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In the global bio-pharmaceutical market and research & development, monoclonal antibody drugs are one of the most important categories. Monoclonal antibodies are characterized by high specificity, strong targeting and low toxic side effects. For this reason, the development of monoclonal antibody drugs is the focus of Global R&D.
As you know, how to optimize the antibody structure and obtain the most ideal antibody with affinity for Fc receptors is the most important factor in the process of the development of therapeutic monoclonal antibodies. And Fc receptors play a crucial role in immune cells activation by antibodies stimulation. Based on this reason, we summarize an article about Fc receptors from the following aspects, involving definition, function, classification, signaling mechanisms and application. We hope these information can give help to your research.
Fc receptors, as its name shows, have an ability of specific binding for a part of an antibody known as the Fc fragment region. In this section, we illustrate the definition and function of Fc region and Fc receptors respectively.
Fc region, also called fragment crystallizable region, is the tail region of an antibody that plays a role in modulating immune cell activity. It is composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. Antibodies are also called immunoglobulins. The type known as IgG is most commonly found in the blood. The Fc region of IgG is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains.
As shown in the figure 1, in an experimental setting, Fc and Fab fragments can be produced in the laboratory. The enzyme papain can be used to cleave an immunoglobulin monomer into two Fab fragments (also called fragment antigen-binding) and one Fc fragment. The Fc region is the base of Y that interacts with cell surface receptors and some proteins of the complement system. This property allows antibodies to activate the immune system. In this way, it mediates different physiological effects of antibodies, including detection of opsonized particles, cell lysis, degranulation of mast cells, basophils, and eosinophils, and other processes. Fab region, the other part of an antibody, contains variable sections that can bind to antibody specifically. If you want know more things about antibody, please click here.
Figure 1. The structure of IgG antibody isotype
Fc receptors are a series of cell surface proteins that recognizes with Fc region of antibody and that is the name of Fc receptor derived from. Fc receptors are found on the membrane of certain immune cells, including B lymphocytes, natural killer cells, macrophages, neutrophils, and mast cells. Fc receptors binding to antibodies that are attached to infected cells or invading pathogens leads to the protective functions of the immune system.
Their activity stimulates phagocytic or cytotoxic cells to destroy microbes, or infected cells by antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity. As shown in the figure 2.
Figure 2. The illustration of an Fc receptor
Generally speaking, there are several types of Fc receptors depending on the kind of antibody that they recognize. Among of these types, the following three types are major. One is Fc receptors recognizing Fc portion of IgG called Fc gamma receptor (FcγR). Other is Fc receptor recognizing C-terminal of IgA called Fc alpha receptor (FcαR). The last is Fc receptor recognizing C-terminal of IgE called Fc epsilon receptor (FcεR).
Fc gamma receptor, also known as FcγR, is divided into three classes based on genetic homology: FcγR1 (CD64), FcγR2 (CD32) and FcγR3 (CD16). Each type is encoded by specific genes located in the same region of the long arm of chromosome 1 [1] [2]. There are several different closely related genes for each FcgR which have different antibody affinities due to their different molecular structure, including FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIc (CD32c), FcγRIIIa (CD16a), FcγRIIIb (CD16b) [3].
Figure 3. The structure of Fc gamma receptors
ITAM: immunoreceptor tyrosine-based activation motif; ITIM: immunoreceptor tyrosine-based inhibitory motif
As the figure 3 shows, FcγRI binds to IgG more strongly than all of other FcγRs (FcγRII or FcγRIII) do. FcγRI also has an extracellular portion composed of three immunoglobulin (Ig)-like domains, one more domain than others. This characteristic allows FcγRI to bind a signal IgG molecule (or monomer). But please note that all of FcγRs must bind multiple IgG molecules within an immune complex to be activated. Among of FcγRs, FcγRI, FcγRIIa, FcγRIIc and FcγRIIIa possess a classic cytoplasmic ITAM signaling domain, and can stimulate immune effector-cell activation. As a counterpoint to the activating FcgRs, FcgRIIb possesses a cytoplasmic ITIM [4]. In the following Table 1, we compare the features of all FcγRs to each other.
