CANCER AND ANTI-ANGIOGENIC AGENTS
In 2004, the humanised version of a monoclonal antibody to vascular endothelial growth factor (VEGFA), bevacizumab, better known as Avastin®, became the first FDA approved anti-angiogenic drug. The antibody recognises all isoforms of VEGFA and has a long circulating half-life of up to 21 days after intravenous infusion. Initial clinical trials showed that when used alone Avastin® was generally ineffective but that it had significant effects when combined with other drugs. It was therefore initially approved for metastatic colorectal cancer in combination with 5-fluorouracil. Subsequently, it has been approved for treatment of unresectable, recurrent or metastatic non-squamous- cell lung cancer, breast cancer and glioblastoma multiforme and is currently undergoing extensive clinical trials for use against many types of tumours including melanoma, ovarian carcinoma, renal cell carcinoma, gastric carcinoma and prostate cancer. Although the precise mechanism of action is unknown, the anti-VEGF antibody may play a role in the ‘normalisation’ of tumour vasculature thereby making it more susceptible to drugs administered subsequently. Despite its promise, Avastin® received a setback in 2010 when the FDA rescinded its approval for use against breast cancer because cumulative evidence has shown that it does not prolong life and has serious side effects. Avastin® retains approval for the treatment of other cancers.
In addition, Avastin® is also being evaluated in phase 3 trials for treating the vascular damage associated with age-related macular degeneration; the recent phase 2 trials have shown an improvement in vision in patients. Ranibizumab (Lucentis®), another monoclonal antibody recognising VEGFA165 (an isoform of VEGF having 165 amino acids) and pegaptanib (Macugen), a single strand nucleic acid aptamer, bind to the heparin-binding domain of VEGFA165. These are in use for treating the ‘wet’ age-related macular degeneration (ARMD).
Currently, 12 anti-angiogenic agents have received FDA approval although their effects have been relatively limited and there is at least anecdotal evidence that recurrence of some cancers after anti-angiogenic treatment may come in the form of more aggressive tumours that arise in patients not so treated. This puzzling observation is beginning to be resolved by studies in immunosuppressed mice of intravenously injected human metastatic breast cancer or melanoma cells as a model for metastatic seeding. Treatment either before or after tumour cell injection with VEGFR inhibitors (sorafenib, sunitinib or SU10944) accelerated the formation of metastases by approximately ten-fold with a corresponding decrease in the median survival time. This occurred notwithstanding the fact that sunitinib, for example, strongly inhibits the growth of established primary tumours in mice. Similarly, anti-angiogenic drugs (anti-VEGFR2 antibody DC101) or tumour cell deletion of Vegfa may inhibit primary tumour growth but increase rates of invasion and metastasis. Another anti-angiogenic drug, cilengitide, which blocks αv integrins and is in phase 3 clinical trials, also has the anomalous effects at low doses of stimulating angiogenesis and tumour growth. Non-specific effects of these inhibitors may contribute to these responses. On the other hand, these findings strongly suggest that VEGF inhibitors can promote signalling events that, for example, stimulate the release of bone marrow cells to act as markers for tumour cell adhesion in pre-conditioned niches. This, in turn, indicates that both drug combinations and their administration regimes will be critical in evolving effective anti-angiogenic strategies. The options for administration include continuous versus discontinuous, adjuvant or neo-adjuvant (i.e. before surgery) therapy, and metronomic chemotherapy (frequent administration of very low doses over prolonged periods).
It may also be noted that most of the currently approved anti-angiogenic are directed at either VEGF or RTKs. That is, they target the growth factors (by antibodies and soluble forms of their receptors, so-called ‘ligand traps’), the ligand binding domains of their receptors (by antibodies), and the activated receptors (by kinase inhibitors). As the complexity of the molecular biology is gradually unveiled other targets present themselves. Thus, for example, HIF1 and PHD proteins, the regulation of VEGF by TIS11B and VHL, and the diverse functions of the four PI3K isoforms have come to the fore as potential targets for the treatment of cancers and ischemic diseases. The major role of RAS in signalling from almost all RTKs makes it a less attractive target for angiogenic therapy and attempts to develop RAS inhibitors have not been notably successful. However, it has recently been shown that NF-κB signalling, promoted by RAS acting via RALGDS, RALB and TBK1, is required for tumour formation in a mouse model of RAS-induced lung cancer. Inhibition of TBK1 selectively kills RAS-mutant cells in a synthetic lethal interaction.
This broad category of drugs damages the vasculature of the circulatory system, which can lead to tumour nutrient starvation. They are administered directly into the circulation so that their target is essentially the monolayer of endothelial cells that line the vessels. Within those cells VTAs bind to cytoskeletal microtubules, destabilising them and thus inhibiting cell division. They do not, therefore, have specificity for tumour cells. Nevertheless, VTAs have been widely used in the treatment of cancer, particularly the taxanes (which include paclitaxel and docetaxel) and combretastatin.
