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A tumour is an abnormal proliferation of cells which exceeds that of normal tissue and persists even after cessation of the stimuli evoking the change. When the proliferating cells become invasive, it is then known as a cancer. Cancer is one of the leading causes of death worldwide. Until recently the main medical treatment options consisted of either chemotherapy or radiotherapy, both of which are particularly non-specific and can cause devastating side effects. However the ability to produce Monoclonal antibodies (mAbs) en masse has made the development of immunotherapeutic drugs that specifically target tumour cells possible.
Antibody Structure and Function
Antibodies constitute the effectors of the adaptive immune system. Recognition of an immunogen by a B cell triggers proliferation and differentiation into plasma cells which secrete antibodies that are specific for that antigen.1 Secreted antibodies circulate in the blood and mediate humoral immunity.1
Two types of tumour antigen have been identified: Tumour-specific antigens as the name suggests are specific to tumour cells. They result from genetic mutations coding for altered cellular proteins.1 Tumour-associated antigens are non-specific to tumour cells. They may for example be proteins that are normally only expressed during fetal development or at extremely low levels.1
Tumour antigens do induce both humoral and cell-mediated immune responses in vivo, and it has been shown that individuals with impaired of NK cell function have increased rates of cancer.1 However it is an ineffective system which is commonly evaded by tumour cells.1
Antibodies are heterodimers consisting of four peptide chains: two light and two heavy. The unique specificity of each Antibody is determined by the amino acid sequence of its CDRs (complementarity-determining regions), which form the antigen binding site.1 The CDR is located within the variable region at the amino terminus of each heavy and light chain.1 There are five different possible heavy chain constant regions, relating to the class of Antibody formed.1
Hybridoma technology allowed for the immortalization of B-cells derived from immunised mice and the production of monoclonal antibodies.2 However these murine mAbs proved ineffective in humans due to immunogenicity of the murine protein, short half-life and reduced effector functions.3 In order to overcome these problems, Chimeric mAbs in which the murine constant region is substituted for a human one, Humanized mAbs in which only the CDRs are of murine origin and finally fully human mABs have been created.3-5 Although it is far rarer, it is worth noting that even fully human mAbs can still cause hypersensitivity reactions.6
Antibodies can be split into two regions: The Fab (Fragment antigen binding) region containing the antigen binding site and the Fc (Fragment, crystallisable) region which mediates effector functions. The easiest way to examine the anti-tumour activity of IgG is to split it into Fab and Fc mediated activities. mAbs are designed around their target specific (Fab-mediated) activities, and these account for much of their efficacy. However it has now become apparent that the Fc region also mediates immune mechanisms contributing to activity, the two major effectors of which, ADCC (antibody-dependent cell-mediated cytoxicity) and CDC (complement-dependent cytoxicity), which will be discussed later.
