1Direct costs; ²data taken from the American Heart Association (click here); ³Including Myocardial infarction and angina; ⁴world-wide costs; ⁵death in these patients usually results from other cardiovascular conditions; ⁶costs associated with amputations alone.

The processes of thrombosis and thrombolysis are in a state of balance known as hemostasis that is reviewed in Merck's online manual of diagnosis & therapy (click here). Thrombosis occurs following the release of mediators from platelets (these bind to damaged vessels) and also the activation of the coagulation cascade through the intrinsic and extrinsic pathways (see below - after the Stroke Center). The intrinsic pathway is initiated by blood coming into contact with sub-endothelial connective tissues or with negatively charged surface that are exposed as a result of tissue damage. This results in the activation of factor XIa that in turn converts IX to IXa. The extrinsic pathway is activated by vascular tissue factors released from the endothelial wall upon injury. Tissue factors in the presence of factor VIIa also converts IX to IXa. Thus both the intrinsic and extrinsic pathways produce IXa which is further converted to Xa in the presence of factor VIII. The accumulation of factor Xa then converts prothrombin to thrombin, which, in turn causes the generation of clots from fibrin. The generation of fibrin is countered through the thrombolytic pathway. The importance of factor VIII is readily demonstrated in hemophilia and also von Willebrand's disease. Hemophilias are genetically inherited diseases which exist in two forms, hemophilia A (a lack of factor VIII) and hemophilia B (a lack of factor IX). Both diseases have similar clinical characteristics. A patient with a factor VIII or IX level < 1% of normal has severe bleeding episodes throughout life. The first episode usually occurs before age 18 months. Minor trauma can result in extensive tissue hemorrhages and hemarthroses, which, if improperly managed, can result in crippling musculoskeletal deformities. Bleeding into the base of the tongue, causing airway compression, may be life threatening and requires prompt, vigorous replacement therapy. Even a trivial blow to the head requires replacement therapy to prevent intracranial bleeding. Factor VIII circulates as a complex with von Willebrand's factor which if insufficiently synthesized causes similar clinical signs to that of hemophilia.

A variety of different cardiovascular treatments options have been developed and these are well reviewed on line (see for example Jones & Robinson, 2000).

Antithrombotic drugs: The earliest example of antithrombotic drugs is aspirin, which prevents thromboxane A2 formation and consequent platelet aggregation and mediator release. Thrombin-induced platelet aggregation remains unaffected however and furthermore, aspirin does not inhibit shear-induced platelet aggregation. Despite sub-maximal inhibition of platelet function, aspirin can reduce the incidence of myocardial infarction in unstable angina patients by 50% (Lewis et al, 1983) and it is recommended for all patients with suspect unstable angina. Likewise aspirin reduces the incidence of stroke in patients with a prior history of transient ischemic attack or stroke by 18% (Forbes, 1998). Aspirin is of little use in preventing the risk of venous thromboembolism in general surgical patients. The newest class of antithrombotics inhibits platelet glycoprotein IIb/IIIa receptors (GRIs), which as mentioned are responsible for platelet aggregation. Studies have shown that GRIs such lamifiban and tirobifan can reduce adverse ischemic events when administered to unstable angina patients in combination with aspirin (Paragon investigators, 1998; Prism Investigators, 1998). Similar studies have not been performed on patients at risk of stroke. In addition to the glycoprotein IIb/IIIa inhibitors, clopidogrel is rapidly becoming one of the most widely prescribed anti-platelets agents.

Anticoagulant drugs: The most common representatives from this class are the heparins which facilitate the action of circulating antithrombin III (AT III in figure above), an enzyme that inhibits thrombin and several other activated factors essential for the clotting cascade. Unlike aspirin, heparin directly blocks thrombin action. Fractionated heparin (in the class known as low molecular weight heparins – LMWH- such as enoxaparin and dalteparin) have gained widespread acceptance as more attractive agents than unfractionated heparin, and have been reported to reduce myocardial infarction when given to patients with unstable angina as an adjunct to aspirin (FRISC study group, 1993) or alone (Antman et al, 1999). Heparins have not been shown to offer improved benefit to patients at risk of stroke. Finaly, it should be mentioned that warfarin remains the most widely prescribed anticoagulant on a global basis.

