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Molecular structure

Antibodies are plasma glycoproteins, called gamma globulins because of their mobility in an electric field and immunoglobulin (Ig) because of their role in immunity. Antibodies were initially characterized using myeloma proteins, homogenous antibodies produced by cancerous plasma cells in individuals with multiple myeloma. Antibodies which are identical with each other at every amino acid (because they have all been produced by the descendants of a single B cell) are called monoclonal antibodies (mAb).

Myeloma proteins are naturally occurring monoclonal antibodies, because the myeloma develops from a single cancerous plasma cell (a clonal tumor). Monoclonal antibodies can also be produced in the lab. Serum antibodies are polyclonal antibodies, because they are produced by the descendants of several B cells that recognize different epitopes on the same antigen.

To produce monoclonal antibodies, one removes B-cells from the spleen or lymph nodes of an animal that has been challenged with the antigen. These B-cells are then fused with myeloma tumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer). This fusion is done by making the cell membranes more permeable. The fused hybrid cells (called hybridomas), being cancer cells, will multiply rapidly and indefinitely and will produce large amounts of antibodies.

All antibodies share a basic structure, each antibody "monomer" is composed of two identical heavy (H) polypeptide chains and two identical light (L) chains, covalently bonded via interchain disulfide (S-S) linkages between cysteine residues. Each H chain is about 440 amino acids long; each L chain is about 220 amino acids long. H and L chains each contain intrachain disulfide bonds which stabilize their folding into 110amino acid domains.

Immunoglobulin domains are a common feature of many soluble molecules and membrane-bound receptors of the immune system, comprising the Ig superfamily. All antibodies have one of two kinds of L chain, kappa (k) or lambda (l); each antibody has two identical k chains or two identical l chains. Five different H chains have been found: alpha (a), gamma (g) , delta (d), epsilon (e), and mu (m). Antibody isotypes (classes) are named IgA, IgG, IgD, IgE, and IgM to correspond to their H chain types, which influence the effector functions of the antibody molecules.

The amino acid sequence of the H and L amino terminal domains vary considerably from one Ig to the next and are responsible for the antigen-binding diversity of antibodies; these make up the variable regions, VH and VL. Each light chain has one VL domain and each heavy chain has one VH domain. One VH and one VL fold together to form an antigen-binding site, so each Ig molecule has two identical antigenbinding sites.

The amino acid sequence of the carboxyl half of the L chain and three-fourths of the H chain show relatively limited variability, and make up the constant regions (CH and CL). Each light chain has one CL domain, and each heavy chain has three (a, g, and d chains) or four (e and m chains) CH domains. The hinge region is a more extended region (not folded into a domain) between H chain CH1 and CH2 that is present in a, g, and d chains. It allows the two antigen-binding regions of each antibody molecule to move independently to bind antigen.

Limited proteolytic digestion with papain cleaves the Ig prototype into three fragments. Two identical amino terminal fragments, each containing one entire L chain and about half an H chain, are the antigen binding fragments (Fab). The third fragment, similar in size but containing the carboxyl terminal half of both H chains with their interchain disulfide bond, is the crystalizable fragment (Fc).

The Fc contains carbohydrates, complement-binding, and FcRbinding sites. Limited pepsin digestion yields a single F(ab')2 fragment containing both Fab pieces and the hinge region, including the H-H interchain disulfide bond. F(ab')2 is divalent for antigen binding. Pepsin digests the carboxyl halves of the H chains.

Within VH and VL there are hypervariable regions which show the most sequence variability from one antibody to another and framework regions which are less variable. Folding brings the hypervariable regions together to form the antigen-binding pockets. These sites of closest contact between antibody and antigen are the complementarity determining regions (CDR) of the antibody.

The antigen-binding site of a typical antibody is a cleft formed by folded VH and VL regions. It can accommodate approximately four to seven amino acids or sugar residues. Contact between large antigens and antibodies probably extends along the surface of the antibody outside as well as inside the antigen-binding pocket. Antigen-binding specificity of antibody resembles that of enzyme binding substrate.

Antibodies bind their specific antigens using hydrogen bonds, ionic bonds, hydrophobic interactions, and Van der Waals interactions. Covalent bonds are not formed between antigen and antibody, so binding is reversible.

An epitope is the portion of an antigen bound by an antibody. Viral capsid proteins and bacterial cell wall components usually have multiple epitopes. Antibody is produced to each epitope in relation to its immunogenicity; epitopes to which the most antibody is produced are called immunodominant epitopes. Antibodies distinguish antigenic differences (serotypes) between members of the same bacterial or viral species.

