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(Circulation. 1995;92:2373-2380.)
© 1995 American Heart Association, Inc.

Articles

Blockade of Platelet GPIIb/IIIa Receptors as an Antithrombotic Strategy

Barry S. Coller, MD

From the Department of Medicine, Mount Sinai School of Medicine, New York, NY.

Correspondence to Dr Barry S. Coller, Department of Medicine, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029.

	Introduction

Top Introduction The GPIIb/IIIa Receptor as... Development of 7E3 as... Reflections References

 
Moving from bench to bedside in the development of the monoclonal antibody 7E3, directed against the platelet GPIIb/IIIa receptor, from a basic science laboratory reagent to an antithrombotic drug was a long, incremental process. From the beginning, we hoped that this powerful new agent would allow us to define better the biochemistry of the GPIIb/IIIa receptor and its role in platelet physiology and pathology, and we dreamed that we might be able to use it to improve the therapy of patients with vascular disease.

I first review the evolution of our understanding of platelet physiology since the 1960s and why the platelet GPIIb/IIIa receptor appeared to be a logical target for antithrombotic therapy. I then describe our studies with 7E3 to block the receptor in experimental animal models of thrombosis. The results of these studies encouraged us to try to develop 7E3 as a human therapeutic agent. The development was a monumental effort conducted by hundreds of people working for nearly a decade and culminated in the approval by the Food and Drug Administration of c7E3 Fab (abciximab [ReoPro]) in December 1994 as conjunctive therapy for use in patients undergoing high-risk coronary artery angioplasty and atherectomy. A detailed description of the developmental process is beyond the scope of this essay, so it is only briefly described. My experience in moving back and forth from the academic medical center research bench to the world of drug development has allowed me to reflect on the processes involved, and at the end of this essay, I share some of my thoughts and concerns.

	The GPIIb/IIIa Receptor as a Therapeutic Target

Top Introduction The GPIIb/IIIa Receptor as... Development of 7E3 as... Reflections References

 
The choice of the GPIIb/IIIa receptor as a therapeutic target rests on an enormous amount of basic and clinical research by many investigators working in what might appear to be quite disparate fields, including platelet-fibrinogen interactions, the rare blood platelet disorder Glanzmann thrombasthenia, platelet membrane glycoproteins, integrin receptors, coronary artery pathology and biochemistry, and experimental thrombosis.

Glanzmann Thrombasthenia
Glanzmann thrombasthenia was first described in 1918,¹ but its modern description as a hereditary disorder causing mucocutaneous hemorrhage, marked prolongation of the bleeding time, and abnormal clot retraction dates from the mid-1960s.^{2 3 4} Studies at that time demonstrated that patients' platelets failed to aggregate at all in response to all of the agonists believed to operate in vivo, including ADP, epinephrine, serotonin, collagen, and thrombin. This profound defect in platelet aggregation contrasted with the much more modest inhibition of platelet aggregation produced by aspirin, whose discovery as an antiplatelet agent occurred at about the same time.^{5 6} Patients with Glanzmann thrombasthenia have serious mucocutaneous bleeding, but despite having essentially infinite bleeding times throughout their lives, spontaneous gastrointestinal and genitourinary hemorrhage occurs only sporadically, and spontaneous central nervous system hemorrhage is exceedingly rare.^{7 8}

One important clue to both the pathogenesis of Glanzmann disease and the mechanism by which normal platelets aggregate was the observation made independently by Zucker et al⁴ and Jackson et al⁹ that Glanzmann patients' platelets are severely deficient in fibrinogen. In the mid-1970s, Nurden and Caen¹⁰ and Phillips et al¹¹ independently identified deficiencies of two different platelet membrane glycoproteins in several kindreds with Glanzmann thrombasthenia. These glycoproteins were designated glycoprotein (GP) IIb and GPIIIa based on their relative electrophoretic mobilities in sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Soon thereafter, it was established that these two glycoproteins exist as a calcium-dependent complex (GPIIb/IIIa) on the platelet surface.¹²

