ADC Internalization Assay

Creative biolabs has years of experiences in developing and analyzing antibody-drug conjugates (ADCs). We provide internalization analysis service using various techniques to help selecting internalizing monoclonal antibody for ADC development and to verify the internalization of an ADC after conjugation.

Antibody-drug conjugates (ADCs), a promising class of antitumor agents, are by design to rely on the use of a monoclonal antibody to selectively deliver a cytotoxic drug into tumor cells. The antibody portion of an ADC functions by specific binding to the target antigen expressed on surface of tumor cells and deliver the cytotoxic drugs into the cells via internalization, a process also known as receptor mediated endocytosis. After internalization, the ADC molecules are transported to subsequent intracellular compartments where the toxic payload drug is released to accomplish the major cytotoxicity of the ADC. Therefore, the rate and extent of ADC internalization is of crucial importance to its effectiveness and need to be characterized extensively. The internalization efficiency of an ADC varies from the unconjugated monoclonal antibody. Previous studies have shown that in some cases, an unconjugated mAb and its corresponding ADC are internalized at the same rate, while in other cases, ADC internalization is affected by the payload conjugation.

Generally, the rate of mAb and ADC internalization is influenced by a number of parameters, including epitope selectivity, binding affinity, and intracellular transportation. A cell surface receptor offers many epitopes for mAbs binding and different antibody-epitope interactions reveal a wide distribution of internalization kinetics. Thus, the selection of the antibody-epitope pair with the most suitable internalization kinetics is an important factor in ADC development. Additionally, earlier studies have revealed an intricate correlation between antibody internalization, tissue penetration, and binding affinity:

  1. antibodies with moderate affinity exhibited the highest levels of tumor accumulation, indicating good tumor penetration, whereas high-affinity antibodies usually exhibited a low level of tumor accumulation.
  2. the internalization kinetics is related to binding affinity, with high-affinity antibodies exhibited greater internalization potential comparing to low-affinity antibodies.

Thus, in order to develop it into an ADC, it is of crucial importance to identify an antibody with optimal binding and internalization potentials. Together with our affinity determination service module, Creative biolabs is experienced and confident to help identify antibodies with good potential as ADC precursors.

The chemical conjugation process leading to the generation of an ADC changes the biochemical characteristics of the antibody molecule and could potentially lead to altered internalization kinetics of the ADC comparing to the unconjugated mAb. Since internalization is a prerequisite for ADC function, the internalization of the ADC should be extensively evaluated to ensure its proper subcellular function.

With years of accumulative work in ADC development, Creative biolabs has adopted and perfected several techniques to study antibody/ADC internalization for the selection of suitable antibodies for ADC development and for the validation of the internalization of the corresponding ADCs. Our approaches include radiolabels, fluorescent microscopy, flow cytometry…, among which antibody/ADC fluorescence labeling is the most common. Various organic dyes, especially some pH-sensitive dyes have been used in this application and the resulted antibody/ADC internalization is characterized using fluorescent or confocal microscopy. For more information regarding “internalizing” antibody selection, please visit our “Internalizing antibody screening” webpage.

ADC Internalization Assay Antibody and ADC internalization analysis. The internalization behavior of ADCs analyzed using pH-sensitive dyes (left, Blood, 2009) and flow cytometry with fluorescent labeling (right, Mol Cancer Ther, 2016).

Creative biolabs is committed to assist you in selecting an internalizing antibody suitable for ADC development and we also provide a great variety of ADC analysis services to help you fully characterize the customized ADCs. Please contact us for more information and a detailed quote.


  1. Gerber, H.S.; et al. Potent antitumor activity of the anti-CD19 auristatin antibody drug conjugate hBU12-vcMMAE against rituximab-sensitive and -resistant lymphomas. Blood. 2009, 113:4352-4361.
  2. Strop, P.; et al. RN927C, a site-specific Trop-2 antibody–drug conjugate (ADC) with enhanced stability, is highly efficacious in preclinical solid tumor models. Mol Cancer Ther. 2016, 15(11): 2698-2708.

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