Antibody-Drug Conjugate (ADC) Internalization Assay

Antibody-Drug Conjugate (ADC) Internalization Assay2021-06-15T00:13:56-07:00
  • Assess internalization of your ADC/antibody with iQ’s ADC internalization assay
  • Personalized experimental design ensures that the most relevant data for your molecule are generated
  • 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
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Service Overview

About iQ Biosciences’ ADC Internalization Assay Service

Antibody-drug conjugates (ADCs) are currently used in targeted therapies against numerous cancer indications. They are a class of molecules that have the specificity of antibodies to target certain cell types while possessing the ability to target intracellular proteins and processes using a broad set of small molecules conjugated to the antibody. This leads to potent therapeutic molecules that have specificity for the cells that express the cognate antigen while also carrying molecules that have cytotoxic effects.

The main components of an ADC are: 1) the antibody, 2) the small molecule (cytotoxic payload), and 3) the linker that connects the antibody to the payload. 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 effects. The last step is dependent on the low pH environment of the lysosomes and/or the presence of proteases within them.
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 intracellular target. At iQ Biosciences, we are experienced in measuring the internalization of antibody/ADCs and are now offering it as a bioservice to our customers. In these assays, the antibody/ADC is conjugated to a pH-sensitive reagent that is non-fluorescent in the extracellular environment and becomes fluorescent at low pH, which is found in lysosomes. The readout, which is fluorescence, can be subsequently measured by flow cytometry. If cells are fluorescent, it suggests the antibody has internalized due to the fluorescent molecule becoming activated in the low pH of lysosomes.

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

Percent of cells internalizing Antibody X.

Figure 1. Percent of cells internalizing Antibody X. 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.

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 is important to assess your ADC across a variety of cell types/lines to determine how well the ADC internalizes across a spectrum of cells.
  2. Antibody isotype – Antibodies can come in different isotypes and the isotype on the backbone of these antibodies 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 antibodies against HER2 with differing isotypes but the same antigen binding domain will internalize at different rates. Therefore, it will be important to assess the effect of your ADC with different Fc backbones.

At iQ, we have developed an antibody internalization assay that employs quality reagents and standards. In addition, with our knowledge of factors that influence internalization, iQ can help design the right experiments for you to determine the potential of your ADC.

The iQ Experience

Tailored Experiments

Your research is important to us. Our scientists will work through each step of the process with you, including assay design, data analysis, and recommendations for future studies.

Streamlined Process

Simplify your workflow. Bypass the middle-man: at iQ Biosciences, you'll get immediate access to our biospecimen inventory, saving you both valuable time and money.

Expertise

We're experts- so you don't have to be. Augmenting years of experience in immunology and working with immune assays, our scientists stay current with the latest publications and technology.

Exceptional Service

We're here to help. We know the challenges you're facing: whether it be through expedited service or our complimentary consulting services, our team is dedicated to helping you reach your goals.

Order Information

Pricing

At iQ Biosciences, we understand that each client is unique. Upon quote request, we will provide complimentary consulting services to determine how to tailor our services to best meet your goals. Based on this preliminary assessment, we will be able to provide a price quote.

Expected Timetable

Fit a timetable appropriate to your research goals. We work with you to determine your expected turn-around on a case-by-case basis and ensure that our services fit your needs. Some experiments may require an expedited service – please inquire for more information.