An alternative approach has been to target matrix metalloproteinases (MMPs), a large family of zinc-dependent endopeptidases that degrade extracellular matrix proteins and cell surface receptors. Because of their capacity for tissue remodelling, they are important in angiogenesis and metastasis and high levels of MMPs correlate with invasion and metastasis in a variety of human cancers. More than 50 MMP inhibitors have undergone clinical trials for the treatment of a variety of cancers but the results have thus far been disappointing.
One of the major problems of chemotherapeutics is that drug delivery to the target cells is inefficient. To compensate, more of the drug is administered, which in turn increases the incidence of severe side effects. To circumvent these problems much effort has been focused on drug delivery by liposomes that can be targeted to specific sites where their drug content is released in a concentrated burst. A variety of ingenious targeting strategies have been used, the most common being the incorporation of receptor-targeting antibodies in liposomal membranes.
A notable example of this method has used liposomes to deliver doxorubicin (an anthracycline antibiotic that intercalates between the bases of DNA). The drug is pegylated, that is, covalently attached to a polyethene glycol polymer to reduce its immunogenicity, generating what has been called ‘stealth’ liposomes. This preparation has been used in the treatment of ovarian cancer and AIDS-related Kaposi’s sarcoma. A pegylated form of cisplatin (Platinol®) is currently being tested in vivo. The same design of vehicle has also been used in a combination of an anti-EGFR monoclonal antibody and either vinorelbine, gemcitabine or paclitaxel.
A method under development uses minute plastic spheres injected into the bloodstream to deliver drugs to tumours. These drug- encapsulating beads are made of polyvinyl alcohol hydrogel modified by the addition of sulphonate groups and are loaded with anthracycline drugs (e.g. doxorubicin) before injection. The administration procedure is called transarterial chemoembolisation (TACE): the loaded beads accumulate in the abnormal vasculature of a tumour, forming an embolism, from which the encapsulated drug slowly elutes.
This term refers to any therapeutic method in which exogenous genes are expressed. This means that the gene concerned has to be administered in a vector. Viral vectors are often used, taking advantage of their capacity to infect cells, following which the inserted therapeutic gene is transcribed. Viruses, of course, do not specifically target tumour cells and targeting remains a problem, although it is possible to engineer viruses to express proteins that bind to specific cell surface receptors. The first gene replacement therapy was carried out in 1990 when the T cells of a four-year-old girl were exposed outside her body to retroviruses containing an RNA copy of a normal adenosine deaminase gene. Injection of the engineered cells allowed her immune system to begin functioning.
An elegant example of the viral strategy is the modified adenovirus ONYX 015 that can destroy tumour cells that have lost the P53 tumour suppressor gene while having no significant effect on normal cells. The promise of approaches based on genetic engineering is enormous but technical difficulties have meant that progress thus far has been slow.
The strategy driving work on these vaccines is to boost the immune response of the patient and they could be used not only to inhibit the growth of tumours but to complement conventional therapies. Although progress has been slow, sipuleucel-T (Provenge®) was approved by the FDA in 2010 for prostate cancer. Sipuleucel-T elicits an immune response and has prolonged the life of patients with the advanced stage of hormone-refractory prostate cancer.
These are designed to prevent the initiation of cancer and work in a similar manner to traditional vaccines by activating an immune response in the recipient, the antibodies thus produced acting to protect against infection. There have FDA approved vaccines for the DNA human papillomaviruses (HPVs) and for hepatitis B virus.
Approximately 70% of cervical cancers and about 5% of all cancers worldwide are caused by HPV and one of the great triumphs of science in the battle against cancer has been the development of vaccines (Cervarix® and Gardasil®) that appear to give almost complete protection against infection by the tumour-promoting HPVs. The vaccines are artificially synthesised, non-infectious, virus-like particles that induce a strong immune response. The antibodies thus produced block binding of HPV to the basal cells of stratified epithelium and hence prevent infection. They have no effect on therapeutic vaccines. Recombivax HB® and Engerix-B® have FDA approved vaccines for HBV.
In principle, vaccination with protein fragments that act as immunogens to prime the immune system simplest method for enhancing natural defences against tumour development. The protein fragments used are derived from tumour-associated antigens, a considerable number of which have now been identified. Because these are small peptides, they can bind directly to MHC class I or II molecules and induce specific effector and memory T cells. A number of peptide vaccines have been tested in small numbers of patients against cancers that include acute myeloid leukaemia, myelodysplastic syndrome, multiple myeloma, breast cancer and colorectal cancer. Usually, such vaccines have been administered together with an ‘adjuvant’ that enhances the T cell response of the recipient. Despite some positive results, peptide vaccination, in general, has been disappointing and the focus of research has shifted to adoptive cell transfer (ACT) therapy. In ACT, lymphocytes that have invaded a tumour, and are therefore antigen-specific, are removed and grown in vitro to generate expanded clones of tumour-reactive T cells. The patient undergoes lymphocyte depletion by chemotherapy before the activated cells are infused back into them. Genetic engineering of the antigen-specific T-cell receptor has also been incorporated in this method. Adoptive cell transfer has been particularly effective in patients with metastatic melanoma.
