the IT is released into the cytosol, causing ADP-ribosylation of elongation factor (EF)-2, which subsequently inhibits protein translation and ultimately leads to cell cytotoxicity. The internalization efficiency of the antibody is assessed by measuring the cell killing effect.
Figure 3: Mechanism of immunotoxin induced cytotoxicity
Strong specificity: only when the mAb-IT conjugate is internalized by target cells, a significant decrease in cell viability can be observed, and free immunotoxins do not cause cell toxicity.
High efficiency: mAb-IT conjugates have a more stable molecular weight and higher internalization efficiency compared to the traditional mAb-ZAP method.
Wide applicability: suitable for early screening of antibodies in hybridoma supernatant ; applicable to different species (such as human, mouse, rabbit, and goat), and different antibody subtypes.
Low cost: easy to prepare while simulating the MOA of ADC drugs.
Figure 4: Toxin-based cytotoxicity assay.
Dose-dependent internalization rate of trastuzumab was determined by the immunotoxin-induced cell toxicity upon binding to SK-BR-3 cells after 24-48 hours of incubation.
pH probe-based assay: antibodies labeled with pH probes sensitive to pH are only able to release fluorescence signals when they are engulfed by target cells and enter acidic intracellular compartments. The fluorescence signals can be captured and analyzed using flow cytometry.
Cell surface fluorescence quenching: the internalization level of the test antibody can be traced by incubating at 37°C and 4°C respectively, and using the secondary antibody labeling method to track the antibodies left on the surface.
pH probe-based assay
These two classical internalization screening methods have been widely applied in ADC naked antibody screening due to their simplicity and flow cytometry-based detection. However, the endpoint detection nature of these methods can limit their ability to capture dynamic changes sequentially and may result in changes in cell status due to a lack of environmental control, thereby affecting the accuracy of the results.
Case study 1: cell surface fluorescence quenching
Analysis of internalization internalization can be performed by quantifying the remaining antibody on the cell surface using secondary antibodies. This indirect method can reflect the internalization efficiency of the cells.
Case study 2: pH probe-based assay
Antibodies labelled with pH probes undergo internalization and sequentially translocate to the lysosomes, where the acidic pH induces fluorescence enhancement of the pH probes.
Cell viability assays can be used to measure the ability of cytotoxic compounds to cause cellular damage or death, and are a critical factor in evaluating the efficacy and intended use of such compounds.
Quantification of cell viability using the CellTiter-Glo® assay kit revealed that the dose-response curve data exhibited suppression of SK-BR-3 cell viability after addition of the tested ADC samples.