WO2005112968A2

WO2005112968A2 - Method of using recombinant human antithrombin for neurocognitive disorders

Info

Publication number: WO2005112968A2
Application number: PCT/US2004/013454
Authority: WO
Inventors: Yann Echelard; Harry M. Meade
Original assignee: Gtc Biotherapeutics, Inc.
Priority date: 2004-04-30
Filing date: 2004-04-30
Publication date: 2005-12-01
Also published as: WO2005112968A3

Abstract

The present; invention provides for the production of recombinant human antithrombin (rhAT) for the treatment or prophylaxis of neurocognitive disorders typically associated with major surgical procedures. The recombinant processes of the current invention as well as more efficient methods of treatment, formulation and production have been developed to treat the incidence of neurocognitive problems associated with visuoconstruction, parieto-occipital watershed area injury, hypoperfusion, microemboli or other larger embolic factors and/or CABG procedures secondary to surgery.

Description

METHOD OF USING RECOMBINANT HUMAN ANTITHROMBIN FOR NEUROCOGNITIVE DISORDERS

FIELD OF THE INVENTION [001 ] The present inv ention relates to the production of recombinant human antithrombm (rhAT) for use in the treatment or pre ention of neurocognitiv e disorders typically associated w ith major surgical procedures In particular the current invention prov ides for the production of transgenic mammal deπv ed AT for the treatment or prophylaxis of neurocogniti e disorders associated w ith surgical procedures

BACKGROUND OF THE INVENTION [002] The frequencv and sev enty of central nerv ous system complications in patients undergoing cardiopulmonary bypass (CPB) hav e been documented in a large number of cases In fact as many as 30% of all major surgery patients may hav e some problems, either temporary or longterm, w ith their neurocognitive abilities These potential patients may also be substantially higher numbers of indiv iduals over 60 years of age For example, the risks of embolic neurologic complications and stroke in the population older than 70 >ears from a sev erely atherosclerotic ascending aorta are well documented Moreov er, w hile the majoπty of CPB patients do not experience peπoperative stroke, a high incidence of more subtle central nerv ous system dysfunction has been demonstrated to persist for up to 5 year after surgery in a high percentage of patients (5 year decline documented in Newman et al New England Journal of Medicine (2001 ) 344 395) [003] Age has also been shown to be one of the strongest predictors of subsequent neurologic complications ("sequelae") in patients undergoing CPB or other major surgical procedures The risk of embolic complications in the brain also increases in proportion to the degree of atherosclerosis in the ascending aorta, which is also age- related Transesophageal echocardiography has been found to be only partly useful in diagnosing these lesions or in guiding follow-on surgical procedures in compaπson w ith epiaortic imaging, hich is more precise and less inv asiv e Transcranial Doppler and retinal fluorescein angiography hav e provided further ev idence of microemboli during surgical procedures [004] Neurological injury is a potentially dev astating complication of surgery that results in a longer hospita zation, increased costs, and a substantially increased likelihood of death Such injury can affect any level of the central nervous s> stem, and its manifestations are broad, ranging from barely registrable neurocognitiv e dysfunction to frank stroke Many variables ha e been found to be indicative or risk for peπoperativ e neurological injury, but the predictive models are more useful for stroke risk than for neurocognitive dy sfunction already in place Strategies aimed at reducing neurological injury during cardiac surgery hav e focused, for the most part, on the technical aspects of cardiopulmonary bypass Similar strategies hav e been discussed for other major surgical procedures Surgical procedures such as carotid endarterectomy and cardiac surgery continues to be controv ersial rclativ e to their potential for initiating neurocognitiv e disfunction [005] Cerebral embolism, including atheroembolism from the ascending aorta, has an important role in the pathogenesis of neurological injury of all types Epiaortic ultrasound imaging of the aorta is a sensiti e technique for the identification of atherosclerosis of the ascending aorta at the time of surgery, w hich can allow it to be av oided and therefore reduce the πsk for atheroembolism Results of laboratory inv estigations hav e pro ided insight into the mechanisms of ischemic neuronal injury and a basis for the dev elopment of neuroprotectiv e drugs Neuroprotection may best be accomplished duπng major surgical procedures, especially heart surgery because, in contrast to nonsurgical situations, potential agents can be administered before the neurological insult occurs Neuroprotective procedures and thereapeutic agents may be effectiv e in reducing the neurocognitive dysfunction associated w ith peπoperativ e or post-operative stroke but it will require the assessment of πsk factors and the determination by doctores and their patients about the need for the delivery of nov el diagnostic and therapeutic strategies [006] The search for clinically-effectiv e neuroprotective agents has received enormous support in recent years-an estimated $200 million by pharmaceutical companies on clinical trials for traumatic brain injury since 1995 In fact, neurological complications are, at the present time, considered among the most important causes of morbidity and mortality after heart surgery At the same time, the pathophysiology of brain injury has proved increasingly complex, rendeπng the likelihood of a single agent to pre ent this damage ev en more remote On the other hand, progress continues with technology that makes surgery less inv asive and less πsky One example is the application of endovascular techniques to treat coronary artery stenosis, where both the invasiveness of sternotomy and the significant neurological complication rate, due to microemboli showering the cerebral vasculature, can be eliminated. [007] According to the current invention many patients who receive cardiopulmonary bypass experience lasting negative postoperative events. Four categories of neurologic outcome are of particular importance: ( 1 ) persistent neurological focal deficits, (2) stupor or coma, (3) temporary neurological focal deficits, and (4) seizures. Predictors of risk or neurobioligical complications were aortic aneurysm and aortic valve surgery, advanced age, female sex, and the use of intra- aortic balloon pump or angioplasty. Moreover, a significantly longer hospitahzation time was noticed among patients with neurological side effects. [008] In addition, the current incidence of stroke during cardiopulmonary bypass is somewhat lower than in the 1980's but remains a significant problem today. Levels of cognitive impairment also are cuπently very high. Recognized predictors enable us to identify patients at particularly high risk of stroke. Hypertensive patients are particularly susceptible to ischemic injury during bypass and should be perfused at mean perfusion pressures higher than those for normotensive patients. [009] Cardiopulmonary bypass has been shown to activate various inflammatory cascades in the body, resulting in pathophysiological changes that may affect patient outcome after cardiac surgery. Many of these inflammatory cascades are enzyme mediated, involving serine proteases. The cuπent invention reviews the mechanisms of bypass-mediated activation of the inflammatory cascades and outlines the role of serine protease inhibitors, particularly antithrombin and more specifically recombinant human antithrombin, in ameliorating the consequences of the inflammatory response. Data are reviewed on the action of aprotinin in inhibiting the intrinsic coagulation system and in limiting the contact activation of blood platelets and leukocytes. Also reviewed is the role of aprotinin in impacting the incidence of perioperative myocardial ischemia and the central nervous system dysfunction and stroke that are not infrequent complications of surgery with cardiopulmonary bypass. [0010] The hemostatic system is an intricate system that maintains the fluidity of blood under normal physiologic conditions, yet reacts instantaneously to vascular injury to prevent blood loss by sealing the defect. Balanced stimulation and inhibition of the various mechanisms involved with hemostasis are fundamental to the physiologic state. Maintenance of this balance, under various pathologic and altered physiologic conditions, including cardiopulmonary bypass (CPB). remains a significant medical challenge Blood contact w ith the extracorporeal circuit used in CPB prov ides a pow erful stimulus to activ ate the hemostatic system Anticoagulation is used duπng CPB to minimize acti ation of the hemostatic system and pre ent thrombus formation, particularly in the extracorporeal circuit Rev ersal of anti-coagulation at the end of CPB is important to minimize pen-operativ e blood loss and return the hemostatic sy stem to its normal phy siologic state Well-controlled hemostatic management including rapid anti-coagulation and subsequent rev ersal is cπtical to the o erall clinical outcomes of CPB [001 1 ] Heparin therapy is used for anticoagulation during CPB, and other major surgical procedures, due to its rapid onset of action, ease of titration, and ability to be rev ersed Hepaπn w orks by accelerating approximately 1000-fold the binding of antithrombin (generally . "AT III" or for the purposes of this inv ention recombinant human antithrombin "rhAT") to thrombin and other coagulation factors The binding of antithrombin to thrombin and other factors induces anticoagulation, prev ents blood clot formation, and markedly inhibits activ ation of thrombin. factor Xa and platelets These interrelationships are illustrated in Figure 2 Antithrombin binds thrombin in a one-to-one stoichiometπc fashion Thus, adequate AT III concentrations are critical to hepaπn's anticoagulant activ ity [0012] According to the current inv ention, when AT III concentrations decline below normal physiologic lev els, decreased hepaπn anticoagulant response or hepaπn resistance may develop Hepaπn resistance, or heparin "dy sfunction" in surgical procedures due to a low level of rhAT, or its consequent pathological consequences may be broadly defined as the lack of normal anticoagulant response to hepann The concept of hepann resistance has been applied to many clinical conditions where heparin is used For example, heparin resistance when treating deep v enous thrombosis (DVT) has been defined as the need for more than 35,000 U/day of hepaπn to maintain a therapeutic activ ated partial thromboplastin time (aPTT) In the setting of CPB, or other major surgical ev ents, most patients develop adequate anticoagulation after 300 U kg of heparin Thus, patients ho do not achieve adequate anticoagulation for CPB after a typical dose of hepann may be refeπed to as heparin resistant and may be in need of treatment ith rhAT [0013] Antithrombin ("AT III") is a senne protease inhibitor w hich inhibits thrombin and the activated forms of factors X, VII, IX, XI, and XII It is normally present in serum at levels of 14-20 mg/dL. Decreased levels of AT III may be found in the serum of individuals who have either a hereditary deficiency of AT III or an acquired deficiency, which can result from a number of pathologic conditions. The conventional treatment for hereditary AT III deficiency is protein replacement therapy, which may also be effective in treating some acquired deficiencies. [0014] Cuπent methods of obtaining plasma-derived AT III involves isolating the protease inhibitor from blood plasma, However, the use of plasma-based AT III presents various problems due to the many components in plasma, including: variation between lots; immunogenicity problems; and biohazardous risks due to viral contamination. Therefore, a need exists to develope a method to produce recombinant antithrombin without the inherent problems and risks of the present process. [0015] Accordingly, the recombinant processes of the cuπent invention as well as more efficient methods of treatment, formulation and production are needed to treat the incidence of neurocognitive problems associated with the coagulation cascade and its associated pathologies. This may be accomplished, according to the cuπent invention, through the use of rhAT in therapeutically effective amounts to reduce the incidence and severity of neurocognitive disorders associated with major surgical procedures preferably CPB.