Drug resistance is a major limitation to the effectiveness of chemotherapy and most cancer deaths occur because of the inadequacy of the available drugs. The effect is very variable with some tumours having generally low levels of resistance (e.g. Hodgkin’s lymphoma, childhood acute leukaemia). Other types of a tumour usually respond to initial eventually acquire resistance (e.g. non-small cell lung cancer) and some types are inherently resistant (e.g. melanoma). Several mechanisms can promote resistance including either increased expression or loss of the target. For example, methotrexate treatment can cause amplification of the dihydrofolate reductase gene so that eventually the maximum dose that can be administered is ineffective against the high levels of the enzyme that are expressed. Conversely, tamoxifen can be rendered ineffective by a loss of the oestrogen receptor from breast or ovarian cells. However, the most significant drug resistance mechanisms are the acquisition of mutations by tumour cells that render the drug ineffective together with an increased capacity for exporting the drug whence it came. These include the effects of imatinib on BCR-ABL1 and KIT referred to earlier but may extend to switching to an alternative receptor-driven pathway. An example of the latter is the amplification of MET in lung cancers that develop resistance to the EGFR inhibitor gefitinib.
Drug efflux is mediated by members of the ABC transporter superfamily of ATP-driven, trans-membrane pumps, notably ABCB1 (also known as MDR1) or the permeability glycoprotein (P-gp). P-gp has broad specificity and it is expressed by a wide variety of cell types. Chemotherapeutic agents may increase the expression of P-gp and, in particular, resistance to doxorubicin, the vinca alkaloids (vinblastine, vincristine, vindesine and vinorelbine) and epipodophyllotoxins (e.g. etoposide) derive from an enhanced P-gp activity.
The problem of drug resistance is one the greatest cancer challenges. Even with new drugs directed against the increasing spectrum of targets revealed by whole genome sequencing, maintaining patients in sustained remission with complex cocktails may prove problematic. These difficulties have been illustrated in the treatment of tuberculosis for which a standard drug combination cures 95% of cases. Resistance to this first-line combination defines multidrug-resistant TB: second-line drugs are available but these are more expensive, require more prolonged administration and have severe side effects. These problems also arise with cancer but the multifactorial nature of these diseases may offer alternative routes for killing tumour cells, as exemplified by recent developments in ovarian carcinoma therapy. These tumours have a poor prognosis because they have generally metastasised by the time they are diagnosed. Treatment is usually by surgery followed by cisplatin chemotherapy. The majority of patients respond to this drug but will eventually succumb as resistance develops. A novel approach has used a sphingosine analogue, FTY720 (fingolimod, Gilenya®), that after phosphorylation, binds to S1PR1 members of the GPCR family, important in angiogenesis. The cells turn on an autophagic response as if to a stress and, although autophagy is a survival mechanism, eventually the cells die. Combination of FTY720 with inhibitors of autophagy accelerates the rate of necrosis. This strategy targets a normal cellular response and may therefore be promising when combined with agents that, for example, promote tumour cell apoptosis.
A further problem is that among the non-specific targets of drugs will be cells of the immune system so that one of the innate defence systems will be compromised by agents designed to kill tumour cells. Nevertheless, under some circumstances, cytotoxic drugs can stimulate an immune response. This occurs by a variety of mechanisms including activation of dendritic cells, inhibition of immunosuppressive cells and modulation of surface protein expression and hence targeting. Consistent with this is accumulating evidence that immunotherapy (i.e. the use of a vaccine or therapeutic antibodies to enhance the activity of the immune system against tumours) can be combined with conventional chemotherapy (e.g. administration of docetaxel) to generate a more effective anti-tumour response that is generated by either method on its own.
Despite these problems, the prospects for patients with a range of cancers have increased significantly over the latter part of the twentieth century, as we noted earlier. The overall five-year survival rate for white Americans diagnosed between 1996 and 2004 with breast cancer was 91%; for prostate cancer, non-Hodgkin’s lymphoma and leukaemia the figures were 99%, 66% and 52%, respectively. These statistics are part of a long-term trend of increasingly effective cancer treatment and there is no doubt that the advances in chemotherapy summarised above are a contributory factor. Nonetheless, the precise contribution of remains controversial and impossible to disentangle quantitatively from other significant factors, notably earlier detection and improved surgical and radiological methods. On the cautionary side we should also note that for metastatic (breast) cancers there has been little change in the survival rates and that for breast cancer, although chemotherapeutic advances have reduced the rate of recurrence, cancer does recur in a substantial proportion of cases.