May 2002

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Abstract: Between 1970 and 1994, cancer claimed the lives of about 500,000 Americans every year. According to the most recent statistics, it is estimated that 1.3 million new cases of cancer will be diagnosed and 555,500 people will die from cancer in the United States in the year 2002. From 1992 to 1998, the incidence of cancer related death was reduced by 1.1%. This modest improvement was related to improved detection and greater therapeutic options. Continued improvement of therapeutic options should further diminish the morbidity and mortality associated with these diseases. One class of pharmaceuticals with particular promise are inhibitors of angiogenesis. This therapeutic class is anticipated to represent a major focus of anti-cancer therapy and indeed by 2006, the market for all products that play a role in angiogenesis is likely to reach $1.75 billion (according to the analysts Business Communications). One of the major segments of this market will be the treatment of diabetic retinopathy. More than 154 million people world-wide suffer from type 2 diabetes, 50% of whom develop diabetic retinopathy. This complication of diabetes has now become the leading cause of blindness in the United States in middle-aged adults and consequently the human and economic costs are burdensome.

In addition, agents with the ability to safely and effectively inhibit inflammation acutely (of which inhibition of angiogenesis is critical), may be of particular importance. Despite the level of research on a global basis, advance of new clinical entities into the market has yet to occur. A number of strategies designed to block the effects of vascular endothelial growth factor (VEGF), one of the key angiogenic growth factors, have however generated promising data. Bevacizumab is one such antibody, and it may offer the advantage of being given as a single administration. One disadvantage of this approach however is that VEGF is widely expressed and plays a role in a number of physiologic pathways. Consequently, it may be difficult to target VEGF antibodies to tumors and a challenge to attain efficacy within reasonable dose ranges.

In contrast, the expression of placental growth factor (PlGF), which acts in concert with VEGF to mediate adaptive response to angiogenesis, is limited principally to diseased or inflamed tissue, and thus may circumvent this problem. Scientists within the Center for Transgene Technology and Gene Therapy, a group closely affiliated with ThromboGenics Ltd, have exploited targeted mutagenesis in animals to elucidate the mechanisms by which antibodies to PlGF may demonstrate therapeutic activity in a number of disease models - including abdominal adhesion, diabetic retinopathy and cancer. Deletion of PlGF is able to limit the growth of tumors and development of retinopathies with similar efficacy to VEGF. The further development of these approaches is thus likely to advance therapeutic antibodies within the context of angiogenesis and acute inflammatory states, and could represent a major therapeutic advance.

1. Background: Between 1970 and 1994, cancer claimed the lives of about 0.5 million Americans every year. According to the most recent statistics (Jemal et al, 2002), it is estimated that approximately 1.3 million new cases of cancer will be diagnosed and 555,500 people will die from cancer in the United States in the year 2002. Although this is about half as much as cardiovascular disease, the human cost is nonetheless a major social and economic burden, and together with cardiovascular disease makes up about 60% of all deaths. Information regarding the incidence of various types of cancer in the US can be found on-line. The data in the table to the left can be taken as indicative of the situation in much of North America and Europe. This data describes the most commonly diagnosed cancers in white Americans and their associated mortality rates. One striking observation that can be made from the table is the variability between survival rates in the different types of cancer. Perhaps the most dramatic example is pancreatic cancer which occurs relatively infrequently, but because the 5-year survival rate is so poor, pancreatic cancer represents one of the most common causes of cancer related death. From 1992 to 1998 (click here for statistics), the overall incidence of cancer diminished by 1.1%; likewise the incidence of cancer related death was also reduced by 1.1%. Despite the continued decline in cancer death rates, the total number of recorded cancer deaths in the United States continues to increase slightly due to the aging and expanding population. The gradual decline in cancer death rates is likely the result of an increase in early diagnosis and a gradual improvement in the spectrum of clinical agents available. As can be seen from the graph below (left) the level of research effort in the pharmaceutical industry regarding the development of anticancer drugs is considerable. Not surprisingly, preclinical development is much higher than for efforts in cardiovascular disease although the latter has a higher impact on mortality statistics. Analysis of pharmaceutical development databases suggests about 25% of all drugs in preclinical development are targeted towards cancer. In contrast however, the number of anti-cancer drugs available to clinicians is relatively small - the cardiovascular arsenal is about double that available to the oncologist. Research effort is sub-divided according to pharmacological efforts in the figure below (right). With regard to anticancer drugs in general, the majority (about 50%) of all drugs (almost 2000 in total) are in preclinical development, while only a small proportion (about 200) have been launched. This again reflects the enormous preclinical research effort dedicated to oncology and the relatively restricted number of marketed anticancer drugs. A closer look at development by pharmacological class shows that the alkylating agents and molecules that prevent cellular metabolism (ie protein synthesis) are well advanced. This represents about 50% of all anti-cancer drugs. Thus, the two major emerging fields of research relevant to cancer are inhibitors of angiogenesis and agonists of apoptosis.