Platelet-Fibrinogen Interactions
It took until the late 1970s to establish that the binding of fibrinogen to the platelet surface is necessary for normal in vitro platelet aggregation induced by all of the agonists believed to operate in vivo.^{13 14 15} Although the GPIIb/IIIa receptor immediately became a prime candidate as the platelet "fibrinogen receptor," controversy persisted as to whether the abnormality in Glanzmann thrombasthenia was due to a deficiency in the platelet activation machinery or in the receptor itself.¹⁶ Studies that I conducted on platelet interactions with immobilized fibrinogen in 1980 supported the view that the defect was in the receptor itself,¹⁷ and data from others showing direct in vitro interactions between GPIIb/IIIa and fibrinogen lent support to this view.¹⁸ The technology for preparing monoclonal antibodies using murine hybridomas was becoming generally available at this time, and we and others began to try to develop such antibodies against platelet glycoproteins.^{19 20 21 22} Because we wanted to conduct functional studies, our strategy was to restrict our search to antibodies that would interfere with the interaction between platelets and immobilized fibrinogen. We were able to produce such antibodies, and studies with one of them (10E5) showed that it could abolish platelet aggregation of normal platelets, block platelet-fibrinogen interactions, and inhibit clot retraction; that is, it could essentially induce a functional thrombasthenic phenotype.²⁰ This antibody, and others with similar properties prepared by other investigators,^{19 22} immunoprecipated both GPIIb and GPIIIa, providing crucial confirmatory evidence that these glycoproteins exist as a complex and are involved in fibrinogen binding. Approximately 40 000 antibody molecules bind to the surface of platelets, indicating that there are probably 40 000 to 80 000 GPIIb/IIIa receptors per platelet, depending on whether the antibodies bind bivalently or monovalently. GPIIb/IIIa thus is probably the most dense adhesion/aggregation receptor present on any cell.

Integrin Receptors
During the 1980s, it became apparent that investigators studying a wide variety of cell-protein and cell-cell interactions involved in developmental biology, cellular differentiation, metastasis formation, leukocyte function, and trafficking of cells in the immune system were studying different members of a large family of receptors, ultimately termed integrins.^{23 24} All of these receptors are composed of two chains, an subunit and a ß subunit, which are held together by noncovalent bonds. Both and ß subunits are transmembrane proteins. The subunits characteristically have three or four divalent cation binding domains, whereas ß subunits contain many disulfide bonds. The cloning of the cDNAs for GPIIb and GPIIIa^{25 26} led to the identification of GPIIb as a typical integrin subunit (_IIb) and GPIIIa as a typical ß subunit (ß₃). The GPIIb/IIIa receptor is platelet specific and thus uniquely adapted for platelet function. GPIIIa (ß₃) however, can form a complex with another subunit, _v, to form the _vß₃ vitronectin receptor,²⁷ which is present on endothelial cells, osteoclasts, and other cells; trace amounts of _vß₃ are also present on platelets (50 to 100 receptors per platelet).²⁸

The RGD Cell Binding Mechanism
Investigators in the early 1980s studying the ₅ß₁ "fibronectin" integrin receptor made a striking discovery concerning the mechanism by which fibronectin binds to this receptor. Using proteolytic digestion fragments of fibronectin and synthetic peptides comprised of amino acids sequences contained within the fibronectin sequence, they were able to identify a three–amino acid sequence, arginine-glycine-aspartic acid (single letter code, RGD), that was crucial for the binding.²³ At approximately the same time, we and others showed that von Willebrand factor, fibronectin, vitronectin, and thrombospondin could all bind to platelet GPIIb/IIIa under appropriate conditions of platelet activation and that the binding could be blocked by small synthetic peptides containing the RGD sequence.^{29 30 31 32 33 34} Also at about the same, the cDNAs of the adhesive glycoprotein ligands were cloned and their amino acid sequences were deduced, yielding the remarkable discovery that RGD sequences were present in all of these GPIIb/IIIa ligands. Thus, the promiscuity of GPIIb/IIIa in binding multiple ligands was explainable on the basis of a common binding mechanism involving the RGD sequence. Ironically, although fibrinogen contains two pairs of RGD sequences in its A chains, it appears that a sequence at the carboxyl terminus of the chain is responsible for its initial binding to GPIIb/IIIa.³⁵ This sequence has similarities to RGD, however, and the binding of peptides based on this sequence to platelets is also inhibited by RGD-containing peptides.³⁶ Thus, it is likely that the chain sequence binds to the RGD site or a related site on GPIIb/IIIa.