CD20 is an antigen expressed by most human B lymphocytes including follicular B-cell lymphomas, but is lost during their final maturation into plasma cells.7 This has made it a suitable target for treatment of B-cell lymphoproliferative disorders. It is thought that anti-CD20 mAbs upon binding transmit intracellular signals that cause apoptosis. The exact mechanism by which this takes place is not fully understood however when rituximab is sufficiently cross-linked, it appears to elicit apoptosis via the intrinsic mitochondrial pathway.8 How great a contribution this has over the efficacy of Rituximab is unknown.8
Vascular Endothelial Growth Factor (VEGF) is a mediator of pathological angiogenesis in the majority of human tumours.9 Bevacizumab (Avastin) binds VEGF, preventing it from interacting with its receptors on endothelial cells and thus inhibiting new vessel growth.10 It was also shown to reduce interstitial pressure and increase blood flow to the tumour, which may improve delivery of drugs given in combination.10 In 2004 Avastin was approved as first line treatment for metastatic colorectal cancer in combination with 5-fluorouracil.4
TGF-β is a pleiotropic cytokine that is often overexpressed in cancers. It plays a role in tumour progression by stimulating cell proliferation and angiogenesis, activating stroma cells and suppressing anti-tumour immunity. It has been shown that anti-TGF-β mAbs reduce tumour growth in mice.11 The pan specific, recombinant, fully human anti-TGF-β mAb, Fresolimumab (GC1008) is currently in phase II clinical trials. Malignant Melanoma, Renal Cell Carcinoma and Mesothelioma have thus far been identified as potential therapeutic targets.12
Another target for mAbs is the erbB family of receptors. erbB-1/ EGFR is a member of the type I tyrosine kinase family. Binding of its ligand induces receptor dimerization, internalisation of the receptor/ligand complex and tyrosine kinase activation.13 Overexpression of EGFR leads to various cellular processes involved in carcinogenesis and is seen in many cancers.13 The chimeric mAb Cetuximab binds EGFR, competitively antagonising its natural ligands and inhibiting the mitogen activating protein kinase signaling pathway. This upregulates p27KIP1 and induces apoptosis.4 In addition it also causes downregulation of EGFR on the cell surface and inhibits production of VEGF.10
Antibodies to CTLA-4
A major influencing factor in selection of IgG for use in therapeutic mAbs is the fact that it expresses binding sites for both C1q and FcRs and thus is able to stimulate ADCC and CDC.
ADCC is the mechanism whereby antibodies direct effector cells of the innate immune system to specific targets.3 Firstly, an IgG antibody binds to its specific receptor on the surface of a tumour cell. The Fc fragment then binds to an FcγR on an effector cell. High affinity FcγRI bind monomeric IgG, whilst the lower affinity FcγRII and FcγRIII bind IgG aggregates and Immnuocomplexes. This binding causes cross linking of receptors and activation of the effector cell.3 NK cells are the principle effectors of ADCC, however monocytes, macrophages and granulocytes bearing the FcγR are also activated. Activated NK cells release of perforin, granulysin and granzymes that induce apoptosis, whilst release of cytokines and chemokines inhibit cell proliferation and angiogenesis.3, 10 The ability of IgG to stimulate ADCC is determined by its affinity for the FcγR. Minor changes in the amino acid sequence of the γ heavy chain give rise to four IgG isotypes of which IgG1 and IgG3 have the greatest affinity for FcγRs.10
In CDC, the classical complement pathway is activated: Formation of the Antibody-antigen complex induces a conformational change in the Fc region allowing C1q to bind. This activates the complement cascade, allowing formation of C3b and eventually a membrane attack complex that can cause lysis of tumour cells.1 Again IgG isotypes 1 and 3 are most effective in stimulating this pathway.1 However a number of membrane-bound complement regulatory proteins which inactivate C3b, thus inhibiting MAC formation are commonly overexpresed on tumour cells.14 This means CDC is unlikely to directly play a significant role in Tumour lysis.10 Instead what it can do is initiate a second mechanism, whereby CR3 binds to iC3b, thus enhancing FcγR-mediated effector cell binding and ADCC.14
Implications for Design
In addition to its apoptotic activity, rituximab has been shown to have CDC and ADCC activity. The high rate of expression of CD20 on cells, the proximity of the mAb-binding epitope to the plasma membrane and the formation of lipid rafts which cluster mAb result in Rituximab having a high propensity for eliciting CDC.8 Again however exactly how substantially if at all this mechanism actually contributes to the drug's efficacy is still in dispute.8
