Schbio Biotech

Antibody Overview


The recognition of foreign material is the hallmark of the specific adaptive immune response in mammals, of which immunoglobulins are an integral part. Immunoglobulins (Ig) are a group of glycoproteins present in the tissue and fluids of all vertebrates (including mammals, birds, reptiles and cartilaginous fish). Some are on the surface of B lymphocytes (or B-cells) and others, known as antibodies, are free in the blood or lymph. This section of the Absolute Antibody resources aims to act as a basic introduction to antibodies covering the following areas:


A brief history of antibodies. 1

Antibody structure. 2

Antibody isotypes and subtypes. 4

Allotypes. 11

Antibody effector function. 13

Other antibody interactions. 15

Antibodies as tools. 17

1. A brief history of antibodies

2. Antibody structure

3. Antibody isotypes and subtypes

4. Allotypes

5. Antibody effector function

6. Other antibody interactions

A brief history of antibodies

The acquisition of immunity to a disease that a patient has already encountered has been documented for many centuries. Arguably some of the earliest work in the field that has now become known as immunology was performed in the period around 1714-1717. Lady Mary Wortley Montagu, Emanuel Timoni and James Pylarini pioneered a smallpox inoculation, a course of action unparalleled in medical advance up to that point. Variolation, as it was known, used live smallpox virus in the liquid taken from a smallpox blister in a mild case of the disease and carried in a nutshell (1). In 1798 the first smallpox vaccination was more notably demonstrated by Edward Jenner. This was performed by inoculating a boy with the fluid from a cowpox pustule giving him immunity to the very similar but much more serious disease smallpox (2, 3).

The earliest reference to antibodies came from Emil von Behring and Shibasabura Kitasato in 1890. In a landmark publication they showed that the transfer of serum from animals immunised against diptheria to animals suffering from it could cure the infected animals (4). The potential for treatment in humans was immediately apparent and Behring was later awarded the Nobel Prize for this work in 1901.

In 1900 Paul Ehrlich, who is regarded as one of the fathers of modern immunology, proposed the side-chain theory, where he hypothesised that side chain receptors on cells bind to a given pathogen. He was the first to propose a model for an antibody molecule in which the antibody was branched and consisted of multiple sites for binding to foreign material, known as antigen, and for the activation of the complement pathway (5). This model agreed with the ‘lock and key’ hypothesis for enzymes proposed by Emil Fischer (6, 7) and still in general terms holds true today.

Astrid Fagraeus in 1948 described that plasma B cells are specifically involved in antibody generation and by 1957 Frank Burnet and David Talmage had developed the clonal selection theory (8). This stated that a lymphocyte makes a single specific antibody molecule that is determined before it encounters an antigen, which was in contrast to the instructive theory developed by Linus Pauling in 1940 where the antigen acted as a template for the antibody (9).

By 1959 Gerald Edelman and Rodney Porter independently published the molecular structure of antibodies
(10, 11), for which they were later jointly awarded the Nobel Prize in 1972. The first atomic resolution structure of an antibody fragment was published in 1973 (12) and this was quickly followed by the invention of monoclonal antibodies in 1975 by Georges Köhler and César Milstein (13) signalling the start of the modern era of antibody research and discovery.

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), recognises the antigen, the other part of the antibody, known as the crystallisable 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 recognise 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).

    Antibody isotypes and subtypes

    In mammals, antibodies are classified into five main classes or isotypes – IgA, IgD, IgE, IgG and IgM. They are classed according to the heavy chain they contain – alpha, delta, epsilon, gamma or mu respectively. These differ in the sequence and number of constant domains, hinge structure and the valency of the antibody. Antibody light chains fall into two classes in mammals, kappa and lambda, with kappa light chains being the more common of the two. Although these are relatively dissimilar in protein sequence they share a similar structure and function.

