General Features of Antibody Structure

Plasma or serum proteins are traditionally separated by solubility characteristics into albumins and globulins and may be more extensively separated by migration in an electric field, a process called electrophoresis. Most antibodies are found in the third fastest migrating group of globulins, named gamma globulins for the third letter of the Greek alphabet. Another common name for antibody is immunoglobulin (Ig), referring to the immunity-conferring portion of the gamma globulin fraction. The terms immunoglobulin and antibody are used interchangeably throughout this book.

All antibody molecules share the same basic structural characteristics but display remarkable variability in the regions that bind antigens. This variability of the antigen-binding regions accounts for the capacity of different antibodies to bind a tremendous number of structurally diverse antigens. There are believed to be a million or more different antibody molecules in every individual (theoretically, the antibody repertoire may include more than 10¹¹ different antibodies), each with unique amino acid sequences in their antigen-combining sites. The effector functions and common physicochemical properties of antibodies are associated with the non–antigen-binding portions, which exhibit relatively few variations among different antibodies.

An antibody molecule has a symmetric core structure composed of two identical light chains and two identical heavy chains (Fig. 5-1). Both the light chains and the heavy chains contain a series of repeating, homologous units, each about 110 amino acid residues in length, that fold independently in a globular motif that is called an Ig domain. An Ig domain contains two layers of β-pleated sheet, each layer composed of three to five strands of antiparallel polypeptide chain (Fig. 5-2). The two layers are held together by a disulfide bridge, and adjacent strands of each β sheet are connected by short loops. It is the amino acids in some of these loops that are the most variable and critical for antigen recognition, as discussed later.

FIGURE 5–1 Structure of an antibody molecule.

A, Schematic diagram of a secreted IgG molecule. The antigen-binding sites are formed by the juxtaposition of V_L and V_H domains. The heavy chain C regions end in tail pieces. The locations of complement- and Fc receptor–binding sites within the heavy chain constant regions are approximations. B, Schematic diagram of a membrane-bound IgM molecule on the surface of a B lymphocyte. The IgM molecule has one more C_H domain than IgG does, and the membrane form of the antibody has C-terminal transmembrane and cytoplasmic portions that anchor the molecule in the plasma membrane. C, Structure of a human IgG molecule as revealed by x-ray crystallography. In this ribbon diagram of a secreted IgG molecule, the heavy chains are colored blue and red, and the light chains are colored green; carbohydrates are shown in gray.

(Courtesy of Dr. Alex McPherson, University of California, Irvine.)

FIGURE 5–2 Structure of an Ig domain.

Each domain is composed of two antiparallel arrays of β strands, colored yellow and red, to form two β-pleated sheets held together by a disulfide bond. A C domain is schematically depicted that contains three and four β strands in the two sheets. Note that the loops connect β strands that are sometimes adjacent in the same β-pleated sheet but that loops sometimes represent connections between the two different sheets that make up an Ig domain. Three loops in each variable domain contribute to antigen binding and are called complementarity determining regions (CDRs).

Both heavy chains and light chains consist of amino-terminal variable (V) regions that participate in antigen recognition and carboxyl-terminal constant (C) regions; the C regions of the heavy chains mediate effector functions. In the heavy chains, the V region is composed of one Ig domain and the C region is composed of three or four Ig domains. Each light chain is made up of one V region Ig domain and one C region Ig domain. Variable regions are so named because they contain regions of variability in amino acid sequence that distinguish the antibodies made by one clone of B cells from the antibodies made by other clones. The V region of one heavy chain (V_H) and the adjoining V region of one light chain (V_L) form an antigen-binding site (see Fig. 5-1). Because the core structural unit of each antibody molecule contains two heavy chains and two light chains, every antibody molecule has at least two antigen-binding sites. The C region domains are separate from the antigen-binding site and do not participate in antigen recognition. The heavy chain C regions interact with other effector molecules and cells of the immune system and therefore mediate most of the biologic functions of antibodies. In addition, heavy chains exist in two forms that differ at their carboxyl-terminal ends: one form of the heavy chain anchors membrane-bound antibodies in the plasma membranes of B lymphocytes, and the other form is secreted when associated with Ig light chains. The C regions of light chains do not participate in effector functions and are not directly attached to cell membranes.

Heavy and light chains are covalently linked by disulfide bonds formed between cysteine residues in the carboxyl terminus of the light chain and the C_H1 domain of the heavy chain. Noncovalent interactions between the V_L and V_H domains and between the C_L and C_H1 domains may also contribute to the association of heavy and light chains. The two heavy chains of each antibody molecule are also covalently linked by disulfide bonds. In IgG antibodies, these bonds are formed between cysteine residues in the C_H2 regions, close to the region known as the hinge (see later). In other isotypes, the disulfide bonds may be in different locations. Noncovalent interactions (e.g., between the third C_H domains [C_H3]) may also contribute to heavy chain pairing.