The receptor FcγRIIB which is present on B-cells, macrophages and monocytes is a potent regulator of ADCC. Rituximab, which non-selectively binds both excitatory FcγRIII and inhibitory FcγRIIB was shown to have increased efficacy in FcγRIIB deficient mice.15 The binding sites on IgG1 for FcγRs can be found in the lower hinge region. By altering residues at this site, it is possible to enhance or reduce binding to specific FcγRs.16 This knowledge has led to engineering mAb's to have high affinity for FcγRIII and decreased affinity for FcγRIIB.4
Since rituximab was approved for use in 1997, many anti-CD20 mAbs have been designed. The latest, 3rd generation reagents such as GA101 are humanized and have an engineered Fc region to enhance FcR binding.8
Modification of carbohydrate moieties in the Fc - engineering
It is possible to increase the affinity of IgG for C1q and thus CDC activity by mutating the four amino acids that make up the binding site on the Fc region.4 Overcoming tumour cell mCRP's is the main obstacle in increasing mAb CDC activity. Changing isotype, or humanisation may increase complement activating capacity.14
In order to activate the complement cascade, antigen density must be relatively high to enable formation of IgG dimers. In tumors with low antigen density, one possible way of boosting the CDC response is to use a cocktail of mAbs that are specific for multiple epitopes of the target antigen.14
One way in which mAbs have been altered to improve their cytotoxic actions is through forming immunoconjugates with toxic substances such as radioisotopes and cytotoxic agents.10 These make it possible to deliver toxic substances specifically to tumour cells.10
In 2000, gemtuzumab ozogamicin - a conjugate of a humanized anti-CD33 IgG linked to colicheamicin was approved for treatment of relapsed acute myelotic leukemia.5 A useful feature of drug immunoconjugates is that the attached drug is essentially detoxified, allowing drugs which would otherwise be too toxic to be used.5 Once interternalized, the drug must be separated from the antibody before it is active. Often these drugs are bound to the antibody using linkages that can only be cleaved in the acidic environment of a lysosome. This helps to ensure maximum potency.5 Once gemetuzumab ozogamicin is hydrolysed, it causes double stranded DNA breaks, and apoptosis.4
In unconjugated mAbs it is beneficial to have a long half-life so that it can continually interact with its target. However in Immunoconjugates, especially those bound to radioisotopes, a short half-life is preferred to minimise exposure of the bone marrow.5 90Y-ibritumomab tiuxetan is a murine anti-CD20 IgG radioconjugate approved for use in transformed forms of NHL.5 The murine mAb is used as it has a short half-life in humans due to its low affinity for the Fc neonatal receptor (FcRn).4
FcRn is expressed in many tissues. IgG enters FcRn expressing cells by pinocytosis where IgG-FcRn complexes can form. IgG-FcRn complexes are salvaged whilst uncomplexed IgGs enter the lysosomal pathway and are degraded. Thus FcRn plays a crucial role in maintaining serum concentration of IgG.4 The affinity of IgG for FcRn has provided a focus for improving pharmacokinetics of mAbs.4 It is possible to decrease the half-life of Immunoconjugates by either altering the IgG FcRn binding site, or by co-administering mutant IgGs (known as FcRn blockers) to which FcRn preferentially binds.17 Likewise it is possible to increase half life of 'naked' mAbs by mutating the Fc binding region to have increased affinity for FcRn.4
One method which has been employed to improve tumour penetration is using mAb's with low molecular weights. F(ab')2 fragments can be created by cleaving IgG with pepsin.The loss of the Fc region results in a short half life and an inability to induce either ADCC or CDC. This would be detrimentary to regular therapeutic antibodies however lends itself to Immunoconjugates. The short half life reduces bone marrow exposure, and the inability to bond cells expressing FcγRs reduces side effects.4
Interestingly, it has been shown that increasing an antibodies affinity for its tumour antigen above a certain level can actually reduce efficacy against solid tumours.18 This is due to the fact that they bind strongly to the first available antigen and are retained in the perivascular regions.18 This is particularly important for cytotoxic agents where distribution throughout the tumour is necessary. 5
In conclusion, mAbs have proven extremely successful in the treatment of cancer. This can be attributed to their unique specificity for tumour antigens and their ability to recruit the innate immune system. Since their conception in 1975, mAbs have undergone constant modification in order to improve their therapeutic effects. In the future, we can expect to see mAbs that have been engineered to recognise a greater range of targets, have improved pharmacokinetics and tumour penetration and increased propensity to recruit the innate immune system.