About Complement-dependent Cytotoxicity (CDC)

Complement-dependent cytotoxicity (CDC) is one mechanism by which antibodies can mediate specific target cell lysis through activation of an organism’s complement system.  The complement system consists of over 20 small proteins synthesized by various tissues and cell types, including immune cells, and is regulated by 3 different pathways. After activation of the pathway, complement components in the serum are recruited to target cells that are opsonized by antibodies. Opsonization is the binding of an antibody to its cognate antigen on the cell surface.  Once bound to the target cell, the complement components undergo multiple enzyme activation and cleavage events that lead to lysis of the antibody-bound target cell. Additionally, activation of the complement system leads to inflammation and subsequent recruitment and stimulation of immune cells.

CDC plays a role in general human health by eliminating foreign pathogens, such as bacteria and extracellular organisms, and aberrant cells that would otherwise promote illness.  In addition, CDC is a mechanism by which therapeutic antibodies for cancers, such as those that target CD20 and CD38, may mediate their effect. Therefore, during drug development, potential therapeutic antibody candidates will be screened for CDC activity to find ones that will have these potent anti-cancer effects.

Factors Important for CDC Activity

iQ has extensive experience with CDC assays and can help you design the best experiment based on key factors that can influence CDC.  Four well-studied factors that can enhance or decrease CDC are:

1. Antibody isotype – Certain antibody isotypes have better CDC abilities than others.  In general, IgG3 > IgG1 >>> IgG2 ≈ IgG4 in terms of CDC capability. Additionally, engineering specific amino acid substitutions into either the hinge region or CH2 domain of the antibody can improve CDC activity.
2. Expression level of antigen on target cells – The target cell line should express sufficient target antigen for the antibodies to bind and recruit complement.  Without appropriate amounts of antigen, complement activation cannot be initiated.
3. Expression of complement regulators on target cells – The target cell line may express surface proteins that inhibit or regulate complement activation.  Some of these membrane bound regulators include CD46, CD55, and CD59.
4. Complement source – Complement is found in the serum and is added to the assays to promote CDC.  Therefore, it is important to know the organismal source of the serum to ensure that it is compatible with the target cells since some cells of a certain origin are not compatible with serum from certain animals.

At iQ, we have various methods to assay for cell death.  We can perform CDC assays by flow cytometry wherein cells are stained with an intercalating dye to identify dead targets.  In these experiments, cells are cultured with antibody and serum, followed by staining with the dye and analysis on the cytometer.  This type of assay provides more sensitivity as it directly interrogates cell death.

We can also load target cells with an intracellular nonradioactive dye called calcein, which is released into the supernatant upon lysis and can be measured using a fluorometer.  This method offers a safe, high-throughput method for performing experiments that incorporate a large number of molecules and conditions.

Another method for assaying cell death that is very similar to calcein is the use of alamar blue, a reagent that is converted into a fluorescent molecule only in live cells. In these experiments, alamar blue is added at the beginning of the experiment with the proper reagents (serum, antibody, etc.), and is subsequently read on a spectrophotometer or fluorometer at the end of the culture period. This method also offers a safe, high-throughput method for measuring CDC activity in experiments with numerous molecules and conditions. See Figure 1 for an example of our CDC assay using alamar blue.

At iQ, we have refined protocols with quality-controlled complement sources, including human, non-rodent, and rodent, that can be used for product characterization or therapeutic candidate screening purposes as described in ICH guideline Q6B – Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products (2.1.2 Biological Activity).  In addition, with our extensive knowledge of factors that influence CDC and the different readouts we support for CDC, iQ can help design the right experiments for you to determine the CDC potential of your therapeutic antibodies.

Figure 1. Percent viability of target cell line treated with Test Article. The percentage of live target cells after treatment with increasing concentrations of the Test Article (green circles) or negative control article (blue squares) in the presence of human serum. Alamar blue was added after the treatment period and viability was measured with a spectrophotometer. The spectrophotometer reading is directly proportional to the number of live target cells, allowing the calculation of percent viability.

Our goal is to provide the most robust and consistent approaches to obtain meaningful results for you. Together, we will design assays and analyze data to functionally characterize your antibody for bioactivity and/or guide preclinical transition from in-vitro to in-vivo studies.