Anti-angiogenic molecules for the treatment of cancer: A considerable effort in basic research has begun to advance our understanding of the key molecules which mediate angiogenesis. This research will undoubtedly open up new approaches to develop new cancer therapies. This field is embodied in an enormous quantity of literature, including almost 1000 reviews in the past 5 years alone. The reader is referred to a number of online resources to learn more about the process of angiogenesis in relationship to tumor progression, and strategies for targeting this process. Selected reviews include articles by Brem (click here to access), Folkman (click here to access) and Carmeliet & Jain (Click here to access). In addition Sugen have a variety of information relating to angiogenesis at their website.

Initiation	Promotion	Progression	Metastasis

Angiogenesis performs a critical role in the development and maintenance of cancer. Solid tumors measuring less than 1 to 2 cubic millimeters are typically not highly vascularized. Beyond the critical volume of 2 cubic millimeters, oxygen and nutrients have difficulty diffusing to the cells in the center of the tumor. This promotes a state of cellular hypoxia that marks the initiation of angiogenesis and enhances the transition from hyperplasia to neoplasia - i.e. the passage from a state of cellular multiplication to a state of uncontrolled proliferation characteristic of tumor cells. During the promotion phase a variety of angiogenic factors are released leading to tumor progression. Inhibition at this stage forms the basis of many anti-angiogenic molecules currently in development. If this process continues unabated, metastasis generally occurs. As early as the 1970s, it was suggested that inhibition of new blood vessel formation may represent a way to fight cancer. Subsequently, a number of key classes of angiogenic targets have now been identified. These include:

Cancer cells, which are genetically unstable, frequently mutate resulting in drug resistance, either due to the expression of multi-drug transporters or due to loss of drug targets. Since this is unlikely to occur in the tumor vasculature per se, drug treatment directed to the expanding tumor vasculature should be possible, thus maintaining tumors in a state of chronic dormancy or enhancing their susceptibility to additional cancer agents (although it is conceivable that tumors could mutate to release alternative angiogenic factors).