SUMMARY OF THE INVENTION [0016] Briefly stated, the present invention relates generally to the production and therapeutic use of rhAT in the neurocognitive disorder field. As previously stated, cerebral injury is among the most common and disabling complications of open heart surgery and other major surgical events. Previous attempts to provide some level of neuroprotection have yielded conflicting and disappointing results. Acording to the cuπent invention the therapeutic use of rhAT may ameliorate the observed neurocognitive problems. [0017] One method of measuring problems with neurocognitive function is the measurement of neuron-specific enolase (NSE) and the S-100 protein. These proteins have been used as markers for major brain damage. Cognitive dysfunction after cardiac surgery represents subtle brain damage that is detected by neuropsychological testing. Therefore, one objective of the cuπent invention is to use rhAT to therapeutically treat neurocognitive disorders that are or may prospectively be caused by major surgical procedures and to monitor such improvements according to the NSE and/or S-100 measurements [0018] The development of coronary artery bypass grafting (CABG) and its effect on angina is the product of a senes of technical and scientific advances Despite these adv ances, how ev er, substantial numbers of adv erse neurobehavioural outcomes continue to occur Stroke is the most serious complication of CABG, but studies that have identified demographic and medical risk factors av ailable before surgery are an important advance Short-term neurocognitive deficits are common after a CABG procedure, but are not be specific to this procedure Deficits in some cognitiv e areas such as v isuoconstruction persist o er time, and may reflect paneto-occipital w atershed area injury secondary to hypoperfusion, microemboli or other larger embolic factors According to the inv ention antithrombin. preferably rhAT, may be used therapeutically to lessen the disabilities suffered by surgical or CABG patients [0019] This inv ention is also directed to pharmaceutical compositions w hich compπse an amount of a transgenically produced protein of interest, a prodrug thereof, or a pharmaceutically acceptable salt of said compound or of said prodrug and a pharmaceutically acceptable v ehicle, diluent or carrier useful in the treatment of neurocognitive disorders [0020] These and other objects w hich w ill be more readily apparent upon reading the follo ing disclosure may be achiev ed by the present inv ention. [0021 ] An additional objective of the cuπent invention is to provide rhAT for the treatment and improvement in pulmonary function as assessed by PaO₂ FiO₂ ratio, reduce the number of ventilator days/%TBSA and reduce the number of ventilator days/length of hospital stay [0022] Another objective of the cuπent inv ention is to decrease the incidence of ARDS and/or pneumonia while a patient is on the ventilator through the use of rhAT [0023] The present invention further relates to a drug composition for thrombotic disorders which contains the human AT III mutant according to the present inv ention and pharmaceutically acceptable earners, a use of the human AT III mutant according to the present invention for the making of a medicament for treating thrombotic disorders, and a method for treating thrombotic disorders which compnses admimstenng a pharmaceutically effective amount of the human AT III mutant according to the present invention to a patient suffenng from the thrombotic disorders [0024] Further scope and the applicability of the present invention w ill become apparent from the detailed descπption gi en hereinafter How ever, it should be understood that the detailed description and specific examples, w hile indicating prefeπed embodiments of the inv ention, are giv en by w ay of illustration only, since v arious changes and modifications w ithin the spint and scope of the inv ention ill become apparent to those skilled in the art from this detailed description

BRIEF DESCRIPTION OF THE DRAWINGS [0025] FIG 1 Show s a Generalized Flowchart of the Process of Creating Cloned Animals through Nuclear Transfer [0026] FIG 2 Show s a Thrombin Generation and/or Acti ation Cascade [0027] FIG 3 Shows Timetable and Dosage for the Administration of Hepann, rhAT. and FFP [0028] FIG 4 Sho s Indiv idual Patient Fibrin Monomer Plasma Lev els Ov er

[0029] FIG 5 Sho s Indiv idual Patient AT III Plasma Activ ity Lev els Ov er

[0030] FIG 6 Show s Indiv idual Prothrombin Fragment 1 2 Plasma Lev els Ov er Time for the rhAT without FFP and Placebo with FFP Cohorts [0031 ] FIG 7 Show s Thrombin-Antithrombin Complex Plasma Lev els Over Time for the rhAT without FFP and Placebo with FFP Cohorts

DETAILED DESCRIPTION [0032] The following abbrev lations hav e designated meanings in the specification

Explanation of Terms: Bovine - Of or relating to vaπous species of cow s Biological Fluid - an aqueous solution produced by an organism, such as a mammal, bird, amphibian, or reptile, w hich contains proteins that are secreted by cells that are bathed in the aqueous solution Examples include milk, unne, saliv a, seminal fluid, v aginal fluid, synovial fluid, lymph fluid, amniotic fluid, blood, sw eat, and tears, as well as an aqueous solution produced by a plant, including, for example, exudates and guttation fluid, xylem, phloem, resin, and nectar Biological-fluid producing cell - A cell that is bathed by a biological fluid and that secretes a protein into the biological fluid

Biopharmaceutical - shall mean any medicinal drug, therapeutic, v accine or any medically useful composition whose ongin. synthesis, or manufacture inv olv es the use of microorganisms, recombinant animals (including, w ithout limitation, chimeric or transgenic animals), nuclear transfer, microinjection, or cell culture techniques

Capnne - Of or relating to v arious species of goats

Encoding - refers generally to the sequence information being present in a translatable form, usually operably linked to a promoter (e g , a beta- casein or beta-lacto globulin promoter) A sequence is operably linked to a promoter when the functional promoter enhances transcription or expression of that sequence Ai anti-sense strand is considered to also encode the sequence, since the same informational content is present in a readily accessible form, especially w hen linked to a sequence w hich promotes expression of the sense strand The information is conv ertible using the standard, or a modified, genetic code

Expression Vector - A genetically engineered plasmid or virus, deπved from, for example, a bactenophage, adenov lrus, retrov uus, poxv lrus. herpesv irus, or artificial chromosome, that is used to transfer an obesity related transgenic protein coding sequence, operably linked to a promoter, into a host cell, such that the encoded recombinant obesity related transgenic protein is expressed within the host cell

Functional Proteins - Proteins which hav e a biological or other activ ity or use, similar to that seen w hen produced endogenously

Homologous Sequences - refers to genetic sequences that, when compared, exhibit similarity The standards for homology in nucleic acids are either measures for homology generally used in the art or hybridization conditions Substantial homology in the nucleic acid context means either that the segments, or their complementary strands, when compared, are identical when optimally aligned, with appropnate nucleotide insertions or deletions, in at least about 60% of the residues, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95 to 98% of the nucleotides Alternativ ely, substantial homology exists when the segments will hybridize under selective hybndization conditions, to a strand, or its complement Selectivity of hybndization exists when hybridization occurs which is more selective than total lack of specificity Typically, selective hybndization ill occur when there is at least about 55% homology ov er a stretch of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90% Leader sequence or a "signal sequence" - a nucleic acid sequence that encodes a protein secretory signal, and, when operably linked to a do nstream nucleic acid molecule encoding a transgenic protein and directs secretion The leader sequence may be the nati e human leader sequence, an artificially-deri ed leader, or may obtained from the same gene as the promoter used to direct transcnption of the transgene coding sequence, or from another protein that is normally secreted from a cell

Milk-producing cell - A cell (e g , a mammary epithelial cell) that secretes a protein into milk

Milk-specific promoter - A promoter that naturally directs expression of a gene in a cell that secretes a protein into milk (e g , a mammary epithelial cell) and includes, for example, the casein promoters, e g , -casein promoter (e g , alpha S- l casein promoter and alpha S2-caseιn promoter), β-casein promoter (e g , the goat beta casein gene promoter (DiTullio, BIOTECHNOLOGY 10 74-77, 1992), γ-casein promoter, and K- casein promoter, the w hey acidic protein (WAP) promoter (Gorton et al , BIOTECHNOLOGY 5 1 183- 1 187. 1987), the β-lactoglobulm promoter (Clark et al , BIOTECHNOLOGY 7 487-492, 1989), and the ct-lactalbumin promoter (Sou er et al . FEBS LE ΓTS 297 13, 1992) Also included are promoters that are specifically activ ated in mammary tissue and are thus useful in accordance with this inv ention, for example, the long terminal repeat (LTR) promoter of the mouse mammary tumor v IΓUS (MMTV)

Nuclear Transfer - This refers to a method of cloning wherein the nucleus from a donor cell is transplanted into an enucleated oocyte

Operably Linked - A gene and one or more regulatory sequences are connected in such a w ay as to permit gene expression w hen the appropπate molecules (e g , transcnptional activator proteins) are bound to the regulatory sequences

Ovine - Of or relating to or resembling sheep

Parthenogenic - The development of an embryo from an oocyte w ithout the penetration of sperm

Pharmaceutically Pure - This refers to transgenic protein that is suitable for unequivocal biological testing as well as for appropnate administration to effect treatment of a human patient Substantially pharmaceutically pure means at least about 90% pure

Porcine - of or resembling pigs or sw ine

Promoter - A minimal sequence sufficient to direct transcnption Also included in the inv ention are those promoter elements w hich are sufficient to render promoter-dependent gene expression controllable for cell type- specific, tissue-specific, temporal-specific, or inducible by external signals or agents, such elements may be located in the 5' or 3' or intron sequence regions of the native gene

Protein - as used herein is intended to include glycoproteins. as w ell as proteins hav ing other additions This also includes fragmentary or truncated polypeptides that retain physiological function

Recombinant - refers to a nucleic acid sequence w hich is not naturally occurring, or is made by the artificial combination of tw o otherw ise separated segments of sequence This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e g . by genetic engineering techniques Such is usually done to replace a codon w ith a redundant codon encoding the same or a conserv ativ e ammo acid, w hile typically introducing or remov ing a sequence recognition site Alternativ ely, it is performed to join together nucleic acid segments of desired functional polypeptide sequences to generate a single genetic entity comprising a desired combination of functions not found in the common natural forms Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e g , promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incoφorated by design A similar concept is intended for a recombinant. e g . a obesity related transgenic protein according to the instant in ention

Therapeutically-effective amount - An amount of a therapeutic molecule or a fragment thereof that, when administered to a patient, inhibits or stimulates a biological activ ity modulated by that molecule

Transformation, "Transfection," or "Transduction" - Any method for introducing foreign molecules into a cell Lipofection. DEAE-dextran- mediated transfection, microinjection, nuclear transfer (see e g , Campbell et al BIOL REPROD 49 933-942, 1993, Campbell et al , NATURE 385 810-813, 1996), protoplast transgenic, calcium phosphate precipitation, transduction (e g , bacteπophage, adenov iral retroviral, or other viral delivery), electroporation, and bio stic transformation are just a few of the methods know n to those skilled in the art w hich may be used

Transformed cell or Transfected cell - A cell (or a descendent of a cell) into w hich a nucleic acid molecule encoding obesity related has been introduced by means of recombinant DNA techniques The nucleic acid molecule may be stably incoφorated into the host chromosome, or may be maintained episomally Transgene - Any piece of a nucleic acid molecule that is inserted by artifice into a cell, or an ancestor thereof, and becomes part of the genome of the animal w hich de elops from that cell Such a transgene may include a gene hich is partly or entirely exogenous (1 e , foreign) to the transgenic animal, or may represent a gene having identity to an endogenous gene of the animal

Transgenic - Any cell that includes a nucleic acid molecule that has been inserted by artifice into a cell, or an ancestor thereof, and becomes part of the genome of the animal w hich dev elops from that cell. Transgenic Organism - An organism into w hich genetic material from another organism has been expeπmentally transferred, so that the host acquires the genetic information of the transfeπed genes in its chromosomes in addition to that already in its genetic complement. Ungulate - of or relating to a hoofed typically herbivorous quadruped mammal including, w ithout limitation, sheep, sw ine, goats, cattle and horses Vector - As used herein means a plasmid. a phage DNA, or other DNA sequence that (1) is able to replicate in a host cell, (2) is able to transform a host cell, and (3) contains a marker suitable for identifying transformed cells

[0033] According to the present inv ention, there is provided a method for the production of a transgenic protein of interest, the process comprising expressing in the milk of a transgenic non-human placental mammal a transgenic protein useful in the treatment of obesity or related pathologies The term "treating", "treat" or "treatment" as used herein includes preventative (e.g , prophylactic) and palliative treatment.