Anti-thrombotics and anti-coagulants are both commonly associated with hemorrhagic side-effects. Consequently dosing must be carefully monitored. Overdosing must be avoided at all costs and hence dose reduction with potentially compromised therapeutic efficacy is frequently necessary. Even with careful use hemorrhage is common and one recent study (Zidane et al, 2000) of heparin usage in deep vein thrombosis have revealed that 4% of patients suffer serious hemorrhage. Thrombocytopenia is also common contributing to the risk of bleeding. The most important adverse effect of heparin, after bleeding, is a reduction in platelet count, known as heparin-induced-thrombocytopenia (HIT). HIT has been reported with small doses and low molecular weight heparin. HIT is an early, mild form of thrombocytopenia and as many as 10% of all patients receiving intravenous heparin experience mild to moderate HIT within the first one to two days of heparin therapy. Of the 12 million US patients treated each year, 360 000 develop HIT. The patient typically remains asymptomatic and requires little or no therapy. Heparin-induced-thrombocytopenia and thrombosis syndrome (HITTS) however, is a delayed and more severe form of thrombocytopenia, occurring in 0.5-5% of patients treated with heparin for >5 days. HITTS results from the binding of heparin-antibody complexes to Fc receptors on the platelet surface membrane and is clinically significant, as it is associated with fatal thromboembolic disorders.LMWHs do not appear to be associated with the occurrence of this syndrome.

The development of a type II antibody to Factor VIII
Data obtained serendipitously from hemophilia patients has led to an alternative approach to anticoagulant therapy. Patients with hemophilia A are generally treated with factor VIII replacement, however 15-50% of patients develop factor VIII antibodies (also known as factor VIII inhibitors) that inhibit the coagulant activity of further factor VIII given to the patient. This inhibition involves the proteolytic degradation of factor VIII (Lacroix-Desmazes et al, 2002) or steric hindrance of the interaction of FVIII either with stabilizing molecules, with molecules essential for its activity or with activating molecules. Patients with a low initial antibody titer may be given a large dose of factor VIII to overcome the inhibition. If this does not control the bleeding, further factor VIII infusion will usually be futile because of the rapid rise in antibody titer. Long-term control of inhibition in hemophilia A is achieved in most patients by inducing immune tolerance through continuous exposure to factor VIII. The development of antibodies is particularly interesting because two types of antibody are produced, type I and type II. Type I antibodies completely block the activity of factor VIII, while this effect is only partial with type II antibodies (Ling et al, 2001). Type II inhibition has been suggested to result from a blockade of factor VIII:von Willebrand factor binding (Gawryl & Hoyer, 1982). Clinically this is important because the problems of factor VIII inhibition are not so severe in the presence of type II antibodies. Scientifically it yields important data showing that normal clotting can occur if factor VIII levels are significantly but not fully reduced. This is confirmed through the investigation of different populations of hemophiliacs. While a patient with a factor VIII or IX level < 1% of normal is severely compromised clinically, patients with levels about 5% of normal have mild hemophilia. They rarely have spontaneous hemorrhages; however, they will bleed severely (even fatally) after surgery if not managed correctly. Occasional patients have even milder hemophilia with a factor VIII or IX level in the 10 to 30% of normal range. This suggests that the controlled neutralization of factor VIII may offer a therapeutic approach to prevention of patients at risk for thrombo-embolic complications.

Development of antibodies for therapeutic use: Even before purified preparations of antibodies were readily available, early workers deduced considerable information about the structure of immunoglobulins. In the 1950, Porter demonstrated using the most abundant antibody species, IgG that antibodies are made up of 2 antigen binding fragments (Fab) and a crystilizable fragment (Fc). The latter has many effector functions including the activation of immune cells. Activation of immune cells results from binding and activation of the Fc region to a variety of cell types including T cell, macrophages, NK cells and neutrophils. On it's own however the binding of antibody to immune cells results in a relatively inefficient immune response. A second function of antibody antigen binding is the cleavage of the complement factor C3. This results in antibody bound cells being coated with iC3b, which in the presence of co-factors boosts immune system activation. Co-factors (such as b-glucan) are commonly expressed by yeast but not by mammalian cells explaining why antibody therapy has been relatively disappointing as an approach to cancer. In the absence of co-factors much of the immune response directed against mammalian cells results from the activation of the complement cascade. The initial production of iC3b by antibody binding evokes multiple responses including cell lysis if the antigen is a cell surface protein, anaphylaxis (an increase in vascular permeability, smooth muscle contraction, and mast cell degranulation), chemotaxis and neutrophil/monocyte activation. Thus complement activation is an important component of antibody directed immunology, mediating both a direct response and an indirect response through the amplification of immune cell responsiveness. In addition to being used to direct immune responsiveness, therapeutic antagonists have been developed to block specific targets. Three key examples of this can be found in the antithrombosis/anticoagulation literature.

