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Purpose of Fc Region

Purpose of Fc Region


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Could someone explain to me the role played by the Fc region of an antibody as well as the purpose of isotype switching? According to Wikipedia, it's to allow the antibody to be usable by different Fc receptors, but I don't get what Fc receptors are for.

For context,

  1. I'm reading about affinity maturation and learning about how ecological and evolutionary processes --- division, selection, and mutation --- drive the maturation of B cells leading to high-affinity plasmocytes that can better target a foreign antigen. Because all this happens on the Fab fragment, it makes sense to me why the Fab region is important in the antibody.
  2. I'm not trained in cell biology.
  3. My goal is to understand the general purpose of the Fc region and how isotype switching may be a major determinant for specific immunological or physiological outcomes, although I'd love to hear anything interesting!

Thanks in advance!


Preamble

From the point of view of understanding I think it is better to talk about the constant and variable regions of antibodies, rather than Fab and Fc, as these are historical designations for fragments of the molecules that were useful in research, but do not exist as separate entities.

Cartoon representation

It is possible to represent the immunoglobulin molecule diagrammatically as shown below. In the left-hand diagram V = variable and C = constant, and the suffixes H and L refer to the heavy and light chains. The right-hand diagram, which purports to be more three-dimensional, shows how the variable regions are responsible for binding the antigen - different manifestations of the variable region have different antigen-specificity.

The different iotypes for an immunoglobulin with a particular antigen-specificity are shown next, with a pre-colour scheme that focuses on the difference between the constant regions of each isotype.

Functions of the constant region of different isotypes

The variable region of an immunoglobulin does the exciting stuff - it binds an antigen, but that is not sufficient for its action. As well as the specific discriminatory action of the variable region, there are roles in presentation and disposal that are the same for all antibodies of a class, but may differ between classes. That is what the constant regions do.

I am not an immunologist (although had to teach the biochemistry at one time in my life - hence the diagrams) so the examples I present are simplistic and incomplete. However here are a few:

  • IgM is made early in the immune response and hence is not very abundant. In order to enhance its response it forms pentamers in which the constant region of the heavy chain interacts with an additional J chain.
  • IgA is involved in immune protection in mucous regions of the body exposed to the exterior. It is directed there by interaction with a receptor on the appropriate epithelial cells, which binds the constant regions of an IgA dimer attached to a J chain.
  • IgE stimulates mast cells to produce histamine. These mast cells have receptors for the constant region of the heavy chains of IgE.

There are many sources on line dealing with this topic, including a section in Berg et al. that has a similar structural approach to that presented here.


The Fc region is composed of the two heavy chain constant regions, and its amino acid composition differs between the different classes of immunoglobulin (antibody). The Fc region of the five immunoglobulin (Ig) classes (IgA, D, E, G, and M)confers upon each class of Ig certain capabilities or properties. For example, IgG can cross the placenta, has a long half-life in serum, and it is opsonic (i.e., binds to Fc-gamma receptors on macrophages, facilitating phagocytosis of microbes), IgE binds to mast cells stimulating hayfever-type allergies, IgA is secreted onto mucosal surfaces, and IgM (and IgG) can activate the complement system, etc.


1.The Fc region is used as binding site for different immune system cells after the opsonisation of a pathogen. 2.Complement protein C1 binds to Fc region of antibody bound to an antigen to trigger classical complement pathway which leads to destruction of that antigen-antibody complex or opsonised pathogen


Harnessing Fc receptor biology in the design of therapeutic antibodies

The antibody Fc domain engages immune-activating, inhibitory, and homeostatic Fc receptors.

Fc domains are largely unaltered in existing therapeutic antibodies, whether to promote or to avoid Fc receptor recruitment.

Recent advances in Fc biology have enabled new antibody and Fc fusion design strategies.

Fc engineering facilitates functional optimization of antibodies for the desired clinical indication.