Generation of the Gene Construct [0034] A mammary gland-specific transgene was constructed by inserting the human Antithrombin III (rhAT) cDNA into the caprine beta casein gene (CSN2). The caprine beta casein gene was cloned as an 18 5 Kb fragment in a lambda EMBL3 vector (Roberts, et al., GENE , ( 1992) 121 255-62) The 6 2 Kb promoter (including exon 1 and part of exon 2) was fused to the rhAT cDNA to direct high level mammary gland-specific expression A 7.2 Kb 3' flanking region (including part of exon 7, exon 8, and exon 9) w as added to the 3' end of the rhAT cDNA to help stabilize the expression lev els The 14 95 Kb transgene w as excised from bactenal sequences and injected into goat embryos for the production of rhAT in goats' milk. Identification of Gene Coding for the Protein of Interest [0035] The rhAT cDNA was received from Dr G. Zettlmeissl on the pBAT6 plasmid The sequence of the cDNA is the same as that published by Bock, et al , NU CLEIC ACIDS RESE ARCH, ((1982)) 10 81 13-25), except for the silent nucleotide changes at bp 1096 (T-C) and bp 1417 (A-G)

Identification of Regulatory Sequences of Interest [0036] To direct high lev el tissue-specific expression of rhAT to the mammary gland of transgenic goats, the goat beta casein gene as cloned from a lambda EMBL3 goat genomic library The goat beta casein gene is a mammary gland-specific gene w hich directs expression of high lev els of beta casein into the milk In goats, beta casein is thought to comprise 25-50% of the total milk proteins (aboutl O-20 mg/ml) The goat beta casein gene w as cloned from a Saanen goat genomic library and characterized in transgenic mice

Goat Species and Breeds [0037] The transgenic goats produced for AT III production are of Sw iss ongin, and are the Alpine, Saanen, and Toggenburg breeds

Evaluation of Expression Levels [0038] The expression level of rhAT in the milk of transgenic animals is determined using a thrombin inhibition assay, hich measures the inhibition of thrombin's ability to remove a small peptide from an artificial substrate (S2238, Kabi, Franklin Ohio) The basis for this assay is described as follows The interaction between AT III and thrombin amounts to rapid irreversible inhibition of the protease by AT III in the presence of hepaπn However, the interaction is very slow in the absence of hepaπn Attempts to extend the range of AT III detectable on a single standard cur e reveal that AT III can only be determined accurately in stoichiometπc titration across the linear range of standard curves At low total thrombin concentration (0 7 times 10 sup -9 M), the effectiv e measuring range for AT III is 0 15-0 75 10^"9 M (e g , about 7 3-36 8 ng/ml) At high total thrombin, the effective measuπng range for AT III is 0 25 to 1 25x 10^'9 M (e g , about 12-60 ng/ml) if the data are fit w ith a first degree polynomial, and 0 25 to 2 5x l0^"9 M ( about 12-120 ng/ml) if the data are fit with a second degree polynomial [0039] Transgenic rhAT contains a significant amount of o gomannose type and hybπd forms at Asn _{1 55} and only a very low lev el of hybπd stmctures at the other locations Ohgomannose type structures are more primitive structures that are remodeled into the complex type ohgosacchandes in the endoplasmic reticulum Ohgomannose structures display masses ranging from Hex5 to HexNAc2. Hex9 These v alues agree with structures compπsed of 5 up to GlcNAc2, Man9, w ith only the number of mannose residues v arying Hybπd o gosacchandes contain elements of complex ohgosacchandes on one antenna of an individual glycosylation site and components of ohgomannose type ohgosacchandes on the other antenna The N-hnked glycosylation for rhAT w as much more heterogeneous than prhAT (plasma derived human antithrombin "prhAT") w ith a higher degree of fucosylation and more v aried sialylation. Sev eral glycoforms of the molecule w ith a mass difference of 41 were observ ed by LC MS w hich can be accounted for by the substitution of a Hexose residue by a HexNAc In v lew of the monosacchaπde composition and the lack of O-linked glycosylation (based on companson of the observed vs theoretical mass for all peptides other than those containing an N-hnked site) this could be accounted for by the substitution of one or more galactose residues by GalNAc [0040] Several glycoforms w ith mass differences of 16 mass units were also identified. The difference is explained by the presence of an oxidized form of sialic acid, N-Glycolyineuraminic acid (NGNA) in place of N-Acetyineuraminic acid (NA A) NGNA is a common form of sialic acid found in goats Approximately 25% of the sialic acids found in rhAT are NGNA Approximately 25% of goat plasma AT III sialic acid is NGNA [0041 ] Thus, we have determined that; (1 ) one of the four glycosylation sites on rhAT has mainly high mannose (ohgomannose) and hybπd type ohgosacchaπde structures, whereas the prhAT has biantenaπy, complex ohgosacchandes on each of the four sites, (2) the complex ohgosacchandes of rhAT are not fully sialylated, whereas the prhAT ohgosacchandes are fully sialylated, (3) the rhAT has a percentage of its sialic acid that is NGNA whereas the prhAT has only NANA, and (4) rhAT contains N- acetylgalactosamine on its N-hnked ohgosacchandes and the prhAT does not; and (5) the rhAT has fucose on its proximal GlcNAc on each of the three sites hav ing complex ohgosacchandes, w hereas the prhAT has only a v ery small amount of fucose on any

[0042] The rhAT exhibits a faster clearance time in rabbits, mice and monkeys than does prhAT Tw enty μg samples of test AT III was injected v ia the tail vein and residual AT III determined using an ELISA assay w hich has little cross reactivity w ith mouse AT III The pattern for clearance in mice mimics the pattern found for the same materials in rabbits The clearance appears to be bimodal and is approximately 10 times faster than for rhAT In v iv o clearance s also examined in a monkey model system Both trace and high lev els of radioiodinated AT III w ere injected and detected in plasma samples by counting in a gamma counter The clearance pattern of rhAT in monkeys indicated only a 4 to 5-fold faster clearance from the circulation than the prhAT and could also be defined by a biphasic mechanism [0043] Early expenments indicate that the rhAT may hav e a stronger affinity for hepaπn than the prhAT This ould be important since AT III inhibits thrombin at inflamation or injury sites by binding to heparan sulfate in the endothelial layer of the vasculature Once bound its affinity for thrombin is enhanced 1000-fold and it binds to and lπev ersibly inhibits thrombin [0044] It is know n that human AT III is a glycoprotein of a molecular w eight of approximately 60 kd w hich is mainly synthesized in the liv er and contained in normal plasma at a concentration of about 150 μg/ml and that human AT III inhibits seπne proteases participating in coagulation and fibπnolysis systems including thrombin and factor Xa The pnmary structure of human AT III has been clarified by the direct determination of its amino acid sequence (see, in Petersen, T E et al , THE PHYSIOLOGICAL INHIBITORS OF BLOOD COAGU L ATION AND FIBRINOLYSIS, ((Elsev ier Science Publishers. Amsterdam,) p 43 et seq , (1979)) and cDNA cloning (see Bock, S C et al , NUCL ACIDS RES , 10 81 13 (1982), and, Prochownik, E V et al , J BIOL CHEM , 258 8389 (1982)) According to these reports, human AT III is a single-chain glycoprotein consisting of 432 amino acids which is secreted and formed by excising a signal peptide of 32 residues from a precursor protein It has four N- hnked glycosylation sites in the molecule The carbohydrate content is about 15% of the molecular w eight [0045] It is an object of the present inv ention to prov ide nov el human rhAT mutants hav ing a high antithrombin activity useful for the treatment of neurocognitiv e disorders caused or initiated by major surgical events It is another object of the present invention to prov ide a method for mass producing said human rhAT mutants by the recombinant DNA technology, most preferably through transgenic technology

Isolation of cDNA coding for rhAT [0046] Since AT III is mainly synthesized in the li er, a commercially av ailable human liv er cDNA library may be used for the isolation of cDNA coding for rhAT Cloning can be effected by a publicly know n method For example, the plaque hybndization method with the use of a synthetic oligonucleotide coπesponding to AT III ammo acid sequence as a probe (see, Sambrook, J et al . MOLECL L AR CLON ING, (Cold Spring Harbor Laboratory (1989)) may be used therefor [0047] The clones thus obtained are subcloned into a plasmid such as pUC 18, if required The nucleotide sequence of cDNA thus obtained can be determined and estimated by the Maxam-Gilbert method (see, Maxam, A M and Gilbert, W , PROC N ATL AC AD SCI USA, 74 560 (1977)) or the dideoxy method (Sanger, F , SCIENCE, 214 1205 (1981 )) The nucleotide sequence of the coding region of AT III cDNA is thus obtained and the amino acid sequence is deduced therefrom [0048] To recombinantly produce a protein of interest a nucleic acid encoding a transgenic protein can be introduced into a host cell, e g , a cell of a primary or immortalized cell line The recombinant cells can be used to produce the transgenic protein, including a cell surface receptor that can be secreted from a mammary epithelial cell A nucleic acid encoding a transgenic protein can be introduced into a host cell, e g , by homologous recombination In most cases, a nucleic acid encoding the transgenic protein of interest is incoφorated into a recombinant expression vector [0049] The nucleotide sequence encoding a transgenic protein can be operatively linked to one or more regulatory sequences, selected on the basis of the host cells to be used for expression The term "operably linked" means that the sequences encoding the transgenic protein compound are linked to the regulatory sequence(s) in a manner that allows for expression of the transgenic protein The term "regulatory sequence" refers to promoters, enhancers and other expression control elements (e g . polyadenylation signals) Such regulatory sequences are descπbed, for example, in

Goeddel, GEN E EXPRESSION TECHNOLOGY METHODS IN ENZYMOLOGY 185,

Academic Press, San Diego, Calif (1990), the contents of which are incoφorated herein by reference [0050] Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, those that direct expression of the nucleotide sequence only in certain host cells (e g , tissue-specific regulatory sequences) and those that direct expression in a regulatable manner (e g , only in the presence of an inducing agent). It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed, the lev el of expression of transgenic protein desired, and the like The transgenic protein expression vectors can be introduced into host cells to thereby produce transgenic proteins encoded by nucleic acids [0051 ] Recombinant expression vectors can be designed for expression of transgenic proteins in prokaryotic or eukaryotic cells. For example, transgenic proteins can be expressed in bacteπal cells such as E coli, insect cells (e g.. in the baculovirus expression system), yeast cells or mammalian cells. Some suitable host cells are discussed further in Goeddel. GENE EXPRESSION TECHNOLOGY : METHODS IN ENZWIOLOGV 185, Academic Press, San Diego, Calif (1990) Examples of v ectors for expression in yeast S cerevisiae include pYepSecl (Baldaπ et al . (1987) EMBO J 6 229-234), pMFa (Kurjan and Herskowitz. ( 1982) Cell 3:933-943), pJRY88 (Schultz et al . ( 1987) Gene 54 1 13-123), and pYES2 (Invitrogen Coφoration, San Diego, Calif ) Baculo irus vectors available for expression of transgenic proteins in cultured insect cells include: the pAc senes (Smith et al , (1983) MOL. CELL. BIOL 3:2156- 2165) and the pVL series (Lucklow, V.A., and Summers, M D., ( 1989) VIROLOGY 170.31 -39). [0052] Examples of mammalian expression vectors include pCDM8 (Seed et al., (1987) N ATURE 3 840) and pMT2PC (Kaufman et al. ( 1987), EMBO J 6: 187-195) When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements For example, commonly used promoters are deπved from polyoma, Adenovirus 2, cytomegalovirus and SV40 [0053] In addition to the regulatory control sequences discussed abov e, the recombinant expression vector can contain additional nucleotide sequences For example, the recombinant expression vector may encode a selectable marker gene to identify host cells that have incoφorated the v ector Moreover, to facilitate secretion of the transgenic protein from a host cell, in particular mammalian host cells, the recombinant expression vector can encode a signal sequence operatively linked to sequences encoding the amino-terminus of the transgenic protein such that upon expression, the transgenic protein is synthesized with the signal sequence fused to its amino terminus. This signal sequence directs the transgenic protein into the secretory pathway of the cell and is then cleaved, allowing for release of the mature transgenic protein (i.e., the transgenic protein without the signal sequence) from the host cell. Use of a signal sequence to facilitate secretion of proteins or peptides from mammalian host cells is known in the art. [0054] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory manuals.