Some epitopes are shared by different antigens, so that antibody made to one also binds the other (is cross-reactive). Crossreactive antibodies are one mechanism by which autoimmunity is induced. For example, antibodies made by some people to Streptococcus pyogenes bind a crossreactive antigen on their heart valves and cause rheumatic heart disease.

The strength of the interaction between a single antigen-binding site on the antibody and its specific antigen epitope is called the binding affinity of the antibody. The higher the affinity, the tighter the association between antigen and antibody, and the more likely the antigen is to remain in the binding site. Antibody affinity generally increases with repeated exposure to antigen because B cells with higher affinity antigen receptors are selected to produce larger clones of antibody-secreting plasma cells.

The affinity constant Ka is the ratio between the rate constants for binding and dissociation of antibody and antigen. Typical affinities for IgG antibodies are 105-109 L/mole. Antibody affinity is measured by equilibrium dialysis. The relationship between bound and free antigen and antibody affinity is expressed by the Scatchard equation, r/c = Kn - Kr, where r = the ratio of [bound antigen] to [total antibody], c = [free antigen], K = affinity, and n = number of binding sites per antibody molecule (valence). Antibodies have the same affinity for antigen (monoclonal antibody), the antibody is heterogeneous (polyclonal).

IgG, IgD, IgE, and "monomeric" IgA have two identical antigen-binding sites (valence = 2). Dimeric IgA has four. Serum IgM has ten, although the observed valence of IgM is five because all the binding sites cannot make contact with antigen simultaneously due to steric hindrance. Avidity is the functional affinity of multivalent antigen binding to multivalent antibody molecules. Avidity strengthens binding to antigens with repeating identical epitopes.

The more antigen-binding sites an individual antibody molecule has, the higher its avidity for antigen. Crosslinking antibody by binding two different Ig molecules to the same antigen, common with pathogens which have many copies of the same epitopes on their surface, is crucial for activating both complement and B cells.

Immunoglobulins can be used as antigens to generate antibodies that distinguish several Ig epitopes: isotypes, allotypes, and idiotypes. Anti-isotype sera differentiate epitopes in the constant regions of H and L chains. All members of a species share the same isotypes. For example, antiisotype made against one cloned human m chain would bind to all human m chains. Humans have four subisotypes of g chain ( g1- g4) and two subisotypes of a chain. Subisotypes differ in amino acid sequence and biological functions but are more closely related to each other than to other isotypes.

Within a species there is some variation in amino acid sequence within an isotype or subisotype; these differentiate Ig allotypes. Allotypic epitopes are in CH and CL. Each individual B cell or plasma cell produces antibody of a single allotype (allotypic exclusion), and both H chains or both L chains of an individual antibody molecule have the same allotype. Each person has antibodies with one (homozygous) or two (heterozygous) allotypes, depending on whether they inherited the same or different allotypes from each parent.

For example, a person who inherited GM1 from one parent and GM3 from the other would have B cells making either GM1 or GM3 H chains, but their serum would contain roughly equal numbers of IgG molecules with each allotype. Idiotypic epitopes are due to sequence differences within VH and VL, so each individual makes antibodies with many idiotypes. Monoclonal antibody molecules share the same idiotype. Polyclonal antibodies, even those made against the same epitope, may have different idiotypes. Antiidiotype antibody and antigen usually compete for the antigen-binding region of the Ig.

Biological Functions

Properties of Antibody Isotypes % of total Ig (adult serum) 11-14 1-4 0.2 0.004 Biological half-life (days) 5.9 Pathogen neutralization in mucosal secretions 4.5 2-8 1-5 Membrane BCR Mast cell histamine release Pathogen neutralization in tissues Classical complement activation Opsonization NK cell ADCC Transplacental transfer Pathogen neutralization in tissues Isotype Biological Functions IgA1 IgA2 IgD IgE










Pathogen neutralization in tissues Classical complement activation Opsonization NK cell ADCC Transplacental transfer Pathogen neutralization in tissues Transplacental transfer Classical complement activation Membrane BCR (monomer)







While antibody VH and VL bind antigen, antibody constant regions determine its biological functions. CH2 domains bind complement and control the rate of Ig catabolism (breakdown). CH2 and CH3 domains bind phagocyte FcR (Fc Receptor) to stimulate antigen uptake. The biological functions of the C domains are independent of the antigen specificity of the molecule.

Antibody is synthesized on membranebound polyribosomes (rough endoplasmic reticulum, RER) in the cytoplasm of the B cell or plasma cell. A signal recognition protein attached to the H and L chain leader sequences sends the chains into the endoplasmic reticulum (ER). H and L chains assemble into H2L2 monomers with formation of the interchain disulfide bonds; carbohydrate is added to the CH regions.

