See also “antibodies” for discussion of affinity  and “isotypes” of antibodies for a discussion of the various effector functions of antibody isotypes.

See also antibody engineering and specifically engineering of the FcyRs to develop more potent antibodies. 

Introduction to Fc Receptors, generally

The Fc receptors, members of the immunoglobulin gene superfamily of proteins, are surface glycoproteins that can bind the Fc portion of immunoglobulin molecules. Fc receptors are named after the capability of binding to the constant Fc part of IgG. Based on binding experiments to IgG and subclasses thereof, Fc receptors have been divided into six types I-VI.

An Fc receptor is a protein found on the surface of certain cells –including NKCs, macrophages, neutrophils, and mast cells –that contribute to the protective funciton of the immune system. Its name is dervied form its binding specificity for a part of an antibody known as the Fc (Fragment-crystallizable) region. Fc recetpros bind to antibodies that are attached to infected cells or invading pathogens. Their activity stimulates phagocytic or cytotoxic cells to destroy microbes or infected cells by antibody medaited cellular phagocytosis (ADCP) or antibody dependent cell mediated cytotoxicity (ADCC). There are several different types of Fc recetpros, which are classified based on the type of antibody that thye recognize: htose that bind IgG are called Fc gamma recetpors, those that bind IgA are called Fc alpha receptors nad those that bind IgE are called Fc epsilon receptors. (HImmler (US 2010/0184615).

The receptor from S. aurea, protein A, for example, definining the type I receptor has been the subeject of immense studies. The structure for a complex between human Fc (IgG1) and a single domain B of SPA has been determined by X-ray crystallography. Based on this structure, the B domain can be viewed as a compact structure consisting of three anti-parallel alpha-helices connected with loops. In the Fc binding, which is of both electrosatic and hydrophobic nature, only side chains of residues from helices 1 and 2 are involved, while the the third helix does not participate. Based on this domain B, a synthetic IgG binding domain Z was constructed. Streptococcal strains of seriological groups C and G display a binding repertoire for mammalina IgGs, including human IgG3, which is even broader than the type I receptor. The name protein G was suggested for this type III receptor from group C streptococci. (Nilsson, US 6,534,628). 

FcgammaR Receptor classes:

There are two well-defined funcitonal classes of mammalian receptors. One class of receptors transports immunoglobulins across epithelial tissues to their main sites of action. This class includes the neonatal Fc receptors (FcRn) (see outline), which transports IgG,, and the polymeric immunoglobulin receptor (pIgR) which transports IgA and IgM. Another class of receptors present on the surfaces of effector cells which triggers various biological response upon binding antibody-antigen complexes. Of these, the IgG receptors (FcygammaR) and IgE receptors, Fceipsilon R, are the best characterized. (Raghavan, “Fc receptors and their interactions with immunoglobulins” Ann. Rev. Cell Dev. Biol. 1996, 12: 181-220). 

Specific FcRs exists for all Ig classes, the ones for IgG being the most abundant with the widest diversity. Together with the high affinity receptor for IgE (FcepsilonRIa), FcgammaRI (CD64), Fcgamma RII (CD32 ) and FcygammaRIIIa (CD16) occur as type I transmembrane proteins or in soluble forms (sFcRs) but also a glycosylphosphatidylinositol anchored form of the FcgammaRIII (FcgammaRIIIb) exists. Furthermore, the FcyRs occur in various isoforms and alleles. In contrast to the overall homologous extraceullar parts, the membrane spanning and the cytoplasmic domains differ. They may be delted entirely or be of a size of 8 kDa. They many contain either a 26 amino acid immunoreceptor tyrosine-based activation motif (ITAM) as in FcyRIIa or a respective 13 amino acid inhibitory motif (ITIM) in FcyRIIb involed in signal transduction. (Sondermann, WO 00/32767). 

FcgammaR are expressed on immune system effector cells (e.g., B cells, neutrophils, natural killer cells, monocytes and macrophages). They consist of 3 distinct classes:

FcgammaR IA (CD64): are epxressed on macrophage, monocyte, eosinophile, neutrophil and DCs.