    Fig. 1. Schematic representation of antibody sites are marked byVariable regions are marked by Ɣ, while constant regions are depicted in Ɣ. Interchain disulphide bridges are shown by Ň. IgG subclasses and IgE antibodies only occur as monomers, while both IgA subclasses exist either as monomers or connected by the Jchain (0) as dimers. Furthermore, IgA may also interact with pIgR to form secretory IgA, e.g. secretory IgA1. In contrast to the other isotypes, IgM antibodies solely exist as multimers, primarily as pentamers. N-glycosylation sites are depicted by  .ڼO-glycosylation

  • Table 1. List of approved monoclonal antibodies for tumor therapy and their isotype/subclass

  • IgG1

    Almost 30 years ago Brüggemann et al. analyzed different human antibody isotypes and subclasses for their potential to activate complement to mediate complement-dependent cytotoxicity (CDC) and to recruit effector cells for antibody-dependent cellular cytotoxicity (ADCC) against human target cells. Based on their observations, IgG1 appeared as the most promising antibody isotype for tumor immunotherapy. In addition to these and many other in vitro results, human IgG1 antibodies were also effective in mouse models, since human IgG1 binds well to activating murine Fcγ receptors on effectors cells. Apart from its promising effector functions, IgG1 antibodies were demonstrated to interact well with the human, but also with the murine neonatal Fc receptor (FcRn).

    Binding to FcRn protects IgG1 molecules from degradation and thereby extends their serum half-life compared to non-FcRn-binding isotypes . Additionally, human IgG1 antibodies demonstrated favorable biotechnological characteristics such as high production rates in transfectoma cells (e.g. Chinese hamster ovary (CHO) cells), easy and cost-effective purification (e.g. by protein A columns), and development of specific storage formulations for increased stability. These characteristics allowed the establishment of Good Manufacturing Practice (GMP) to obtain optimal therapeutic agents. From an economical point of view, these industrial procedures contributed to the prominent role of IgG1 antibodies in the clinic. Importantly, human IgG1 antibodies are often used as backbone for Fc engineering strategies which aim to further improve effector functions, stability, or pharmacokinetic properties of therapeutic antibodies.From the initial clinical studies, it took almost 10 years until rituximab as the first therapeutic antibody for oncological treatment received FDA approval in 1997. Rituximab is a chimeric CD20-targeting antibody, which paved the way for the development of so far almost 20 chimeric, humanized or human IgG1monoclonal antibodies being approved for different oncological indications during the following two decades (table 1). When ipilimumab was approved for the treatment of metastatic melanoma in 2011, the era for the so-called immune checkpoint blockers began. However, also antibodies against these types of antigens may not merely act by ‘blocking’ cellular interactions. For example, the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody ipilimumab also is of human IgG1 isotype and triggers Fc-mediated T regulatory cell depletion, at least in mouse models.


    The human IgG2 isotype is predominantly selected when neutralization of antigens (e.g. soluble cytokines) or inhibition of receptor-ligand interactions are targeted, while Fc-mediated effector functions (e.g. ADCC and CDC) appear undesired. Currently, the epidermal growth factor receptor (EGFR) antibody panitumumab is the only approved IgG2 antibody for cancer immunotherapy. However, with the emergence of immune checkpoint blockade as therapeutic principle, many more IgG2 antibodies are currently in clinical trials – with some of them short before approval. IgG2 has limited C1q binding activity, but can trigger CDC at high target antigen and high antibody concentrations. Furthermore, human IgG2 is only capable of binding to FcγRIIa (CD32a), but not to other activating Fcγ receptors. Importantly, human IgG2 binding to FcγRIIa is significantly affected by a functional single nucleotide polymorphism (SNP) in this receptor, leading to a single amino acid change at position 131 (histidine or arginine). This polymorphism impacts the functional activity of human IgG2 antibodies. Thus, the high affinity variant (131-His) of FcγRIIa was demonstrated to induce anti-CD3-IgG2-mediated T-cell activation and proliferation after cross-linking via myeloid cell engagement. Furthermore, the human IgG2 EGFR antibody panitumumab was demonstrated to mediate ADCC by myeloid effector cells against EGFR-positive tumor cells. A randomized phase III study showed that panitumumab was non-inferior to the EGFR IgG1 antibody cetuximab in regard to overall survival. Further studies need to address the impact of the FcγRIIa polymorphism on the efficacy and potential toxicity of human IgG2 antibodies in cancer immunotherapy across different target antigens. Recently, White et al. analyzed the activity of immune stimulatory CD40 monoclonal antibodies of IgG2 isotype. In contrast to other IgG subclasses, IgG2 antibodies displayed Fc receptor-independent stimulatory activity. Furthermore, they showed that this activity was provided by an IgG2 subfraction, IgG2B, but not by the IgG2A isoform. IgG2B is characterized by a unique arrangement of disulfide bonds in the hinge region. Other studies investigating Fc-independent activity of rituximab reported that an IgG2 version of this antibody triggered programmed cell death more effectively than its IgG1 version. Further studies in the structural and functional relationships of these effects may enable a new field of antibody engineering and can increase the potential of IgG2-related monoclonal antibodies for cancer immunotherapy.


