Antibodies In Cancer Therapy Biology Essay

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Antibody-based therapy for cancer has become estab­lished over the past 15 years and is now one of the most successful and important strategies for treating patients with haematological malignancies and solid tumours.

Antibodies are produced by plasma B-lymphocytes and function as a part of the immune system in mammals in the battle against disease. The fundamental basis of antibody-based therapy of tumours dates back to the original observations of antigen expres­sion by tumour cells through serological techniques in the 1960s1. The definition of cell surface antigens that are expressed by human cancers has revealed a broad array of targets that are overexpressed, mutated or selectively expressed compared with normal tissues2. A key challenge has been to identify antigens that are suitable for anti­body-based therapeutics. Such therapeutics can function through mediating alterations in antigen or receptor func­tion (such as agonist or antagonist functions), modulat­ing the immune system (for example, changing Fc function and T cell activation) or delivering a specific drug that is conjugated to an antibody that targets a specific anti­gen2-5.

Antibody structure

Antibodies were first identified in 1939 by Kabat and Tiselius (Tiselius et al., 1939). Subsequently, their structure was discovered by Porter and Edelman in the 1950s (Edelman 1958; Porter, 1958). The main architecture of most immunoglobulins consist of two heavy chains (50-70 kDa) and two light chains (~ 30 kDa) (Porter et al., 1962; Edelman et al., 1975), which have constant domains (IgG and IgD have CH1, CH2, CH3 and CL, whereas IgE and IgM have an extra heavy domain, CH4) and variable domains (VH and VL). The most abundant of the immunoglobulins is IgG, generally depicted as a Y shape format (Fig. 1.2), it is the main immunoglobulin structure used in the design of therapeutic antibodies. The special structure of the domains is termed the immunoglobulin fold and is composed of two β-sheets stacking against each other by the interaction of hydrophobic amino acids on each sheet (figure 1.1 A). Heavy chains are bound together by disulfide bridges and non-covalent bonds, such as salt-bridges, hydrophobic bonds and hydrogen bonds. In addition one light chain is attached to each heavy chain by the same kind of covalent and non-covalent bonds (figure 1.1 B). Within the first 110 amino acids of the L and H chain there are large variations between immunoglobulins, this region was therefore denoted the variable region (V) and constitutes the antigen-binding site of the immunoglobulin 6 (Riechmann et al., 1988; Skerra & Pluckthun, 1988). The variation in amino acid sequence allows for different binding specificities, creating a diverse population of antibodies. Immunoglobulins can be divided into five different classes (IgG, IgM, IgA, IgE and IgD), based on differences in the amino acid sequences in the constant region of the heavy chains. Within the different immunoglobulin classes subtypes can occur (for example mouse IgG1, IgG2a, IgG2b and IgG3). IgM is the first immunoglobulin class produced in response to antigen, but class switching later results in expression of IgG, IgA and IgE with the same antigenic specificity. All immunoglobulins within a given class have very similar heavy chain constant regions. IgG, IgD and IgE have a general Y-shaped structure, whereas IgM has a pentameric structure (figure 1.1 B). IgA is a dimer of two Y-shaped structures linked together (not shown in figure 1.1). The active regions of immunoglobulins are the two antigen-binding fragments (Fab) and the constant region (Fc). Both heavy and light chains contribute to the Fab-regions, while the Fc region consists of the heavy chains only (CH2 and CH3). The Fc region is attached to Fabs via the linker region (figure 1.1). Antibodies are bivalent and the antigen binding part (i.e. complimentary determining regions, CDR) of the molecule is located on the tip of the two Fab domains, whereas the stem Fc domain mediate effector functions (27). Characterisation of the Fab fragment by sequence analysis revealed the difference in the amino acid sequence within the V domains to be confined to six regions, 3 within the VH and 3 within the VL. These regions are termed the hypervariable regions, also known as complementarily determining regions (CDRs). The CDRs are responsible for the antibodies vast array of antigen binding specificity (Wu & Kabat, 1970). The region within the VH and VL chains where there is less amino acid variation is termed the framework regions. The conserved sequence in the framework region forms a β sheet structure, which acts as a scaffold to the three hypervariable loops within the VH and VL regions. The rest of the Fab arm contains the CH and CL regions, these have a number of roles including: assisting in the antigen interaction, increasing the

maximum rotation of the Fab arms and holding the VH and VL chains together by an inter-chain disulfide bond.