The human FcyRI (CD64) is expressed on monocytes nad macrophages and a number of myeloid cell lines and binds to IgG1 and IgG3 with similar affinity at the lower hinge region around residues 234-238 and residues in the hinge proximal region of the CH2 domain. (Lobner, “Engineered IgG1-Fc–one fragment to bind them all” Immunological Reviews, 2016)

Fcgamma IIA (CD32): are expressed on macrophage, platelet, neutrophil, monocyte and DCs.

In humans, two forms of FcyRII (CD32) are found, namely FcyRIIa and FcyRIIb. Both are widely expressed on multiple cell types constitutively and/or following induction or upregulation and bind to IgG1 and IgG3 with similar affinity. While their external domains are highly homologus they transduce opposite signals (activating versus inhibitory) via their intracytoplamatic domains. The binding site at the Fc is similar to that of FcyRI. (Lobner, “Engineered IgG1-Fc–one fragment to bind them all” Immunological Reviews, 2016)

In humans, the FcgammaRII class is further divided into FcgammaRIIa (an activating receptor)  and FcgammaRIIb (an inhibitory receptor).  Engaging the activity of FcgammaRIIa, FcgammaRIIIa and FcgammaRb is further complicated by differential cell expression patterns and receptor polymorphisms. For example, FcgammaRIIIa-V158 binds human IgG1 with about 10 fold higher affinity than the FcgammaRIIIa-F158 polymorph (Presta, 20008, Curr. Opin. Immunol. 20: 460-470).

FcyR IIC (CD32c): are expressed on NK cells

FcgammaRIII (CD16A): are expressed on NK cells, macrophage, monocyte, DCs

FcyRIII (CD16) is a low-affinity receptor either expressed as an intrinsic (FcyRIIIa) or glycosphingolipid-linked protein (FcyRIIIb). Binding of natural killer (NK) cell expressed FcyRIIIa to the lower hinge region activates ADCC. (Lobner, “Engineered IgG1-Fc–one fragment to bind them all” Immunological Reviews, 2016)

In humans the FcgammaRIII class is further divided into FcgammaRIIIa (activating) and FcgammaRIIIb (unique in being GPI linked and not containing an intracellular domain as do the others). Engaging the activity of FcgammaRIIa, FcgammaRIIIa and FcgammaRb is further complicated by differential cell expression patterns and receptor polymorphisms. For example, FcgammaRIIIa-V158 binds human IgG1 with about 10 fold higher affinity than the FcgammaRIIIa-F158 polymorph (Presta, 20008, Curr. Opin. Immunol. 20: 460-470).

FcgammaR structure

Ig superfamily members are efined as molecules that contain domains with sequence similarity to the variable or constant domains of antibodies. (Raghavan, “Fc receptors and their interactions with immunoglobulins” Ann. Rev. Cell Dev. Biol. 1996, 12: 181-220). 

The FcgammaR receptors are all members of the immunoglobulin superfamily, haivng an IgG binding alpha chain with an extracellular portion comprised of either two (FcgammaRII and FcgammaIII) or three (FcgammaRI) Ig-like domains. FcgammaRI and FcgammaRIII also have accessory chains associated with the alpha chain which function in signal transduction. Signaling by FcgammaR is either through an immunoreceptor tyrosine based activaiton motif (ITAM) or immunoreceptor tyrosine based inhibitory motif (ITIM) resident in the intracellular domain of the receptor; the exception is FcgammaRIIIB, a glycosyl-phosphatidylinositol (GPI) linked receptor, which may function through assocation with other membrane boudn proteins. (Presta, Advanced Drug Delivery Reviews 58 (2006) 640-656). 

Affinity of Fc gamma R for IgG

The FcgammaR are also differentiated by their affintiy for IgG and their interaction with the 4 subclasses of human IgG. FcgammaRI exhibits a high affinity for monomeric IgG. The order of affinity is IgG1, IgG3, IgG4, IgG2. In contrast, monomeric IgG binds poorly to FcgammaRII and FcgammaRIII and thus these receptors can only interact effectively with multimeric IgG complexes (e.g., with multiple mAbs bound to a cell). The complexity of the human FcgammaR system is amplified by the existence of polymorphic forms of FcgammaRIIA, FcgammaRIIIA, and FcgammaRIIIB. (Presta, Advanced Drug Delivery Reviews 58 (2006) 640-656). 