The antibody Fc domain engages the small family of Fc receptors, expressed on cells of the immune system and beyond, to stimulate a rich diversity of positive and negative cell-mediated effector functions. The emergence of monoclonal antibodies for the treatment of various pathologic conditions has provided additional insights into Fc receptor biology, and has suggested new strategies to exploit Fc receptor interactions to create improved therapeutics. While most therapeutic IgGs approved to date have retained a native IgG Fc domain, the knowledge gained over the last decades has provided the opportunity to design tailored and more efficacious immunotherapies exhibiting fewer side effects and longer half-life. This review summarizes recent advances made in the design of biologics that modulate or exploit Fc receptor-IgG interactions, and describes innovative drugs currently under investigation in clinical trials that have been precisely tuned to achieve a desired therapeutic effect.


Antibody Structure

In simplistic terms antibodies perform two main functions in different regions of their structure. While one part of the antibody, the antigen binding fragment (Fab), recognizes the antigen, the other part of the antibody, known as the crystallizable fragment (Fc), interacts with other elements of the immune system, such as phagocytes or components of the complement pathway, to promote removal of the antigen.

Figure. Schematic representation of an IgG.
An antibody consists of two heavy chains (blue) and two light chains (green) folded into constant and variable domains. The enlargement of the variable domain shows a ribbon representation of the β-sheet framework and CDR loops.

Antibodies all have the same basic structure consisting of two heavy and two light chains forming two Fab arms containing identical domains at either end attached by a flexible hinge region to the stem of the antibody, the Fc domain, giving the classical ‘Y’ shape. The chains fold into repeated immunoglobulin folds consisting of anti-parallel β-sheets (1), which form either constant or variable domains. The Fab domains consist of two variable and two constant domains, with the two variable domains making up the variable fragment (Fv), which provides the antigen specificity of the antibody (2) with the constant domains acting as a structural framework. Each variable domain contains three hypervariable loops, known as complementarity determining regions (CDRs), evenly distributed between four less variable framework (FR) regions. It is the CDRs that provide a specific antigen recognition site on the surface of the antibody and the hypervariability of these regions enables antibodies to recognize an almost unlimited number of antigens (3).

Figure. Structural representations of an IgG.
The heavy chain is shown in blue, light chain in green and glycosylation in orange. On the left is a ribbon representation showing the secondary structure elements and on the right hand side is a space-filled model of the same molecule. PDB accession number of the mouse IgG1 is 1IGY.

Antibodies are glycosylated proteins, with the position and extent of glycosylation varying between isotypes. As displayed in the image above the Fc region of an IgG consists of two paired CH3 domains and, in contrast, two CH2 domains that are separated and do not interact but have two oligosaccharide chains interposed between them. These chains cover the hydrophobic faces that would normally lead to domain pairing. The N-glycans contain a common core region of two N-acetyl-glucosamine residues (GlcNAc) linked to an asparagine (N297 in human IgG1) via an amide bond and three mannose residues. This core structure may contain additional terminal sugars, such as mannose, GlcNac, galactose, fucose and sialic acid, generating a large amount of heterogeneity (4).


Fc receptors

Fc receptors (FcRs) are key immune regulatory receptors connecting the antibody mediated (humoral) immune response to cellular effector functions. Receptors for all classes of immunoglobulins have been identified, including FcγR (IgG), FcεRI (IgE), FcαRI (IgA), FcμR (IgM) and FcδR (IgD). There are three classes of receptors for human IgG found on leukocytes: CD64 (FcγRI), CD32 (FcγRIIa, FcγRIIb and FcγRIIc) and CD16 (FcγRIIIa and FcγRIIIb). FcγRI is classed as a high affinity receptor (nanomolar range KD) while FcγRII and FcγRIII are low to intermediate affinity (micromolar range KD) (1).