    The interest in IgG3 as a therapeutic antibody isotype was stimulated following observations that an anti-HIV-specific IgG3 response was correlated with improved disease control and longer survival. Antibodies of the IgG3 subclass were long known to exhibit strong Fc-mediated effector functions in vitro. For example, hapten-directed IgG3 antibodies were able to induce ADCC as well as CDC very effectively. Interestingly, IgG3 antibodies showed an increased ability to induce C1q binding and were more effective in Fc receptor binding than their IgG1 counterparts. Detailed analyses of IgG antibodies and their ability to activate the complement system demonstrated the superior efficacy of complement activation by the IgG3 isotype, particularly when epitope densities were lower. Furthermore an IgG3 version of cetuximab was able to activate complement in contrast to the parental IgG1 antibody – with CD55 being the main regulator of IgG3- induced complement activation.Despite these promising biological activities, no IgG3 antibody has entered the clinic so far. This is probably explained by manufacturing issues: IgG3 antibodies cannot be purified by protein A chromatography, have a tendency to form aggregates, and carry Olinked glycans in their extended hinge region. Furthermore, most IgG3 allotypes are not recycled by FcRn – leading to a serum half life of approximately 7 days for IgG3, compared to 21 days for other IgG isotypes. However, Stapleton et al. characterized an IgG3 allotypic variant, which contains an amino acid exchange at position 435 (arginine to histidine), which lead to effective FcRn transport and increased serum half-life.Recently, Bournazos et al. generated an engineered bispecific antibody for broad HIV neutralization. For improvement of the neutralization efficacy, the hinge region of an IgG3 was grafted onto the backbone of an IgG1 antibody. To further increase the flexibility of this molecule, all but the lowest two cysteines in the hinge region were exchanged by serine, thereby leading to an open conformation of this new molecule. In comparison to the parental unmodified antibody, the IgG3 open-hinge molecule was significantly more effective in HIV-1 neutralization. Further studies need to address the impact of these hinge engineering approaches for the generation of novel antibodies, especially under the aspect of neutralization or Fc-mediated effector functions.


    IgG4 is commonly regarded as a non-activating antibody isotype in immunotherapy. Experiments performed by Brüggemann et al. displayed the low activity of IgG4 to induce CDC as well as ADCC. Nevertheless, antibodies of the IgG4 subclass bind to activating Fcγ receptors – in particular to FcγRI (CD64). While the affinity of IgG4 to FcγRI was similar to that of IgG1 and IgG3, the affinities for FcγRII (a/b/c) and FcγRIIIa (CD16a) were lower. Thus, a human IgG4 antibody against CD20 was able to induce ADCC against human B cells by engaging mononuclear effector cells.In contrast to other antibody isoforms, the biology of IgG4 is characterized by a unique process called Fab arm exchange. During this process half-molecules are formed – consisting of one heavy and one light chain, which are able to recombine with other half-molecules. Thereby, natural monovalent bispecific antibodies are formed, which may explain the biology and pathophysiology of IgG4 in health and disease.This Fab arm exchange was also documented to occur with a therapeutic IgG4 antibody, which exchanged Fab arms with natural IgG4 antibodies from the serum. Fc engineering studies demonstrated that this Fab arm exchange can be prevented by a serine 228 proline (S228P) mutation, which stabilizes the IgG4 hinge and which is employed in most currently approved or evaluated therapeutic IgG4 antibodies. Forexample, two monoclonal IgG4 antibodies (pembrolizumab and nivolumab) were recently approved for immune checkpoint blockade. Both of these programmed cell death protein 1 (PD-1) monoclonal antibodies are mediating their therapeutic efficacy by blocking PD-1 and programmed death-ligand 1/2 (PD-L1/L2) interactions, leading to increased anti-tumor T-cell responses. Since PD-1 antibodies, in contrast to PD-L1 antibodies, were shown to mediate their efficacy independently from Fcγ receptors, IgG4 appeared to be a reasonable IgG subclass for this therapeutic strategy.While uncontrolled Fab arm exchange constituted a problem for IgG4 antibodies as therapeutic agents, it also opened up a new way to design bispecific antibodies. Introduction of two different matching mutations (K409R/F405L) into the Fc backbones of two parental IgG1 antibodies targeting different antigens resulted in the controlled formation of bispecific antibodies. These mutations are located in the CH3 domains and are responsible for the efficient and directed Fab arm exchange between the two parental IgG1 antibodies.