FcgammaR Functions

Various mechanisms can be invovled in providing therapeutic efficacy of antibodies such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and apoptosis. For example, evidence inducates that the anti-CD20 mAb rituximab oeprates through conventional effector mechanisms measured by CDC and ADCC. (Glennie, “Antibody specificity controls in vivo effector mechanisms of anti-CD20 reagent” Blood, 2004, 103(7))

Neutralization:

Neutralization is the most sought-after property of vaccine-induced antibodies and is a property that is measured in vitro as the ability of antibodies to inhibit viral entry into target cells in the absence of other anti-viral functions. Generally, neutralizaiton results from inhibition of viral attachment, fusion events, or both. In vivo, neutralizing antibodies can also exert anti-viral effects by other mechanisms, including effector functions medited by the crystalizable fragment (Fc) part of the antiboyd moelcule, such as complement activaiton and antibody-dependent cellular cytotoxicity (ADCC). These effector functions can act agaisnt both free virions and virus-infected cells. (Burton”Fc receptor but not complement binding is important in antibody protection against HIV” 449 (2007). 

Apotosis: 

The important of apoptosis in the therapeutic effect of some antibodies can be seen with comparing various anti-CD20 antibodies. The anti-CD20 mAb rituximab for example does not evoke apoptosis very well but it does evoke CDC and ADCC. The anti-CD20 mAb B1 in contrast shows very weak CDC acitivity yet protected mice from tumors very effectively, even in the absence of complement. The B1 mAb was still effective even in the absence of NK cells, arguing that it was not evoking potent potent ADCC type mechaisms. Even when (F9ab’)2 fragments of B1 were used, rendering the molecule inert in terms of Fc dependent effector mechanisms, a substanital proportion of therapeutic activity remained. However, B1 was able to induce high levels of apoptosis. (Glennie, “Antibody specificity controls in vivo effector mechanisms of anti-CD20 reagent” Blood, 2004, 103(7))

FcRs in antibody transport:

Transfer of materanl IgG molecuels form teh mother to the fetus or infant is a mechanism by which mammalian neonates acquire humoral immunity to antigens encountered by the motehr. The protein responsbiel for teh transfer of IgG is called FcRn (see outline). 

The transport of IgA from the circulation into secretions has also been well studies. IgG is found in epithelial secretions such as the lumen of the gut, the salivary and tear glands, respiratory secretions and breat milk, where the neutralizing capacity of IgA forms the first line of defense against entering pathogens. IgA is synthesized by plasma cells beneathed the basement membranes of surface epithelia. Subsequent to synthesis, the IgA molecuels must cross the eptihelial cell barrier to enter into scretions. Transcytosis of IgA across polarized epithelial cells first invovles the binding of IgA to pIgR prtoeins on the basolateral surface. pIgR-IgA complexes are interanlzied and sorted into endosomes destined for the apical surface. At the apical surface, pIgR is cleaved and the extracellular portein of the receptors is released into secretions as a complex with IgA. Theis complex, called secretory IgA, prects against pathogens in the digestive, respiratry and genital tracts. (Raghavan, “Fc receptors and their interactions with immunoglobulins” Ann. Rev. Cell Dev. Biol. 1996, 12: 181-220). 

FcRs in antibody-mediated effector responses: 

FcRs for IgG and IgE are present on the surface of several accessory cells of the immune system. These receptors, designed FcgammaRs for those that bind IgG and FcepisolonRs for those that bind IgE, interact with antibody-antigen complexes to activate various biological resposnes. (Raghavan, “Fc receptors and their interactions with immunoglobulins” Ann. Rev. Cell Dev. Biol. 1996, 12: 181-220). 

FcRs play a key role in defending the organisms against infections. After pathogens have gained access to the blood, they are opsonized by Igs. The resulting immunocomplexes bind due to their multivalency with high avidity to FcR bearing cells leading to clustering of the FcRs which triggers several effector functions. These include, depending on the expressed FcR type and associated proteins endocytosis with subsequent neutralization of the pathogens and antigen presentation, ADCC, secretion of mediators or the regulations of antibody production. (Sondermann, US 7,074,896))

Engagement of Fcy receptors (FcyRs) with the Fc region of IgG elicit immune respones by leukocytes. (Mimura, J. Biol. Chemistry, 276(4), 2001)

–Antibody dependent cell mediated cytotoxicity (ADCC):