Although there are multiple opportunities for the development of anti-angiogenic molecules, the most advanced targets are the growth factors. The principal growth factors driving angiogenesis are VEGF, bFGF, and hepatocyte growth factor/scatter factor. Discovered in the 1980's, VEGF is one of the archetypal angiogenic growth factors and has received considerable attention (see MacMahon, 2000; Kerbel, 2000 for reviews). VEGF is a homodimeric 45kDa glycoprotein that specifically acts on endothelial cells binding to a growing number of endothelial tyrosine kinase receptors including Flt-1 (VEGFR-1), KDR/flk-1 (VEGFR-2) and VEGFR-3/Flt-4. VEGFR-2 is exclusively expressed in endothelial cells and appears to play a pivotal role in endothelial cell differentiation and vasculogenesis (Millauer et al, 1993; Quinn et al, 1993; Folkman et al, 1995). Like all cytokine receptors, VEGF receptor binding activates intracellular mechanisms through receptor dimerization, which in the case of VEGF receptors increases receptor tyrosine kinase activity. VEGFR-2 receptor activation induces angiogenesis, endothelial cell proliferation and the elongation, network formation, and branching of nonproliferating endothelial cells (Millauer et al, 1994; Waltenberger et al, 1994; Helmlinger et al, 2000). VEGFR-1 activation produces fewer direct responses, however cell migration is thought to be one effect (Waltenberger et al, 1994; Clauss et al, 1996; Barleon et al, 1996). Recent evidence suggests that VEGF may not only play a role in inducing angiogenesis but also is important in promoting the survival of new vessels formed in tumors (Benjamin & Keshet, 1997). The expression of VEGF, VEGFR-1 and possibly VEGFR-2 is enhanced during hypoxia - although the latter is disputed (Gerber et al, 1997; Waltenberger et al, 1996). VEGF over-expression during hypoxia involves both an increase in the rate of gene transcription, mediated by the transcription factor hypoxia-inducible factor-1 (Forsythe et al, 1996), and an enhancement of the stability of VEGF mRNA (Ikeda et al, 1995). Transcription of VEGF mRNA is also induced by a variety of growth factors and cytokines, including PDGF, EGF, tumor necrosis factor alpha, TGF-ß1, and interleukin 1-beta. In addition to its role in the paracrine stimulation of angiogenesis, VEGF may also have an autocrine stimulatory effect on tumor cells (Leu et al, 1995). VEGF and its receptors have been implicated in the angiogenesis that occurs in many solid tumors including breast cancer (Kurebayashi et al,1999), colon cancer (Shaheen et al, 1999), pancreatic (Knoll et al, 2001), non-small cell lung cancer (Han et al, 2001) and prostate cancer (Balbay et al, 1999).

Since VEGF may play a role in cancer progression a number of molecules have been advanced into clinical development that block its action:

From the above trials it would appear that, at doses used, blocking VEGF or VEGFR-1 represents a safe and effective option, while blocking VEGFR-2 may produce undesired hemodynamic side effects. Since VEGF binds both receptors it is possible that targeting of VEGFR-1 could represent the strategy of choice.

In 1991, Maglione et al isolated a cDNA encoding a novel protein, named placental growth factor (PlGF), from a human placental library. PlGF is a 149 amino acid protein and is highly homologous (53% identity) to the platelet-derived growth factor-like region of human VEGF. Conditioned medium from COS-1 cells containing PlGF is capable of stimulating endothelial cells in vitro. Subsequent studies showed that angiogenic activity was also activated in vivo (Ziche et al, 1997). In 1993, a second PlGF isoform, PlGF-2 was identified. This isoform has a 21-amino acid insertion not present in PlGF-1 coding for a highly basic region near the C-terminus (Hauser & Weich). This isoform is unique in that it fails to bind heparin. More recently, a further isoform of PlGF, PlGF-3 has been identified Cao et al, 1997. PlGF-1 and PlGF-2 do not interact directly with VEGFR-2 but are able to bind to VEGFR-1. This suggests that although they may play a direct role in VEGFR-1-mediated endothelial migration (Waltenberger et al, 1994; Clauss et al, 1996; Barleon et al, 1996), any angiogenic, proliferative, or permeabilization activity is indirect (Park et al, 1994; Kroll & Waltenberger, 1999). This indirect activity has been proposed to involve the potentiation of VEGFR-2-mediated effects of VEGF including blood vessel permeabilization, mitogenesis, and angiogenesis. A number of explanations have been suggested to explain these observations.

Perhaps two of the most credible explanations (depicted in the figure to the right) are that: 1. membrane-bound VEGFR-1 and VEGF-2 can form synergistic heterodimers (Park et al, 1994; DiSalvo et al, 1996) and 2. that soluble VEGF-1 acts as a "sink" for VEGF (see Carmeliet et al, 2001). With respect to the latter, PlGF is proposed to bind soluble VEGFR-1 displacing VEGF, which is then able to activate VEGFR-2 receptors. VEGF is a key modulator of a number of physiological processes such as angiogenesis during the menstrual cycle and bone growth (Ferrara, 1999; Ferrara, 199b) and the direct blockade of VEGF could have significant side-effects. Indeed, as mentioned above, blocking VEGFR-1 with SU5416, was associated with an unacceptable level of serious thromboembolic events (Kuenen et al, 2002). In contrast the expression of PlGF and VEGFR-1, in particular membrane bound VEGFR-1, is restricted to pathophysiological conditions. Consequently the exogenous administration of PlGF may only be expected to enhance the activity of endogenous VEGF (either through synergistic heterodimerization or by increasing the availability of VEGF for VEGFR-2) in pathologic tissue thus avoiding the hemodynamic side effects of VEGF. Likewise the administration of PlGF blockers is only likely to have an effect in diseased tissue over-expressing PlGF and VEGFR-2 (eg tumors or diabetic retinas) and hence the deleterious effects of blocking normal physiological effects of VEGF can be limited.