    AntibodiesDespite the success of therapeutic antibodies of the IgG isotype, research on alternative antibody isotypes for tumor immunotherapy continues. Antibodies of the IgA isotype were particularly effective in recruiting myeloid effector cells (monocytes/macrophages and granulocytes) for ADCC [38–40] while natural killer cells (NK cells) are not activated by IgA antibodies. Physiologically, IgA antibodies are the first line of immune defense against pathogens at mucosal surfaces [41]. Two different isoforms – IgA1 and IgA2 – are characterized in men (fig. 1), which share many key characteristics with IgG antibodies. However, IgA antibodies differ in the number of glycosylation sites, the length of their hinge regions, and the number and position of disulfide bridges within the molecules (fig. 1). Furthermore, IgA can form dimeric and secretory isoforms. Dimeric IgA is produced by mucosal plasma cells by connecting the tailpiece cysteines of two monomeric IgA molecules covalently with the so-called joining (J) chain. Dimeric IgA can then bind to the polymeric immunoglobulin receptor (pIgR) on the basolateral surface of mucosal epithelial cells. Bound dimeric IgA is then transported by transcytosis through epithelial cells to the luminal site of mucosal surfaces, where secretory IgA is released by proteolytic cleavage of pIgR. Thus, secretory IgA consists of J-chain-connected dimeric IgA and the associated secretory component (SC), which is an extracellular part of the pIgR.In addition to their potent activity in recruiting myeloid effector cells for ADCC, IgA antibodies against CD20 triggered CDC against lymphoma cells [40]. Fab-mediated effector functions of monomeric IgA antibodies were similar to IgG antibodies, but were enhanced when dimeric IgA antibodies were compared with monomeric molecules [38]. In vivo experiments in different xenoand syngeneic human FcαRI transgenic mouse models demonstrated significant anti-tumor activity of an EGFR-directed human IgA2 antibody, although its serum half-life was short [42]. Additional studies suggested that the short serum half-life was triggered by asialo-glycoprotein receptor(ASGPR)-mediated elimination of the therapeutic antibody in the liver. To overcome this limitation, an Fc-engineered IgA2 molecule was developed, which demonstrated improved stability and a longer serum half-life translating into higher in vivo efficacy of the engineered compared to the parental IgA molecule [43]. Also IgA antibodies against human epidermal growth factor receptor 2/neu (HER2/neu) or CD20 demonstrated in vitro and in vivo efficacy [40, 44]. Despite these promising preclinical activities, IgA antibodies have currently not beenintroduced into clinical studies.


    Like other immunoglobulin isotypes, monomeric IgM molecules are composed of heavy and light chain heterodimers, which are covalently connected via disulfide bridges. However, serum IgM antibodies are predominantly pentameric molecules which are interconnected by the J-chain (fig. 1). As described for IgA, binding of pentameric IgM to the pIgR and its transport through epithelial cells leads to the formation of secretory IgM at mucosal surfaces. Nevertheless, IgM antibodies are mostly found as pentameric IgM in the circulation. IgM is produced by either B1 lymphocytes as ‘natural antibodies’ without being exposed to antigenic stimuli or by B2 lymphocytes after immunization as a defense mechanism against invading pathogens. Natural antibodies recognize a variety of pathogenic molecules such as nucleic acids, lipids and proteins,

