Antibody-Dependent Cellular Cytotoxicity (ADCC) – Definition, Mechanism & Application

Antibody therapy has been instrumental in developing highly effective immunotherapy approaches for cancer treatment. Cancer immunotherapy leverages the immune system to target and eliminate malignant cells. Monoclonal antibodies induce cytotoxicity through antibody effector functions, which are mediated by the antibody’s fragment crystallizable (Fc) region.

This includes antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The action of the effector functions can be enhanced by engineering the Fc portion of antibodies to improve ADCC.

In this blog, we will explore the ADCC process, the role of ADCC antibodies and the innovative antibody engineering solutions provided by evitria.

Immunotherapy

Therapeutic monoclonal antibodies (mAbs) have been invaluable tools in cancer treatment. Recognised as the “Breakthrough of the Year” in Science in 2013¹, cancer immunotherapy aims to harness the patient’s own immune system to target and eliminate malignant cells².

These antibodies effectively slow tumour growth by targeting specific antigens expressed on tumoural cells or within the tumour microenvironment. The unique structural features of antibodies facilitate the induction of tumour cell death via direct or indirect mechanisms (see targeting therapies section). 

Antibody structure

Antibodies are classified into five categories according to the structure of their heavy chain constant regions. These immunoglobulin (Ig) isotypes include IgM, IgD, IgG, IgE, and IgA. Cancer immunotherapy frequently employs IgG antibodies because of their versatility, efficacy, specificity and ability to use their structure to engage with and induce immune responses.

The IgG structure comprises two heavy and two light chains, which include constant and variable regions. These regions play a crucial role in the function of the antibody, which can be divided into two main parts. The first is the fragment antigen binding (Fab) region, which contains the antigen binding site that confers specificity. The second part of the antibody is responsible for its effector functions as the constant fragment (Fc) is able to interact with immune cells, as detailed in the targeting therapies section.

Furthermore, certain isotypes can be subdivided into subclasses, which is the case for IgG and IgA (IgG1, IgG2, IgG3, IgG4 and IgA1, and IgA2)³. Isotypes and subclasses vary in their ability to initiate immune responses, such as antibody-dependent cellular cytotoxicity (ADCC), due to differences in the structure of their Fc regions, which affects their binding efficiency to Fcγ receptors (FcγRs).

Targeting therapies

Antibodies can induce tumour cell death by both direct and indirect mechanisms. Direct mechanisms involve binding to growth factors to block receptor signalling. Indirect mechanisms involve recruiting host immune system cells to induce the destruction of tumoural cell death. To achieve this, three main mechanisms are involved:

• Complement-dependent cytotoxicity (CDC)
• Antibody-dependent cell-mediated phagocytosis (ADCP)
• Antibody-dependent cellular cytotoxicity (ADCC)

Complement-dependent cytotoxicity (CDC)

The binding of the Fc region of monoclonal antibodies to the serum protein (C1q) can activate the complement pathway, which is part of the innate immune system⁴. Subclasses of IgG, specifically IgG1 and IgG3, are potent activators of the classical complement pathway.

This pathway is triggered when one or two IgG molecules bind to a target cell, which facilitates the high-affinity binding of C1q via the Fc domain. This interaction activates the enzymatic activity of C1q, initiating a series of proteolytic cleavage events, which leads to the deposition of complement component 3b (C3b). The accumulation of membrane-bound C3b on the cell surface of target cells results in the formation of pores by the membrane attack complexes (MAC) on the tumour cells, ultimately leading to cell lysis of tumour cells.

Additionally, the production of C3a and C5a, which are chemotactic complement proteins, aid in recruiting and activating immune cells, including macrophages, neutrophils, basophils and eosinophils (reviewed in^{4 5}).

Rituximab is an example of a mAb that activates the human complement system. Rituximab is an mAb directed against the CD20 on B-cells and approved by the FDA for B-cell lymphoma and chronic lymphocytic leukaemia, as well as autoimmune diseases⁶.

The critical role of CDC in the anti-tumoural activity of rituximab has been demonstrated by previous preclinical studies, where knocking out the complement cascade component C1q completely abolished its effects⁷.

Furthermore, clinical data provided by Racila et al. highlighted that specific variants in the C1qA gene correlated with prolonged responses to rituximab in follicular lymphoma patients⁸, reinforcing the importance of CDC in the efficacy of rituximab.

Antibody-dependent cell-mediated phagocytosis (ADCP)

Antibody-dependent cell-mediated phagocytosis (ADCP) is another major mechanism of action of antibodies. Here, antibodies target cells and coat them – a term known as opsonization. This causes activation of Fc receptors on the surface of phagocytes (macrophages, monocytes, dendritic cells and neutrophils). This results in the internalization of cells via phagosomes, which later fuse with lysosomes for protein degradation.

