• Assess internalization of your ADC or antibody with iQ’s pH-indicator or fluorescence quenching internalization assay
• Personalized experimental design ensures that the most relevant data for your molecule are generated, including adaptations for primary cells or cell lines
• All assays are performed with high-quality reagents and strict controls by experienced immunologists
• Data are provided in appropriate formats, including PowerPoint slides and/or direct data files

About iQ Biosciences’ ADC or Antibody Internalization Assay Service

Antibody-drug conjugates (ADCs) are currently used as targeted therapies against cancer and autoimmune indications. ADCs consist of an antibody plus a cytotoxic payload (e.g., chemotherapeutic or immunomodulatory stimulus) attached through a linker conjugate. This design results in therapeutic molecules with increased efficacy due to their specificity and potency toward cells that express the antigen of interest.

Mechanistically, there are 3 main steps for the ADC to properly promote its effects: 1) ADC binding to cells that express its cognate antigen, 2) ADC/antigen complex internalization through receptor-mediated endocytosis, and 3) release of the small molecule into the cytoplasm of the cell to promote its cytotoxic or immunomodulatory effects. The release of the payload is dependent on the low pH environment of the lysosomes and/or the presence of proteases within them that facilitate cleavage of the linker.

As indicated above, internalization of the ADC is a key step in its mechanism of action. Without internalization, the antibody cannot deliver the small molecule to its target. At iQ Biosciences, we are experienced in measuring the internalization of ADCs (or “naked” antibodies) and are now offering it as a service to our customers in two assay formats.

Option 1: pH-Indicator Conjugation Internalization Assay

In these assays, the antibody or ADC is conjugated to a pH-sensitive reagent that is non-fluorescent in the extracellular environment and becomes fluorescent at the low pH maintained in lysosomes. After treating cells of interest with a dose-response of pH-indicator labeled antibody or ADC, the resulting fluorescent signal, can be measured by flow cytometry such that a high degree of fluorescent signal suggests that the antibody has been internalized into the lysosomes. This assay can be performed at a single timepoint or at multiple timepoints.

In the example study shown below, Antibody X specific for Antigen X was added to Antigen X-expressing cells at various concentrations and incubated for 2 hours. After the incubation, a dose-dependent increase in the percentage of fluorescent cells relative to the lack of signal from cells cultured with an isotype control was observed (Figure 1). These data suggest that the antibody has been internalized at the 2 hour time point.

Figure 1. Percent of cells internalizing Antibody X, Conjugated to a pH-sensitive Dye. Antigen X-expressing cells were incubated with increasing concentrations of Antibody X, which is specific for Antigen X, for 2 hours. After the incubation period, cells were analyzed by flow cytometry to assess internalization.

Option 2: Fluorophore Quenching Internalization Assay

In these assays, the antibody or ADC is conjugated to a standard fluorophore or is provided in a fluorophore-labeled format by the client. Cells of interest are treated with a dose-response of the fluorescently-labeled antibody or ADC. One set of antibody or ADC-treated cells is acquired directly by flow cytometry, while another set of cells are incubated with a fluorescence quenching reagent prior to acquisition. This assay is performed across multiple timepoints so that the resulting values can be applied to a series of calculations, taking into account the total versus quenched fluorescence, in order to generate an internalization half-life. Internal fluorescence values are also generated. Overall, this can indicate comparative levels of internalization between test articles and controls, as well a ‘rate’ of internalization.

Factors Important for Internalization

iQ has experience with internalization assays and can help you design the best experiment based on key factors that can influence internalization. Some factors that can influence internalization are:

1. Cell type – Cell types that express the same antigen do not necessarily internalize the antigen/antibody at the same rate. Internalization by the cell can be dependent on antigen expression level as well as the endocytosis mechanism as there are multiple pathways for receptor-mediated endocytosis. Therefore, it may be important to assess your ADC across a variety of primary cell types or cell lines to determine how well the ADC internalizes across a spectrum of cells.
2. Antibody isotype – Antibodies can come in different isotypes that can influence internalization. For example, a CD19-specific antibody with a human IgG1 backbone internalizes 100-fold faster than the same antibody with an IgG2a isotype. Similarly, mouse anti-HER2 antibodies with differing isotypes but the same antigen binding domain will internalize at different rates. Therefore, it may be important to assess the effect of your ADC with different Fc backbones.

At iQ, we have developed antibody internalization assays that employ quality reagents and standards. With our knowledge of factors that influence internalization, iQ can help design the right experiments for your therapeutic program.