Although many different proteins can bind to GPIIb/IIIa in vitro under appropriate conditions of platelet activation, during ex vivo platelet aggregation in normal plasma, fibrinogen appears to be the dominant protein binding to the receptor.³¹ Studies with ex vivo flow chambers designed to simulate better in vivo conditions, however, indicate that von Willebrand factor plays a more important role than fibrinogen.³⁷ No direct data on the relative contributions of these glycoproteins to platelet aggregation in vivo are available, and the roles, if any, of the other adhesive glycoproteins in platelet adhesion and aggregation remain to be established.

A Model of Platelet Adhesion and Aggregation
Collectively, the data presented provide the basis for our current views of platelet physiology (Fig 1). Thus, damage to a normal blood vessel or an atherosclerotic plaque results in exposure of adhesive glycoproteins such as von Willebrand factor and collagen. Platelets have receptors for these glycoproteins (Table) that result in platelet adhesion. These receptors are present on the surface of platelets in their active states so that they can effect adhesion immediately after vascular injury. Thus, platelet adhesion is controlled by the normal endothelial lining "hiding" the adhesive glycoproteins from the circulating platelets.

View larger version (29K): [in this window] [in a new window]   Figure 1. Schematic of platelet adhesion, activation, and aggregation. A, Damage to an atherosclerotic plaque or normal blood vessel results in exposure of subendothelial proteins such as von Willebrand factor and collagen, among others. Platelets have on their surface receptors that bind these proteins (Table), and this results in platelet adhesion. After adhesion, a variety of agonists (Fig 2) can induce platelet activation, resulting in a conformational change in the GPIIb/IIIa receptor that allows it to bind fibrinogen, von Willebrand factor, and perhaps other glycoproteins with high affinity. B, Fibrinogen and von Willebrand factor are multivalent, so they can bind to more than one platelet at a time, resulting in crosslinking. This results in platelet thrombus formation. From Braunwald E, ed. Inhibitors of Platelet Aggregation: GPIIb/IIIa Antagonists: Heart Disease, Update 4. Philadelphia, Pa: WB Saunders; in press. Reproduced with permission.

View this table: [in this window] [in a new window]   Table 1. Platelet Adhesion and Aggregation Receptors

After platelet adhesion, the platelet undergoes an activation process that produces a conformational change in the GPIIb/IIIa receptor so that it has a high binding affinity for fibrinogen, von Willebrand factor, and perhaps other glycoproteins. Binding of fibrinogen and/or von Willebrand factor to GPIIb/IIIa receptors is followed by other events that probably involve receptor clustering. Because both fibrinogen and von Willebrand factor are multivalent molecules, they can bind to GPIIb/IIIa receptors on two different platelets simultaneously, resulting in platelet crosslinking and platelet aggregation.

From a teleological standpoint, the aggregation mechanism probably reflects the need for a rapid and effective response to hemorrhage and, thus, the high density of GPIIb/IIIa on the surface of platelets and the high concentration of circulating fibrinogen. If the resting GPIIb/IIIa receptor had a high affinity for fibrinogen and/or von Willebrand factor, however, platelet thrombus formation would occur continuously and produce inappropriate thrombosis. Thus, control over platelet aggregation is exercised by requiring the presence of one or more platelet-activating agents, most of which are synthesized and/or released only at sites of vascular injury (Fig 2). There are positive feedback loops in the system; activated platelets synthesize and/or release the platelet activators thromboxane A₂, ADP, and serotonin, and they also facilitate thrombin formation. It is notable that thrombolytic agents can also activate platelets, either directly or indirectly via thrombin generation,^{38 39} suggesting that the net clinical effect of these agents involves competition between their fibrinolytic effects and their ability to enhance platelet deposition. Finally, high shear forces have been shown to activate platelets ex vivo,⁴⁰ and it is possible, but not certain, that such forces can be achieved in vivo.