In addition to intact immunoglobulins, antibody fragments (Fab, Fab2, scFv) with antigen binding ability can be generated by proteolysis or by antibody engineering (figure 1.1 B). Further sequence analysis found the light chain family to consist of two basic amino acid sequences denoted lambda (λ) and kappa (κ); a single antibody can only contain one type of light chain (Cook & Tomlinson, 1995; Tonegawa, 1983). In comparison, the heavy chain family contains five different amino acid sequences, with each of these isotypes being denoted μ, δ, γ, ε and α. The five different heavy chain isotypes gives rise to the five different subgroups of immunoglobulins, IgM (μ), IgD (δ), IgG (γ), IgE (ε) and IgA (α), which ultimately determines the function of the immunoglobulin. The length of the isotypes amino acid sequence also varies, with δ, γ and α having 330 amino acids, which constitutes three C regions CH1, CH2, CH3 and a hinge region. Where as, the isotypes μ and ε have 440 amino acids, constituting four C regions CH1, CH2, CH3 and CH4 and no hinge region. The hinge region is a

flexible proline rich region, found between the CH1 and CH2 domain, giving the Fab arm extra flexibility when binding to antigens (Harris et al., 1997). The understanding of how diverse antibodies are produced in vivo through combinational rearrangement (Tonegawa, 1983) and somatic hypermutations (Neuberger, 2008), has allowed the development of genetic engineering technology (Hoogenboom et al., 2005). Antibody engineering has advance at a fast rate; enabling the generation and construction of large-scale amounts of high specificity and affinity monoclonal antibodies to be produced, for diagnostic and therapeutic applications.

1.1.2 Polyclonal and monoclonal antibodies

Antibodies are made by immunising a suitable mammal with the antigen. The host immune system will react with the antigen and B-lymphocytes will produce antibodies against the target. Several different Blymphocyte clones produce antibodies, which are therefore termed polyclonal antibody (pAb). pAbs can easily be purified from the blood of the mammal by chromatographic techniques. A pAb raised against an antigen bind different epitopes on the target, which gives an increased risk that pAbs cross-react with biomolecules containing similar epitopes. Furthermore, the supply of pAbs is limited as the mammal is eventually killed. To circumvent these limitations Kohler and Milstein produced monoclonal antibody (mAb) producing cells in 1975 (51). This Nobel Prize winning work (1984, Physiology and medicine) revolutionised antibody production and today it forms the basis of many diagnostic applications, disease therapy and basic research (27). mAbs are antibodies of a single idiotype produced by immortalised Blymphocytes recognising a single epitope on the antigen. Normal B-lymphocytes are fully differentiated and cannot be maintained in culture. Kohler and Milstein fused antibody producing B-lymphocytes with myeloma cells, thereby creating immortal antibody producing cell lines (hybridoma cells). As in pAb production, a suitable animal is immunised (usually mice or rats) with the antigen and after a sufficient serum antibody titer is detected, the animal is sacrificed and the splenocytes recovered. The splenocytes are fused with myeloma cells using polyethylene glycol (PEG) (32). The fusion is random and fused hybridoma cells must be selected and isolated from unfused B-lymphocytes and myeloma cells. The selection process is performed in medium which only allows for hybridoma survival. The cells are cultured in hypoxanthineaminopterin-thymidine (HAT) medium. Aminopterin (A) blocks the de novo biosynthesis of purines and pyrimidines essential for DNA synthesis. When this pathway is blocked, cells use the salvage pathway utilising Hypoxanthine (H) and Thymidine (T), and this requires the activity of the enzymes Thymidine Kinase (TK) and Hypoxanthine-Guanine Phosphoribosyl Transferase (HGPRT). The myelomas selected for the fusion lack the HGPRT, so that unfused myeloma cells and myeloma cells fused to other myeloma cells cannot proliferate in HAT medium. The unfused splenocytes do possess HGPRT but have a limited lifetime and the culture will die within two weeks. The hybridoma cells grow effectively in the HAT media. Many different hybridomas are developed during the fusion and every cell type produces a specific antibody towards a wide range of antigens and not only the antigen used. To identify the correct hybridomas, cells are distributed in 96-wells plates and hybridoma supernatant is used in Enzyme-linked immunosorbent assay (ELISA) to detect the positive wells for subsequent cloning by limiting dilution (54). The method essentially consists of diluting the cells and growing them at very low densities, often in the presence of feeder cells, which supply growth factors, see figure 1.2 for an overview of mAb production.