With this in mind, data reported in 1998 implicating for the first time PlGF may be involved in the etiology of tumor angiogenesis is of considerable interest (Nomura et al, 1998). In this study, PlGF mRNA was found in all hypervascular primary brain tumors investigated. Furthermore, anoxic conditions were reported to drive the production of PlGF by U-251MG human glioma cells. In addition to brain cancer, PlGF is of apparent importance to the progression of melanoma (Lacal et al, 2000). PlGF-1 and PlGF-2 are both expressed by human melanoma cells. Likewise, both primary and metastatic melanoma cells were also found to express the mRNAs encoding for PlGF receptors. Finally, exposure of melanoma cells to PlGF resulted in a specific proliferative response. On the other hand blocking VEGFR-1 conferred anti-tumor activity and provocatively, the effect was greater than blocking VEGFR-2 (Weng & Usman, 2001).

In addition to tumor vascularization, pathological angiogenesis contributes to a number of other disease states including diabetic retinopathy and wound healing. It is therefore of interest that PlGF is also associated with both of these conditions.

Despite the potential of targeting PlGF as a therapeutic strategy, few studies have been able to establish a proof of concept for exploiting this target. To address this issue, the (therapeutic) consequences of eliminating PlGF were established in genetically modified mice.

Anti-PlGF antibody as a candidate anti-angiogenic molecule with potential for the treatment of cancer and other diseases associated with pathological angiogenesis.

Key points (for further details see Carmeliet et al, 2001): The effect of deleting the PlGF gene was investigated in models of cancer and diabetic retinopathy.

Patent position: The use of PlGF inhibitors for conditions including (but not restricted to) the treatment of pathological angiogenesis, pathological arteriogenesis and tumor formation is protected by WO 01/85796 A2 which has been sub-licensed to ThromboGenics. A number of other patents are pending which will further strengthen the proprietary position of this concept. Patents include EP 00201714.3 and PCT/EP01/05478.

Market size: According to the market consultants, BCC, the combined market for angiogenesis regulating products was estimated at $100 million in 2001. Two marketed inhibitors of angiogenesis and one stimulator of angiogenesis were identified. In the year 2006, the market for all products regulating angiogenesis is likely to reach $2.4 billion as it grows at an average annual growth rate of over 88% during the forecast period. The approved inhibitors of angiogenesis, thalidomide and Glivec, are used off-label for the treatment of cancer. Of interest, Glivec inhibits platelet-derived growth factor. It was approved in May 2001 for the treatment of chronic myelogenous leukemia (CML). Currently estimated at $70 million, the market for products which inhibit angiogenesis is likely to reach $1.75 billion in 2006.

Market competition: A search of pharmaceutical databases was performed to identify the level of activity focused on angiogenesis inhibitors in general. This search included those molecules listed as being VEGF or PlGF inhibitors even though angiogenesis may not have been specifically mentioned. As can be seen from the graph to the left, of the 169 molecules in development, the vast majority of molecules are in preclinical stages of development. None are on the market. Not surprisingly, nearly all of the inhibitors of angiogenesis listed as being in development include cancer as an indication. In addition a small number are also being developed for the treatment of retinopathy. None of the angiogenesis inhibitors are targeted towards surgical adhesion. The angiogenesis inhibitors in advanced clinical development are listed in the table to the left. Each of the molecules listed is described as being, in part or exclusively, a growth factor or a growth factor receptor blocker. The majority interfere with VEGF signaling, while none list PlGF inhibition as a pharmacological property. In fact the pharmaceutical databases are remarkably devoid of PlGF inhibitors.

