Application Note: Complement-Dependent Cytotoxicity with Streamlined, Miniaturized Advanced Flow Cytometry
Overview
In this application note, we present a simple, streamlined, miniaturized workflow for quantifying monoclonal antibody induction of Complement-Dependent Cytotoxicity (CDC) activity using the iQue® Advanced Flow Cytometry Platform. Combining the high-throughput and low sample volume requirements of the iQue® with rapid data analysis using the integrated iQue Forecyt® software, has the potential to simplify and speed up antibody characterization and drug discovery processes, including the quality of generated hits.
This facilitates rapid screening of large antibody libraries along with the capacity to increase replication to enhance robustness of data. Combining CDC data with Fc function analysis using the iQue® Human ADCP Kit and iQue® Natural Killer Cell Killing Kit can provide full profiling of the three key Fc receptor mediated functions of antibodies using a single instrument.
Page count: 11
Read time: 10 minutes
Target audience: Immunologists, Immuno-oncology, virology researchers, neuroscientists and cancer immunotherapy drug discovery researchers, in biotechnology, pharmaceuticals, biopharma, research institutes and academia.
Key Takeaways
Key topics detailed in this application note, include:
- Serum Donor and Concentration May Impact Levels of CDC
- Levels of CD20 Expression on Target Cells Correlate with CDC Activity
- Multiplexed Measurement of Complement Protein Binding and CDC Activity
- Induction of Fc Receptor Mechanisms Is Dependent on Antibody Isotype
- Adherent Cells are Highly Resistant to CDC
Background to this application note
Research and development of monoclonal antibody (mAb) therapeutics is a vast and expanding area of drug discovery, largely due to mAb potential for high specificity and affinity towards target molecules. Approved mAb-based therapeutics span a wide range of disease areas, although most are indicated for treatment of cancers or for immune-mediated disorders.
There are several mechanisms of action (MoAs) through which mAbs are cytotoxic towards target cells, such as cancer cells. These MoAs often harness the power of the body’s own immune system to exert anti-tumor effects. Examples of this include the three key Fc-mediated functions: antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). ADCC and ADCP rely on a mAb simultaneously engaging with Fc receptors on immune cells and the antigen on target cells. This brings the target cells into proximity with cytotoxic cells (e.g., natural killer cells) or phagocytic cells (e.g., monocytes or macrophages), leading to enhanced immune clearance.
Contrastingly, in CDC, the antigen-bound mAb recruits proteins present in the blood to induce lysis. This process begins with binding of the C1q protein to the mAb Fc region, which triggers activation of the complement pathway. A complex molecular cascade follows, which eventuates in the formation of the membrane attack complex (MAC). The MAC creates a pore in the target cell membrane, resulting in cell death.
During early stage mAb development, in vitro assays are often used to profile activity towards several MoAs, including CDC, to ensure mAbs with desirable characteristics are selected for clinical evaluation. Conventional techniques for measuring CDC activity often:
- Require large volumes of precious antibody and serum samples
- Use instrumentation with low-throughput acquisition (e.g., traditional flow cytometry)
- Are laborious and time-consuming, requiring steps such as protocol optimization, fixation, and multiple washes
