Page 67 - Live-cellanalysis handbook

Kinetic Antibody Internalization Assays

Visualization and quantification of antibody internalization, in real time

Introduction

The growing focus on antibodies as therapeutics makes it essential There is an urgent need to have an efficient, affordable method
to be able to study their dynamics in a realistic cell environment. that can be used throughout the entire antibody screening and
Antibodies are used both as therapeutic agents that themselves functional characterization process to fully and rapidly quantify
block the activity of certain molecules, or for the targeted delivery antibody internalization using real time live-cell analysis.
of treatments to sites or cells of interest. This includes the delivery
of highly toxic drugs to cancer cells via antibody drug conjugates In this chapter, we will illustrate how novel pH-sensitive Fc-
(ADCs), removal or degradation of surface receptors from cancer targeting antibody labeling reagents allow for real-time rapid,
cells (i.e. EGFR), and antibody immunotherapies used to identify kinetic, high-throughput analysis of antibody internalization in
tumor cells for immune cell killing (i.e. ADCC or ADCP). living cells. This is an ideal tool for optimal antibody engineering
early in the biologic discovery process.
Each of these therapeutic strategies hold the promise of increased
efficacy and reduced side effects, and each requires a series of IncuCyte FabFluor antibody internalization
®
antibody features, for example, to enable maintenance on the cell
surface for identification of tumor cells, or for rapid internalization assays at a glance
when delivering ADCs. To realize their potential, it is important
to understand the uptake profile and clearance of antibody The IncuCyte® antibody internalization assay uses IncuCyte®
candidates, and to be able to measure and optimize functional FabFluor-pH Red reagent, a novel pH-sensitive Fc-targeting
responses to antibodies for optimal antibody engineering and antibody fragment, isotype-matched to the antibody of interest.
internalization characteristics. For example, pinocytosis, which is The IncuCyte® FabFluor-pH Red reagent and antibody of interest
one of the main elimination routes of antibodies, requires antibody are mixed in a one-step, no wash, labeling protocol. At pH 7.0,
optimization for qualitative pharmacokinetic measurements during the FabFluor-Ab complex has little or no fluorescence. When
therapeutic antibody development. labeled antibodies are added to cells, a fluorogenic signal is seen
as the antibody is internalized and processed in acidic (pH 4.5-
Current methods for measuring antibody internalization have several 5.5) lysosomes and endosomes. The full time-course of antibody
limitations. These include the requirement to label each antibody, internalization can then be followed with the IncuCyte Live-Cell
conduct multiple washing steps, and the ability to only do single- Analysis System for real-time analysis of internalization rates. An
time point analysis. This can lead to unnecessary and labor-intensive overview of the workflow is shown on the next page. (Figure 1)
processes, loss of cells through washing, and missed and incorrect
measurements due to limitations of single time-point analysis.

Shortcomings of Traditional Assays Live-Cell Imaging and Analysis Approaches

• Requires labor-intensive labeling of each • Rapid, single-step labeling allows efficient testing of panels of antibodies.
antibody.
• Real-time, kinetic measurements of antibody responses.
• Only provides single-time point analysis.
• High-throughput, reproducible antibody screening validated with individual
• Low throughput due to combination of labeling images.
approach and end-point analysis.

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