The vesicle containing antibody moves via the Golgi apparatus to the plasma membrane and exocytosis releases secreted antibody from the plasma cell. Membrane-bound antibody has an additional transmembrane sequence on its carboxyl terminal CH region which anchors the molecule to the lipid bilayer.

IgM is the first antigen receptor (BCR) made during B cell development and the first antibody secreted during an immune response. Membrane IgM is a four-chain "monomer" of two m chains and two light chains (either both k or both l). Serum IgM is a "pentamer" containing five four-chain monomers held together by interchain disulfide bridges in the CH3 and CH4 regions plus an extra polypeptide chain called J chain.

Pentameric IgM is the most efficient antibody for activating complement because the five adjacent C regions easily bind two complement (C1) molecules. IgM is too large to efficiently leave the circulation, reducing its effectiveness in the tissues. Low levels of IgM are present in mucosal secretions.

IgG is the predominant serum antibody with the longest half-life. Four subisotypes of IgG in humans have somewhat varied biological functions. IgG is made later in a primary response than IgM, but it is produced more rapidly in a memory response. IgG crosses the placenta to transfer maternal immunity to the fetus and leaves the circulation to neutralize virus and toxin binding to host cells. Two molecules of IgG are required to activate complement. IgGantigen complexes bound to FcR stimulate phagocytosis (opsonization).

IgA is present in serum and predominates in mucosal secretions: breast milk, saliva, tears, and respiratory, digestive, and genital tract mucus. Secretory IgA provides a first-line defense where pathogens enter the body. More IgA is made than any other isotype. Serum IgA is usually monomeric, although dimers, trimers and tetramers are present. Secretory IgA is dimeric or tetrameric and contains one J chain and one additional chain called secretory component (SC), which protects it from proteolytic degradation.

Plasma cells make IgA and J chain and assemble and secrete polymeric IgA. IgA then travels through the circulation to the mucosal epithelial cells, which have binding molecules called poly-Ig receptor on their apical membranes. Poly-Ig receptor binds to J chain and allows IgA (and some IgM) to enter the epithelial cell, cross the cytoplasm, and exit on the luminal side with part of the poly Ig receptor still attached as secretory component.

IgE is produced in response to helminth parasites and to allergens. Epsilon chain binds very efficiently to mast cell FceR. Antigen cross-linking of IgE on FceR signals the mast cell to release histamine, which increases fluid entry into the tissues and mucus production. IgE also helps eosinophils destroy helminth (worm) parasites.

IgD, with IgM, is the BCR for antigen. Its presence on the B cell membrane signals that the B cell is mature and ready to leave the marrow and respond to antigen in the secondary lymphoid organs. IgD is present in serum in low amounts; no effector functions have been identified for serum IgD.

Isotype Distribution and Function

Microbes usually enter the body through the epithelial cells of the respiratory, digestive, or genital tracts or through skin broken by a scrape, cut, insect bite or hypodermic needle. Once inside, the microbe may begin replicating locally or get into the circulation and move throughout the body. Bacterial toxins also travel from the initial infection site. The Fc regions of the Ig isotypes allow them to bind Fc receptors and cross tissue barriers to reach pathogens throughout the body.

IgM is secreted first in a primary response. No somatic hypermutation has yet occurred, so it is low affinity antibody. IgM avidity is high, however, because it is a pentamer, and IgM fixes complement very efficiently to promote inflammation and pathogen lysis. Because IgM is so large, it cannot enter the tissues very efficiently; but it is effective in controlling pathogens in the circulation.

Once isotype switching occurs, IgG predominates in serum and in tissues. IgG both neutralizes pathogens and their toxins and opsonizes them for phagocytosis by neutrophils and macrophages. IgG can also activate complement on the pathogen surface once concentrations are high enough for two IgG molecules to bind nearby epitopes.

IgA is the predominant antibody that is secreted across epithelial cells of the respiratory, digestive and genital tracts to block pathogen entry into the body. IgE binds FceR on mast cells lining the blood vessels throughout the body. When pathogen binds to the mast cell IgE, the mast cells immediately release inflammatory mediators that trigger coughing, sneezing or vomiting to expel pathogens from the body.

The selective transport of various Ig isotypes to particular regions of the body occurs because of isotype-specific Fc receptors on different tissues. Dimeric Ig A (and, to a lesser extent, pentameric IgM) bind to the poly Ig receptor on the body side of epitheilial cells in the intestines, respiratory tract, tear and salivary glands, and lactating mammary gland. The antibody-poly Ig receptor complex is endocytosed into the epithelial cell and travels in an endocytic vesicle across the cytoplasm (transcytosis) to be secreted on the outer surface of the epithelium (into the intestine or lung surface or tears, saliva, or milk).