The associations between the chains of antibody molecules and the functions of different regions of antibodies were first deduced from experiments done by Rodney Porter in which rabbit IgG was cleaved by proteolytic enzymes into fragments with distinct structural and functional properties. In IgG molecules, the unfolded “hinge” region between the C_H1 and C_H2 domains of the heavy chain is the segment most susceptible to proteolytic cleavage. If rabbit IgG is treated with the enzyme papain under conditions of limited proteolysis, the enzyme acts on the hinge region and cleaves the IgG into three separate pieces (Fig. 5-3A). Two of the pieces are identical to each other and consist of the complete light chain (V_L and C_L) associated with a V_H-C_H1 fragment of the heavy chain. These fragments retain the ability to bind antigen because each contains paired V_L and V_H domains, and they are called Fab (fragment, antigen binding). The third piece is composed of two identical, disulfide-linked peptides containing the heavy chain C_H2 and C_H3 domains. This piece of IgG has a propensity to self-associate and to crystallize into a lattice and is therefore called Fc (fragment, crystallizable). When pepsin (instead of papain) is used to cleave rabbit IgG under limiting conditions, proteolysis occurs distal to the hinge region, generating a F(ab′)₂ antigen-binding fragment of IgG with the hinge and the interchain disulfide bonds intact (see Fig. 5-3B).

FIGURE 5–3 Proteolytic fragments of an IgG molecule.

IgG molecules are cleaved by the enzymes papain (A) and pepsin (B) at the sites indicated by arrows. Papain digestion allows separation of two antigen-binding regions (the Fab fragments) from the portion of the IgG molecule that binds to complement and Fc receptors (the Fc fragment). Pepsin generates a single bivalent antigen-binding fragment, F(ab′)₂.

The results of limited papain or pepsin proteolysis of other isotypes besides IgG, or of IgGs of species other than the rabbit, do not always recapitulate the studies with rabbit IgG. However, the basic organization of the Ig molecule that Porter deduced from his experiments is common to all Ig molecules of all isotypes and of all species. In fact, these proteolysis experiments provided the first evidence that the antigen recognition functions and the effector functions of Ig molecules are spatially segregated.

Many other proteins in the immune system as well as numerous proteins that have nothing to do with immunity contain domains with an Ig fold structure—that is, two adjacent β-pleated sheets held together by a disulfide bridge. Although such a domain structure evolved long before the development of vertebrates, all molecules that contain this type of domain are said to belong to the Ig superfamily, and all the gene segments encoding the Ig domains of these molecules are believed to have evolved from one ancestral gene. Ig domains are classified as V-like or C-like on the basis of closest homology to either Ig V or Ig C domains. V domains are formed from a longer polypeptide than are C domains and contain two extra β strands within the β sheet sandwich. A third type of Ig domain, called C2 or H, has a length similar to C domains but has sequences typical of both V and C domains. Examples of Ig superfamily members of relevance in the immune system are depicted in Figure 5-4.

FIGURE 5–4 Examples of Ig superfamily proteins in the immune system.

Examples included a membrane-bound IgG molecule, the T cell receptor, an MHC class I molecule, a coreceptor on T cells, the CD4 molecule, CD28, a costimulatory receptor on T cells, and the adhesion molecule ICAM-1.

Structural Features of Antibody Variable Regions

Most of the sequence differences and variability among different antibodies are confined to three short stretches in the V region of the heavy chain and to three stretches in the V region of the light chain. These diverse stretches are known as hypervariable segments, and they correspond to three protruding loops connecting adjacent strands of the β sheets that make up the V domains of Ig heavy and light chain proteins (Fig. 5-5). The hypervariable regions are each about 10 amino acid residues long, and they are held in place by the more conserved framework sequences that make up the Ig domain of the V region. The genetic mechanisms leading to amino acid variability are discussed in Chapter 8. In an antibody molecule, the three hypervariable regions of a V_L domain and the three hypervariable regions of a V_H domain are brought together to form an antigen-binding surface. The hypervariable loops can be thought to be like fingers protruding from each variable domain, three fingers from the heavy chain and three fingers from the light chain coming together to form an antigen-binding site (Fig. 5-6). Because these sequences form a surface that is complementary to the three-dimensional structure of the bound antigen, the hypervariable regions are also called complementarity-determining regions (CDRs). Proceeding from either the V_L or the V_H amino terminus, these regions are called CDR1, CDR2, and CDR3. The CDR3s of both the V_H segment and the V_L segment are the most variable of the CDRs. As we will discuss in Chapter 8, there are special mechanisms for generating more sequence diversity in CDR3 than in CDR1 and CDR2. Sequence differences among the CDRs of different antibody molecules contribute to distinct interaction surfaces and therefore to specificities of individual antibodies. The ability of a V region to fold into an Ig domain is mostly determined by the conserved sequences of the framework regions adjacent to the CDRs. Confining the sequence variability to three short stretches allows the basic structure of all antibodies to be maintained despite the variability among different antibodies.

FIGURE 5–5 Hypervariable regions in Ig molecules.

A, Kabat-Wu plot of amino acid variability in Ig molecules. The histograms depict the extent of variability, defined as the number of differences in each amino acid residue among various independently sequenced Ig light chains, plotted against amino acid residue number, measured from the amino terminus. This method of analysis, developed by Elvin Kabat and Tai Te Wu, indicates that the most variable residues are clustered in three “hypervariable” regions, colored in blue, yellow, and red, corresponding to CDR1, CDR2, and CDR3, respectively. Three hypervariable regions are also present in heavy chains.