In antibody dependent cellular cytotoxicity (ADCC), FcvRs on the surface of effector cells (natural killer cells, macrophages, monocytes and eosinophils) bind to the Fc region of an IgG which itself is bound to a target cell. Upon binding a signalling pathway is triggered which results in the secretion of various substances, such as lytic enzymes, perforin, granzymes and tumour necrosis factor, which mediate in the destruction of the target cell. The level of ADCC effector function various for IgG subtypes. Although this is dependent on the allotype and specific FcvR in simple terms ADCC effector function is high for human IgG1 and IgG3, and low for IgG2 and IgG4. See the table below for IgG subtype variation in effector functions, ranked in decreasing potency.

Effector Function Species IgG Subtype Potency
ADCC Human IgG1≥IgG3>>IgG4>IgG2
Mouse IgG2b>IgG2a>IgG1>>IgG3
C1q Binding Human IgG3>IgG1>>IgG2>IgG4
Mouse IgG2a≥IgG2b>IgG3>IgG1

As shown in the model below FcγRs bind to IgG asymmetrically across the hinge and upper CH2 region. Knowledge of the binding site has resulted in engineering efforts to modulate IgG effector functions – see Fc engineering section for more detail.

Figure. Human IgG1-FcγRIII complex.
A model of human IgG1 in complex with Fcγ receptor III, which binds asymmetrically across the hinge and upper CH2 region of the antibody. The left hand image shows a ribbon representation and the right hand side a space-filled model. The antibody heavy and light chains are shown in blue and green respectively, glycosylation in orange and FcγRIII in red. Model produced from PDB accession numbers 1IGY and 1E4K.


The role of IgG in the immune response

IgG is the major immunoglobulin in blood, lymph fluid, cerebrospinal fluid and peritoneal fluid and a key player in the humoral immune response. Serum IgG in healthy humans presents approximately 15% of total protein beside albumins, enzymes, other globulins and many more.

The Fc portion of IgG, but not F(ab´)2 or Fab fragments, can cross the placenta of a mother and enter fetal circulation, providing the fetus with postpartum protection. IgG molecules are able to react with Fcγ receptors that are present on the surface of macrophages, neutrophils and natural killer cells, and can activate the complement system.

The binding of the Fc portion of IgG to the receptor present on a phagocyte is a critical step in the opsonization. Phagocytosis of particles coated with IgG antibodies is a vital mechanism that cells use to cope with microorganisms.

IgG is produced in a delayed response to an infection and can be retained in the body for a long time. The longevity in serum makes IgG most useful for passive immunization by transfer of this antibody. Detection of IgG usually indicates a prior infection or vaccination.


Antibody potency, effector function and combinations in protection from SARS-CoV-2 infection in vivo

SARS-CoV-2, the causative agent of COVID-19, is responsible for over 24 million infections and 800,000 deaths since its emergence in December 2019. There are few therapeutic options and no approved vaccines. Here we examine the properties of highly potent human monoclonal antibodies (hu-mAbs) in a mouse adapted model of SARS-CoV-2 infection (SARS-CoV-2 MA). In vitro antibody neutralization potency did not uniformly correlate with in vivo activity, and some hu-mAbs were more potent in combination in vivo. Analysis of antibody Fc regions revealed that binding to activating Fc receptors is essential for optimal protection against SARS-CoV-2 MA. The data indicate that hu-mAb protective activity is dependent on intact effector function and that in vivo testing is required to establish optimal hu-mAb combinations for COVID-19 prevention.

Competing Interest Statement

The authors have declared no competing interest.


Structure of Antibody

150 kDa) globular plasma proteins. The basic structure of all antibodies are same.

Structure of Antibody.
Source: Kyowa Hakko Kirin Co., Ltd

There are four polypeptide chains: two identical heavy chains and two identical light chains connected by disulfide bonds. Light Chain (L) consists polypeptides of about 22,000 Da and Heavy Chain (H) consists larger polypeptides of around 50,000 Da or more. There are five types of Ig heavy chain (in mammal) denoted by the Greek letters: α, δ, ε, γ, and μ. There are two types of Ig light chain (in mammal), which are called lambda (λ) and kappa (κ).