    which are phylogenetically conserved and which were not encountered previously. Thus, IgM antibodies close the gap that arises after the first contact of potential pathogens and the first adaptive response of the immune system [, 45]. IgM antibodies and their pentameric structure are ideal activators of the complement system. Recently, Michaelsen and colleagues [46] showed not only that serum and pentameric IgM are potent CDC inducers but also that secretory IgM, which is transported via transcytosis to mucosal tissues, induced comparable levels of CDC. Furthermore, IgM-induced effector functions do not seem to be influenced by the association of the molecule with either J-chain or SC. The Fc receptor for IgM is called FcμR and is found exclusively on lymphocytes (B, T and NK cells) in men and only on B cells in mice. FcμR shows a unique immunoreceptor tyrosine-based inhibition motif (ITIM) and immunoreceptor tyrosine-based activation motif (ITAM) pattern suggesting that the receptor may have the ability to act as a dual signal transmitter [7]. However, rather little is known about the IgM FcR and its role in immunity. Only few tumor-directed IgM antibodies have been moved into clinical trials. For example, a human IgM antibody (called PATSM6) is directed against a tumor-specific variant of the glucoseregulated protein 78 (GPR78) / heat shock 70 kDa protein 5 (HSPA5). This antibody-mediated induction of apoptosis and, to a lower extent, CDC against multiple myeloma cells and was evaluated in a phase I trial regarding safety and tolerability in patients with relapsed or refractory multiple myeloma. PAT-SM6 demonstrated good tolerability and modest activity in this phase I study [47]. Another monoclonal IgM antibody, which was introduced into a phase I trial, was MORAb-028. This GD2 antibody was administered intra-tumorally in patients with metastatic melanoma (NCT01123304).


    IgE antibodies are commonly associated with allergic or parasitic diseases. Monomeric IgE antibodies bind with high affinity to FcεRI, which is predominantly expressed on mast cells, basophils, monocytes, and macrophages making them favorable effector cell populations for IgE-mediated tumor growth control. Upon antigen binding, FcεRI is cross-linked and triggers degranulation and mediator release to induce acute allergic reactions or tumor cell killing. Complexed or target-bound IgE can bind to the low-affinity IgE receptor FcεRII (CD23), which is expressed on dendritic cells, macrophages, and eosinophils. Unlike for IgG antibodies, inhibitory Fc receptors have not been described for IgE. Compared with IgG1, IgE has a short serum half-life of 1.5 days, whereas in tissues the half-life is prolonged to approximately 2 weeks due to binding to Fcε receptor-expressing immune cells (reviewed in [48]). Bridging allergy and cancer IgE antibodies were demonstrated to contribute to the natural immune surveillance of tumors. For example, elevated IgE levels were observed in serum samples of pancreatic cancer patients. Some of these IgE antibodies were found to be tumor antigen-specific and induced ADCC in pancre- atic cancer cell lines [49]. Thus, IgE antibodies against different target antigens such as HER2/neu, CD20, 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP), or mouse Ly2 (CD8a) were generated, and their immunotherapeutic features were analyzed in vitro and in vivo. For example, Karagiannis and colleagues [50] investigated an IgE isoform of the MOv18 antibody (directed against the folate binding protein) in a human ovarian carcinoma xenograft model.They observed that the IgE antibody was not only superior to its IgG1 counterpart in prolonging survival of mice (together with human peripheral blood mononuclear cell as effector cells), which was demonstrated to be mediated by monocytes acting as effector cells for IgE-mediated ADCC and phagocytosis of tumor cells. The critical involvement of IgE in allergy and its role in inducing systemic type I hypersensitivity reactions (anaphylactic shock) raised concerns about the IgE antibody format for therapeutic use in clinical applications. However, several studies addressed this issue and concluded that systemic hypersensitivity reactions caused by tumor antigen-directed IgE antibodies may not occur in a therapeutic setting. Additionally, no evidence for systemic hypersensitivity reactions due to IgE antibody therapy was seen in various in vivo models (reviewed in [51]). As a result of these in vitro and in vivo observations, an ongoing phase I clinical trial evaluates the potential of IgE antibodies in cancer immunotherapy (NCT02546921).