(Courtesy of Dr. E. A. Kabat, Department of Microbiology, Columbia University College of Physicians and Surgeons, New York.)

B, Three-dimensional view of the hypervariable CDR loops in a light chain V domain. The V region of a light chain is shown with CDR1, CDR2, and CDR3 loops, colored in blue, yellow, and red, respectively. These loops correspond to the hypervariable regions in the variability plot in A. Heavy chain hypervariable regions (not shown) are also located in three loops, and all six loops are juxtaposed in the antibody molecule to form the antigen-binding surface (see Fig. 5-6).

FIGURE 5–6 Binding of an antigen by an antibody.

A, A schematic view of complementarity-determining regions (CDRs) generating an antigen-binding site. CDRs from the heavy chain and the light chain are loops that protrude from the surface of the two Ig V domains and in combination create an antigen-binding surface. B, This model of a globular protein antigen (hen egg lysozyme) bound to an antibody molecule shows how the antigen-binding site can accommodate soluble macromolecules in their native (folded) conformation. The heavy chains of the antibody are red, the light chains are yellow, and the antigen is blue.

(Courtesy of Dr. Dan Vaughn, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.)

C, A view of the interacting surfaces of hen egg lysozyme (in green) and a Fab fragment of a monoclonal anti–hen egg lysozyme antibody (V_H in blue and V_L in yellow) is provided. The residues of hen egg lysozyme and of the Fab fragment that interact with one another are shown in red. A critical glutamine residue on lysozyme (in magenta) fits into a “cleft” in the antibody.

(Reprinted with permission from Amit AG, RA Mariuzza, SE Phillips, and RJ Poljak. Three dimensional structure of an antigen antibody complex at 2.8A resolution. Science 233, 747-753, 1986.

Antigen binding by antibody molecules is primarily a function of the hypervariable regions of V_H and V_L. Crystallographic analyses of antigen-antibody complexes show that the amino acid residues of the hypervariable regions form multiple contacts with bound antigen (see Fig. 5-6). The most extensive contact is with the third hypervariable region (CDR3), which is also the most variable of the three CDRs. However, antigen binding is not solely a function of the CDRs, and framework residues may also contact the antigen. Moreover, in the binding of some antigens, one or more of the CDRs may be outside the region of contact with antigen, thus not participating in antigen binding.

Structural Features of Antibody Constant Regions

Antibody molecules can be divided into distinct classes and subclasses on the basis of differences in the structure of their heavy chain C regions. The classes of antibody molecules are also called isotypes and are named IgA, IgD, IgE, IgG, and IgM (Table 5-2). In humans, IgA and IgG isotypes can be further subdivided into closely related subclasses, or subtypes, called IgA1 and IgA2 and IgG1, IgG2, IgG3, and IgG4. (Mice, which are often used in the study of immune responses, differ in that the IgG isotype is divided into the IgG1, IgG2a, IgG2b, and IgG3 subclasses; certain strains of mice, including C57BL/6, lack the gene for IgG2a but synthesize a related isotype called IgG2c). The heavy chain C regions of all antibody molecules of one isotype or subtype have essentially the same amino acid sequence. This sequence is different in antibodies of other isotypes or subtypes. Heavy chains are designated by the letter of the Greek alphabet corresponding to the isotype of the antibody: IgA1 contains α1 heavy chains; IgA2, α2; IgD, δ; IgE, ε; IgG1, γ1; IgG2, γ2; IgG3, γ3; IgG4, γ4; and IgM, µ. In human IgM and IgE antibodies, the C regions contain four tandem Ig domains (see Fig. 5-1). The C regions of IgG, IgA, and IgD contain only three Ig domains. These domains are generically designated C_H domains and are numbered sequentially from amino terminus to carboxyl terminus (e.g., C_H1, C_H2, and so on). In each isotype, these regions may be designated more specifically (e.g., Cγ1, Cγ2 in IgG).

TABLE 5–2 Human Antibody Isotypes

Different isotypes and subtypes of antibodies perform different effector functions. The reason for this is that most of the effector functions of antibodies are mediated by the binding of heavy chain C regions to Fc receptors on different cells, such as phagocytes, NK cells, and mast cells, and to plasma proteins, such as complement proteins. Antibody isotypes and subtypes differ in their C regions and therefore in what they bind to and what effector functions they perform. The effector functions mediated by each antibody isotype are listed in Table 5-2 and are discussed in more detail later in this chapter and in Chapter 12.

Antibody molecules are flexible, permitting them to bind to different arrays of antigens. Every antibody contains at least two antigen-binding sites, each formed by a pair of V_H and V_L domains. Many Ig molecules can orient these binding sites so that two antigen molecules on a planar (e.g., cell) surface may be engaged at once (Fig. 5-7). This flexibility is conferred, in large part, by a hinge region located between C_H1 and C_H2 in certain isotypes. The hinge region varies in length from 10 to more than 60 amino acid residues in different isotypes. Portions of this sequence assume an unfolded and flexible conformation, permitting molecular motion between the C_H1 and C_H2 domains. Some of the greatest differences between the constant regions of the IgG subclasses are concentrated in the hinge. This leads to different overall shapes of the IgG subtypes. In addition, some flexibility of antibody molecules is due to the ability of each V_H domain to rotate with respect to the adjacent C_H1 domain.