Table 1. The feature of Fc gamma receptors
| Name | FcγRI | FcγRIIa | FcγRIIb | FcγRIIc | FcγRIIIa | FcγRIIIb | |
|---|---|---|---|---|---|---|---|
| Function | Activating | Activating | Inhibitory | Activating | Activating | Activating | |
| Expression | Lymphoid | Not expressed | Not expressed | B cell and Plasma cell | NK cell | NK cell | Not expressed |
| Myeloid | Monocyte, DC and macrophage | Monocyte, DC and macrophage | Monocyte, DC, and macrophage | Not expressed | Monocyte, DC and macrophage | Not expressed | |
| Granulocyte | Neutrophil and eosinophil | Neutrophil | Neutrophil, mast cell, and basophil | Not expressed | Not expressed | Neutrophil, mast cell, and eosinophil | |
| Affinity | High | Low | Low | Low | Low | Low | |
Fc-alpha receptor, also known as FcαR, is the receptor specific for IgA which binds IgA with low affinity. Only one type Fc receptor belongs to the FcαR subgroup, which is called FcαRI (CD89) [5]. FcαRI is expressed on neutrophils, eosinophils, monocytes/macrophages, dendritic cells, and Kupffer cells [6] [7] [8]. FcαRI expression is constitutive and independent of the presence of IgA ligand because the receptor is expressed at similar levels on cells from patient deficient in IgA [9]. The structure of Fc alpha receptor is shown in the figure 4.
Figure 4. The structure of Fc alpha receptor and Fc epsilon receptors
A single gene encoding FcαRI has been isolated, which is located within the leukocyte receptor cluster on chromosome 19. The FcαRI lacks canonical signal transduction domains but can associate with two FcRγ signaling chains that bear an activation motif (ITAM) in the cytoplasmic domain. FcαRI expressed alone mediates endocytosis and recycling of IgA [10].
Table 2. The feature of Fc alpha receptor
| Receptor name | Principal antibody ligand | Affinity for ligand | Cell distribution | Effect following binding to antibody |
|---|---|---|---|---|
| FcαRI (CD89) | IgA | Low (Kd > 10−6 M) | Monocytes Macrophages Neutrophils Eosinophils | Phagocytosis Induction of microbe killing |
*The content of Table 2 is derived from Wikipedia.
Fc epsilon receptor, also known as FcεR, is an Fc receptor that binding IgE. Currently, two types of FcεR are known.
The high-affinity receptor FcεRI is capable of binding monomeric IgE (it has two Ig-like domains). The classical Fc epsilon receptor I is tetrameric. FcεRI is found on epidermal Langerhans cells, eosinophils, mast cells and basophils [11] [12]. Based on its cellular distribution, FcεRI plays a crucial role in controlling allergic responses. The low-affinity receptor FcεRII (CD23) interacts preferentially with complex IgE. FcεRII has multiple functions as a membrane-bound or soluble receptor; it controls B cell growth and differentiation and blocks IgE-binding of eosinophils, monocytes, and basophils. The structure of FcεRs are shown in the figure 4.
Table 3. The feature of Fc epsilon receptors
| Receptor name | Principal antibody ligand | Affinity for ligand | Cell distribution | Effect following binding to antibody |
|---|---|---|---|---|
| FcεRII (CD23) | IgE | Low (Kd > 10−7 M) | B cells Eosinophils Langerhans cells | Possible adhesion molecule IgE transport across human intestinal epithelium Positive-feedback mechanism to enhance allergic sensitization (B cells) |
| FcαRI (CD89) | IgA | Low (Kd > 10−6 M) | Monocytes Macrophages Neutrophils Eosinophils | Phagocytosis Induction of microbe killing |
*The content of Table 2 is derived from Wikipedia.
Beside the above Fc receptors, there are several other Fc receptors, including Fcα/μR, and neonatal Fc receptor (FCGRT). Fcα/μR is an Fc receptor for IgA and IgM. FCGRT is kind of MHC class-I receptor and plays a role in monitoring IgG turnover.