Comparison of anti-PlGF antibodies with other inhibitors of angiogenesis: Data from knock-out mice show that deletion of PlGF is as effective as VEGF at preventing tumor progression. Much of the market competition described above involves the blockade of VEGF. Such a blockade is relatively non-specific, and as a result signal transduction through both VEGFR-1 and VEGFR-2 receptors is poorly understood. VEGFR-2 mediates important physiological roles and hence its blockade can have unwanted side-effects as demonstrated by Semaxanib. Thus blocking VEGFR-1, growth factors which activate VEGFR-1 but not VEGFR-2 (ie PlGF) or the blockade of VEGF is preferable. The latter case scenario will depend on there being sufficient VEGF available to satisfy its physiological role. One particularly exciting approach has been through the use of anti-VEGF antibodies (Bevacizumab). This concept is perhaps more attractive than targeting small molecule inhibitors of VEGF since antibodies have a considerably longer half-life - resulting in the possibility of a single dosing regimen. Bevacizumab is safe and without major side-effects (Gordon et al, 2001; Margolin et al, 2001; Ryan et al, 1999), however to date, a therapeutic effect has yet to be reported, although it should be stressed that completed clinical trials were not designed to identify efficacy. One particular problem with targeting VEGF is that it has a number of physiological roles and its expression is associated with healthy tissue. This contrasts to PlGF which is expressed at much lower levels by healthy tissue, yet its overexpression in pathological tissue can exceed that of VEGF. The effective use of VEGF antibodies could thus be precluded by the inability to target VEGF antibodies to tumors. In contrast, it is possible that PlGF antibodies may be naturally targeted towards PlGF producing tumors resulting in a significant efficacy advantage.

Summary & strategic analysis: Between 1970 and 1994, cancer claimed the lives of about 0.5 million Americans every year. According to the most recent statistics, it is estimated that approximately 1.3 million new cases of cancer will be diagnosed and 555,500 people will die from cancer in the United States in the year 2002. From 1992 to 1998, the incidence of cancer related death was reduced by 1.1%. This modest improvement was related to improved detection and greater therapeutic options. Continued improvement of therapeutic options should drive mortality figures down further and inhibitors of angiogenesis have thus attracted considerable attention. This therapeutic class is anticipated to represent a major arm of anti-cancer therapy and indeed by 2006, the market for all products targeting regulation of angiogenesis is likely to reach $1.75 billion. A portion of this market will result from the treatment of diabetic retinopathy and abdominal adhesions, two areas that are largely devoid of pharmaceutical options at present. Despite the level of research, advances towards the market have yet to occur. A number of strategies designed to block the effects of vascular endothelial growth factor (VEGF), one of the key angiogenic growth factors, have however generated promising data. Bevacizumab is one such molecule, and since it is an antibody it carries the advantage of being given as a single administration. One disadvantage of this approach however is that VEGF is widely expressed and is responsible for a number of key physiological effects. Consequently it will be difficult to target VEGF antibodies to tumors and it may therefore be difficult to attain efficacy within reasonable dose ranges. In contrast the expression of PlGF, which potentiates the angiogenic effects of VEGF, is limited to pathological tissue. Deletion of PlGF is able to limit the growth of tumors and development of retinopathies with similar efficacy to VEGF. Scientists associated with ThromboGenics have exploited this approach by developing antibodies to PlGF which have already shown therapeutic activity in a number of disease models associated with pathological angiogenesis - including abdominal adhesion, diabetic retinopathy and cancer. The further development of this technology is thus likely to allow the advantages of therapeutic antibodies to be placed within the context of angiogenesis, and could represent a major advance in the unfilled market for inhibitors of angiogenesis. ThromboGenics is currently completing process development work in house and plans to move this into GMP production. In addition, the company’s scientists are evaluating proposals for high-throughput screening (HTS) of small molecular weight inhibitors of PlGF. Considering the promise of PlGF-related therapies, ThromboGenics has prioritized their PlGF program despite its early stage of development. . The Company will welcome proposals for partnering, from co-development of additional pre-clinical work, to full out-licensing for clinical development. The company is prepared to present confidential information following a completed CDA and initial discussions.