FIGURE 5–7 Flexibility of antibody molecules.

The two antigen-binding sites of an Ig monomer can simultaneously bind to two determinants separated by varying distances. In A, an Ig molecule is depicted binding to two widely spaced determinants on a cell surface, and in B, the same antibody is binding to two determinants that are close together. This flexibility is mainly due to the hinge regions located between the C_H1 and C_H2 domains, which permit independent movement of antigen-binding sites relative to the rest of the molecule.

There are two classes, or isotypes, of light chains, called κ and λ, that are distinguished by their carboxyl-terminal constant (C) regions. An antibody molecule has either two identical κ light chains or two identical λ light chains. In humans, about 60% of antibody molecules have κ light chains and about 40% have λ light chains. Marked changes in this ratio can occur in patients with B cell tumors because the many neoplastic cells, being derived from one B cell clone, produce a single species of antibody molecules, all with the same light chain. In fact, a skewed ratio of κ-bearing cells to λ-bearing cells is often used clinically in the diagnosis of B cell lymphomas. In mice, κ-containing antibodies are about 10 times more abundant than λ-containing antibodies. Unlike in heavy chain isotypes, there are no known differences in function between κ-containing antibodies and λ-containing antibodies.

Secreted and membrane-associated antibodies differ in the amino acid sequence of the carboxyl-terminal end of the heavy chain C region. In the secreted form, found in blood and other extracellular fluids, the carboxyl-terminal portion is hydrophilic. The membrane-bound form of antibody contains a carboxyl-terminal stretch that includes a hydrophobic α-helical transmembrane anchor region followed by an intracellular juxtamembrane positively charged stretch that helps anchor the protein in the membrane (Fig. 5-8). In membrane IgM and IgD molecules, the cytoplasmic portion of the heavy chain is short, only three amino acid residues in length; in membrane IgG and IgE molecules, it is somewhat longer, up to 30 amino acid residues in length.

FIGURE 5–8 Membrane and secreted forms of Ig heavy chains.

The membrane forms of the Ig heavy chains, but not the secreted forms, contain transmembrane regions made up of hydrophobic amino acid residues and cytoplasmic domains that differ significantly among the different isotypes. The cytoplasmic portion of the membrane form of the µ chain contains only three residues, whereas the cytoplasmic region of IgG heavy chains contains 20 to 30 residues. The secreted forms of the antibodies end in C-terminal tail pieces, which also differ among isotypes: µ has a long tail piece (21 residues) that is involved in pentamer formation, whereas IgGs have a short tail piece (3 residues).

Secreted IgG and IgE and all membrane Ig molecules, regardless of isotype, are monomeric with respect to the basic antibody structural unit (i.e., they contain two heavy chains and two light chains). In contrast, the secreted forms of IgM and IgA form multimeric complexes in which two or more of the four-chain core antibody structural units are covalently joined. IgM may be secreted as pentamers and hexamers of the core four-chain structure, whereas IgA is often secreted as a dimer. These complexes are formed by interactions between regions, called tail pieces, that are located at the carboxyl-terminal ends of the secreted forms of µ and α heavy chains (see Table 5-2). Multimeric IgM and IgA molecules also contain an additional 15-kD polypeptide called the joining (J) chain, which is disulfide bonded to the tail pieces and serves to stabilize the multimeric complexes and to transport multimers across epithelia from the basolateral to the luminal end. As we shall see later, multimeric forms of antibodies bind to antigens more avidly than monomeric forms do, even if both types of antibody contain Fab fragments that individually bind the antigen equally well.

Antibodies of different species differ from one another in the C regions and in framework parts of the V regions. Therefore, when Ig molecules from one species are introduced into another (e.g., horse serum antibodies or mouse monoclonal antibodies injected into humans), the recipient mounts an immune response and makes antibodies largely against the C regions of the introduced Ig. The response often creates an illness called serum sickness (see Chapter 18) and thus greatly limits the ability to treat individuals with antibodies produced in other species. Much effort has been devoted to overcoming this problem with monoclonal antibodies, and this issue is discussed in more depth later. Smaller sequence differences are present in antibodies from different individuals even of the same species, reflecting inherited polymorphisms in the genes encoding the C regions of Ig heavy and light chains. When a polymorphic variant found in some individuals of a species can be recognized by an antibody, the variants are referred to as allotypes, and the antibody that recognizes an allotypic determinant is called an anti-allotypic antibody. The differences between antibody V regions map to CDRs and constitute the idiotypes of antibodies. An antibody that recognizes some aspect of the CDRs of another antibody is therefore called an anti-idiotypic antibody. There have been interesting theories that individuals produce anti-idiotypic antibodies against their own antibodies that control immune responses, but there is little evidence to support the importance of this potential mechanism of immune regulation.