View larger version (20K): [in this window] [in a new window]   Figure 2. Table of platelet aggregation. A variety of agonists, including adhesion itself, and agents either synthesized or released at the site of injury, can initiate platelet aggregation. Thrombolytic agents and shear forces can also cause platelet activation. All of the agonists operate through several transducing mechanisms, including arachidonic acid metabolism and protein kinase C, but it is almost certain that other unknown transducing mechanisms exist. These transducing mechanisms ultimately result in activation of the GPIIb/IIIa receptor, which is the final common event. The transducing mechanisms can be inhibited by prostacyclin (PGI2), which increases cAMP, or nitric oxide (endothelium-derived relaxation factor [EDRF]), which increase cGMP. From Coller BS. Antiplatelet agents in the prevention and therapy of thrombosis. Ann Rev Med. 1992;43:171-180. Reproduced with permission.

It is likely that combinations of agonists are present in vivo at the sites of vascular injury and that the mixture of agonists differs in different thrombi, or even differs over time in a single thrombus. It is notable, therefore, that combinations of agonists can show additive or synergistic effects on platelet activation.⁴¹ Thrombin appears to play a central role in some models of platelet thrombus formation but not others.^{42 43 44} Its precise role in thrombus formation in humans with vascular injury to an atherosclerotic plaque remains to be defined.

The membrane signals induced by the agonists are transduced by several mechanisms, including arachidonic acid metabolism and protein kinase C activation, but our understanding of these mechanisms remains incomplete, and it is certain that other mechanisms are also involved.⁴¹ Because aspirin blocks only arachidonic acid metabolism, it is only a partial inhibitor of platelet aggregation⁴⁵ and offers only incomplete protection from platelet-mediated thrombosis.⁴⁶ The final common pathway for platelet aggregation, regardless of agonist, is the conformational change in the GPIIb/IIIa receptor that results in it developing high affinity for its adhesive glycoprotein ligands.

Platelets in Human and Experimental Vascular Disease
An abundance of evidence derived from examination of pathological specimens from patients with ischemic vascular events implicated platelet aggregation as a major initiating factor,^{47 48} and biochemical evidence of platelet activation during ischemic vascular events provided independent support for this view.⁴⁹ Animal models of ischemic vascular disease also incriminated platelets in initiating, supporting, and exacerbating the thrombotic process.^{42 43 44 50 51 52} They also identified that virtually no vaso-occlusion or ischemic damage results from just a monolayer of platelets, and thus platelet adhesion in the absence of platelet aggregation is not likely to produce ischemic damage.

GPIIb/IIIa as a Therapeutic Target
Analysis of all of these data suggested that blockade of GPIIb/IIIa receptors might be a desirable therapeutic strategy because the monoclonal antibodies to GPIIb/IIIa are more potent inhibitors of platelet function than aspirin, GPIIb/IIIa is platelet specific, inhibition of GPIIb/IIIa still leaves platelet adhesion largely intact and platelet adhesion may contribute to hemostasis without leading to ischemic damage, and the hemorrhagic diathesis produced by the inherited deficiency of GPIIb/IIIa receptors found in Glanzmann thrombasthenia only rarely produces spontaneous central nervous system hemorrhage, the most feared complication of anticoagulant and antiplatelet therapy.

	Development of 7E3 as a Therapeutic Agent

Top Introduction The GPIIb/IIIa Receptor as... Development of 7E3 as... Reflections References

 
Unfortunately, the antibody we initially selected for study (10E5) did not react with canine platelets, and several of the best characterized animal models of arterial thrombosis were in dogs. We then tested other antibodies for their reactivity with canine platelets. Antibody 7E3 showed cross-reactivity, and so the 7E3 cells, which had previously been frozen, were thawed and the cloned cell line was reestablished.⁵³

Preliminary Canine Studies
As a prelude to conducting in vivo studies, we had to produce sizable amounts of purified 7E3 free of endotoxin. We also decided to cleave off its Fc region [producing an F(ab')₂ fragment] so as to eliminate the possibility that platelets coated with the intact antibody would be rapidly cleared in the spleen and elsewhere by macrophages bearing Fc receptors. Armed with only a limited supply of 7E3-F(ab')₂, we treated three dogs at the State University of New York at Stony Brook with increasing doses, ultimately achieving virtually complete inhibition of platelet aggregation, a feat that had not been reported previously for any antiplatelet agent administered in vivo.⁵⁴ The animals did not develop spontaneous hemorrhage, nor did they demonstrate any other gross evidence of toxicity.