Methods [0055] This experiments that formed the basis of this specification consisted of multi-center, randomized, double-blind, placebo-controlled studies in which 54 assessable heparin resistant patients undergoing elective cardiac surgery requiring sternotomy and CPB were enrolled in two dosing cohorts. One dosing cohort received a single bolus intravenous injection (75 U kg) of rhAT, and the other cohort received a single bolus intravenous injection of a normal saline placebo. [0056] Heparin resistance was defined as those patients failing to achieve an ACT of >480 seconds after receiving a total dose of 400 U/kg heparin intravenously after anesthesia induction and surgical incision but just prior to CPB. The study was conducted at 1 1 study centers (8 in the U.S., 2 in Germany, and 1 in The Netherlands). Patients were monitored throughout the study for heparin resistance using ACT 11^ (the activated clotting time device). Patients were considered treatment failures if fresh frozen plasma (FFP) was required to achieve an ACT of >480 seconds after having received their randomized study medication. All patients were closely monitored for adverse events by the study investigator during their hospitahzation period and again at the 4-week postoperative follow-up assessment. Test Product Dose, Mode of Administration and Batch Number:

rhAT: [0057] Supplied in clear, single-dose glass 20-mL vials. Each vial contained a lyophilized cake of 250 mg rhAT. The vial was reconstituted with 10 mL of sterile water for injection (WFI), USP, to provide a 25 mg/mL solution (150 units per mL). Reconstituted rhAT was administered at a dose of 75 U/kg using a dosage/volume/vveight table. Mode of Administration: rhAT administration was achieved by bolus (slow intravenous push) injection.

Batch (Lot) Number: Patients were dosed using a single lot of rhAT during the study: lot # T7010

EFFICACY: [0058] The primary efficacy analysis was based on an Intent-to-Treat (ITT) population that included all randomized patients and received the blinded study drug per protocol (n=53). The criterion for the primary analysis was the difference between the proportions of patients requiring FFP in the rhAT group compared to placebo. [0059] Secondary evaluations included the inhibition of thrombin activity as measured by changes in levels of Fibrin Monomer, the inhibition of fibrinolysis as measured by changes in D-Dimer levels, and the changes in plasma AT III activity levels at two time points (30 minutes after initiation of CPB, and preprotamine administration). Differences between the treatment groups in mean values for these parameters were assessed at each time point.

Primary Efficacy Endpoint: [0060] The proportion of rhAT patients (19%) who required administration of two units of fresh frozen plasma (FFP) to achieve an activated clotting time > 480 seconds was significantly less (p<0.001) than the proportion of placebo patients (81 %) who required FFP. This was observed for both the Intent-To-Treat (ITT) and the Per

Protocol populations. Secondary Efficacy Endpoints [0061 ] AT III plasma activ ity lev els w ere significantly higher (p<0 001 ) in the rhAT treatment group compared w ith placebo at both the 30 minutes on CPB and at the preprotamine administration time points and for both the ITT and the Per Protocol populations A cohort analysis (rhAT w ithout FFP v ersus placebo w ith FFP) also show ed a statistically significant difference in AT III plasma activity lev el [0062] The mean change from baseline in the production of D-Dimer. a marker of fibπnolysis and indirectly of thrombin activity, w as not statistically significantly different between the treatment groups at the 30 minutes post CPB initiation or the preprotamine time points for the ITT or Per Protocol populations How ev er, there w as an apparent trend to ard a smaller increase in D-Dimer le els from baseline at the preprotamine time point among the rhAT patients compared to the placebo patients A cohort analysis (rhAT w ithout FFP versus placebo w ith FFP) show ed a trend tow ard smaller increases in D-Dimer lev els that approached statistical significance [0063] Inhibition of fibnn monomer, another secondary efficacy endpoint of the study . is a marker for tlirombin activ ity There w ere no significant differences betw een treatment groups in fibnn monomer lev els at any of the time points in either the ITT or the Per Protocol populations Ho ev er, fibrin monomer is v anably affected by thrombin and fibπnolytic activity, and the assay procedure used was not specific for fibnn monomer Therefore, changes in the absolute amount of fibnn monomer over time may not have been accurately measured in either treatment group duπng this study A cohort analysis (rhAT w ithout FFP versus placebo with FFP) also showed no difference in fibnn monomer level [0064] Additional exploratory evaluations were performed that further support the efficacy results discussed above

SAFETY RESULTS [0065] No adverse event was considered by the study investigators to be definitely or probably related to rhAT administration Two deaths occuπed dunng the study, both in the rhAT group Ten rhAT patients and seven placebo patients reported one or more senous adv erse events (SAEs) each Among the adverse events associated with bleeding, hemoπhage NOS was the most frequently reported ev ent in both treatment groups Mean chest tube drainage volume was significantly greater among patients who received rhAT compared to patients who received placebo during both the 12-hour and the 24-hour postoperative periods. Adverse events associated with bleeding were reported as SAEs for four rhAT patients (3 hemoπhage NOS, 1 coagulation disorder) and for three placebo patients (3 hemoπhage NOS). One SAE (hemoπhage NOS) was considered possibly related to rhAT administration. Additional safety results did not appear to reveal any unexpected safety concerns associated with rhAT treatment, compared with a placebo control. [0066] The results of these experiments support the safety and efficacy of intravenous administration of rhAT (75 U/kg) for the restoration of heparin responsiveness and prevention of neurocognitive dysfunctions realized as a result of surgical procedures.

Abbreviations and Terms

Abn, Abnormal

ACT Activated Clotting Time

Admin, Administration

AE Adverse Event

ALAT (SGPT) Alanine Aminotransferase

ANOVA Analysis of Variance

APPT Activated Partial Thromboplastin Time

ASAT (SGOT) Aspartate Aminotransferase bpm Beats per minute

BUN Blood Urea Nitrogen

°C Degrees centigrade

Ca++ Calcium

CABG Coronary Artery Bypass Graft

CFR Code of Federal Regulation

CICU Cardiac Intensive Care Unit

CPB Cardiopulmonary Bypass

CPK Creatinine Phosphokinase

CPMP Committee for Proprietary Medicinal Products

CRF Case Record Form

CT Computenzed Tomography

Ct. Count

CV Curriculum Vitae

Dev. Deviation

Dis. Disorders dL Deciliter

Δ delta (change)

DSMB Data Safety Monitoπng Board

DVT Deep Vein Thrombosis

EC European Community

ECG Electrocardiogram ECOG Eastern Cooperative Oncology Group

ELISA Enzyme-linked Immunosorbant Assay et al. et alπ (and others) e g exampli gratia (for example) etc etcetera

FDA Food & Drug Administration

FFP Fresh Frozen Plasma

Func. Function g Gram(s)

GCP Good Clinical Practice g% Grams percent

GTC GTC Biotherapeutics Incorporated

H„ Null Hypothesis

HCT Hematocrit

HDR Hepaπn Dose Response

Hep Hepann

Hgb Hemoglobin

HIV Human Immunodeficiency Virus

HMS Hemostasis Management System

Hosp. Hospital

Hx History

ICH International Conference on Harmonization

ICU Intensive Care Unit

IEC Independent Ethics Committee

Intra-op I tra-operative

Inv. Investigator

IRB Institutional Review Board

ITT Intent-To-Treat

IU International Unit rv/iv Intravenous kg Kilogram

L Liter

LDH Lactate Dehydrogenase

LLC Limited Liability Corporation

MA Massachusetts

Max. Maximum

MCH Mean Coφuscular Hemoglobin

MCHC Mean Corpuscular Hemoglobin Concentration

MCV Mean Corpuscular Volume

MD Medical Doctor mg milligrams μl microhter mm. Mmute(s)

Min. Minimum mL millihter mmHg Millimeters of mercury

N/A Not Applicable

Neutroph. Neutrophil

N Number n Number ng Nanogram(s) nmol nanomole(s)

NMR Nuclear Magnetic Resonance

NOS Not Otherw ise Specified

NT Not Provided

NR Not Related

OR Operating Room

Post-op Post-operative

PPSP Factor Substitution

Pre-op Pre-operative

PRBC Packed Red Blood Cells

Pt. Patient

PT Prothrombin Time

PTT Partial Thromboplastin

RBC Red Blood Cells

RBC/HPF Red Blood Cells/High Powered Field rhAT Recombinant Human Antithrombin III

SAE Serious Adverse Event

SAS Statistical Analysis Software

Sec. Second(s)

Seg. Segmented

Sept. September

SICU Surgical Intensive Care

Std. Standard

Stnd. Standard

Surg. Surgery

TLA Transient Ischemic Attack tPA Tissue Plasminogen Activator

U Units

U k Units per kilogram

U.S. United States

USP United States Pharmacopeia

WBC White Blood Cell(s)

WBC/HPF White Blood Cells/High Powered Field

WFI Water for Injection

WHO World Health Organization

WHOART WHO Adverse Reaction Thesaurus yr Year(s)