Monoclonal Antibodies

A tumor of plasma cells (myeloma or plasmacytoma) is monoclonal and therefore produces antibodies of a single specificity. In most cases, the specificity of the tumor-derived antibody is not known, so the antibody cannot be used to specifically detect or bind to molecules of interest. However, the discovery of monoclonal antibodies produced by these tumors led to the idea that it may be possible to produce similar monoclonal antibodies of any desired specificity by immortalizing individual antibody-secreting cells from an animal immunized with a known antigen. A technique to accomplish this was described by Georges Kohler and Cesar Milstein in 1975 and has proved to be one of the most valuable advances in all of scientific research and clinical medicine. The method relies on fusing B cells from an immunized animal (typically a mouse) with a myeloma cell line and growing the cells under conditions in which the unfused normal and tumor cells cannot survive (Fig. 5-9). The resultant fused cells that grow out are called hybridomas; each hybridoma makes only one Ig. The antibodies secreted by many hybridoma clones are screened for binding to the antigen of interest, and this single clone with the desired specificity is selected and expanded. The products of these individual clones are monoclonal antibodies that are each specific for a single epitope on the antigen or antigen mixture used to identify antibody-secreting clones.

FIGURE 5–9 The generation of monoclonal antibodies.

In this procedure, spleen cells from a mouse that has been immunized with a known antigen or mixture of antigens are fused with an enzyme-deficient partner myeloma cell line, with use of chemicals such as polyethylene glycol that can facilitate the fusion of plasma membranes and the formation of hybrid cells that retain many chromosomes from both fusion partners. The myeloma partner used is one that does not secrete its own Igs. These hybrid cells are then placed in a selection medium that permits the survival of only immortalized hybrids; these hybrid cells are then grown as single cell clones and tested for the secretion of the antibody of interest. The selection medium includes hypoxanthine, aminopterin, and thymidine and is therefore called HAT medium. There are two pathways of purine synthesis in most cells, a de novo pathway that needs tetrahydrofolate and a salvage pathway that uses the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Myeloma cells that lack HGPRT are used as fusion partners, and they normally survive using de novo purine synthesis. In the presence of aminopterin, tetrahydrofolate is not made, resulting in a defect in de novo purine synthesis and also a specific defect in pyrimidine biosynthesis, namely, in generating TMP from dUMP. Hybrid cells receive HGPRT from the splenocytes and have the capacity for uncontrolled proliferation from the myeloma partner; if they are given hypoxanthine and thymidine, these cells can make DNA in the absence of tetrahydrofolate. As a result, only hybrid cells survive in HAT medium.

Monoclonal antibodies have many practical applications in research and in medical diagnosis and therapy. Some of their common applications include the following:

TABLE 5–3 Monoclonal Antibodies of Therapeutic Significance

One of the limitations of monoclonal antibodies for therapy is that these antibodies are most easily produced by immunizing mice, but patients treated with mouse monoclonal antibodies may make antibodies against the mouse Ig, called a human anti-mouse antibody (HAMA) response. These anti-Ig antibodies eliminate the injected monoclonal antibody and can also cause serum sickness. Genetic engineering techniques have been used to expand the usefulness of monoclonal antibodies. The complementary DNAs (cDNAs) that encode the polypeptide chains of a monoclonal antibody can be isolated from a hybridoma, and these genes can be manipulated in vitro. As discussed before, only small portions of the antibody molecule are responsible for binding to antigen; the remainder of the antibody molecule can be thought of as a framework. This structural organization allows the DNA segments encoding the antigen-binding sites from a mouse monoclonal antibody to be “stitched” into a cDNA encoding a human myeloma protein, creating a hybrid gene. When it is expressed, the resultant hybrid protein, which retains the antigen specificity of the original mouse monoclonal but has the core structure of a human Ig, is referred to as a humanized antibody. Humanized antibodies are far less likely than mouse monoclonals to appear “foreign” in humans and to induce anti-antibody responses.

Half-life of Antibodies

Different antibody isotypes have very different half-lives in circulation. IgE has a very short half-life of about 2 days in the circulation (although cell-bound IgE associated with the high-affinity IgE receptor on mast cells has a very long half-life; see Chapter 19). Circulating IgA has a half-life of about 3 days, and circulating IgM has a half-life of about 4 days. In contrast, circulating IgG molecules have a half-life of about 21 to 28 days.

The long half-life of IgG is attributed to its ability to bind to a specific Fc receptor called the neonatal Fc receptor (FcRn), which is also involved in the transport of IgG from the maternal circulation across the placental barrier as well as the transfer of maternal IgG across the intestine in neonates. FcRn structurally resembles MHC class I molecules but lacks a peptide-binding groove, and in specific cell types, such as the placenta and the neonatal intestine, it transports IgG molecules across cells without targeting them to lysosomes. In adult vertebrates, FcRn is found on the surface of endothelial cells (and other cell types) and binds to micropinocytosed IgG in acidic endosomes. FcRn does not target bound IgG to lysosomes but sequesters the IgG for a while and then returns it to the circulation, when it recycles to the cell surface and releases the IgG at neutral pH (Fig. 5-11). This intracellular sequestration of IgG for significant periods prevents it from being targeted for degradation as rapidly as most other serum proteins, including other antibody isotypes, and as a result, IgG has a relatively long half-life. This long half-life of IgG has been used to provide a therapeutic advantage for certain infused proteins by producing fusion proteins containing the biologically active part of the protein and the Fc portion of IgG. One therapeutically useful fusion protein is TNFR-Ig, which consists of the extracellular domain of the type II TNF receptor fused to an IgG Fc domain; it is used to treat certain autoimmune disorders, such as rheumatoid arthritis and psoriasis, in which it blocks the inflammatory actions of TNF. Another therapeutically useful fusion protein is CTLA4-Ig, which contains the extracellular domain of the CTLA-4 inhibitory receptor fused to the Fc portion of human IgG; it has also been used in the treatment of rheumatoid arthritis and may serve more broadly as an immunosuppressive therapeutic (see Fig. 9-7, Chapter 9).