GPIIb/IIIa Receptor Blockade Assay
To correlate the functional affects of 7E3 treatment with the percentage of GPIIb/IIIa receptors blocked, we developed an ex vivo assay using the binding of radiolabeled 7E3 to platelets obtained before and after 7E3 treatment to directly assess the percentage of receptors blocked. Using this assay, we were able to show that at antibody doses that decrease the number of available receptors to less than 50% of normal, platelet aggregation shows significant inhibition. At 80% GPIIb/IIIa receptor blockade, platelet aggregation is nearly completely eliminated, but the bleeding time is only mildly affected. It is only with >90% receptor blockade that the bleeding time becomes >15 to 30 minutes.^{55 56}

Quantitative analysis of 7E3-F(ab')₂ binding was also helpful in deciding whether to proceed with development of 7E3 as a therapeutic agent as opposed to trying to obtain another antibody of higher affinity. When doses of 7E3-F(ab')₂ that produced near-saturation of GPIIb/IIIa receptors were administered to primates, 60% of the injected antibody actually became bound to platelets. Compared with other drugs, this percentage binding to the target receptor is remarkably high, indicating that the effective dose is less than twice the theoretical minimum required to occupy the available receptors. Thus, it was unlikely that we could produce a "better" antibody, regardless of differences in affinity measured in vitro.

Animal Models of Thrombosis
The greater potency of 7E3 in both vitro and in vivo in inhibiting platelet function compared with aspirin made us eager to test it in animal models of thrombosis. Deciding on which animal models to test was difficult since a large number of models existed and our supply of antibody was limited. We considered two criteria to be most important: (1) the model had to convincingly simulate a human vascular disease, and (2) aspirin had to have failed to show complete protection. The latter criterion guaranteed that we would be able to assess whether 7E3-F(ab')₂ treatment was more effective than aspirin.

Dr John Folts had developed a model of cyclical flow reductions in the coronary artery of the dog and carotid artery of the monkey designed to simulate the vascular abnormalities found in patients with unstable angina and transient ischemic attacks, respectively.⁴² The model involves creating a partial stenosis of the vessel with an external cylindrical device and damaging the vessel with a hemostat. Platelet thrombi then form, resulting in a decrease in blood flow. The thrombi subsequently embolize several minutes later, either spontaneously or as a result of shaking the cylinder, and blood flow is restored. A new cycle is then initiated, and the cycles keep repeating, causing cyclical flow reductions. Aspirin can diminish or abolish the cyclical flow reductions, but the cyclical flow reducations can be restored by infusing a low dose of epinephrine or increasing the stenosis.⁴⁶ Willerson and colleagues^{43 57} made substantial contributions to this model, defining the roles of serotonin, thromboxane A₂, and thrombin in platelet thrombus formation. Most important, they demonstrated the clinical relevance of the model by showing that cyclical flow reductions occur in humans with atherosclerotic vascular disease, in particular, after coronary angioplasty.^{58 59}

One of the most exciting days of my research career was my first visit to Dr Folts' laboratory when I was able to directly watch the effect of the first dose of 7E3-F(ab')₂ in this open-chest model. The rapid improvements in the color and contractility of the heart after 7E3-F(ab')₂ injection were dramatic, offering hope that similar phenomena might occur in humans with acute ischemic events.

7E3-F(ab')₂ was the most potent agent that Dr Folts had tested in his model, inhibiting cyclical flow reductions despite severe prothrombotic provocations such as infusing epinephrine, increasing the vascular stenosis, increasing the vascular damage, and even passing current through the stenosing cylinder.^{56 60} Electron microscopic examination of blood vessels from treated animals showed only a single monolayer of platelets, despite the presence of extensive vascular damage. Dose-titration studies demonstrated that to achieve an antithrombotic effect in this model, it was not necessary to obtain very high grade GPIIb/IIIa receptor blockade, and thus prolongation of the bleeding time was only modest.⁵⁵ Our studies with Dr Folts anticipated the later human studies by Anderson et al,^{58 59} who demonstrated that 7E3 eliminates the cyclical flow reductions that occurr in some patients after angioplasty.