> Greater than

< Less than

> Greater than or equal to

< Less than or equal to [0067] In normal phy siological states, the vascular endothe um and multiple circulating factors play important roles in prev enting thrombosis Endothelial factors and circulating plasma proteins instrumental in prev enting thrombosis include prostacyc n, endothe um-deπ ed relaxing factor, tissue plasminogen activ ator, endothelial heparan sulfate. proteins C and S, hepann cofactor III, and antithrombin III (AT III) AT III is a naturally occurring plasma protein that inhibits thrombin (as well as other circulating coagulation factors) and binds to heparan sulfate moieties on the v ascular endothehum to help maintain homeostasis of the hemostatic system How ev er, in non-physiologic states such as cardiopulmonary bypass (CPB), dunng w hich blood interfaces w ith an extracoφoreal circuit, a tremendous prothrombotic stimulus is initiated Potent anticoagulation. achiev ed pnmaπly w ith unfractionated intrav enous heparin administration, is the most important therapeutic interv ention used to facilitate extracoφoreal circulatory support and prev ent catastrophic thrombotic ev ents [0068] Hepann attains anticoagulation in the presence of the powerful prothrombotic stimulus of CPB by causing a conformational change in the AT III tertiary structure catalyzing the reaction betw een AT III and thrombin approximately 1.000 fold higher than in the absence of heparin Thus, hepann's anticoagulant effect is closely tied to AT III activity Therefore, it follows that abnormally low AT III plasma activ ity can lead to an altered anticoagulant response to hepaπn [0069] In the setting of CPB, the degree of anticoagulation is usually monitored by the acti ated clotting time (ACT), a simple and rapidly performed "point of care" test The decision to initiate CPB after hepaπn administration hinges on attainment of an adequate ACT reflecting adequate anticoagulation Limited expeπmental data exist that define the optimal ACT for initiation of cardiopulmonary bypass (CPB) Young, et al , in a primate study, observed fibnn monomer production in 5 of 6 animals w hose initial ACT w as less than 400 seconds Histoncally, values less than 300 seconds w ere associated with grossly visible clots in the bypass circuit In a survey of members of the Society of Cardiovascular Anesthesiology and Ameπcan Society of Extracoφoreal Circulation, Michelson, et al , found that the target ACT used by 82% (816/1000) of responders w as >400-480 seconds, w ith an additional 4 5% (45/1000) of responding members targeting a higher ACT [0070] Hepann resistance can be considered as the failure to achieve the desired ACT after a typical dose of heparin Failure to achiev e an acceptable ACT (reflecting inadequate anticoagulation) for CPB is usually managed by administration of additional hepaπn Since the etiology of hepaπn resistance dunng CPB is often caused by low plasma AT III activ ity, clinicians may choose to administer an exogenous source of AT III (e g , fresh frozen plasma (FFP) or a plasma-deriv ed AT III) to restore heparin sensitiv ity The ov erall incidence of the hepann resistant state is not w ell defined and w ill v ary as a function of hepann dose and target ACT For example, George Despotis, MD, from Washington Univ ersity , determined that 9 9% (13/131 ) of patients w ho w ere not on preoperative hepaπn infusions and 12 8% ( 14/109) of patients w ho had been on preoperativ e hepaπn infusions w ere heparin resistant In this setting, hepann resistance w as defined as a failure to achiev e an ACT >480 seconds after 400 U/kg of hepann Elhse Delphin, MD, from the Department of Anesthesiology. Columbia Univ ersity , identified 75 out of 1600 (4 7%) patients undergoing CPB considered to be hepaπn resistant In this patient population, heparin resistance as defined as the failure to achiev e an ACT >480 seconds after >500 U/kg of hepaπn. or >600 seconds if the patient w as on aprotinin Finally, Williams, et al , found that 3 7% (85/2279) of patients were heparin resistant, as they did not achieve an ACT >480 seconds after 450 U/kg of hepann [0071 ] The study endpoint for ev aluation of efficacy was to be whether or not the patient met the requirement for administration of FFP An ACT <480 seconds fiv e minutes after injection of study treatment was to w aπant administration of two units of FFP Patients w ho met this requirement but did not receive FFP were to be included in the population of patients who receiv ed FFP It w as anticipated that most hepann resistant patients who receiv ed rhAT would not require FFP administration, w hile most receiving placebo would require the administration of FFP The schema that w as to be used for heparin dosing, randomization, and FFP administration is provided in Figure 3 [0072] The choice of a prospective, randomized, double-blind, placebo- controlled study design w as intended to minimize the potential for subjective bias Overall, the study design attempted to capture and isolate from placebo, the effects of rhAT therapy in heparin resistant patients [0073] The study population for both treatment cohorts w as to consist of hepann resistant patients scheduled for electiv e cardiac surgery requiring sternotomy and cardiopulmonary bypass (CPB). This patient population was chosen to study the safety and efficacy of rhAT because AT III given in the form of fresh frozen plasma is a therapeutic approach sometimes used to facilitate anticoagulation with heparin in heparin resistant patients prior to and during extracoφoreal circulation. Patients who are considered to be heparin resistant represent only a small fraction of the potential patient population (approximately 5-10%).

Treatments [0074] There were two treatment cohorts in this study. Approximately 26 patients were to be randomized to receive rhAT as a single intravenous dose of 75 U/kg, and approximately 26 patients were to be randomized to receive a single intravenous dose of normal saline placebo.

Identity of lnvestigational Product [0075] RhAT, was supplied in clear glass, single-dose 20 mL vials and was refrigerated at 2-8 °C until reconstituted. The The Inventors Clinical Supply Unit located at the Allston Landing Facility in Allston, MA supplied investigational product to each study site. Each vial contained a lyophilized cake of 250 mg rhAT. The vial of lyophilized rhAT was reconstituted with 10 mL of sterile water for injection (WFI), USP, to provide a 25 mg mL solution. Sterile WFI was injected into the vial, being careful to add the liquid along the side of the vial to prevent foaming. The vial was swirled gently (not shaken) to allow adequate dissolution of the product. [0076] The research pharmacist working in the institutional pharmacy department at each study site was to prepare all study materials. The pharmacist, upon notification by the study team, prepared study drug according to the Study Drug Randomization Code. Only the research pharmacist had access to the code. A drug inventory record for monitoring drug accountability was kept, which included the total number of vials shipped and the number of vials remaining. [0077] The volume of reconstituted product drawn for administration to each patient was to be determined using the Study Drug Dosage/Volume/Weight Table (see study protocol). The dose was drawn into a 60 mL syringe, after which the pharmacist added normal saline such that the total volume was equal to 60 mL. Only one production batch of rhAT, Lot # T7010, was used in this study. Selection of Doses in the Study [0078] The rhAT dose, 75 U/kg, administered to patients in this study was based on results obtained in a previous pharmacology study, entitled "A Phase I - II Open, Dose Escalation Study of the Safety of Recombinant Human (transgenic) Antithrombin III in Patients Scheduled for Primary Cardiac Surgery Requiring Cardiopulmonary Bypass." In this study of patients whose endogenous AT III was depleted due to preoperative heparin-induced consumption, it was established that dosing with >50 U/kg of rhAT achieved plasma AT III levels that were approximately 100% of normal (baseline) and were maintained for the duration of CPB. In addition, at rhAT doses up to 200 U/kg where plasma AT III levels reached 600% of normal, no adverse effects were observed. In view of these data, selection of the 75 U/kg was felt to be adequate to achieve the desired efficacy goal of the cuπent study while posing little, if any, safety concern,

Selection and Timing of Dose for Each Patient [0079] The 52 patients were to be randomly assigned to receive either rhAT or normal saline placebo from a randomization schedule generated by The Inventors Coφoration's Biometrics Department. Twenty-seven patients were randomized to the rhAT study group, and 27 patients were randomized to the placebo study group, totals that exceeded the target number of patients for each treatment group. [0080] The timing of dosing (time of day) of study medication and the relationship of dosing to meals was not specified in the study protocol. The relationship of dosing to meals was not an issue in view of intravenous administration of study medication. The study population was to consist of patients scheduled for cardiac surgery requiring CPB. The timing of food consumption and the timing of dosing of study medication were to be determined according to standard preoperative hospital procedures and the surgical schedule of the hospital.

Blinding [0081 ] This was a double-blind, randomized, and controlled trial. Neither the patient nor the investigator knew which treatment was administered. In addition, key clinical, medical, regulatory, and biostatistical personnel of the Sponsor were to remain blinded throughout the study. The blind was not to be broken for the puφose of data analysis until all decisions regarding the evaluability of patients had been made and the database had been finalized. [0082] A blinded randomization schedule was generated for the study sites. Each study site (institutional pharmacist) was to be given an initial assignment for 8 patients. Additional assignment sets were to be given to sites that enrolled 8 patients prior to achieving the study target enrollment of 52 patients. [0083] The institutional pharmacist at each study site performed randomization according to the predetermined blinded randomization schedule. The pharmacist was the only person who had access to the blinded randomization schedule and was responsible for keeping the integrity of the blind and for preparing the blinded study drug. The pharmacist was to keep a drug inventory record that included the total number of vials shipped and the number of vials remaining. [0084] The blind for this study was not to be broken unless the patient experienced a Serious Adverse Event for which, in the opinion of the attending physician, unblinding was deemed absolutely necessary for the patient's treatment. In this situation, the principal or sub-investigator was to first attempt (if time allowed) to call the appointed pharmacovigilance contact at The Inventors to obtain permission to break the code.

Prior and Concomitant Therapy [0085] Patients were not to receive any antifibrinolytic agent [i.e., aprotinin, tranexamic acid, epsilon aminocaproic acid (Amicar®)] within 24 hours prior to administration of the blinded study drug or prior to the preprotamine blood draw at the end of bypass. Patients were excluded from the study if they had recently received or were receiving any of the following medications at the time of randomization: warfarin (within three days); streptokinase (within 48 hours); tPA (within 48 hours); or abciximab (Reopro® (within one week)). [0086] Allowed concomitant therapy was not expected to affect the outcome of the study; however, such effects could not be entirely discounted. At study initiation there were no known drug interactions or direct effects of any concomitant medications, other than those excluded in the protocol, with rhAT. [0087] Any new medications or medications that had their dose or frequency changed during the hospitahzation period of the study were to be recorded on the patient's case record form (CRF). Efficacy Measurements Treatment Period [0088] The follow ing blood/plasma samples w ere obtained for efficacy/bioactiv ity parameters just pπor to study drug administration ("0 min "), at 30 minutes after the initiation of CPB, and just pnor to hepaπn rev ersal with protamine • Kaolin ACT w ithout hepaπnase • Fibrin Monomer to assess thrombin activ ity • D-Dimer to assess inhibition of fibπnoly sis • AT III lev els

[0089] Medtronic's Hepcon^κ Hemostasis Management System (HMS) for measunng the heparin dose response w as used to aid in patient ev aluation for possible hepaπn resistance and to standardize this multi-centered study as much as possible The ACT II^Ji (Automated Coagulation Timer) dev ice as used for measuring the acti ated clotting time dunng the treatment period ACTrac^&, an electronic calibration dev ice, w as to be used in conjunction w ith the ACT II* de ice to insure that evaluation v alues using Hepcon^ and study v alues using ACT II w ere consistent between the tw o dev ices Additionally, a calibration log w as to be kept at each study site [0090] These dev ices w ere used in conjunction ith the follow ing cartπdges • Hepann Dose Response Cartridge to measure the patients' sensitiv ity to hepann • Kaolin ACT cartridge to measure Activated Clotting Times • Hepaπn Protamine Titration cartridge to measure whole blood hepaπn concentration dunng CPB

Heparin Loading Doses and Study Drug Administration [0091 ] Anesthesia induction and surgical incision were performed per institutional protocol When bypass was ready to commence, the patient was hepannized as descnbed below and outlined herein in Figure 1 Patients w ere considered "enrolled" into the study at this point Data collected for "enrolled" patients included patient initials, demographics, hepaπn doses, and resulting ACT responses [0092] Those patients meeting the final inclusion cπtenon as defined by an ACT <480 seconds after a total hepann loading-dose of 400 U/kg were randomized. Only patients randomized to treatment ere follo ed for safety and efficacy/bioacti ity parameters as per protocol Baseline ACT: An ACT w as obtained pnor to hepaπn administration

First Hepaπn Loading Dose Porcine Hepaπn (300 U/kg) w as administered intrav enously and 5 minutes later, the ACT w as determined If the ACT was > 480 seconds, the patient was withdrawn from further study participation Surgery proceeded per institutional protocol If the ACT w as < 480 seconds, a second dose of hepaπn w as administered.

Second Heparin Loading Dose The second dose of heparin (100 U/kg) was administered intravenously and 5 minutes later, the ACT w as determined If the ACT w as > 480 seconds, the patient was w ithdrawn from further study participation Surgery proceeded per institutional protocol. If the ACT w as < 480 seconds, the patient w as randomized to receiv e one of two intravenous study drug treatments (placebo or 75 U/kg rhAT).