FIGURE 5–11 FcRn contributes to the long half-life of IgG molecules.

Micropinocytosed IgG molecules in endothelial cells bind the FcRn, an IgG-binding receptor in the acidic environment of endosomes. In endothelial cells, FcRn sequesters IgG molecules and releases them when vesicles fuse with the cell surface, exposing FcRn-IgG complexes to neutral pH.

Features of Biologic Antigens

An antigen is any substance that may be specifically bound by an antibody molecule or T cell receptor. Antibodies can recognize as antigens almost every kind of biologic molecule, including simple intermediary metabolites, sugars, lipids, autacoids, and hormones, as well as macromolecules such as complex carbohydrates, phospholipids, nucleic acids, and proteins. This is in contrast to T cells, which mainly recognize peptides (see Chapter 6).

Although all antigens are recognized by specific lymphocytes or by antibodies, only some antigens are capable of activating lymphocytes. Molecules that stimulate immune responses are called immunogens. Only macromolecules are capable of stimulating B lymphocytes to initiate humoral immune responses because B cell activation requires either the bringing together (cross-linking) of multiple antigen receptors or protein antigens to elicit T cell help. Small chemicals, such as dinitrophenol, may bind to antibodies and are therefore antigens but cannot activate B cells on their own (i.e., they are not immunogenic). To generate antibodies specific for such small chemicals, immunologists commonly attach multiple copies of the small molecules to a protein or polysaccharide before immunization. In these cases, the small chemical is called a hapten, and the large molecule to which it is conjugated is called a carrier. The hapten-carrier complex, unlike free hapten, can act as an immunogen (see Chapter 11).

Macromolecules, such as proteins, polysaccharides, and nucleic acids, are usually much bigger than the antigen-binding region of an antibody molecule (see Fig. 5-6). Therefore, any antibody binds to only a portion of the macromolecule, which is called a determinant or an epitope. These two words are synonymous and are used interchangeably throughout this book. Macromolecules typically contain multiple determinants, some of which may be repeated and each of which, by definition, can be bound by an antibody. The presence of multiple identical determinants in an antigen is referred to as polyvalency or multivalency. Most globular proteins do not contain multiple identical epitopes and are not polyvalent, unless they are in aggregates. In the case of polysaccharides and nucleic acids, many identical epitopes may be regularly spaced, and the molecules are said to be polyvalent. Cell surfaces, including microbes, often display polyvalent arrays of protein or carbohydrate antigenic determinants. Polyvalent antigens can induce clustering of the B cell receptor and thus initiate the process of B cell activation (see Chapter 7).

The spatial arrangement of different epitopes on a single protein molecule may influence the binding of antibodies in several ways. When determinants are well separated, two or more antibody molecules can be bound to the same protein antigen without influencing each other; such determinants are said to be nonoverlapping. When two determinants are close to one another, the binding of antibody to the first determinant may cause steric interference with the binding of antibody to the second; such determinants are said to be overlapping. In rarer cases, binding of one antibody may cause a conformational change in the structure of the antigen, positively or negatively influencing the binding of a second antibody at another site on the protein by means other than steric hindrance. Such interactions are called allosteric effects.

Any available shape or surface on a molecule that may be recognized by an antibody constitutes an antigenic determinant or epitope. Antigenic determinants may be delineated on any type of compound, including but not restricted to carbohydrates, proteins, lipids, and nucleic acids. In the case of proteins, the formation of some determinants depends only on the primary structure, and the formation of other determinants reflects tertiary structure, or conformation (shape) (Fig. 5-12). Epitopes formed by several adjacent amino acid residues are called linear determinants. The antigen-binding site of an antibody can usually accommodate a linear determinant made up of about six amino acids. If linear determinants appear on the external surface or in a region of extended conformation in the native folded protein, they may be accessible to antibodies. More often, linear determinants may be inaccessible in the native conformation and appear only when the protein is denatured. In contrast, conformational determinants are formed by amino acid residues that are not in a sequence but become spatially juxtaposed in the folded protein. Antibodies specific for certain linear determinants and antibodies specific for conformational determinants can be used to ascertain whether a protein is denatured or in its native conformation, respectively. Proteins may be subjected to modifications such as glycosylation, phosphorylation, ubiquitination, acetylation, and proteolysis. These modifications, by altering the structure of the protein, can produce new epitopes. Such epitopes are called neoantigenic determinants, and they too may be recognized by specific antibodies.

FIGURE 5–12 The nature of antigenic determinants.