An antibody is made up of a variable region and a constant region, and the region that changes to various structures depending on differences in antigens is called the variable region, and the region that has a constant structure is called the constant region.

Structure of Antibody
Source: Sino Biological Inc.

Each heavy and light chain in an immunoglobulin molecule contains an amino-terminal variable (V) region that consists of 100 to 110 amino acids and differ from one antibody to another. The remainder of each chain in the molecule – the constant (C) region exhibits limited variation that defines the two light chain subtypes and the five heavy chains subclasses. Some heavy chains (α, δ, γ) also contain a proline-rich hinge region. The amino terminal portions, corresponding to the V regions, bind to antigen effector functions are mediated by the carboxy-terminal domains. The ε and μ heavy chains, which lack a hinge region, contain an additional domain in the middle of the molecule. CHO denotes a carbohydrate group linked to the heavy chain.


IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions

Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare competing financial interests: details are available in the online version of the paper.


Purpose of Fc Region - Biology

Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.44 Å
  • R-Value Free: 0.339 
  • R-Value Work: 0.278 
  • R-Value Observed: 0.278 

wwPDB Validation   3D Report Full Report

Structural Analysis of Human Igg-Fc Glycoforms Reveals a Correlation between Glycosylation and Structural Integrity

(2003) J Mol Biol 325: 979

  • PubMed: 12527303  Search on PubMed
  • DOI: 10.1016/s0022-2836(02)01250-0
  • Primary Citation of Related Structures:  
    1H3Y, 1H3X, 1H3W, 1H3V, 1H3U, 1H3T
  • PubMed Abstract: 

Antibodies may be viewed as adaptor molecules that provide a link between humoral and cellular defence mechanisms. Thus, when antigen-specific IgG antibodies form antigen/antibody immune complexes the effectively aggregated IgG can activate a wide range of effector systems .

Antibodies may be viewed as adaptor molecules that provide a link between humoral and cellular defence mechanisms. Thus, when antigen-specific IgG antibodies form antigen/antibody immune complexes the effectively aggregated IgG can activate a wide range of effector systems. Multiple effector mechanisms result from cellular activation mediated through a family of IgG-Fc receptors differentially expressed on leucocytes. It is established that glycosylation of IgG-Fc is essential for recognition and activation of these ligands. IgG antibodies predominate in human serum and most therapeutic antibodies are of the IgG class. The IgG-Fc is a homodimer of N-linked glycopeptide chains comprised of two immunoglobulin domains (Cgamma2, Cgamma3) that dimerise via inter-heavy chain disulphide bridges at the N-terminal region and non-covalent interactions between the C-terminal Cgamma3 domains. The overall shape of the IgG-Fc is similar to that of a "horseshoe" with a majority of the internal space filled by the oligosaccharide chains, only attached through asparagine residues 297.To investigate the influence of individual sugar (monosaccharide) residues of the oligosaccharide on the structure and function of IgG-Fc we have compared the structure of "wild-type" glycosylated IgG1-Fc with that of four glycoforms bearing consecutively truncated oligosaccharides. Removal of terminal N-acetylglucosamine as well as mannose sugar residues resulted in the largest conformational changes in both the oligosaccharide and in the polypeptide loop containing the N-glycosylation site. The observed conformational changes in the Cgamma2 domain affect the interface between IgG-Fc fragments and FcgammaRs. Furthermore, we observed that the removal of sugar residues permits the mutual approach of Cgamma2 domains resulting in the generation of a "closed" conformation in contrast to the "open" conformation which was observed for the fully galactosylated IgG-Fc, which may be optimal for FcgammaR binding. These data provide a structural rationale for the previously observed modulation of effector activities reported for this series of proteins.

Organizational Affiliation

Max-Planck-Institut für Biochemie, Abteilung Strukturforschung, Am Klopferspitz 18a, D-82152, Martinsried, Germany.


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