The structure of an antibody is well adapted to its function as an antigen-specific immunoglobulin. Antibody molecules are built up from discrete units of genes encoding for variable segments, which determine the antibody specificity, and constant segments, which determine the basic antibody structure and its interaction with effector mechanisms. The basic structure of an antibody molecule, especially of the IgG class, is schematically represented in Figure 2.1.
Figure 2.1 illustrates that the antibody is a Y-shaped tetramer of polypeptides composed of two immunoglobulin heavy chains (H) and two immunoglobulin light chains (L) covalently associated by disulphide bonds. The chains are folded into compact domains that have homologous sequences. The light chains have two such domains: a variable region domain ( Vl) and a constant region domain (Cl). In each
heavy chain there is a variable domain ( Vh) followed by three constant domains (ChI , Ch2 , Ch3 ). Between the first two constant domains lies a less homologous
region called the hinge. The disulphide bridges (Di-S) between the two heavy chains and the heavy and light chains are usually found within the hinge region. Glycosylation usually occurs in the constant domains of the heavy chains.
Variability in both heavy and light chains is thus found at the ends of the arms of the Y-shaped antibody (the amino terminal end). These variable region domains each
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contain three areas of hypervariable sequence known as complementarity determining regions (CDRs) which principally form the antigen-binding site.
L
CDR
Hinge
D i- S
Figure 2.1 Schematic representation of an antibody molecule. It consists of two identical heavy (H) chains and two identical light (L) chains, covalently bonded by disulphide bonds (Di-S). Each chain has a variable (V) domain and one or more constant (C) domains. The complementarity-determining regions (CDR) are shown in grey.
2.2.2 Antibody fragments
Various antibody fragments can be derived that are proving to be of practical use in therapy and diagnosis. These are shown in Figure 2.2. Some of these antibody fragments were originally derived from whole antibodies by enzyme proteolysis. However, recent developments in recombinant DNA technology mean that these
fragments can also now be produced using expression hosts such as E.coli (Harrison
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All of these small fragments are useful because they are still capable of binding to antigen. Their smaller size can, in certain situations, improve their diffusion or penetration properties (Harrison & Keshavarz-Moore, 1996; Clark, 1995).
Fab fragment
Figure 2.2 Schematic representation of the antibody fragments derived from the basic antibody molecule.
2.2.3 Chimeric and humanised monoclonal antibodies
As mentioned earlier, one of the problems associated with the use of murine monoclonal antibodies in human therapy is their immunogenicity or ‘foreignness’ that results in the human anti-mouse antibody (HAMA) response. Through the use of recombinant DNA techniques, genetic manipulation of rodent monoclonal antibodies began in the 1980s to reduce their immunogenicity. These techniques were used to generate chimeric and humanised antibodies as illustrated in Figure 2.3.
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Murine
Chimeric Humanised
Figure 2.3 Comparison of sequence content of a murine monoclonal antibody, a human-murine chimeric antibody and a humanised antibody. W hite and grey represent the parts of the structure derived from the murine sequence and black represents the human content. (Adapted from Clark, 1995.)
Chimeric antibodies are formed by partially humanising murine monoclonal antibodies by the replacement of murine constant domains with human constant domains. A refinement of the production of chimeric antibodies is the generation of humanised, or CDR-grafted, antibodies. These antibodies only contain the CDR loops from the murine variable regions responsible for the antigen binding. Such antibodies have been shown to be significantly less immunogenic than murine antibodies, and with a longer half-life. More recently, transgenic mice have been genetically engineered to generate fully human antibodies (Birch, 1999a; Chadd & Chamow, 2001).
2.2.4 Application of monoclonal antibodies
Monoclonal antibodies hold a firm place in biomedical science and their applications, either in the form of whole, intact antibodies or fragments, can be classified into the four main areas indicated below.
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1. Therapeutic:
There have been attempts to use antibodies as therapy for diverse human diseases, including coronary artery disease and infarctions, organ transplant failure, autoimmune diseases and cancer.
Different mechanisms are employed to induce a therapeutic effect using monoclonal antibodies. The monoclonal antibody can be used to induce a localised inflammatory response against a target cell or to act as a carrier to deliver another small molecule to a specific site. Alternatively, it can be used to interfere with growth or regulation by binding to critical hormones, growth factors or other regulatory molecules, or to act as vaccines by using anti-idiotypic antibodies which generate an active immune response (Scheinberg & Chapman, 1995).
2. Diagnostic:
Monoclonal antibodies are used as imaging agents for radioimmunodiagnosis of solid tumours in cancer, of infectious and inflammatory sites and of cardiovascular diseases. The monoclonal antibodies are conjugated to a radioactive isotope and serve as carriers to deliver the radionuclide to a specific site in vivo.
3. Assays:
Monoclonal antibodies have been used to develop a new generation of rapid and highly sensitive radioimmunoassays (Perry, 1995).
4. Purification:
Immunoaffinity purification with monoclonal antibodies is used widely in research laboratories for rapid extraction and purification of antigens. Immunoaffinity chromatography also has wider applications as a manufacturing unit operation since extremely high purities can be achieved in a single step.
2 .3 In v e s t ig a t in gm a r k e t e d m o n o c l o n a la n t ib o d ie s
Following the important development of hybridoma technology for producing monoclonal antibodies by Kohler and Milstein in 1975, immediate breakthroughs in the treatment of human diseases were expected. The early monoclonal antibodies, originally labelled as ‘magic bullets’, faced clinical disappointments owing to their
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murine origin which induced the human anti-mouse antibody (HAMA) response. However, monoclonal antibodies currently represent the second largest single category of biopharmaceutical substances under investigation as therapeutic drugs (Holmer, 2000). This can be attributed to the recent genetic engineering methods for constructing chimeric and humanised monoclonal antibodies that circumvent the HAMA response. Consequently, the full potential of monoclonal antibodies, with their unique binding specificity and potential to be produced in unlimited quantities, is rapidly becoming recognised in biomedical research, diagnosis and therapy.