Antigenic determinants (shown in orange, red, and blue) may depend on protein folding (conformation) as well as on primary structure. Some determinants are accessible in native proteins and are lost on denaturation (A), whereas others are exposed only on protein unfolding (B). Neodeterminants arise from postsynthetic modifications such as peptide bond cleavage (C).

Structural and Chemical Basis of Antigen Binding

The antigen-binding sites of many antibodies are planar surfaces that can accommodate conformational epitopes of macromolecules, allowing the antibodies to bind large macromolecules (see Fig. 5-6). The six CDRs, three from the heavy chain and three from the light chain, spread out to form a broad surface. Similar broad binding surfaces are characteristic of the binding sites of T cell receptors. In contrast, MHC molecules contain antigen-binding clefts that bind small peptides. In a number of antibodies specific for small molecules, such as monosaccharides and drugs, the antigen is bound in a cleft generated by the close apposition of CDRs from the V_L and V_H domains.

The recognition of antigen by antibody involves noncovalent, reversible binding. Various types of noncovalent interactions may contribute to antibody binding of antigen, including electrostatic forces, hydrogen bonds, van der Waals forces, and hydrophobic interactions. The relative importance of each of these depends on the structures of the binding site of the individual antibody and of the antigenic determinant. The strength of the binding between a single combining site of an antibody and an epitope of an antigen is called the affinity of the antibody. The affinity is commonly represented by a dissociation constant (K_d), which indicates how easy it is to separate an antigen-antibody complex into its constituents. A smaller K_d indicates a stronger or higher affinity interaction because a lower concentration of antigen and of antibody is required for complex formation. The K_d of antibodies produced in typical humoral immune responses usually varies from about 10⁻⁷ M to 10⁻¹¹ M. Serum from an immunized individual will contain a mixture of antibodies with different affinities for the antigen, depending primarily on the amino acid sequences of the CDRs.

Because the hinge region of antibodies gives them flexibility, a single antibody may attach to a single multivalent antigen by more than one binding site. For IgG or IgE, this attachment can involve, at most, two binding sites, one on each Fab. For pentameric IgM, however, a single antibody may bind at up to 10 different sites (Fig. 5-13). Polyvalent antigens will have more than one copy of a particular determinant. Although the affinity of any one antigen-binding site will be the same for each epitope of a polyvalent antigen, the strength of attachment of the antibody to the antigen must take into account binding of all the sites to all the available epitopes. This overall strength of attachment is called the avidity and is much greater than the affinity of any one antigen-binding site. Thus, a low-affinity IgM molecule can still bind tightly to a polyvalent antigen because many low-affinity interactions (up to 10 per IgM molecule) can produce a high-avidity interaction. This is because an antibody with multiple binding sites will have at least one binding site physically bound to the antigen for a longer time than an antibody with just two binding sites; the latter is more likely to “fall off” the antigen and therefore has less avidity for the antigen, even though each Fab fragment on both forms may possess an equivalent affinity for the antigen.

FIGURE 5–13 Valency and avidity of antibody-antigen interactions.

Monovalent antigens, or epitopes spaced far apart on cell surfaces, will interact with a single binding site of one antibody molecule. Although the affinity of this interaction may be high, the overall avidity may be relatively low. When repeated determinants on a cell surface are close enough, both the antigen-binding sites of a single IgG molecule can bind, leading to a higher avidity bivalent interaction. The hinge region of the IgG molecule accommodates the shape change needed for simultaneous engagement of both binding sites. IgM molecules have 10 identical antigen-binding sites that can theoretically bind simultaneously with 10 repeating determinants on a cell surface, resulting in a polyvalent, high-avidity interaction.

Polyvalent antigens are important from the viewpoint of B cell activation, as discussed earlier. Polyvalent interactions between antigen and antibody are also of biologic significance because many effector functions of antibodies are triggered optimally when two or more antibody molecules are brought close together by binding to a polyvalent antigen. If a polyvalent antigen is mixed with a specific antibody in a test tube, the two interact to form immune complexes (Fig. 5-14). As discussed in Chapters 12 and 18, immune complexes can also contain complement fragments. At the correct concentration, called a zone of equivalence, antibody and antigen form an extensively cross-linked network of attached molecules such that most or all of the antigen and antibody molecules are complexed into large masses. Immune complexes may be dissociated into smaller aggregates either by increasing the concentration of antigen so that free antigen molecules will displace antigen bound to the antibody (zone of antigen excess) or by increasing antibody so that free antibody molecules will displace bound antibody from antigen determinants (zone of antibody excess). If a zone of equivalence is reached in vivo, large immune complexes can form in the circulation. Immune complexes that are trapped or formed in tissues can initiate an inflammatory reaction, resulting in immune complex diseases (see Chapter 18).

FIGURE 5–14 Antigen-antibody complexes.

The sizes of antigen-antibody (immune) complexes are a function of the relative concentrations of antigen and antibody. Large complexes are formed at concentrations of multivalent antigens and antibodies that are termed the zone of equivalence; the complexes are smaller in relative antigen or antibody excess.

Features Related to Antigen Recognition

Antibodies are able to specifically recognize a wide variety of antigens with varying affinities. All the features of antigen recognition reflect the properties of antibody V regions.