Antibody-dependent cellular cytotoxicity (ADCC)

One crucial mechanism employed by cancer immunotherapy antibodies is antibody-dependent cellular cytotoxicity (ADCC).

This phenomenon was first discovered in 1965 by Erna Möller, who demonstrated that immune cells could only induce cytotoxic cell death in tumour cells in the presence of anti-serum from rabbits previously inoculated with the tumour cells⁹. Five years later, in 1970, immunoglobulins were discovered to be the factor in the anti-serum responsible for the cytotoxicity, and the term “ADCC” was coined shortly thereafter¹⁰.

The process of ADCC occurs when antibodies bind to specific antigens on the cell surface of target cells via their antigen-binding fragment (Fab) portions. The antibodies can deliver targeted therapy by, on the one hand, binding to specific target cells, then acting as a bridge and engaging with immune cells via their Fc portions.

The IgG class of antibodies is most commonly used for cancer immunotherapies. However, other classes of human antibodies can also induce ADCC – IgA and IgE¹¹. The prerequisite for effector cells is the expression of an Fc receptor (FcR) capable of binding to the antibody. The FcγR are a key class of receptors and bind IgG. There are three classes of FcγR — I (CD64), II (CD32) and III (CD16).

The FcγR on effector cells transduce signals by forming complexes that can regulate either activating or inhibitory signals. The FcγRIIIA (CD16A) is the major activating receptor on natural killer cells, dendritic cells (DCs), macrophages and mast cells¹². The consequent formation of intracellular signalling cascade downstream of the FcγRIIIA causes activation of NK cells to induce ADCC.

Upon activation, NK cells release granules filled with cytotoxic factors, granzymes and perforin onto the target cell. Perforin inserts into the target cell’s membrane and forms pores through which granzymes can enter the inside of the target cell. Granzymes are protease proteins that attack target cells from the inside, leading to cell death. Ultimately, the NK cell lyses the pathogenic target cell into harmless fragments that are digested and recycled by the body.

1. Antibodies bind via their Fab portion to the target cell 
2. Effector cells like NK cells recognize the Fc portion of antibodies through the FcγRIIIA (CD16A) receptor 
3. FcγRIIIA (CD16A) receptor cross-link, leading to NK cell activation
4. Release of cytotoxic granules (containing perforin and granzymes) towards the target cell. Perforin inserts into the target cell’s membrane and forms pores. Consequently, granzymes enter the target cell and activate cytotoxic pathways, leading to cell death.
5. Detachment of the NK cell from the targeted cell

Application of Antibody-dependent cell-mediated cytotoxicity (ADCC)

Numerous monoclonal therapeutic antibodies have been shown to mediate their effects partially or predominantly via NK cell activation through antibody-dependent cell cytotoxicity (ADCC):

HER-2 targeting antibodies

Breast cancers overexpressing human epidermal growth factor receptor 2 (HER2) have a high metastatic potential, which is associated with a poor prognosis in patients. ADCC has been shown to be the critical mechanism behind the efficacy of the HER2 targeting antibodies, trastuzumab and pertuzumab.

Daratumumab: multiple myeloma

Multiple myeloma (MM) is a condition characterized by the abnormal production of monoclonal immunoglobins by plasma cells.

The CD38 transmembrane glycoprotein is highly and uniformly expressed in MM cells. It is the ideal target as it has low expression levels in normal lymphoid and myeloid cells¹³. Daratumumab targets CD38 and binds with a high affinity. Daratumumab induces cell death through multiple mechanisms, including ADCC¹³.

Enhancing ADCC activity

In recent years, novel therapeutic recombinant antibodies have been engineered to exhibit enhanced ADCC activity. Two available engineering methods include amino acid substitution and afucosylation.

Enhancing ADCC with amino acid substitutions

Enhancement of ADCC activity can be achieved by increasing binding capacities to FcγRIIIA.

One strategy is protein engineering where amino acid substitution in the Fc portion of IgG1 has generated multiple variants that display increased affinity of Fc binding to FcγRIIIA^{14 15 16}. The HER2-targeting monoclonal antibody, margetuximab, has five mutations in the Fc portion of the antibody — including (Phe243Leu, Arg292Pro, Tyr300Leu, Val305Ile, Pro396Leu). These optimized mutations allow the antibody to strengthen its interaction with the FcγRIIIa receptor on immune effector cells, boosting the activity of natural killer (NK) cells and increasing ADCC compared to standard-of-care antibody trastuzumab¹⁷.

Afucosylation & afucosylated antibodies

Afucosylation refers to the removal or absence of fucose in the Fc region of antibodies. Such afucosylated antibodies are typically manufactured by recombinant technology in mammalian cell lines.

Compared to antibodies containing fucose, ADCC-enhanced antibodies have a modified glycosylation pattern that lacks fucose. It has been shown in vitro that the lack of fucose in the Fc region leads to stronger binding of FcγRIIIA (CD16A) receptors, hence reducing non-specific competition with other antibodies and increasing the ADCC response.