Perhaps the best example of neutralizing antibodies being used to treat cardiovascular disorders is that of Abciximab (ReoPro, c7E3). This chimeric antibody consists of a murine Fab fragment antibody 7E3 grafted on to human constant regions (chimeric technology was developed to reduce immunogencity associated with murine antibodies). Following injury or rupture of atherosclerotic plaques, platelet glycoprotein Ib receptors bind to von Willebrand factor on the exposed subendothelium of the damaged vascular wall causing adhesion. In addition, platelet aggregation occurs as a result of platelet surface glycoprotein IIb/IIIa receptors binding fibrinogen. Both mechanisms play a key role in thrombus formation. Hence the blockade of platelet GPIIb/IIIa by Abciximab was shown in the EPIC study to significantly reduce the risk of ischemic complications and clinical restenosis after coronary artery angioplasty or atherectomy (The EPIC Investigators, 1994; Topol et al, 1994). In addition to preventing platelet aggregation and adhesion it has recently been suggested that Abciximab also slows coagulation (Ammar et al, 1997). Despite its association with an increased risk of bleeding, Abciximab has been approved for percutaneous coronary intervention. Although ReoPro is a foreign peptide, it does not cause hypersensitivity or anaphylactic reactions in patients. Additionally, no signs or symptoms of local or systemic allergic reactions to ReoPro were noted. A second risk is the development of ReoPro antibody responses. This is seen in 5.8% of patients examined.

Lagging behind the development of GPIIb/IIIa antibodies has been the development of GPIb antibodies. Where GPIIb-IIIa blockers mainly prevent platelet aggregation, interruption at an earlier stage by a GPIb blocker is expected not only to limit the platelet plug that is formed but also to reduce additional platelet-dependent effects. This is likely to reduce granule release, thought to play a role in the development of arteriosclerosis and restenosis (Cauwenberghs et al, 2001). Antibodies to GP1b may therefore offer a significant advantage over GPIIb/IIIa antibodies (see accompanying dossier "TGX-6B4, a therapeutic antithrombotic Fab anti-GP1b antibody") .

A final example of monoclonal antibodies being developed for cardiovascular diseases is the chimerized form of the murine antibody D3, D3H44 (Presta et al, 2001). In vitro, the humanized D3 antibodies displayed potent inhibition of plasma clotting and tissue factor:factor VIIa-mediated activation of factors IX and X. In addition, D3H44-F(ab')2 completely prevented fibrin deposition in a human ex vivo thrombosis model under venous blood flow conditions.

Type II anti-factor VIII mAbs for anticoagulation treatments

Key Findings and conclusions: In 2000, University of Leuven researchers succeeded in immortalizing B lymphocyte cell lines from a patient that produced antibodies to factor VIII. One particular cell line produced an anti-factor VIII IgG4kappa antibody, TGX-mAbFVIII-1 (LE2E9) which inhibited fVIII cofactor activity, following type 2 kinetics (Jacquemin et al, 2000). Even at large excess TGX-mAbFVIII-1 was incapable of fully inhibiting factor VIII (see insert). This was because the factor VIII expressed by the patient producing TGX-mAbFVIII-1 carried a mutation such that its antigenicity was different to wild-type factor VII. A second antibody produced in a similar fashion but from a patient free of this mutation produced complete factor VIII inhibition. Mechanistically, TGX-mAbFVIII-1 was shown to prevent factor VIII association with von Willebrand's factor.

Antibodies and antibody derivatives constitute twenty five percent of therapeutics currently in development, and a number of therapeutic monoclonal antibodies have recently reached the market. All antibodies approved by the US FDA, however, contain mouse protein sequences (Abciximab for example). These partially murine antibodies, therefore, have the potential to elicit allergic or other complications when used in human patients. Recent developments aim to reduce or eliminate murine components, and fully human antibodies are rapidly becoming the norm (for a review see van Dijk & van de Winkel). TGX-mAbFVIII-1 is fully human having been derived from a hemophilia patient and hence it should meet future preferences of the FDA due to reduced risks of developing hypersensitivity or anaphylactic reactions or mounting an anti-TGX-mAbFVIII-1 antibody response.

Antibodies evolved to activate the immune system. This can result from activation of the complement cascade or it can arise from binding to Fcg receptors on immune cells. An inherent risk of using whole antibodies is therefore that an unwanted immune response could be mounted. The therapeutic use of IgG4 antibodies largely avoids this problem since this subclass does not bind to Fcg receptors. Furthermore, in contrast to other IgG subclasses IgG4 also fails to bind complement.

Patent position:The use of TGX-mAbFVIII-1 is protected by patent application WO0104269 assigned to Leuven Research & Development which has sub-licensed the technology to ThromboGenics. Further patents, each assigning the technology to Leuven Research & Development are pending including PCT/EP00/06677, JP 509473/2001, US 60/143,891 and UK 9916450.1.