Study Drug Administration [0093] All patients receiv ing blinded study drug were considered as "Treated & Randomized" and were to complete all study requirements Vital signs and all safety and efficacy/bioactivity laboratory studies ere obtained pnor to study drug administration. Blinded study drug, either rhAT or placebo, was administered by bolus (slow intravenous push). Vital signs were taken one minute after administration of study medication. Vital signs were to be taken again at 5 minutes following study drug administration in addition to determination of the ACT. • If the ACT was > 480 seconds, aortic cannulation and CPB commenced. • If the ACT was < 480 seconds, patient w as to receiv e 2 units of FFP [0094] Vital signs w ere obtained again at 10 minutes post study drug administration Post FFP Administration The ACT was determined 5 minutes after FFP administration • If the ACT w as > 480 seconds, aortic cannulation and CPB commenced • If the ACT was < 480 seconds, the patient w as treated according to standard hospital practice

Surgery [0095] The surgical procedure w as to be performed according to the standard of care at each of the treating institutions Bioactiv ity laboratory studies and the ACT ere obtained at "30 minutes on CPB" and "preprotamine" time points (see Table 1 Study Flow Chart) All pen-operati e blood products administered w ere to be recorded up to the time of discharge from the hospital

Heparin Maintenance [0096] Dunng bypass additional hepann w as administered as needed to maintain whole blood hepann concentrations at 3 4 U/mL, as guided by the automated protamine titration method (Hepcon ) and ACT v alues In hospitals that had an institutional protocol for intra-operati e hepann maintenance, the method for hepann maintenance descπbed abo e was waived, and additional hepann w as administered during the bypass according to institutional standard practice

Heparin Reversal [0097] Hepaπn rev ersal w ith protamine w as to be per institutional protocol The time and dose of all protamine administrations were recorded

Post-operative Period [0098] Chest tube drainage was recorded until the tube w as remov ed Safety laboratory studies and ECG w ere obtained 24-48 hours post-operatively for all "treated and randomized" patients Four w eeks post-operatively, a blood sample for rhAT antibodies and viral studies w as to be obtained, in addition to a post-operati e review to identify any Senous Ad erse Ev ents that may hav e occuπed during the four-w eek period

Primary Efficacy Variables [0099] The primary efficacy endpoint of the study w as a compaπson of the difference betw een rhAT and placebo in the av oidance of the use of fresh frozen plasma (FFP) to achiev e an ACT >480 seconds in heparin resistant patients This was accomplished by comparing the proportion of patients requinng FFP in the tw o treatment groups (rhAT ersus placebo) All patients w ho warranted FFP administration based on the protocol requirements (failure to achiev e an ACT of >480 seconds as measured 5 minutes after administration of blinded study medication) w ere considered to hav e receiv ed FFP w hether or not it w as actually administered Specifically , patients w ho failed to complete the study after being randomized (I e , w ho w ere unable to hav e an ACT assessment subsequent to receiving blinded study drug or w ho failed to receiv e FFP despite meeting the requirements for FFP) w ere included in the group of patients who receiv ed FFP

Secondary Efficacy Endpoints [00100] Secondary efficacy endpoints were to compare the follow ing laboratory parameters on an Intent-To-Treat basis in patients randomized to rhAT v ersus placebo • The inhibition of thrombin activity (as assessed by change in Fibnn Monomer plasma levels) • The inhibition of fibrin formation and fibnnolysis (as assessed by changes in D-Dimer plasma levels) • The change in plasma AT III activity level

[00101 ] Blood plasma samples were obtained just pnor to study drug administration, at 30 minutes after the initiation of CPB, and just prior to hepaπn reversal with protamine for determination of the secondary efficacy endpoints (see Table 1 - Flow Chart) Statistical Methods Planned in the Protocol and Determination of Sample Size Efficacy Analysis [00102] To assess the comparability of treatment groups, clinical and demographic variables at baseline are to be presented using descriptive statistics. The primary efficacy analysis is to be based on an Lntent-To-Treat population (ITT), which was to include all randomized patients. The primary efficacy analysis — the difference between the proportion of patients requiring FFP in the rhAT group compared to placebo — is to be assessed using Pearson Chi-Square test or Barnard's Unconditional Exact test, depending on the data distribution. Patients who do not complete the study, including, but not limited to, those who are not evaluated for an ACT after receiving blinded study drug, or who fail to achieve an ACT of > 480 seconds after receiving blinded study drug but do not receive FFP, are to be included in the ITT population and considered to have received FFP for the primary efficacy analysis. In the event that important demographic or prognostic variables (i.e., age, weight, or surgical procedure) differ between the groups, a secondary analysis may be performed. Differences in the treatment effect on the odds of requiring FFP will be assessed using logistic regression. Odds ratios, comparing rhAT to placebo, and 95% confidence intervals will be presented. [00103] The secondary efficacy endpoints — levels of Fibrin Monomer,

D-Dimer, and plasma AT III activity will be examined at two time points: 30 minutes after initiation of CPB and preprotamine administration. Differences between the group means will be assessed at each study time point using an analysis of variance (ANOVA) including treatment and study center as main effects. Should distribution requirements not be satisfied, non-parametric approaches may be used. A subset analysis may also be performed to compare the patients receiving rhAT only and those receiving FFP only.

Determination of Sample Size [00104] An estimate of 24 assessable patients in each treatment group would provide 80% power to detect a 40% absolute reduction (65% versus 25%) in the proportion of patients requiring fresh frozen plasma (FFP). To account for a dropout rate of approximately 10%, a total of 52 randomized patients, 26 in each treatment group, were recommended for the trial. Table 2 provides the number of patients that would be required per treatment group at various proportions of patients in each treatment group requiring FFP (two-sided, α=0.05),

Table 2. Estimated Sample Size Requirements

Changes in the Conduct of the Study or Planned Analysis [00105] Five amendments to the protocol (approved January 28. 1998) w ere made after the study was initiated in May 1998. The effective dates for the fiv e amendments w ere as follow: September 10, 1998 (Amendment 1 ); October 28, 1998 (Amendment 2); January 18, 1999 (Amendment 3); May 26. 1999 (Amendment 4); and July 29. 1999 (Amendment 5). Seven, 15. 18, 22. and 24 patients had been screened and signed informed consent forms prior to implementation of the first, second, third, fourth, and fifth amendments, respectiv ely. None of the changes in any of the five amendments w as felt to have an impact on the inteφretation of the study data.

Analysis of Efficacy

Primary Efficacy Endpoint and Disposition of Patients A flow chart summarizing patient recruitment beginning w ith all patients screened is prov ided in Table 3 below. Table 3. Patient Recruitment Flow Chart Number of Patients HDR Evaluated n = 329 I Number of Patients Enrolled (Consented) n = 296

Number o Jf Patients Eligible for Randomization (Not Recorded)

Number of Patients Number of Patients Not Randomized Randomized &. (Not Recorded) Treated n = 54

Placebo Treated rhAT Treated Patients Patients n = 27 n = 27

[00106] A total of 329 patients were HDR screened for entry into the study The number of patients found to have met all eligibility criteria except for the final randomization criteria (determined in the operating room) was not recorded In addition, the total number of patients that met the randomization criteria w as not recorded but of those that did, 54 patients ere randomized into the study Reasons for w hy the other eligible patients were not randomized cannot be prov ided Tw enty -sev en of the patients w ere randomized into the placebo treatment group and 27 ere randomized into the rhAT treatment group All placebo patients completed the trial (n=27), w hile 24 of the rhAT patients completed the study One rhAT patient (Patient 02-035) received only a partial dose of study drug ( 1 mL) after w hich administration w as stopped in view of the surgeon deciding to perform the surgery "off-pump" T o other rhAT patients (Patients 04-024 and 13- 197) died, 2 an 18 days, respectiv ely, post- operativ ely due to complications associated w ith their underlying disease The study blind w as broken for one patient prior to study completion and fina zation of the database, the code for Patient 13- 197 in the rhAT treatment group w as unblinded for the DSMB. [00107] The disposition of study patients is summarized by study phase in Table 4 below

Table 4. Patient Disposition

[00108] Patient recruitment into each treatment group by study site is prov ided in Table 4 [00109] Study site 12 contributed the most patients (n = 10) to the study population Importantly across all of the study centers, the distribution of patients randomized to receive rhAT or placebo w as w ell balanced [001 10] The primary efficacy endpoint of the study w as the compaπson of the proportion of patients in each treatment group requiring the infusion of fresh frozen plasma (FFP) after administration of study medication but prior to initiation of CPB Analy sis of the primary endpoint w as performed on the Intent-To-Treat (ITT) and the Per Protocol populations Table 5 summarizes the proportion of patients in each treatment group w ho receiv ed FFP, as observ ed in each of the tw o patient populations

Table 5. Primary Efficacy Results: Patients' FFP Administration Status (Intent-To-Treat and Per Protocol Populations)

FFP = Fresh Frozen Plasma (2 units) P-v alues based on Barnard ^'s Unconditional Exact test for difference betw een tv, o binomial proportions. Note. The percentages are based on the total number of patients in each treatment group, Note. Patients requiring FFP are defined as al l patients fai l ing to achiev e an ACT >4S0 seconds 5 minutes after administration of stud medication regardless o the actual status of FFP administration. Note' Patients 01 -069 (rhAT) and 01 -071 (placebo) did not hav e ACTs taken and w ere considered to have received FFP

[001 1 1 ] In the Lntent-To-Treat population, 22 of 27 placebo treated patients required the infusion of FFP, while only five of 26 rhAT treated patients required FFP prior to initiation of CPB. The proportion of patients requiring the administration of two units of FFP prior to proceeding to CPB was significantly smaller (p<0.001 ; Barnard's Unconditional Exact Test) in the rhAT treatment group (19%) compared to the proportion among placebo treated patients (81 %). The same level of statistical significance was observed when the analysis was performed using the Per Protocol population. [001 12] The distribution of the primary efficacy endpoint outcomes across study centers was reviewed for both treatment groups, These data are displayed in Table 6.

Table 6. Outcome (FFP or No FFP) Distribution by Study Center (Intent-To-Treat Population)

FFP = Fresh Frozen Plasma * Patients 01 -069 (rhAT) and 01 -071 (placebo) did not hav e ACTs taken and w ere considered to hav e receiv ed FFP

[001 13] Although no formal statistical comparisons were made, the primary efficacy outcomes appear to be similarly distributed across study centers for both treatment groups. In no case was a single center responsible for a particular outcome in either treatment group.

Secondary Efficacy Endpoints [001 14] The secondary efficacy endpoints were measures of the comparative effects of each treatment on laboratory measures that could potentially be affected by rhAT or FFP administration. These "bioactivity" measures included fibrin monomer, D-Dimer, and AT III blood plasma activity levels taken just prior to and 30 minutes after administration of study medication and just prior to heparin reversal with protamine at the end of CPB. Table 7. D-Dimer: Actual Value Descriptive Statistics (nmol/L) (Intent-To-Treat Population)

Std Dev = Standard Dev lation Note Subject counts mav v arv due to missed sampl ing or samples that could not be assav ed Note The normal range for D-Dimer is less than or equal to 100 ng mL

[001 15] For the Intent-To-Treat population, there were no statistically significant differences in mean D-Dimer levels between the two treatment groups at baseline, 30 minutes after initiation of CPB, or prior to protamine administration. The mean change from baseline to 30 minutes post initiation of CPB and the mean change from baseline to the preprotamine administration time point for D-Dimer is provided in Table 8 for both treatment groups.

Table 8. D-Dimer Mean Change from Baseline (nmoles/L) (Intent-To-Treat Population)

Std = Standard Deviation Note Subject counts mav v arv due to missed sampl ing or samples that could not be assav ed, Note The normal range for D-Di mer is less than or equal to 100 ng'mL Note Subject 04-023 's preprotamine measure has been excluded

[001 16] For the Intent-To-Treat population, there were no statistically significant differences in mean change in D-Dimer levels between the two treatment groups at 30 minutes after initiation of CPB or prior to protamine administration.

