Advanced Flow Cytometry for Complement-Dependent Cytotoxicity (CDC) Assay
Development of monoclonal antibody (mAb) therapeutics has grown substantially over the past decade, with more than 100 products currently approved for use in the United States1. These therapeutics span a wide range of disease areas, from treatments for cancer (such as anti-Her2 mAb Trastuzumab) to agents against infectious diseases like HIV (e.g., Ibalizumab). mAbs can enhance the immune response against cancer through different mechanisms - including Fc-mediated functions - which involve specific recruitment of immune cells to targets through Fc receptor interactions. The three key Fc-mediated functions of antibody therapeutics are antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). For CDC, the antibody binds specifically to the over-expressed target on the tumor cell, which triggers the complement cascade and leads to recruitment of the membrane attack complex on target cells and ultimately results in target cell lysis.
During mAb development, it is essential to profile the CDC activity of a novel mAb candidate to build up a full picture of its Fc-mediated anti-tumor function. Conventional techniques for measuring CDC activity often:
- Use instrumentation with low-throughput acquisition (e.g., traditional flow cytometry)
- Necessitate the use of large volumes of precious antibody and serum samples
- Are laborious and time-consuming, requiring steps such as protocol optimization, fixation and repetitive washes
Here we present a simple, streamlined assay for measuring CDC activity of monoclonal antibodies against live target cells using the iQue® Advanced Flow Cytometry Platform. Target cells are cultured with mAbs of interest in the presence of human serum, which contains the proteins required to initiate the complement cascade and for subsequent formation of the membrane attack complex on cells. Cells are labeled using iQue® Cell Membrane Integrity (R/Red) Dye to enable quantification of cell death as an indicator of CDC activity. Combining the high throughput of iQue® with rapid data analysis using integrated iQue Forecyt® software will streamline your antibody characterization and drug discovery processes.
CDC Assay Workflow
Figure 1. Workflow schematic for CDC assay using high-throughput flow cytometry - Combine target cells (optionally labelled with iQue® Cell Proliferation and Encoding (V/Blue) Dye if required to distinguish multiple cell types in a single well) and mAbs of interest (10 µL of each per well, in serum free media) in a 96 or 384-well plate and incubate for 15 minutes at room temperature (RT) to promote antibody binding to targets. Add human serum (15%, 10 µL/well) and incubate for a further 30 mins (37°C). Wash then label with iQue® Cell Membrane Integrity Dye (choice of V/Blue, R/Red, B/Green, B/Red) for 30 mins (RT). Wash then collect data using the iQue® Advanced Flow Cytometer
- Enhance Productivity - Screen Fc function of antibody libraries in minimal time as part of a high-throughput antibody discovery process
- Maximize Use of Precious Samples - Miniaturize your assay with low volume requirements and simultaneously assess CDC of distinct cell types co-cultured in a single well
- Flexible Assay Format - Characterize multiple therapeutic antibodies in your choice of suspension or adherent cell models
- Streamline Data Acquisition and Analysis - Collect and analyze complex data quickly to rapidly drive decisions
Enhance productivity - Quickly screen Fc function of antibody libraries integrating high-throughput antibody discovery processes.
Figure 2. Efficiently screen mAbs for CDC activity in a 96 or 384-well format
Ramos cells (5K/well) were incubated with a range of concentrations (3 replicates, 12 concentrations per mAb) of anti-CD20 antibodies (listed in Table 1) for 15 minutes prior to addition of human serum (15%) for 30 minutes to induce CDC. Cells were then washed and labelled with iQue® Cell Membrane Integrity (R/Red) Dye and the heat map represents the % live Ramos per well (lighter grey = lower proportion cells are live).
Heating Rituximab and Truxima caused a large decrease in their induction of CDC, with EC50 > max for the heated mAbs. Comparatively, the non-therapeutic anti-CD20 was more resistant to heating with a 2-fold decrease in EC50 after heat treatment. The non-fucosylated form of the non-therapeutic anti-CD20 displayed increased CDC activity over the native antibody.
Maximize use of precious samples - Miniaturize your assay with low volume sample requirements and simultaneously evaluate CDC of distinct cell types co-cultured in a single well.
Figure 3. Compare CDC activity across multiple cell types in a single well
(A) Three suspension cell types were profiled for their CD20 expression. Expression on Ramos was highest, followed by Rajis, while Jurkats were CD20 negative, displaying similar expression to the IgG background control.
(B) Raji cells (brightly labeled with iQue® Cell Proliferation and Encoding (V/Blue) Dye), Ramos cells (dimly labeled with encoder dye) and Jurkat cells (unlabeled) were combined (5K/well) for a CDC assay and separated based on their encoder dye fluorescence, as displayed in the histogram.
(C) Induction of CDC by Rituximab (anti-CD20-IgG1) varied depending on cell type and correlated with the CD20 expression, for example the Ramos expressed the highest levels of CD20 and were most susceptible to CDC by Rituximab.
Flexible assay format - Characterize multiple therapeutic antibodies using either suspension or adherent cell models.
Figure 4. Measure CDC activity with a range of mAbs using both suspension and adherent cell models
Non-adherent cells (CD20 positive Ramos and CD20 negative Jurkats) or adherent cells (Her2 positive AU565 or Her2 negative MDA-MB-468) were combined in CDC assays. In both cases the antigen negative cell type was labeled with iQue® Cell Proliferation and Encoding (V/Blue) Dye to enable the two cell types to be distinguished in each well (as shown in the plots).
(A,B) Anti-CD20 mAbs (Rituximab, Truxima, non-therapeutic anti-CD20) induced an antigen positive cell type-specific increase in CDC. (C,D) None of the anti-Her2 antibodies (Trastuzumab, Kadcyla, Pertuzumab) impacted CDC. (E,F) Lack of CDC activity against adherent cells is likely due to enhanced expression of complement regulatory markers on adherent cell types e.g., CD46, CD55 and CD59.
Streamline data acquisition and analysis - Collect and analyze large amounts of complex data quickly to rapidly drive decisions.
Figure 5. Compare CDC activity across multiple serum donors using iQue Forecyt® software with novel data visualization features
Induction of CDC by Rituximab was compared across 4 different human serum donors. CDC was comparable across donors 1-3, with EC50 values ranging from 0.29 to 0.36 µg/mL. CDC in the presence of donor 4 serum was slightly reduced with an EC50 value of 0.53 µg/mL measured for Rituximab.
No, as the assay time is so short, adherent cells don’t have time to adhere to the plate and therefore can be treated in the same way as suspension cells.
Yes, we have only used a maximum of two channels (one for the iQue® Cell Membrane Integrity Dye and one for the iQue® Cell Proliferation and Encoding Dye [optional] ), leaving 11 detection channels free for addition of other fluorophores. For example, we included a FITC anti-C4c+C4b antibody to confirm formation of the complement complex on cells (data not shown).
|iQue® Cell Membrane Integrity Dye (V/Blue)|
5 x 384
5 x 384
5 x 384
5 x 384
5 x 384
5 x 384
5 x 384