Afucosylated antibodies for ADCC enhancement from evitria

The scientists at evitria specialize in recombinant antibody expression services, including the engineering and production of afucosylated antibodies to enhance ADCC.

One of the company’s most recent advancements is the state-of-the-art GlymaxX® technology, licensed from ProBioGen. This technology is used to generate afucosylated antibodies through transient expression in CHO cells.

evitria holds an exclusive licensing partnership to offer afucosylation via ProBioGen’s GlymaxX® technology for your transient antibody expression needs. This technology facilitates the expression of both native and afucosylated variants. The technology can be applied to your standard host cell line. Alternatively, we can offer our own optimized GlymaxX host cell line or re-engineer existing producer clones. GlymaxX cell lines are stable and compatible with the highest titer production processes. Learn more about our Afucosylated Antibody Expression Service.

Contact us for more information on the expression of afucosylated antibodies with enhanced ADCC activity

Further readings:

• How are afucosylated antibodies developed?
• Applications of afucosylated antibodies

References

1. Couzin-Frankel, J. Breakthrough of the year 2013. Cancer immunotherapy. Science 342, (2013). ↩︎
2. Calvillo-Rodríguez, K. M., Lorenzo-Anota, H. Y., Rodríguez-Padilla, C., Martínez-Torres, A. C. & Scott-Algara, D. Immunotherapies inducing immunogenic cell death in cancer: insight of the innate immune system. Frontiers in Immunology vol. 14 Preprint at https://doi.org/10.3389/fimmu.2023.1294434 (2023). ↩︎
3. Petar, P. , Dubois, D. , Rabin, B. S. , & Shurin, M. R. Chapter 12 – Immunoglobulin Titers and Immunoglobulin Subtypes. in Measuring Immunity – Basic Biology and Clinical Assessment 158–171 (2005). ↩︎
4. Weiner, L., Surana, R. & Wang, S. Antibodies and cancer therapy: versatile platforms for cancer immunotherapy. Nat Rev Immunol 10, (2010). ↩︎
5. Bondza, S. et al. Complement-Dependent Activity of CD20-Specific IgG Correlates With Bivalent Antigen Binding and C1q Binding Strength. Front Immunol 11, (2021). ↩︎
6. FDA. Highlights of prescribing information. (2012). ↩︎
7. Di Gaetano, N. et al. Complement Activation Determines the Therapeutic Activity of Rituximab In Vivo. The Journal of Immunology 171, (2003). ↩︎
8. Racila, E. et al. A polymorphism in the complement component C1qA correlates with prolonged response following rituximab therapy of follicular lymphoma. Clinical Cancer Research 14, (2008). ↩︎
9. Möller, E. Contact-Induced Cytotoxicity by Lymphoid Cells Containing Foreign Isoantigens. Science (1979) 147, (1965). ↩︎
10. Maclennan, I. C. M., Loewi, G. & Harding, B. The Role of Immunoglobulins in Lymphocyte-Mediated Cell Damage, in Vitro I. COMPARISON OF THE EFFECTS OF TARGET CELL SPECIFIC ANTIBODY AND NORMAL SERUM FACTORS ON CELLULAR DAMAGE BY IMMUNE AND NON-IMMUNE LYMPHOCYTES. Immunology vol. 18 (1970). ↩︎
11. Teillaud, J. Antibody-dependent cellular cytotoxicity (ADCC). Encyclopedia of Life Sciences (2012). ↩︎
12. Nimmerjahn, F. & Ravetch, J. V. Fcγ receptors: Old friends and new family members. Immunity vol. 24 19–28 Preprint at https://doi.org/10.1016/j.immuni.2005.11.010 (2006). ↩︎
13. Sanchez, L., Wang, Y., Siegel, D. S. & Wang, M. L. Daratumumab: A first-in-class CD38 monoclonal antibody for the treatment of multiple myeloma. Journal of Hematology and Oncology vol. 9 Preprint at https://doi.org/10.1186/s13045-016-0283-0 (2016). ↩︎
14. Stavenhagen, J. B. et al. Fc optimization of therapeutic antibodies enhances their ability to kill tumor cells in vitro and controls tumor expansion in vivo via low-affinity activating Fcγ receptors. Cancer Res 67, (2007). ↩︎
15. Shields, R. L. et al. High Resolution Mapping of the Binding Site on Human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and Design of IgG1 Variants with Improved Binding to the FcγR. Journal of Biological Chemistry 276, (2001). ↩︎
16. Lazar, G. A. et al. Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci U S A 103, (2006). ↩︎
17. Nordstrom, J. L. et al. Anti-tumor activity and toxicokinetics analysis of MGAH22, an anti-HER2 monoclonal antibody with enhanced Fcγ receptor binding properties. Breast Cancer Research 13, (2011). ↩︎