Dr Chip Gold and his colleagues at the Massachusetts General Hospital were interested in the mechanism of coronary artery reocclusion after successful thrombolysis with recombinant tissue-type plasminogen activator (rTPA) and had developed a dog model of this phenomenon by placing a severe stenosis in series with a fresh thrombus in the left anterior descending coronary artery.⁵⁰ Thrombolysis with rTPA could be achieved in nearly all animals, but reocclusion usually occurred within minutes, with many animals going on to develop cyclical flow reductions like those seen in the Folts model. Aspirin provided at best only a partial benefit in a fraction of the animals. My first visit to Dr Gold's laboratory was another landmark event for me since the coronary artery of a dog pretreated with 7E3-F(ab')₂ did not reocclude after treatment with rTPA, much to the amazement of the skeptical onlookers.

Subsequent studies by us and other researchers further demonstrated that pretreatment with 7E3-F(ab')₂ not only led to full protection against vascular reocclusion but actually shortened the time to reperfusion and allowed for dramatic reductions (up to 75%) in the dose of rTPA needed to achieve reperfusion; in fact, reperfusion occurred in some animals after just receiving 7E3-F(ab')₂, even without getting rTPA.^{50 61} Moreover, platelet-rich thrombi that could not be lysed by rTPA alone were readily lysed when animals were treated with 7E3 before the rTPA was given.⁵² To explain these observations, we hypothesized that because thrombolytic agents can activate platelets,³⁸ perhaps potent inhibition of platelet aggregation by 7E3 allows the thrombolytic effects of both exogenous rTPA and endogenous TPA to act unopposed by increased platelet deposition.⁶²

These series of animal experiments and the independent confirmation of the antithrombotic effects of 7E3 treatment in other models by Lucchesi, Mickelson, Bates, Rote, and their colleagues,^{51 63 64 65} as well as other laboratories,^{66 67 68} formed the theoretical basis for our decision to move forward with the development of 7E3 as a therapeutic agent. Our data suggested that it may be of benefit in treating patients with unstable angina, transient ischemic attacks, and myocardial infarction, as well as in the prevention of complications of angioplasty. What remained unknown was whether the animal models of thrombosis really simulated the human disease and whether the hemorrhagic risks in humans would be acceptable.

Licensing 7E3 to Centocor and Development of 7E3 as a Drug
The next steps in drug development could not be performed in my laboratory because it would require resources far in excess of those in my grant from the National Heart, Lung, and Blood Institute to study basic platelet physiology. As a result, in 1986 the Research Foundation of the State University of New York licensed 7E3 to Centocor, Inc, a new biotechnology company specializing in the diagnostic and therapeutic application of monoclonal antibodies.

The subsequent development of 7E3 required extensive collaboration among myself, a large number of outstanding scientists at Centocor, and many leading academic cardiologists, resulting in the preparation for, the design, and the conduct of the clinical trials (see "Acknowledgments"). Space, unfortunately, does not permit discussing the many decisions and hurdles that remained for us, including the choice to use the 7E3 Fab fragment and ultimately to develop a mouse/human chimeric 7E3 Fab (c7E3 Fab)⁶⁹ ; the design and execution of the toxicology studies; the assessment of 7E3 crossreactivity with _vß₃ and perhaps activated MAC-1 receptors⁵⁶ ; the development of sensitive and specific assays to assess immune responses to c7E3 Fab; the design, execution, and analysis of the phase I, II, and III studies; and the preparation, submission, and defense of the Product Licensing Application to the Food and Drug Administration and comparable documents to European and Scandinavian agencies.

Based on the results of the 2099 patient EPIC trial,^{70 71} in which conjunctive treatment with a bolus plus infusion of c7E3 Fab significantly reduced the risk of developing an ischemic complication (death, myocardial infarction, or need for urgent intervention) after coronary artery angioplasty or atherectomy in patients at high risk of such complications, the Food and Drug Administration approved the conjuctive use of c7E3 Fab (generic name, abciximab; trade name, ReoPro) in high-risk angioplasty and atherectomy on December 22, 1994. We are continuing to explore the basic mechanisms by which abciximab works, including potential effects on thrombin formation⁷² and restenosis; we are also assessing changes in the heparin dosing to decrease the hemorrhagic risk associated with abciximab treatment,⁷³ potential new indications for abciximab, and the safety and efficacy of repeat administration of abciximab.

