Antibody Internalization Assay Reagents

ADC Antibody Internalization

Antibody-drug conjugates (ADCs) are a powerful class of biopharmaceuticals designed for targeted drug delivery, particularly in cancer therapy. ADCs consist of monoclonal antibodies (mAbs) linked to potent cytotoxic agents, allowing selective targeting of cancer cells while sparing healthy tissues. For ADCs to be effective, antibody internalization plays a crucial role — enabling the antibody-receptor complex to be internalized into the cell and directing the cytotoxic payload to its intracellular target, typically the lysosome, for drug release and subsequent tumor cell death.

The efficiency of ADC internalization can vary significantly even when antibodies recognize the same target epitope. Therefore, evaluating antibody internalization is a critical step in ADC development, helping identify the most suitable candidates for drug conjugation early in the discovery process.

DIMA’s Reagents

DIMA Biotech offers two types of internalization assay reagents optimized for high sensitivity, stability, cost-effectiveness, and broad IgG subtype compatibility:

pH-Sensitive IgG Labeling Reagents: Uses a pH-sensitive fluorescent dye to monitor antibody internalization in real-time with increased fluorescence in acidic compartments like endosomes.
Payload-Conjugated IgG labeling Reagent: Mimics the ADC mechanism by labeling IgG molecules with commonly used ADC payloads, combining internalization detection with cytotoxicity evaluation.

DiTag™ pH-sensitive IgG labeling reagents offer a convenient way to measure antibody internalization. These reagents use a pH-sensitive fluorescent dye conjugated Fc binding protein, which forms a complex with IgG antibodies. Upon internalization, the acidic environment enhances the fluorescence signal, indicating internalization activity. By measuring fluorescence intensity, researchers can assess internalization efficiency and gain insights into antibody uptake mechanisms, helping optimize antibody-based therapies and drug delivery systems.

The fluorescent signal from GPRC5D ADC BMK-AME100002 conjugate is only detected in GPRC5D positive cells (K562-GPRC5D stable expression cell line) and not in parental K562 cells, indicating specific internalization.

Comparison of internalization efficiency between AME100002 and a competitor reagent on GPRC5D-positive cells (K562-GPRC5D stable expression cell line). AME100002 requires less reagent and shorter incubation time while demonstrating higher sensitivity. It is the most cost-effective pH-sensitive fluorescence IgG labeling reagent available. This pH-sensitive IgG labeling reagent is compatible with human IgG1, IgG2, IgG3, IgG4, rabbit IgG, and mouse IgG subtypes (IgG1, IgG2a, IgG2b, IgG3).

Payload-conjugated IgG labeling reagents use a payload-conjugated Fc binding protein to label various IgG antibodies. Once the IgG-payload complex is added to the cells, the cell inhibition rate for each sample is determined using the CCK8 method. This approach mimics the real ADC mechanism, where the antibody delivers the payload into the cells, resulting in cell death. By measuring the cell inhibition, researchers can evaluate the effectiveness of the candidate mAbs and gain insights into the potential of ADCs for therapeutic applications.

reagents-ame100003 internalization of labeled b7h3 adc antibody cck8

Cell inhibition rate of HeLa cells detected using the CCK8 method. B7H3 ADC BMK was mixed with DiTag™ MMAE IgG labeling reagent (Cat. No. AME100003) to form a complex, which was applied to HeLa cells for the inhibition test. The results were compared to the control, where Isotype Control IgG was mixed with AME100003 and applied to the cells. The IC50 of B7H3 ADC BMK is 66 ng/ml.

Accelerated stability test of AME100003. Following lyophilization, samples were stored at -20°C (black), 37°C for 1 day (red), 37°C for 2 days (blue), and 37°C for 3 days (green), separately. Upon reconstitution, the cell inhibition rate of each sample was determined using the CCK8 method. The data indicate excellent stability for all samples.