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Your Position: Home > Insights > Critical importance for the evaluation of the Fc domain binding activity
Critical importance for the evaluation of the Fc domain binding activity
Release time: 2021-07-14 Source: ACROBiosystems Read: 1767

Special Fc function domain

Antibody drugs has quickly become the focus of global drug research and development due to high specificity and potency against intended targets. Antibody drugs are based on engineered immunoglobulin which are composed of the fragments antigen binding (Fab) domain, mainly responsible for recognizing and binding antigens, and the fragment crystallizable (Fc)-fusion domain that binds to effector molecules or interacts with cells to exert toxic effects. Once Fab domain has been successfully engineered against a particular antigen, the focus of antibody drug development and research centers mainly on Fc domain optimization.

Fc crystallizable (Fc)-fusion molecules are often engineered with a specific immunoglobulin G (IgG) isotype and subtype in order to impart particular functionalities. These include half-life extension and induction of cytotoxic activity[1]. Alternatively, a specific isotype and subtype may be selected to mitigate, or lessen, potential induction of unwanted biological activities. These modalities consist of specific structural/functional domains that result in the intended effect and improve therapeutic benefit.


Figure 1. Basic structure of an IgG1 antibody. All the function domain available for engineering is indicated including antigen binding via the fragment antigen binding (Fab), Fc gamma receptor (FcγR) binding, complement component 1q (C1q) binding, and neonatal Fc receptor (FcRn) binding domains.[2]


Fc-mediated effector function activities

The efficacy of a therapeutic antibody not only depends on the activity of the Fab fragments, but also on the interaction of the Fc fragments with cellular Fc receptors. The Fc fragment of the antibody affects the pharmacokinetics and drug toxicity in-vivo by binding to Fc receptors and Clq complex. The functional effects mediated by Fc mainly include the following:


1.  Antibody-dependent cell-mediated cytotoxicity (ADCC)

ADCC is a cell-mediated innate immune mechanism whereby an effector cell of the immune system (e.g., Natural Killer (NK) cells, monocytes, macrophages, or eosinophils) bind to antibody/target cell complex inducing cell lysis through release of cytotoxic factors as shown in Fig. 2. The NK cells contain FcγR and most commonly bind the antibody Fc region with FcγRIII/CD16 receptors.


Figure 2. Process of ADCC begins with binding of antibodies to the intended target on a cell surface followed by immune effector cells like NK cell or other peripheral blood mononuclear cells (PBMCs) binding to the complex and initiating cell lysis and death.[2]


2.  Complement-dependent cytotoxicity (CDC)

CDC represents another mechanism by which a therapeutic monoclonal antibody can destroy target cells depending on the complex formation with C1q resulting in a membrane attack complex which induced target cell lysis (Fig. 3).


Figure 3. CDC begins first with antibody binding to target antigen expressing cells followed by C1q binding, MAC formation and cell lysis.[2]


3.  Antibody-dependent cellular phagocytosis (ADCP)

ADCP is an Fc-dependent, cell-mediated, and innate immunity mechanism in which antibody-opsonized target cells, or in some cases, specific fibrillar proteins, engage the FcγR on the surface of immune effector cells like macrophages or monocytes to induce phagocytosis as shown above (Fig. 4).



Figure 4. ADCP results from macrophage binding of antibody/target cell complex resulting in phagocytosis and cell lysis.[2]


4.  Cross linking antibody-dependent apoptosis

Cross linking secondary antibody-dependent apoptosis is another Fc effector function in which binding of target cell surface antigen by antibodies in the presence of cross-linking secondary antibodies or FcγR-expressing cells initiates signal transduction events that lead to apoptosis of the cells (Fig. 5).


Figure 5. Cross-linking antibody dependent apoptosis occurs after primary antibody bound to target antigen containing cells are polymerized by a secondary antibody binding to the Fc domain leading to cell death.[2]


Non-cell-based binding assay Formats for evaluating the function of the Fc region

Since the binding of Fc receptors can affect Fc-mediated functional effects, engineering of the Fc domain is very important for the characterization of the activity of Fc containing antibody drugs to evaluate its activity, effectiveness and safety. The main role of Fc-containing therapeutic antibody drugs is to engage the FcγR subclass. Since FcγRs contain many different subtypes binding to specific subtypes of Fc domains, the Fc-FcγR interaction is highly complex and in-vitro detection of Fc-Fc receptor binding can predict the functional potency of these antibody drugs. According to the United States Pharmacopoeia, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) and biological layer interference (BLI) are all valid assays to assess Fc-Fc receptor binding.