	Reflections

Top Introduction The GPIIb/IIIa Receptor as... Development of 7E3 as... Reflections References

 
I view my scientific investigation as serving my commitment as a physician to improve the diagnosis and therapy of patients suffering from illness, and thus being able to go from bench to bedside has been extraordinarily satisfying. I realize that I have been extremely fortunate in being able to travel this path, so I am eager to help others have the same opportunity. In this context, I would like to share some of my thoughts about selected aspects of the process.

1. The role of the National Institutes of Health. All of the basic work in my laboratory and much of the work conducted in other laboratories that led to the choice of the GPIIb/IIIa receptor as a therapeutic target was funded by the National Heart, Lung, and Blood Institute. This funding was based on the importance of the scientific questions, including the study of a very rare platelet disorder, and not the immediate prospects of new therapy for the most common cause of death in the United States. It is crucial that the public appreciate the vital role that National Institutes of Health funding of basic research has had on the development of new therapies.

2. The role of the State University of New York at Stony Brook. The investment by the State of New York in developing Stony Brook as a medical school and a research university of excellence made it possible for me to perform my research as a junior faculty member in an enriched scientific environment. By funding a hybridoma core facility in the Department of Microbiology under the direction of Dr Arnold Levine, junior faculty like myself were able to gain early access to this technology at a reasonable cost.

3. The role of the biotechnology and pharmaceutical industries. If there were no mechanism to license 7E3 to a biotechnology company, c7E3 Fab would never have been developed as a therapeutic agent. Thus, technology transfer policies and experts play a crucial role in the process of translating basic research into diagnostic and therapeutic agents. Similarly, many fewer drugs will be developed if the investors in biotechnology and pharmaceutical companies do not believe that at the end of the 10 to 12 years it takes to develop a new drug that they will profit from their exceedingly expensive (estimated at $359 million for research and development per drug) and risky (only 1 of 10 novel agents that reach phase I studies become approved drugs) investment.⁷⁴

4. Bottlenecks in translation of basic knowledge into new therapies. The daunting economic statistics cited above result in biotechnology and pharmaceutical companies choosing only a small number of agents to develop. Thus, it may be very difficult for investigators to develop licensing agreements for agents that may have limited markets or seem very risky. Without such agreements it is difficult for individual investigators to get over the hurdles of preparing purified materials suitable for in vivo use and developing suitable animal models to assess potential human safety and efficacy. Thus, support of resources designed to help investigators overcome these hurdles would likely lead to an extraordinary amount of important pathophysiological information and perhaps new drugs. A commitment to supporting individuals trying to develop and assess clinically relevant animal models through the techniques of experimental pathology is also likely to speed the process of translating basic knowledge into clinical benefit. The explosion in transgenic mouse models of disease reinforces this point.

5. Scale-up from the academic environment to the biotechnology and pharmaceutical level is breathtaking and exceedingly difficult. The first functional screening assay performed with 7E3 required approximately 1 millionth of a gram (ie, 1 µg) of protein (100 µL of hybridoma culture supernatant containing 10 µg/mL of antibody). Abciximab is now produced in lots of more than 1000 g of protein, which is more than 1 billion times the amount that was initially used. Academic scientists rarely have the chance to learn just how difficult it is to develop scale-up technology and meet the stringent criteria established by the Food and Drug Administration for good manufacturing practices of a pharmaceutical.

6. Need for physician and public education. Despite extensive courses in pharmacology during medical school, most medical students, trainees, and practicing physicians learn very little about the process of drug development. It is not surprising then that the public also knows so little. If the public debate about the costs of medications and the appropriate incentives for developing new drugs is to be reasoned, public information is crucial. Finally, it is vital that the public understand the mission of the Food and Drug Administration, the rigor required to meet the standards for safety and efficacy required for new drug approval, and the Food and Drug Administration–imposed restraints on marketing practices.^{75 76} Armed with this knowledge, the public will be better able to compare the quality and quantity of the safety and efficacy data available on Food and Drug Administration–approved drugs with those available on "natural remedies."