Upon the Fc region of antibody binding to special Fc receptor, a series of signaling mechanisms are triggered in cell. Based on recent researches, the signaling mechanisms of Fc gamma receptor, Fc-alpha receptor and Fc epsilon receptor are more lucid than other Fc receptors. In this division, we briefly summarize the mechanism of Fc gamma receptor, Fc-alpha receptor and Fc epsilon receptor respectively.
Fc gamma receptors generate signals within their cells through an important activation motif, ITAM. All of the Fc gamma receptors (FcγR) belong to the immunoglobulin superfamily. Fc gamma receptor is essential participant in many immune system effector functions, such as phagocytosis of opsonized, release of inflammatory mediators and antibody-dependent cellular cytotoxicity. Here, we primary illustrate the mechanism of Fc gamma R mediated phagocytosis.
Phagocytosis plays a crucial role in host-defense mechanisms by the uptake and destruction of infectious pathogens. Specialized cell types take part in this process in higher organisms, including macrophages, neutrophils, and monocytes. Upon opsonization with antibodies (IgG), foreign extracellular materials are recognized by Fc gamma receptors. Cross-linking of Fc gamma receptors initiates a variety of signals mediated by tyrosine phosphorylation of multiple proteins, which lead through the actin cytoskeleton rearrangements and membrane remodeling to the formation of phagosomes. Nascent phagosomes undergo a process of maturation that involves fusion with lysosomes. The acquisition of lysosomal proteases and release of reactive oxygen species are crucial for digestion of engulfed materials in phagosomes.
Figure 5. The picture of Fc gamma R mediated phagocytosis
FcαRI is a member of the multichain immune recognition receptor (MIRR) family. As mentioned in the section of Fc alpha receptor, the FcαRI lacks canonical signal transduction domains known as ITAM signaling motif. For this reason, the signaling transduction of FcαRI is dependent on association with the FcR γ−chain subunit, forming the trimer FcαRIα/γγ
As the Figure 6 shows, FcRγ-ITAM are initially phosphorylated by the Src kinase lyn, which leads Recruitment and phosphorylation of Syk and Btk are modulated by cell stimulation with interferon-γ and/or phorbol ester, indicating that activation of these tyrosine kinases through FcαRI may depend on the level of cell priming at inflammatory sites [14].
FcαRI cross-linking triggers calcium release from intracellular stores in neutrophils and induction of NADPH oxidase activity that is sensitive to inhibition by PI 3-kinase inhibitors. FcαRI can also associate with Grb2, Shc, SHIP, and SLP-76, suggesting the formation of adaptor complexes to regulate signaling [15].
Figure 6. Signaling pathways triggered by FcαRI
Fc epsilon RI signaling pathways in mast cells are initiated via the interaction of antigen with IgE combined to the extracellular domain of the alpha chain of Fc epsilon RI. The activation pathways are regulated both positively and negatively by the interactions of numerous signaling molecules. Mast cells that are thus activated release preformed granules which contain biogenic amines (especially histamines) and proteoglycans (especially heparin). The activation of phospholipase A2 causes the release of membrane lipids followed by development of lipid mediators such as leukotrienes (LTC4, LTD4 and LTE4) and prostaglandins (especially PDG2). There is also secretion of cytokines, the most important of which are TNF-alpha, IL-4 and IL-5. These mediators and cytokines contribute to inflammatory responses.
Figure 7. The picture of Fc epsilon RI signaling pathway
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FcγRs play an important role in determining the therapeutic activity of monoclonal IgG antibodies (mAbs) by their ability to activate the cytotoxic activity of FcγR-positive cells such as NK cells, monocytes, macrophages and neutrophils and by increasing antigen presentation by DC when ligated by the Fc portion of therapeutic antibodies. Recent studies in Fc receptor-deficient nude mice show that the anti-tumor effects of mAbs such as those directed at-CD20 and HER2 require the presence of the signal transducing Fcγ chain that is involved in the activation of FcγRI and FcγRIII receptors that are expressed on monocytes, macrophages, and NK cells.