Affinity Maturation

The ability of antibodies to neutralize toxins and infectious microbes is dependent on tight binding of the antibodies. As we have discussed, tight binding is achieved by high-affinity and high-avidity interactions. A mechanism for the generation of high-affinity antibodies involves subtle changes in the structure of the V regions of antibodies during T cell–dependent humoral immune responses to protein antigens. These changes come about by a process of somatic mutation in antigen-stimulated B lymphocytes that generates new V domain structures, some of which bind the antigen with greater affinity than did the original V domains (Fig. 5-15). Those B cells producing higher affinity antibodies preferentially bind to the antigen and, as a result of selection, become the dominant B cells with each subsequent exposure to the antigen. This process, called affinity maturation, results in an increase in the average binding affinity of antibodies for an antigen as a humoral immune response evolves. Thus, an antibody produced during a primary immune response to a protein antigen often has a K_d in the range of 10⁻⁷ to 10⁻⁹ M; in secondary responses, the affinity increases, with a K_d of 10⁻¹¹ M or even less. The mechanisms of somatic mutation and affinity maturation are discussed in Chapter 11.

FIGURE 5–15 Changes in antibody structure during humoral immune responses.

The illustration depicts the changes in the structure of antibodies that may be produced by the progeny of activated B cells (one clone) and the related changes in function. During affinity maturation, mutations in the V region (indicated by red dots) lead to changes in fine specificity without changes in C region–dependent effector functions. Activated B cells may shift production from largely membrane-bound antibodies containing transmembrane and cytoplasmic regions to secreted antibodies. Secreted antibodies may or may not show V gene mutations (i.e., secretion of antibodies occurs before and after affinity maturation). In isotype switching, the C regions change (indicated by color change from purple to green or yellow) without changes in the antigen-binding V region. Isotype switching is seen in membrane-bound and secreted antibodies. The molecular basis for these changes is discussed in Chapter 11.

Features Related to Effector Functions

Many of the effector functions of immunoglobulins are mediated by the Fc portions of the molecules, and antibody isotypes that differ in these Fc regions perform distinct functions. We have mentioned previously that the effector functions of antibodies require the binding of heavy chain C regions, which make up the Fc portions, to other cells and plasma proteins. For example, IgG coats microbes and targets them for phagocytosis by neutrophils and macrophages. This occurs because the antigen-complexed IgG molecule is able to bind, through its Fc region, to γ heavy chain–specific Fc receptors (FcRs) that are expressed on neutrophils and macrophages. In contrast, IgE binds to mast cells and triggers their degranulation because mast cells express IgE-specific FcRs. Another Fc-dependent effector mechanism of humoral immunity is activation of the classical pathway of the complement system. The system generates inflammatory mediators and promotes microbial phagocytosis and lysis. It is initiated by the binding of a complement protein called C1q to the Fc portions of antigen-complexed IgG or IgM. The FcR- and complement-binding sites of antibodies are found within the heavy chain C domains of the different isotypes (see Fig. 5-1). The structure and functions of FcRs and complement proteins are discussed in detail in Chapter 12.

The effector functions of antibodies are initiated only by molecules that have bound antigens and not by free Ig. The reason that only antibodies with bound antigens activate effector mechanisms is that two or more adjacent antibody Fc portions are needed to bind to and trigger various effector systems, such as complement proteins and FcRs of phagocytes (see Chapter 12). This requirement for adjacent antibody molecules ensures that the effector functions are targeted specifically toward eliminating antigens that are recognized by the antibody and that circulating free antibodies do not wastefully, and inappropriately, trigger effector responses.

Changes in the isotypes of antibodies during humoral immune responses influence how and where the responses work to eradicate antigen. After stimulation by an antigen, a single clone of B cells may produce antibodies with different isotypes that nevertheless possess identical V domains and therefore identical antigen specificity. Naive B cells, for example, simultaneously produce IgM and IgD that function as membrane receptors for antigens. When these B cells are activated by foreign antigens, typically of microbial origin, they may undergo a process called isotype (or class) switching in which the type of C_H region, and therefore the antibody isotype, produced by the B cell changes, but the V regions and the specificity do not (see Fig. 5-15). As a result of isotype switching, different progeny of the original IgM- and IgD-expressing B cell may produce isotypes and subtypes that are best able to eliminate the antigen. For example, the antibody response to many bacteria and viruses is dominated by IgG antibodies, which promote phagocytosis of the microbes, and the response to helminths consists mainly of IgE, which aids in the destruction of the parasites. Switching to the IgG isotype also prolongs the effectiveness of humoral immune responses because of the long half-life of IgG antibodies. The mechanisms and functional significance of isotype switching are discussed in Chapter 11.

The heavy chain C regions of antibodies also determine the tissue distribution of antibody molecules. As we mentioned earlier, after B cells are activated, they gradually lose expression of the membrane-bound antibody and express more of it as a secreted protein (see Fig. 5-15). IgA can be secreted efficiently through mucosal epithelia and is the major class of antibody in mucosal secretions and milk (see Chapter 13). Neonates are protected from infections by IgG antibodies they acquire from their mothers through the placenta during gestation and through the intestine early after birth. This transfer of maternal IgG is mediated by the neonatal Fc receptor, which we described earlier as the receptor responsible for the long half-life of IgG antibody.