Fibrin Monomer Level [001 17] Table 9 provides the actual value descriptive statistics for fibrin monomer levels at baseline, 30 minutes post initiation of CPB, and at the preprotamine time-point for both treatment groups.

Table 9. Fibrin Monomer - Actual Value Descriptive Statistics (ng/mL) (Intent-To-Treat Population)

Std Dev = Standard De iation N ote Subject counts mav v arv due to missed sampling or samples that could not be assav ed Note Values reported as < or > hav e been excluded Note The normal range for fibrin monomer is less than or equal to I 5 nmol L

[001 18] For the Intent-To-Treat population, there were no statistically significant differences in mean fibrin monomer level between the two treatment groups at baseline, 30 minutes after initiation of CPB, or prior to protamine administration. The mean change from baseline to 30 minutes post initiation of CPB and the mean change from baseline to the preprotamine administration time point for fibrin monomer level is provided in Table 10 for both treatment groups.

Table 10. Fibrin Monomer Mean Change from Baseline (ng mL) (Intent-To-Treat Population)

Std Dev = Standard Dev iation Note Subject counts mav v arv due to missed sampl ing or samples that could not be assav ed Note Values reported as < or > hav e been excluded Note The normal range for fibnn monomer is less than or equal to 1 5 nmol L

[001 19] For the Intent-To-Treat population, there w ere no statistically significant differences in mean change in fibrin monomer level between the two treatment groups at 30 minutes after initiation of CPB or prior to protamine administration.

AT III Plasma Activity Level [00120] Table 1 1 provides the actual value descriptive statistics for AT III plasma activity level at baseline, 30 minutes post initiation of CPB, and at the preprotamine time-point for both treatment groups.

Table 11. AT III Plasma Activity Level - Actual Value Descriptive Statistics (%) (Intent-To-Treat Population)

Std Dev = Standard Dev iation Note Subject counts mav varv due to missed sampling or samples that could not be assav ed Note The normal range for plasma AT I II acti ity is 70- 1 30%.

[00121 ] Using the Intent-To-Treat population, there was no statistically significant difference between the rhAT and the placebo treatment groups in the mean plasma AT III activity level at baseline. The mean AT III activity level was above the lower limit of normal physiologic activity (70%) at baseline for patients in both cohorts (78% and 74%, respectively). However, there were statistically significant differences (p < 0.001) between the two treatment groups with respect to plasma AT III activity at 30 minutes post CPB initiation and preprotamine reversal. In the rhAT treatment group, the mean plasma AT III activity level increased to toward the upper limit of normal physiologic activity (130%) at both 30 minutes post initiation of CPB (122%) and at the preprotamine time point (1 13%). In contrast, the AT III activity levels among the placebo patients requiring FFP were further reduced from baseline (74%) at both 30 minutes after initiation of CPB (52%) and at the preprotamine time point (51 %). [00122] The mean change from baseline to 30 minutes post initiation of CPB and the mean change from baseline to the preprotamine administration time point for AT III plasma activity level is provided in Table 12 for both treatment groups. Table 12. AT III Plasma Activity Level - Mean Change from Baseline (%) (Intent-To-Treat Population)

Std. Dev = Standard Dev iation Note. Subject counts may v ary due to missed sampl ing or samples that could not be assayed. Note' The normal range for plasma AT I I ! acti ity is 70- 1 30%,

[00123] A mean decrease from baseline in plasma AT III activity level was observed for the placebo plus FFP patients at both time points. In contrast, for patients who received rhAT without FFP, there was a mean increases in AT III activity level at both time points. The difference between the two treatment groups in the mean change from baseline was statistically significant at both time points (pO.OOl). These observations and the large disparity in maximum AT III plasma activity levels between the two treatment groups as noted in Table 12 demonstrates that the administration of two units of FFP is not effective for increasing plasma AT III activity.

Cohort Analysis of Secondary Endpoints [00124] There were four possible groupings of patients across the two study treatment groups. These included patients randomized to receive placebo that either required FFP or did not require FFP, and patients randomized to receive rhAT who either required or did not require FFP. [00125] In terms of secondary efficacy endpoints, the study objective was to compare the effects of rhAT and FFP on AT III activity, thrombin activity, and fibrinolysis. Therefore, for each of these parameters, the relevant comparison is the group of placebo patients w ho required FFP administration to the group of rhAT patients w ho did not require FFP administration In the Intent-To-Treat population, a total of 22 patients received placebo and subsequent FFP administration prior to the initiation of CPB, w hile 21 patients receiv ed rhAT without requiring FFP administration prior to initiation of CPB The preprotamine time point for one placebo patient (Patient 04-023) has been excluded from the ev aluation of secondary efficacy endpoints because the preprotamine blood samples for this patient w ere taken from pump blood after protamine administration Tables 13 through 1 1 -19 prov ide the descnptiv e statistics, the change from baseline by sampling time point for each of these tw o outcome cohorts, and the p-v alues for the between-group compaπsons for D- Dimer. fibrin monomer, and AT III plasma activ ity lev els Intent-to-Treat actual v alue results are provided for each parameter, hile change from baseline results are prov ided for both the Intent-To-Treat and the Per Protocol populations for each secondary efficacy endpoint

D-Dimer Lev el [00126] The effects of rhAT and FFP on thrombin activ ity w ere compared, as measured by the change in D-Dimer lev els pre- and post-treatment Table 13 prov ides the actual v alue descπptiv e statistics for D-Dimer levels at baseline, 30 minutes post initiation of CPB, and at the preprotamine time point for the placebo with FFP and the rhAT without FFP outcome cohorts

Table 13. D-Dimer: Actual Value Descriptive Statistics (ng/mL) (Intent-To-Treat Population)

FFP = Fresh Frozen Plasma; Std. Dev. = Standard Dev iation.

alucs based on Rank Sum test Note. The normal range for D-Dimer is < 100 ng mL. Note: Patient counts may v arv due to unobtainable assav results

[00127] Using the Intent-To-Treat population, there were no statistically significant differences in mean D-Dimer levels between the two outcome cohorts at baseline, 30 minutes after initiation of CPB, or prior to protamine administration. Although not statistically significant (p = 0.103), there was an apparent trend toward a smaller increase in D-Dimer levels from baseline at the preprotamine time point in the rhAT patients not requiring FFP compared to the placebo patients requiring FFP. The mean change in D-Dimer level from baseline to 30 minutes post initiation of CPB and to the preprotamine administration time point are summarized in Table 14.

Table 14. Mean Change from Baseline in D-Dimer Level (ng mL) (Intent-To-Treat Population)

FFP = Fresh Frozen Plasma. Std Dev = Standard Dev iation. P-v al ues based on Rank Sum test Note The normal range for D-Dimer is ≤ 100 ng, ml_ Note Patient counts may v ary due to unobtainable assay results

[00128] Comparison of the mean change in D-Dimer level from baseline at 30 minutes post CPB and preprotamine administration also did not show any statistically significant differences between the placebo plus FFP and the rhAT without FFP cohorts using the Intent-To-Treat population. However, the mean D-Dimer level was lower in the rhAT without FFP cohort at the preprotamine time point, and the medians do demonstrate a consistent trend toward larger changes from baseline in D-Dimer levels at both time points for the placebo with FFP cohort [281 ng/ml versus 215 ng/ml (30 minutes post-CPB) and 1207 ng/ml versus 643 ng/ml (preprotamine)]. Graphic presentation of individual D-Dimer plasma level profiles for the two cohorts using the Intent-To-Treat Population is provided in Figure 1 1 -1.

Fibrin Monomer Level [00129] The effects of rhAT and FFP on fibrinolysis were compared, as measured by the change in fibrin monomer levels pre- and post-treatment. Table 14 provides the actual value descriptive statistics for fibrin monomer levels at baseline, 30 minutes post initiation of CPB, and at the preprotamine time-point for the placebo with FFP and rhAT without FFP cohorts using the Intent-To-Treat population. Table 14. Fibrin Monomer - Actual Value Descriptive Statistics (nmol/L) (Intent-To-Treat Population)

FFP = Fresh Frozen Plasma; Std Dev . = Standard Deviation ; P-v al ues based on ANOV A Note The normal range for fibrin monomer is < 1 5 nmol- L Note. Values reported as < or > have been excluded Note. Patient counts mav v arv due to unobtainable assav results.

[00130] There was no statistically significant difference between the two outcome cohorts in mean fibrin monomer levels at baseline. Mean fibrin monomer levels were similar for each cohort at the 30 minutes after initiation of CPB and at the preprotamine time points. The mean changes in fibrin monomer levels from baseline for both cohorts at these two time points are summarized in Table 15.

Table 15. Mean Change from Baseline in Fibrin Monomer Level (nmol/L) (Intent-To-Treat Population)

FFP = Fresh Frozen Plasma, Std Dev, = Standard Dev iation , P-v alues based on ANOVA Note The normal range for fibrin monomer is < 1 5 nmol 'L. Note Values reported as < or > hav e been excluded Note Patient counts may v ary due to unobtainable assay results.

[00131 ] No statistically significant differences were noted between the two outcome cohorts in the mean change in fibrin monomer level from baseline at 30 minutes after initiation of CPB or at the preprotamine time point, Graphic presentation of individual fibrin monomer plasma level profiles for the two cohorts using the Intent- To-Treat population is provided in Figure 4. [00132] Mean change from baseline in fibrin monomer level at 30 minutes after initiation of CPB and at the preprotamine time point for the two outcome cohorts in the Per Protocol population are summarized in Table 16.

Table 16. Mean Change from Baseline in Fibrin Monomer Level (nmol/L) (Per Protocol Population)

FFP = Fresh Frozen Plasma; Std. Dev . = Standard Dev iation; P-values based on ANOVA Note. The normal range for fibrin monomer is < 15 nmol L, Note Values reported as < or > have been excl uded. Note. Patient counts mav arv due to unobtainable assav results.