	Acknowledgments

 
This work was supported by grant 19278 from the National Heart, Lung, and Blood Institute. In addition, I give thanks to the following: Drs Pankaj Ganguly, Carol Letendre, and Claude Lenfant of the National Heart, Lung, and Blood Institute for supporting my basic research. Dr Arnold Levine of Princeton University provided early support in sharing his facilities and encouraging my studies of monoclonal antibodies to platelets. Eugene Schuler of the State University of New York developed the licensing agreements between the Research Foundation of the State University of New York and Centocor. Lesley Scudder, my long-time coworker, made many contributions to the development of 7E3, including resuscitating the mouse used in the preparation of 7E3 after it had an apparent cardiac arrest after the last intravenous injection. Dr John Folts, Dr Chip Gold, and Dr Benedict Lucchesi and their coworkers designed and conducted the experimental animal studies with 7E3. Dr Hubert Schoemaker and Michael Wall of Centocor, Inc, and Dr Harvey Berger of ARIAD Pharmaceuticals, made the executive decision to license and develop 7E3. Dr Berger led the development of 7E3 from 1986 until 1990. David Holveck, Bobba Venkatadri, Dr James Woody, and Dr Richard McCloskey of Centocor provided strategic planning. Dr Harlan Weisman of Centocor led the development of 7E3 since 1990. Denise McGinn oversaw the operational management of the 7E3 project. Dr Robert Jordan, Dr Carrie Wagner, Dr Mary Ann Mascelli, Mary-Frances McAleer, Allen Schantz, and Dr Murian Nakada developed and assessed the purification and cleavage techniques for 7E3 and c7E3, as well as the assays to characterize the 7E3 antibody and to detect immune responses to 7E3 and c7E3. Dr Jeff Mattis and Dr Richard Siegel designed and oversaw the production process for 7E3 and c7E3. Dr Keaven Anderson, served as the senior statistician for the clinical trials of 7E3. Dr David Knight and Dr John Ghrayeb produced the chimeric 7E3 molecule. Dr John Iuliucci, Mark Nedelman, Dr Mark Cunningham, Dr Joy Cavagnaro, George Treacy, Dr Ken Mace, and Ellen Lance, designed and conducted the toxicology and pharmacology studies on 7E3 and c7E3. Ann Wang, Dr Thomas Schaible, Maura Musco, and Dr Catherine Cabot oversaw the conduct of the clinical trials. Martin Page and Dr Sally Bolmer directed the interaction with regulatory agencies for 7E3. The TAMI and EPIC study groups, led by Dr Eric Topol and Dr Robert Califf, conducted a series of clinical trials, including the Phase III EPIC study. Dr A. Lahiri conducted a Phase I study of 7E3. The European Cooperative Study group, led by Dr Maarten Simoons, conducted a series of clinical trials, including the Phase III CAPTURE study. Dr H. Vernon Anderson and Dr James Willerson conducted studies of the effect of 7E3 in reversing cyclical flow reductions in the coronary arteries of humans after angioplasty. Eli Lilly provided financial support and marketed abciximab (ReoPro). Suzanne Rivera, Melanie Alvarez, and Shirley Murray provided outstanding secretarial support.

	References

Top Introduction The GPIIb/IIIa Receptor as... Development of 7E3 as... Reflections References

 
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3. Caen JP, Castaldi PA, Leclerc JC, Inceman S, Larrieu JJ, Probst M, Bernard J. Congenital bleeding disorders with long bleeding time and normal platelet count, I: Glanzmann's thrombasthenia. Am J Med.. 1966;41:4-26.

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				S. D. Berkowitz, R. A. Harrington, M. M. Rund, and J. E. Tcheng Acute Profound Thrombocytopenia After c7E3 Fab (Abciximab) Therapy Circulation, February 18, 1997; 95(4): 809 - 813. [Abstract] [Full Text]

				E. J. Weiss, P. F. Bray, M. Tayback, S. P. Schulman, T. S. Kickler, L. C. Becker, J. L. Weiss, G. Gerstenblith, and P. J. Goldschmidt-Clermont A Polymorphism of a Platelet Glycoprotein Receptor as an Inherited Risk Factor for Coronary Thrombosis N. Engl. J. Med., April 25, 1996; 334(17): 1090 - 1094. [Abstract] [Full Text] [PDF]

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