Enzyme-linked immunosorbent assay (ELISA) is an immunoassay technology developed by combining the specificity of the antigen-antibody reaction with the high-efficiency catalysis of detection enzymes. It can be used to detect macromolecular antigens and specific antibodies.

Surface Plasmon Resonance (SPR) technology is a biosensing analytical technology based on the physical optical phenomenon of surface plasmon resonance, centered on biosensing gold plated chips, and is also included in the Chinese, American and Japanese Pharmacopoeias.

Bio-Layer Interferometry (BLI) technology is an optical analysis method (optical interferometry) that relies on fiber optic biosensors to measure changes in light wavelength interference patterns based on binding events.


Table 1. Introduction of ELISA, SPR and BLI

Assay Format



Points to Consider


Label-free formats; precise kinetic data; can measure a   wide range of binding affinities; automated data analysis possible.

Medium throughput; requires specialized equipment and   technical expertise; harder to transfer.

Need high quality of critical reagents such as FcγR, FcRn, and   Fc-containing reagents;

types of biosensor chip and alternate approaches to   immobilization of ligand;

minimize steric hindrance and non-specific   interactions;

avoid avidity artifacts due to overloading ligand on   the chip;

flowing a higher concentration of analyte over the chip   surface to mitigate low-affinity interactions (e.g., FcγRII, FcγRIII).


Label-free format;   high throughput; can provide kinetic and quantitation data; semi-automatable.

More difficult to   transfer; less sensitive than SPR.

Selection of the   appropriate biosensor and assay orientationloading of ligand molecules on the biosensors;

selection of buffer conditions to promote optimal and   reduce non-specific binding;

optimize length of the association step for secondary   interaction consideration.


High throughput,   automatable; economical, easily transferred.

Multiple wash steps;

requires plate   precoating; binding limitations.

Presence of   aggregates may complicate the interpretation of results;

when the binding interactions are weak (e.g., FcγRII and FcγRIII), consider   cross-linking to improve sensitivity; critical reagent quality and   concentration such as FcγR and FcRn.


Fast and reliable services---supporting full-cycle of drug development

ACROBiosystems is a global brand focusing on protein technology, products and services in the development of biological drugs. ACRO is committed to providing target antigens and other key reagents and related services required in the development of targeted therapeutic drugs.

Relying on a professional technical team and validated Biacore and ForteBio Octet platforms, ACRO is committed to providing our customers with high-quality integrated molecular interaction analysis and testing services, including antibody (mAb/BsAb/ADC) screening, characterization, and consistency evaluation, Fc receptor affinity detection and analysis, and biomolecules interactions and small molecule affinity testing, etc., to accelerate development of powerful antibody drugs.

Our advantages:

1. Comprehensive protein pipeline support⸺Thousands of high-quality antigens available for experimental selection

2. Experienced R&D technical team - Multiple proven methods to choose

3. Efficient project operations team ⸺Next day reports at the earliest

4. Personalized solution design and report format ⸺Data quality meets filing requirements, supports the Sino-US dual reporting

5. Quality Certification⸺ High standard quality management control system

6. Multi-testing platforms--Multi-platforms can provide testing services


ACROBiosystems Testing and Analysis Service Center launches long-term preferential activities

Provide samples and receive results within two weeks.

Affinity testing experiments covering all genotypes of human Fc receptor proteins required for antibody drug development (as shown below)

All Fc receptor proteins needed for binding experiments available free of charge!

Click here to view Fc receptor proteins


Figure 6. All genotypes of human Fc receptor protein

Case Study

SPR based affinity analysis based on non-mutated IgG1 and human FcγR molecules of different genotypes



Cat No.


Analysis method



Fc gamma RIIB /   CD32b



His Capture

10.1 μM


Fc gamma RIIA /   CD32a



His Capture

3.12 μM


Fc gamma RIIA /   CD32a



His Capture

1.45 μM


Fc gamma RIIIA /   CD16a



His Capture

163 nM


Fc gamma RIIIB /   CD16b (NA2)



His Capture

2.88 μM


Fc gamma RIIIA /   CD16a



His Capture

0.63 μM


Fc gamma RIIIB /   CD16b (NA1)



His Capture

8.17 μM


Fc gamma RI /   CD64



His Capture

 5.45 nM

Click here to view more cases



[1] Noubia Abdiche Y, et al. The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity. MAbs 2015;7(2):331–343.

[2] USP<1108>Assay to evaluate fragment crystallizable (Fc)-mediated effector function.

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