Additionally, antibody-dependent cellular cytotoxicity (ADCC) is a major mechanism of action of therapeutic monoclonal antibodies (mAbs) such as cetuximab, rituximab and trastuzumab. Fc gamma receptors (FcgR) on human white blood cells are an integral part of the ADCC pathway [16] [17]. Differential response to therapeutic mAbs has been reported to correlate with specific polymorphisms in two of these genes: FCGR2A (H131R) and FCGR3A (V158F). These polymorphisms are associated with differential affinity of the receptors for mAbs.
References
[1] Gessner JE, Heiken H, et al. The IgG Fc receptor family [J]. Ann Hematol 1998, 76(6):231 – 248.
[2] Ravetch JV, Perussia B. Alternative membrane forms of Fc gamma RIII (CD16) on human natural killer cells and neutrophils [J]. J Exp Med 1989, 170(2):481 – 497.
[3] James D Mellor, Michael P Brown, et al. A critical review of the role of Fc gamma receptor polymorphisms in the response to monoclonal antibodies in cancer [J]. J Hematol Oncol. 2013, 6:1-10.
[4] Sondermann P, Szymkowski DE, et al. Harnessing Fc receptor biology in the design of therapeutic antibodies [J]. Curr Opin Immunol. 2016, 40:78-87.
[5] Otten MA, van Egmond M. The Fc receptor for IgA (FcalphaRI, CD89) [J]. Immunology Letters. 2004, 92 (1–2): 23–31.
[6] Geissmann F, Launay P, et al. A subset of human dendritic cells expresses IgA Fc receptor (CD89), which mediates internalization and activation upon cross-linking by IgA complexes [J]. J. Immunol. 2001, 166:346–52.
[7] van Egmond M, van Garderen E, et al. FcαRI-positive liver Kupffer cells: reappraisal of the function of immunoglobulin A in immunity [J]. Nat. Med. 2000, 6:680–85.
[8] Heystek HC, Moulon C, et al. Human immature dendritic cells efficiently bind and take up secretory IgA without the induction of maturation [J]. J. Immunol. 2002, 168:102–7.
[9] Chevailler A, Monteiro RC, et al. Immunofluorescence analysis of IgA binding by human mononuclear cells in blood and lymphoid tissue [J]. J. Immunol. 1989, 142:2244– 49.
[10] Monteiro RC, Van De Winkel JG. IgA Fc receptors [J]. Annu Rev Immunol.2003, 21:177-204.
[11] Ochiai K, Wang B, et al. A review on Fc epsilon RI on human epidermal Langerhans cells [J]. International Archives of Allergy and Immunology. 1994, 104 Suppl 1 (1): 63–4.
[12] Prussin C, Metcalfe DD. IgE, mast cells, basophils, and eosinophils [J]. The Journal of Allergy and Clinical Immunology. 2006, 117 (2 Suppl Mini-Primer): S450–6.
[13] Pfefferkorn LC, Yeaman GR. Association of IgA-Fc receptors (FcαR) with FcεRI γ2 subunits in U937 cells. Aggregation induces the tyrosine phosphorylation of γ2 [J]. J. Immunol. 1994, 153: 3228–36.
[14] LaunayP, LehuenA, et al. IgA Fc receptor (CD89) activation enables coupling to syk and Btk tyrosine kinase pathways: differential signaling after IFN-γ or phorbol ester stimulation [J]. J. Leukoc. Biol. 1998, 63:636–42.
[15] Lang ML, Shen L, et al. Fcα receptor cross-linking causes translocation of phosphatidylinositol-dependent protein kinase 1 and protein kinase B alpha to MHCclassII peptide-loading-like compartments [J]. J. Immunol. 2001, 166:5585– 93.
[16] Alderson KL, Sondel PM: Clinical cancer therapy by NK cells via antibody-dependent cell-mediated cytotoxicity [J]. J Biomed Biotechnol 2011, 2011:379123.
[17] Ravetch JV, Bolland S. IgG Fc receptors [J]. Annu Rev Immunol 2001, 19:275 – 290.
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