Antibody-dependent cellular phagocytosis
Antibody-dependent cellular phagocytosis (ADCP) is an immunological mechanism of elimination whereby tumour cells are targeted with monoclonal antibodies (mAbs) to promote their clearance from the body by phagocytic immune cells. This makes tumour cell-specific mAbs an attractive candidate for use as anti-cancer therapeutics, with several therapies already approved, such as Trastuzumab: a common treatment for Her-2 positive breast cancers.
The mechanism of action (MoA) of these therapeutics relies on both their ability to engage Fc-receptors (FcR) on macrophages or monocytes and their specific binding to antigens over-expressed on tumour cells. During antibody development it is essential to characterize the molecule’s ability to induce the various MoAs, including ADCP, through the use of in vitro assays to give an indication of their therapeutic potential. Traditional techniques for measuring ADCP often:
- Use instrumentation with low-throughput acquisition that necessitate large sample volumes (e.g. traditional flow cytometry)
- Are laborious and time-consuming, requiring steps such as protocol optimization, fixation and repetitive washes
- Provide only qualitative readouts or require manual data analysis
- Measure artefacts rather than quantifying a true ADCP signal
- Rely on genetically engineered reporter cell lines
The iQue® Antibody-Dependent Cellular Phagocytosis application utilizes the iQue® Human Antibody-Dependent Cellular Phagocytosis Kit for high-throughput measurement of ADCP via quantification of co-localization of live target cells with CD14+ primary effector cells using a simplified, streamlined workflow. Leverage the power of the iQue® Advanced High-throughput Flow Cytometer and automated, integrated analysis for instantaneous pharmacological readouts for the effects of targeted, test antibodies on ADCP.
Detect and quantify ADCP events using a simple assay
Figure 1. Illustration of the iQue® Human Antibody-Dependent Cellular Phagocytosis Kit assay principles.
Target cells labeled with iQue® Proliferation and Encoding (B/Green) Dye are incubated with test antibody in 96- or 384-well plates prior to the addition of unlabeled effector cells (either PBMCs or an enriched population of monocytes or macrophages).
Live and dead cells are separated using the iQue® Cell Membrane Integrity (R/Red) Dye. ADCP is quantified as the percentage of live, CD14+ effector cells that are positive for the target cell encoder.
The combination of fluorophores used in this kit means it is compatible with both the iQue® BR and VBR configurations.
Obtain quantitative data
Generate reproducible and specific measurements of ADCP suitable for pharmacological analysis.
Figure 2. Efficient data analysis using Forecyt allows drug potency ranking using automatic calculation of EC50
B/Green labelled Ramos cells (2.5K/well) were seeded in a 96-well plate with PBMCs (20:1 effector-to target ratio). ADCP was stimulated with a range of different Rituximab mAb isotypes, including: IgG1; IgG1NQ (non-glycosylated); IgG2 and IgG4. Cells were labelled using the iQue® Antibody-Dependent Cellular Phagocytosis Kit and ADCP was analysed using the iQue® 3. ADCP (%) is defined as the % CD14 and B/Green Encoder+ cells in the live population.
1. Invivogen: Reagents And Tools For Cell Biology Research.
Perform high-throughput analysis
Facilitate your discoveries using validated reagents for rapid quantification of ADCP in 96 or 384-well plates.
Figure 3. Analyze ADCP response from PBMCs from multiple donors, at several E:T ratios in a single 384-well assay plate.
Non-adherent Ramos target cells (2.5K/well) were incubated with Truxima (CD20-IgG1) and PBMCs at 5:1, 10:1 and 20:1 effector-to-target ratios. Wells with no antibody were included as a negative control. ADCP was assessed using the iQue® Human Antibody-Dependent Cellular Phagocytosis Kit and the iQue® 3.
(A) Heat map of ADCP (%) (B) and (C) Concentration-response curves with ADCP (%) by PBMCs from Donor 1 and 2, respectively.
Analyze physiologically relevant cell models
Assess ADCP by primary effector cells on a variety of adherent or non-adherent target cells.
Figure 4. Distinguish phagocytic effects of therapeutic drugs on adherent target cells using the flexible co-culture model.
B/Green encoded adherent AU565 cells (10K/well) were incubated with Trastuzumab (Her2-IgG1 mAb) and isolated monocytes (5:1 effector-to-target ratio). Cells were labelled using the iQue® Human Antibody-Dependent Cellular Phagocytosis Kit and analysed with the iQue®3.
(A) Plate view of histograms from Forecyt display the increase in ADCP events (CD14+ B/Green+ Live cells) with Trastuzumab concentration.
(B) Increase in ADCP (%) in response to Trastuzumab compared to a control antibody (BGal-IgG1).
Maximize your productivity
Reduce your time to answer with real time data analysis and novel visualization tools
Figure 5a. Preset gating template auto-populates as sample is acquired by the iQue®.
Template provided with the iQue® Human Antibody-Dependent Cellular Phagocytosis Kit is imported into iQue® Forecyt to provide instant quantification of co-localized B/Green encoded target cells with CD14+ monocytes. Omission of a single cell gate combined with a simple 3-step gating strategy ensures measurement of a true ADCP signal.
Figure 5b. Automated data analysis by iQue® Forecyt software provides instantaneous ADCP readouts.
Once the gating template has been applied, the easy-to-use iQue® Forecyt analysis platform can be used for pharmacological analysis with simultaneous presentation of results in simple, clear formats. Examples of data visualizations include:
(A) Contour plot with clear gating of the CD14 positive population (B) Heat map of ADCP (%) response to Truxima by PBMCs with Ramos or Raji target cells (C) Data from (B) represented as a concentration response curve. EC50 values exported directly from iQue® Forecyt are 29.1 and 24.8 ng/mL for Ramos and Raji, respectively.
Platform: Compatible with iQue® 3/iQue® Screener Plus - VBR and BR Configurations
1 x 96 well
5 x 96 wells
|1 x 384 wells|
|5 x 384 wells|