  • Anna Kretschmer Ralf Schwanbeck Thomas Valerius Thies R ner Antibody Isotypes for Tumor ImmunotherapyTransfus Med Hemother 2017;44:320326

  • Allotypes

    In addition to antibody isotypes and subtypes, allelic variation is found among the antibody subtypes. These polymorphic epitopes of immunoglobulins that can differ between individuals and ethnic groups are known as allotypes. Exposure of an individual to a non-self allotype can induce an anti-allotype response (1, 2). However, not all variations are immunogenic because this sequence may be found in other isotypes or subtypes and so these are known as isoallotypic variants. In fact, a recent study suggests that allotypic differences in human IgG1 do not represent a significant risk for induction of immunogenicity (3) and to date little evidence has been found for significant anti-allotype responses to therapeutic antibodies, e.g. adalimumab (4) or infliximab (5).

    Allotpyes have been identified on the g1, g3 and a2 heavy chains (designated G1m, G3m and A2m allotypes respectively) and on the kappa light chain (Km allotypes). Although variants of g2 and g4 exist these are isoallotypic as the amino acids present are also found in other subclasses.

    The remainder of this page will focus on G1m allotypes. See IMGT for further information on G3m, A2M or Km allotypes.

    G1m17 and G1m3

    G1m17, also known as G1m(z), corresponds to Lys (K) at position 214 in the CH1 domain (EU numbering).G1m3, also known as G1m(f), corresponds to Arg (R) at position 214 in the CH1 domain.

    G1m1 and nG1m1

    G1m1, also known as G1m(a), corresponds to Asp (D) and Leu (L) at positions 356 and 358 in the CH3 domain (EU numbering).nG1m1, also known as nG1m(a) corresponds to Glu (E) and Met (M) at positions 356 and 358.

    G1m2 and nG1m2

    G1m2, also known as G1m(x), corresponds to Gly (G) at position 431 in the CH3 domain (EU numbering).nG1m2, also known as nG1m(x), corresponds to Ala (A) at position 431 in the CH3 domain.

    Figure. Sequence alignment of human G1m allotypes.

    The main allelic forms for IgG1 are G1m (z,a), G1m (f), and G1m (f,a) (6,7). The G1m (f) allele is only found in Caucasians, whereas the G1m (f,a) allele is common in Orientals, but other variants, G1m (z,a,x) and G1m (z,a,v), have also been described (8,9).

    Antibody effector function

    Antibodies act by a number of mechanisms, most of which engage other arms of the immune system. Antibodies can simply block interactions of molecules or they can activate the classical complement pathway (known as complement dependent cytotoxicity or CDC) by interaction of C1q on the C1 complex with clustered antibodies. Critically antibodies also act as a link between the antibody-mediated and cell-mediated immune responses through engagement of Fc receptors.

    Figure. Antibody modes of action.Antibodies have several modes of action: i) they can block ligand-receptor interactions; ii) cause cell lysis through activation of complement dependant cytotoxicity (CDC); iii) interact with Fc receptors on effector cells to engage antibody dependent cellular cytotoxicity; iv) signal for ingestion of a pathogen by a phagocyte.

    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 human 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. 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.

    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.

    Other antibody interactions


    It was recognised in the 1960s that the two processes of IgG transport from mother to her young and the protection of IgG from catabolism were mediated by receptors that share many features (1). Originally these were referred to as the neonatal transport receptor (FcRn) and the IgG protection receptor (FcRp) respectively. It wasn’t until 1996 that it was conclusively shown that these were the same receptor (2-4). These have since been unified under the term Brambell receptor (FcRB) in honour of their discoverer, although the receptor is still more commonly referred to as FcRn.

    Figure. Human IgG1-FcRn complex.A model of human IgG1 in complex with the FcRn-B2M heterodimer, which binds in the CH2 and CH3 regions of the antibody. The upper images show space-filled models and the lower images are ribbon representations. The antibody heavy and light chains are shown in blue and green respectively, glycosylation in orange, FcRn heavy chain in red and β2-microglobulin (B2M) in yellow. Model produced from PDB accession numbers 1IGY and 1I1A.