Maternal IgA in milk can neutralize pathogen in the infant's digestive tract until the infant's immune system is mature enough to take over that task. IgG which has crossed the placenta into the fetal circulation offers additional protection during the first few months of life. An FcRn has been identified on placental cells for transport of IgG across the placenta, and a similar molecule has been identified on intestinal cells of some mammals that may allow uptake of IgG in colostrum, the first fluid secreted by the mammary gland after birth.

Numerous bacteria cause disease by releasing toxins that damage cells. Like viruses, bacterial toxins must enter cells via specific receptors in order to damage the cells. Neutralizing antibodies, usually IgG, block binding to the target cells. Toxins may kill us before we can produce neutralizing antibodies, so they are a natural target for vaccination. We are immunized as infants with inactivated diphtheria and tetanus toxins (toxoids); we may become infected with Corynebacterium diphtheriae or Clostridium tetani, but any toxin they produce will be neutralized before it can harm our cells. For snake venoms, passive immunization with antitoxins produced in horses can neutralize the venom.

Bactericidal agents released in response to FcR binding include oxygen radicals and peroxides, nitric oxide, defensins, and lysozyme. Respiratory burst is the process by which phagocytes generate the toxic oxygen compounds that inactivate key microbial enzymes and structural proteins by oxidizing them. Phagocytes also acidify their phagocytic vesicles to activate degradative enzymes and release molecules such as lactoferrin and vitamin B12-binding protein that compete with microbes for essential nutrients.

For antibody-coated microbes which are too large to phagocytose, phagocytes excrete these bactericidal agents into the extracellular space. Helminth parasites generally induce secretion of IgE, which on eosinophil FceR1 signals eosinophils to kill the parasite .

Cells infected with enveloped viruses express the envelope proteins on their membranes before the viruses take pieces of membrane for their envelope as they but from the cell. Antibodies to those viral proteins can bind to FcgRIII (CD16) on NK cells and activate them to kill the virusinfected cell, a process called ADCC (Antibody-Dependent Cell-mediated Cytotoxicity). NK cells use perforin and granzymes, present in their granules (remember NK cells are also called Large Granular Lymphocytes), to kill their target cells.

In response to some antigen challenges, especially helminth parasites and allergens, the body responds by producing IgE. Mast cells with their membrane FceR1 are found just below the skin and respiratory and digestive epithelia. Mast cell cytoplasm is packed with granules containing histamine and other mediators of inflammation. Unlike other FcR, FceR1 binds free IgE (without bound antigen), so most IgE in the body is found on mast cell surfaces and on circulating basophils.

When antigen enters the body, it cross-links the IgE on mast cells and ITAMs signal the mast cells to immediately secrete their granule contents. Mast cells also secrete cytokines, including IL-4 that stimulates IgE synthesis by B cells. Eosinophils express FceR1 when activated at an infection site. Experiments with mice deficient in mast cells show that these mice have difficulty eliminating helminth parasites compared to their normal counterparts. Mast cells, IgE, basophils and eosinophils have been implicated in resistance of mice to certain bloodsucking ticks, such as those that transmit Lyme disease.

FcgRII-B1 (CD32) on B cells and mast cells has cytoplasmic ITIMS instead of ITAMS, and inhibits cell function. On B cells, CD32 binds Ig with a lower affinity than FcgRI, so high amounts of IgG antibody inhibit activation of naïve B cells (late in immune responses) and mast cells.

Chimeric and humanized antibodies

One problem in medical applications is that the standard procedure of producing monoclonal antibodies yields mouse antibodies. Although murine antibodies are very similar to human ones there are differences. The human immune system hence recognizes mouse antibodies as foreign, rapidly removing them from circulation and causing systemic inflammatory effects.

A solution to this problem would be to generate human antibodies directly from humans. However, this is not easy primarily because it is clearly not ethical to challenge humans with antigen in order to produce antibody. Furthermore, it is not easy to generate human antibodies against human tissues.

Various approaches using recombinant DNA technology to overcome this problem have been tried since the late 1980s. In one approach, one takes the DNA that encodes the binding portion of monoclonal mouse antibodies and merges it with human antibody producing DNA. One then uses mammalian cell cultures to express this DNA and produce these half-mouse and halfhuman antibodies. Depending on how big a part of the mouse antibody is used, one talks about chimeric antibodies or humanized antibodies. Another approach involves mice genetically engineered to produce more human-like antibodies.



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