Market size: In 1996, cardiovascular drugs had a global market of US$ 35 billion. Among the top 20 selling drugs, 6 were for cardiovascular indications. The annual market for anticoagulant therapies is around US$6 billion. Worldwide, around 30 million patients receive heparin each year for the treatment or prevention of various thrombotic conditions, acute coronary syndromes and prophylaxis of thrombosis during surgery. Low molecular weight heparin generates sales of US$1.3 billion. The sales of Aspirin account for a further US$ 900 million.

Market competition: TGX-mAbFVIII-1 can be considered an anticoagulant agent and consequently an analysis of representatives from this class was performed. In addition a review of antithrombotics was also conducted. For the purpose of this analysis, antithrombotics were defined as molecules that prevented platelet aggregation or function, while anticoagulants were defined as molecules that block the coagulation cascade. The graph to the left suggests that a below average proportion of antithrombotics are in preclinical development. A more dramatic decline not shown in the graph is obvious from data showing that 50% less compounds are in preclinical development than 4 years ago. Anticoagulant development is more promising and although fewer (51) molecules are in development or on the market, this field is however resurgent. An above average proportion of products are on the market while few molecules are in the clinic - those which were in the clinic have now either been abandoned or launched. The table below indicates that most anticoagulants on the market or in development are thrombin/fibrinogen inhibitors, many of which are heparins. In addition, factor X- and factor VII-related molecules have been developed with significant success. In contrast factor VIII has not been targeted other than by TGX-mAbFVIII-1. This is likely due to the fears of provoking hemorrhagic side-effects. On the other hand significant preclinical activity is observed. This trend largely reflects the development of new molecules related to the blood factors.

ART-123
Heparin, Emisphere
V19 
PEG-r-hirudin

Comparison of TGX-mAbFVIII-1 with other targets: LWMHs have offered a significant advance in this regard, since they are not associated with excessive bleeding and like wise do not require monitoring. However, in many instances they must be administered by twice daily injections, an inconvenience for many patients and of particular importance with respect to patient compliance. Since preliminary work with the antibody to Factor VIII suggest overdosing may be impossible with TGX-mAbFVIII-1, even at supra-therapeutic molar doses, the level of residual factor VIII may prove to be sufficient to prevent spontaneous bleeding.

Several new anticoagulant agents targeting factor IX have recently been developed (Choudri et al, 1999, Benedict et al, 1991). Anti-factor IX antibodies also proved to be efficient in animal models of thrombosis while inducing only minor bleeding (Refino et al, 1999, Feuerstein et al, 1999). Likewise factor X and factor VII have both been targeted by drugs in advanced stages of development or on the market. Targeting factor VIII, however, has significant advantages since of all the coagulation factors, factor VIII has the lowest plasma concentration. This predicts that only relatively low amounts of TGX-mAbFVIII-1 antibody may be required for its inhibition. Furthermore since TGX-mAbFVIII-1 has a long half-life (3 weeks), effective and long-term therapeutic intervention will be possible with a single bolus.

Summary and strategic analysis: Together, preventing the occurrence and reoccurrence of myocardial infarction, stroke, PAOD and pulmonary embolism remain a pressing clinical priority. Well over 12.5 million people in the US suffer from one or more of these conditions resulting in around 1 million deaths each year and a health care expenditure of over $US100 billion each year. First line therapy for these conditions includes the use of anticoagulants, a market worth around $US6 billion per year. Unfortunately anticoagulants in current use suffer a number of limitations including a high incidence of serious or dose limiting hemorrhage, and in the case of heparin, heparin-induced-thrombocytopenia and thrombosis syndrome. The development of anti-coagulants with a lower risk of hemorrhage is thus required. ThromboGenics, one of the most advanced biotech companies to focus on cardiovascular disease, has developed a novel approach to this problem, exploiting neutralizing anti-factor VIII antibodies which are frequently raised by hemophilia A patients in response to therapeutic factor VIII. Factor VIII has traditionally been avoided as an anti-coagulant target due to the perceived risk of hemorrhage and indeed anti-factor VIII antibodies raised by hemophiliacs, if they follow type I kinetics can result in serious hemorrhage even during factor VIII therapy. However, the antibody developed by ThromboGenics, TGX-mAbFVIII-1 avoids such a problem since it follows type II kinetics and therefore reduces but does not abolish factor VIII levels even at massive doses. In effect, in contrast to other anti-coagulants, overdosing may be impossible. B cells producing this antibody have been immortalized, and this has allowed the cloning, initial characterization, and quite likely the low cost production of this human antibody. These antibodies partially neutralize factor VIII, although under conditions of maximal inhibition, levels are sufficient to avoid hemorrhage. Antibody treatment thus protects against thrombosis in a model of deep vein thrombosis without causing bleeding. The antibodies are of the IgG4 kappa sub-class and therefore have a long half-life supporting a long-acting single bolus formulation.