    FcRn is a heterodimer of a β2-microglobulin (B2M) light chain and a major histocompatibility complex (MHC) class I-like heavy chain. As shown in the image above, FcRn binds IgG at the interface between the CH2 and CH3. Critically the binding site contains a number of histidine residues which results in a pH dependent binding that is crucial to its function (5, 6). Endocytosis of IgG is followed by binding of IgG to FcRn in the acidic environment (pH 6.0) of the endosome. The IgG-FcRn complex is then trafficked through cellular conduits to bypass lysosomal degradation and finally IgG is released back into the serum at physiological pH. This cycle is depicted in the image below. FcRn-mediated recycling results in IgG1, 2 and 4 having the longest serum half-life of all proteins, at approximately 21 days. Although IgG3 binds FcRn recycling of this sub-class is less efficient due to a single amino acid change at the binding site (7), resulting in a half-life of only 7 days.

    Figure. FcRn mediated recycling.In the early endosome IgG interacts with FcRn at pH 6.0. The FcRn-IgG complex is then recycled back to the cell surface and IgG released at neutral pH thus rescuing IgG from lysosomal degradation.

    Protein A and G

    Although not a function of antibodies per se, two bacterial proteins, Protein A and Protein G, bind to the Fc region of some antibodies. Protein A is a 56 kDa surface protein originally found in the cell wall of Staphylococcus aureus and Protein G is a 65 kDa protein from Streptococcal bacteria. In a similar manner to FcRn, protein A and G bind at the interface between the CH2 and CH3 domains, as depicted in the image below. Although overlapping the three epitopes are distinct and this results in differential binding of Protein A and G to different isotypes, subtypes and species of immunoglobulin.
    Due to their unique ability to bind pH dependently with high affinity to IgG in particular, these proteins have become widely used for the purpose of purifying antibodies.

    Figure. A model of human IgG1 in complex with both Protein A and Protein A.The upper images show space-filled models and the lower images are ribbon representations. The antibody heavy and light chains are shown in blue and green respectively, glycosylation in orange, a Protein A fragment in purple and a Protein G fragment in yellow. Model produced from PDB accession numbers 1IGY, 1L6X and 1FCC.

    Antibodies as tools

    Due to their high specificity and selectivity antibodies have always had the potential to be of great use as biochemical tools for a range of applications including selection, identification, purification and as therapeutics. Broadly speaking antibodies are categorised into two groups (polyclonal or monoclonal) and are utilised in three main areas (research, diagnostics and therapeutics).

    Figure. Categories of antibodies.Comparison of polyclonal antibodies, which bind to the same antigen but different epitopes, with monoclonal antibodies which all bind to the same epitope on a target antigen.

    Polyclonal antibodies (pAbs) are a heterogeneous mixture of antibodies directed against various epitopes on the same antigen. The antibodies are generated by different B-cell clones of the animal and as a consequence are immunochemically dissimilar, with different specificities and affinities.

    The true potential of antibodies as specific targeting agents was not realised until ground breaking work by Köhler and Milstein in 1975 resulted in the production of monoclonal antibodies or mAbs (1). These were produced by the fusing of antibody producing mouse spleen cells with an immortal mouse myeloma cell line, resulting in the formation of an immortal cell line, known as a hybridoma, expressing a single antibody with specificity for one particular epitope on an antigen, i.e. a monoclonal antibody. Once produced hybridomas can be cultured in vitro indefinitely allowing the relatively easy purification of large quantities of monoclonal antibody.


    Antibodies are vital tools in many of the laboratory techniques that are used to answer basic research questions. Due to their outstanding specificity they make exquisite tools that allow researchers to identify molecules that cannot be seen by the naked eye and thus enable conclusions to be drawn about the target molecule and pathway of interest. Routine procedures such as western blot, flow cytometry, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA) and many others all rely antibodies.


    Antibodies have become a critical component of many diagnostic assays. Uses included but are not limited to the detection of infections, recognition of allergies and the measurement of hormones and other biological markers in blood.


    The ability of antibodies to bind an almost unlimited number of target proteins with high specificity always meant they were destined to be used as therapeutics. As early as 1900 Paul Ehrlich coined the term ‘magic bullets’ in reference to antibodies. Following the ground breaking publication on the production of monoclonal antibodies (1) the early success of the first therapeutic antibody OKT3 (muromonab) as a treatment for transplant rejection was not immediately followed by the wave of approvals that many anticipated. However, since the mid to late 1990s therapeutic antibodies have become one of the fastest growing classes of therapeutics in the biological drugs market (2). Some of the work that led to this breakthrough will be discussed in more detail in the antibody engineering section.