ADCC is the killing of antibody coated target cells by cell with Fc receptors that recognize the constant region of the bound antibody. Most ADCC is mediated by NK cells that have the Fc recetpor FcgammaRIII (CD16) on their surface. Typicaly assays emply target cells, like Ramos cells, incubated with serially diluted antibody prior to the addition of freshly isolated effector cells. The ADCC asay is then further incubated for several hours and % cytotoxicity detected. Usually the target:effector atio is about 1:16. (Himmler US 2010/0184615)

The evdience that ADCC and FcygammaR are imporatnt in the therapeutic activity of for example the anti-CD20 mAb rituximab comes from several soruces, both clinical and experimental. It has been shown that patients with non-Hodgkin lympohma (NHL) expressing the high-affinity 158V variant of the FcyRIIIa gene have better resposne rates after rituximab treatment than those carrying the low-affinity allotype. Similar results have been shown for the depletion of B cells with rituximab in systemic lupus erthyematosus (SLE). The importance of FcyR on macophages have also been shown to be crittical to the ability of mAb to control subcutanous B-cell lymphoma. Thus, there are clearly data to support a role of FcgammaR bearing effectors in mediating the activity of anti-CD20 mAb. (Glennie, “Antibody specificity controls in vivo effector mechanisms of anti-CD20 reagent” Blood, 2004, 103(7))

–Complement dependent Cytotoxicity (CDC): Complement Activation and Fc-gamma receptors (CDC):

CDC is a mechanisms of dilling cells in which antibody bound to the target cell surface fixes complement, which results in assembly of the membrane attack complex that punches holes in the target cell membrane resulting in subsequent cell lysis. The commonly used DCD assay faollws the same procedure as for ADCC determination, hwoever, with complement containing serum instead of effector cells. (Himmler US 2010/0184615)

If elimination of complement activation is an advantage for a therapeutic antibody, either human IgG2 or IgG4 is commonly chosen since these two subclasses are inefficient at complement fixation, with IgG4 lacking all complement activation. 

The anti-CD20 monoclonal antibodies rituximab and 1F5, which redistribute CD20 into membrane rafts, are bound efficiently by C1q, deposit C3b, and result in complement-dependent cytotoxicity (CDC). This activity is important in vivo because cobra venom factor markedly reduces the efficiacy of rituximab and 1F5 in lympohoma xenograft models. However, complement depletion has no effect on a different anti-CD20 mAb, B1 which does not redistribute CD20 into membrane rafts, bind C1q, or cause efficient CDC. Instead, B1 more likely functions through its ability to signal apoptosis. (Glennie, “Antibody specificity controls in vivo effector mechanisms of anti-CD20 reagent” Blood, 2004, 103(7))

–Antibody dependent Cellular Phagocytosis (ADCP or ADPC):

Phagocytosis by phagocytes usually human monocytes or monocyte dervied marophages ars mediated by an antibody can be dtermined as follows: Purified monocytes may be cultured with cytokines to enhance expression of FcyRs or to induce differententiation into macrophages. ADCP and ADCC assay are then performed with target cells. Phagocytosis is detemriend as the percetnage of positive cells measrued by flow cytometry. The positive ADCP activity is proven with a significant uptake of the antibody-antigen complex by the phagocytes. (Himmler US 2010/0184615)

In a typical assay PBMC or monocytes or monocyte dervied macrophages are resuspended in RF2 medium (RPMI 1640 supplemented with 2% FCS) in 96 well plates. Appropriate target cells, expressing the target antigen e.g., Her2/neu antigen and SKBR3 cells) are stained with PKH2 green fluorescnece dye. Subsequently PKH2 labeled target cells and an Her2 specific (IgG1) antibody or mouse IgG1 isotype control are added to the well of PBMCs in different concentraiton and inubated. Following the incubation, PBMCs or monocytes or monocyte derived marcrophages and target cells are harvested with EDTA-PBS and transfered to plates. Cells are coutnerstained with RPE-conjugated anti-CD11b, ant-CD14 and human IgG, mixed and incubated. FACS is perforemed. PKH2 labeled target cells (green) are detected in teh FL-1 channel and RPE labeled PBMC or onocytes or monocytes derived macrophages (red) are detected in the FL2 channel. (Himmler US 2010/0184615)

Neonatal Fcr (FcRn) (aka “MHC Class I-related receptor”) (see outline)