Antibody-based immunotherapies have transformed treatment for cancer, autoimmune diseases, and infections. Key to their action are Antibody-Dependent Cellular Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC). This blog explores the science behind these mechanisms and how CBA supports their testing and validation in India.
From the "Only One Analytical Technique" Talk Series at Venture Center. Watch the Full Video Here:
From the "Only One Analytical Technique" Talk Series at Venture Center
What Are Functional Assays?
Functional assays evaluate the biological activity of a therapeutic molecule. Unlike analytical
methods that measure structure or purity, functional assays reveal how a drug behaves
inside the body—its mechanism of action, potency, and efficacy. In the case of monoclonal
antibodies (mAbs), ADCC and CDC are critical mechanisms and must be carefully studied,
especially during biosimilar development.
ADCC: Antibody-Dependent Cellular Cytotoxicity
ADCC is a mechanism where immune cells kill target cells that are coated with antibodies.
Natural killer (NK) cells, macrophages, and neutrophils recognize these antibodies via their
Fc receptors and unleash cytotoxic molecules that lead to cell death.
How ADCC Works:
- A therapeutic antibody binds to a specific antigen on a target cell (e.g., a cancer cell).
- Effector cells like NK cells recognize the antibody’s Fc region via FcγRIIIa (CD16).
- This binding activates the effector cells to release cytotoxic granules, killing the target
cell.
Key Notes:
- ADCC is only triggered when antibodies are bound to cell surface antigens—not to
soluble targets. - Glycosylation (especially afucosylation) enhances ADCC activity.
- Mutant or engineered antibodies can reduce or enhance ADCC, depending on
therapeutic goals.
CDC: Complement-Dependent Cytotoxicity
CDC involves the complement system—proteins in the bloodstream that, when activated,
form membrane attack complexes (MAC) to lyse target cells.
CDC Mechanism of Action:
- A mAb binds to antigens on a target cell.
- The Fc region activates the complement cascade via C1q binding.
- A MAC forms, creating pores in the membrane and killing the cell.
Key Considerations:
- Complement proteins are always present in circulation.
- Glycosylation also influences CDC.
- CDC is heavily influenced by antigen density and accessibility.
- IgG1 and IgG3 isotypes are more efficient in activating CDC.
Applications of ADCC and CDC Assays
These functional assays are essential across a wide range of therapeutic areas:
- Cancer Therapy: mAbs like rituximab and trastuzumab rely heavily on ADCC and
CDC for tumor cell killing. - Autoimmune Disorders: Used to deplete B cells in diseases like rheumatoid athritis.
- Transplantation: Reduce graft rejection by eliminating immune cells via ADCC/CDC
- Biosimilar Development: Required by regulators to demonstrate similarity in
mechanism of action to reference biologics.
Challenges and Best Practices
- Functional assays are inherently variable due to biological components (cell lines,
donor cells, reagents). - Assay robustness, repeatability, and linearity are essential for regulatory acceptance.
- Glycosylation must be controlled throughout product development as it directly
impacts effector function. - Choosing the right Fc variant and isotype (IgG1 or IgG3) is crucial for assay
relevance.
Therapeutic Antibodies and Effector Function
| Antibody | Isotype | ADCC | CDC |
| Rituximab | IgG1 | ✓ | ✓ |
| Trastuzumab | IgG1 | ✓ | — |
| Ofatumumab | IgG1 | ✓ | ✓ |
| Adalimumab | IgG1 | — | — |
| Ranibizumab (Fab) | — | — | — |
| Denosumab | IgG2 | — | — |
| Natalizumab | IgG4 | — | — |
How to Choose the Right Assay?
When selecting between ADCC and CDC assays, consider:
- Mechanism of action of your therapeutic antibody.
- Glycosylation pattern (e.g., afucosylated for higher ADCC).
- Target antigen location (membrane-bound vs. soluble).
- Desired immune engagement (e.g., avoid CDC for safety).
- Regulatory requirements for biosimilar or biologic approval.
Functional Assay Development in India at CBA
CBA offers advanced functional assay development for both ADCC and CDC. Here's how
we do it:
ADCC Assays:
- Use engineered Jurkat effector cells expressing FcγRIIIa and luciferase reporter.
- Target cells express cancer-specific markers.
- Results show a dose-dependent increase in luminescence correlating with ADCC
activity. - Alternative approaches involve PBMC-derived NK cells, but variability due to donor
polymorphism makes engineered cells preferable.
CDC Assays:
- Use target cells and rabbit serum as a complement source.
- Fluorescence-based viability readout indicates cytotoxic effect.
- Relative potency is calculated by comparing samples against a reference standard.
- Lot-to-lot variability in complement sources is minimized through careful comparison.
Why Choose Us
CBA specializes in functional bioassays tailored to the biotech and pharma ecosystem. We
offer:
- End-to-end support for GLP-compliant ADCC and CDC assays.
- Expert assay design and validation.
- State-of-the-art equipment and engineered cell lines.
- Regulatory-aligned documentation for IND/BLA submissions.
Whether you'are developing a new mAb or working on a biosimilar, we can help with robust
and scalable functional assay development.
Contact us at at cba@venturecenter.co.in for your assay needs
Learn more at - https://bioanalysis.in/
Book our best in class services today