[00133] In the Per Protocol population, there were no statistically significant differences between the two outcome cohorts in the mean change in fibrin monomer levels from baseline to 30 minutes after initiation of CPB or at the preprotamine time point. [00134] Additionally, shifts (normal vs. abnormal) in fibrin monomer were also assessed so as to include trim values that were not incorporated into the analysis of actual values (Provided in Section 14.2). For both the Intent-To-Treat and Per Protocol populations, there were no significant differences between the two outcome cohorts with respect to shifts from baseline at either the 30 minutes post CPB initiation or preprotamine administration time points. [00135] The meaning of these findings is not self-evident and must be considered with caution for the following reasons. When fibrin activates fibrinogen it forms a single fibrin molecule or fibrin monomer. During normal hemostasis, the fibrin monomer immediately polymerizes with other fibrin to form a clot and is not detectable in plasma. If fibrin is formed at a relatively low rate away from a polymerizing clot, fibrin can be released into the plasma where it forms a semi-stable complex with two fibrinogen molecules, resulting in plasma fibrin monomers or soluble fibrin. In addition, the lev el of fibnn measured in the plasma is dependent on the type of assay used to measure it [00136] The assay used in the cuπent study w as the Behnng fibnn monomer assay The pnnciple of the assay is detection of fibπn-hke fragments by measuπng their ability to accelerate the activ ation of plasminogen by tissue plasminogen activ ator (TPA) Increased fibnn leads to increased plasmin, w hich produces increased yellow color in the reaction Notably , the assay detects any fibnn that accelerates TPA This w ould include true fibnn monomer as w ell as other materials such as fibnn oligomers (small polymers) and small fragments of degraded polymerized fibnn As a result, the assay cannot be considered a true fibrin monomer assay [00137] Next, the effect of thrombin activ ity and fibπnolysis on the fibnn monomer assay must be considered Increased thrombin activ ity can be the result of an increase in either hemostatic thrombin or non-specific thrombin An increase in thrombin activ ity as the result of an increase in hemostatic thrombin yields little or no increase in fibnn monomer An increase in thrombin activity due to an increase in nonspecific thrombin increases fibnn monomer lev els if the increase is low or moderate If there is an increase in non-specific thrombin to high lev els, fibnn monomer forms polymeπzed fibnn, which is unstable in plasma and not detectable [00138] When the fibnn lev el increases due to clot, increased fibπnolytic activ ity initially increases fibrin fragments, but later degrades fibrin, thus removing it from plasma If the pπmary source of fibnn is soluble fibnn, increased fibnnolytic activ ity degrades fibnn and removes it from plasma [00139] In summary, fibrin monomer level in plasma is dependent on the level of hemostatic versus non-specific thrombin, the relative amount of fibnnolytic activity, the timing of sampling versus fibnnolytic acti ity, and methods used to stabilize the sample Overall, because fibrin monomer is v aπably affected by thrombin and fibnnolytic activ ity and the assay procedure used w as not specific for fibrin monomer, changes in the absolute amount of fibnn monomer ov er time may not hav e been accurately measured in either treatment group dunng the cuπent study

AT III Plasma Activity Lev el [00140] The effect of rhAT and FFP on plasma AT III activity lev el were compared Table 17 prov ides the actual value descπptive statistics for mean AT III plasma activity lev el at baseline, 30 minutes post initiation of CPB, and at the preprotamine time point for the placebo with FFP and the rhAT without FFP outcome cohorts using the Lntent-to-Treat population.

Table 17. AT III Plasma Activity Level - Actual Value Descriptive Statistics (%) (Intent-To-Treat Population)

FFP = Fresh Frozen Plasma, Std Dev = Standard Dev lation * P-value based on ANOV.A * *P-v alue based on Rank Sum test Note¹ The normal range for plasma AT III activity is 70% - 130% activ ity Note Patient counts may vary due to unobtainable assay results

[00141 ] Using the Intent-To-Treat population, there was no statistically significant difference between the rhAT without FFP and the placebo with FFP cohorts in the mean plasma AT III activity level at baseline. The mean AT III activity level was above the lower limit of normal physiologic activity (70%) at baseline for patients in both cohorts (76% and 74%, respectively). However, there were statistically significant differences (p < 0.001) between the two cohorts with respect to plasma AT III activity at 30 minutes post CPB initiation and preprotamine reversal. In the rhAT without FFP cohort, the mean plasma AT III activity level increased to toward the upper limit of normal physiologic activity (130%) at both 30 minutes post initiation of CPB (123%) and at the preprotamine time point ( 1 14%). In contrast, the AT III activity levels among the placebo patients requiring FFP were further reduced from baseline (74%) at both 30 minutes after initiation of CPB (51 %) and at the preprotamine time point (52%), [00142] Graphic presentation of individual AT III plasma activity level profiles for the two cohorts using the Intent-To-Treat population is provided in Figure 5. [00143] Mean change from baseline in plasma AT III activity level at 30 minutes after initiation of CPB and at the preprotamine time point for the two outcome cohorts using the Intent-To-Treat population are summarized in Table 18.

Table 18. AT HI Plasma Activity Level - Mean Change from Baseline (%) (Intent-To-Treat Population)

FFP = Fresh Frozen Plasma; Std. Dev. = Standard Deviation * P-value based on ANOVA •*P-value based on Rank Sum test Note: The normal range for plasma AT III activity is 70% - 130%. Note: Patient counts may vary due to unobtainable assay results.

[00144] A mean decrease from baseline in plasma AT ED activity level was observed for the placebo plus FFP patients at both time points. In contrast, for patients who received rhAT without FFP, there was a mean increases in AT III activity level at both time points. The difference between the two cohorts in the mean change from baseline was statistically significant at both time points (p<0.001). T ese observations and the large disparity in maximum AT HI plasma activity levels between the two cohorts as noted in Table 18 demonstrate that the administration of two units of FFP is not effective for increasing plasma AT III activity. [00145] Mean change from baseline in plasma AT III activity level at 30 minutes after initiation of CPB and at the preprotamine time point for the two outcome cohorts using the Per Protocol population is summarized in Table 19. Table 19. AT III Plasma Activity Level - Mean Change from Baseline (%) (Per Protocol Population)

FFP = Fresh Frozen Plasma; Std Dev = Standard Deviation *P-value based on Rank Sum test ** P-value based on ANOVA Note The normal range for plasma AT III activity is 70% - 130% Note Patient counts may vary due to unobtainable assay results.

[00146] Results of the analysis of plasma AT III activity level using the Per Protocol population were similar to those using the Intent-To-Treat population.

Analyses: Other Measures of Thrombin Activation [00147] Two additional indicators of thrombin activation — prothrombin fragment 1.2 and thrombin-antithrombin complex — were evaluated after completion of the study. These two parameters were measured at baseline (Time 0), at 30 minutes after initiation of CPB, and at the preprotamine time point in retained plasma samples. Measurement of prothrombin fragment 1.2 is an indicator of prothrombin consumption that occurs during its conversion to thrombin in the presence of factor Xa, factor Va, Ca^"^, and a source of phospholipid (PF 3). A low level of prothrombin fragment 1.2 would correlate with reduced thrombin production. [00148] The results of the measurement of prothrombin fragment 1.2 plasma levels for the rhAT and the placebo treatment groups using the Intent-To-Treat population are provided in Table 20. Table 20. Mean Prothrombin Fragment 1.2 Plasma Levels (nmol/L) (Intent-To-Treat Population) -

FFP = Fresh Frozen Plasma, Std Dev = Standard Deviation, Mm Max = Minimum/Maximum, ND = Not Done, CPB = Cardiopulmonary Bypass, P-values based on Rank Sum test Note The normal range for prothrombin fragment I 2 is 0 4 - I 8 nmol/L Note Patient counts may vary due to unobtainable assay results

[00149] Using the Intent-To-Treat population, there were no statistically significant differences between the two treatment groups for mean prothrombin fragment 1.2 levels at baseline or at 30 minutes post CPB initiation. However, the mean change from baseline at the preprotamine time point, although not a statistically significant difference (p = 0.134), showed less of an increase in the rhAT treatment group compared to the group that received placebo (4.5 nmoles/L versus 6.4 nmoles L, respectively). [00150] The results of he measurement of prothrombin fragment 1.2 plasma levels for the rhAT without FFP and the placebo with FFP cohorts using the Intent-To- Treat population are provided in Table 21.

Table 21. Mean Prothrombin Fragment 1.2 Plasma Levels (nmol/L) (Intent-To-Treat Population)

FFP = Fresh Frozen Plasma, Std Dev = Standard Deviation, Min. Max = M inimum Maximum; ND = Not Done; CPB = Cardiopulmonary Bypass, P-values based on Rank Sum test Note The normal range for prothrombin fragment 1 2 is 0 4 - I 8 nmol/L. Note Patient counts may vary due to unobtainable assay results. [00151 ] Using the Intent-To-Treat population, there were no statistically significant differences between the two outcome cohorts for mean prothrombin fragment 1.2 levels at baseline or at 30 minutes post CPB initiation. However, there was a statistically significant difference (p = 0.015) between the outcome cohorts in the mean change in prothrombin fragment 1.2 levels from baseline to the preprotamine time point. The rhAT without FFP cohort demonstrated a significantly smaller increase in prothrombin fragment 1.2 levels when compared to the placebo with FFP cohort. The data also suggest that the effect observed in the rhAT without FFP group increased with time over the course ofthe surgery. Figure 6 displays the individual prothrombin fragment 1.2 plasma levels over time for the rhAT without FFP cohort and the placebo with FFP cohort using the Intent-To-Treat population. [00152] The level of thrombin-antithrombin complex will increase as thrombin is produced in the presence of antithrombin. The level of thrombin-antithrombin complex should be lower in the rhAT treatment group compared to the placebo treatment group if thrombin production was reduced. The results of the measurement of thrombin-antithrombin complex plasma levels for the placebo and the rhAT treatment groups using the Intent-To-Treat population are provided in Table 22.

Table 22. Mean Thrombin-Antithrombin Complex Plasma Levels (ng/mL) (Intent-To-Treat Population)

FFP = Fresh Frozen Plasma, Std Dev = Standard Deviation, Min Max = Minimum Maximum, CPB ^: Cardiopulmonary Bypass, P-value based on Rank Sum test Note The normal range for thrombin-antithrombin complex is 0 0 - 5 0 ng mL Note Patient counts may vary due to unobtainable assay results

[00153] Using the Intent-To-Treat population, there was no statistically significant difference between the two treatment groups in mean thrombin-antithrombin complex plasma levels at baseline, 30 minutes after initiation of CPB, or at the preprotamine time point. However, at the preprotamine time point, the mean value and the mean increase in thrombin-antithrombin complex were less in the rhAT treatment group compared to the placebo group. [00154] The results of the measurement of thrombin-antithrombin complex plasma levels for the placebo with FFP and the rhAT without FFP outcome cohorts using the Intent-To-Treat population are provided in Table 23. Table 23. Mean Thrombin-Antithrombin Complex Plasma Levels (ng/mL)

FFP = Fresh Frozen Plasma, Std Dev = Standard Deviation, Min. Max. = Minimum/Maximum, CPB = Cardiopulmonary Bypass, P-value based on Rank Sum test Note The normal range for thrombin-antithrombin complex is 0 0 - 5 0 ng mL. Note Patient counts may vary due to unobtainable assay results [00155] Using the Intent-To-Treat population, there was no statistically significant difference between the two outcome cohorts in mean thrombin-antithrombin complex plasma levels at baseline. Similar to the results for prothrombin fragment 1.2 outcome cohort levels, there was a statistically significant difference (p = 0.019) between the outcome cohorts in the mean change from baseline to the preprotamine time point for the thrombin-antithrombin complex levels. The rhAT without FFP cohort demonstrated a significantly smaller increase in thrombin-antithrombin complex levels than the placebo with FFP cohort. The mean change at the 30 minutes post CPB initiation time point was less in the rhAT without FFP group, but the change was not statistically significant (p = 0.417); this may be due to a lack of sufficient time from the administration of study medication to see an effect. The data also suggest that the effect observed in the rhAT without FFP cohort increased with time over the course of the surgery. [00156] Figure 7 displays the individual thrombin-antithrombin complex plasma levels over time for the rhAT without FFP cohort and the placebo with FFP cohort using the Intent-To-Treat population.

Effect of Study Medication on Subsequent Heparin Dosing [00157] Although not a prospectively defined study endpoint, some interesting observations can be made regarding the administration of additional heparin during the study among the placebo with FFP patients and the rhAT without FFP patients (Section 14.2). In the Intent-To-Treat population, the mean total amount of heparin given while patients were on CPB was significantly greater (p = 0.005; Rank Sum test) among placebo patients (n = 22) who received FFP (6890 IU per hour) than among rhAT patients (n = 21) who did not require FFP (3884 IU per hour). [00158] However, it is arguably more relevant to look at the number of additional doses of heparin that were required in each of the two treatment groups (rhAT and placebo). Time frames of particular interest include 1) after administration of study medication but before CPB initiation, and 2) after administration of study medication up to the time of heparin reversal with protamine. These data are summarized in Table 24.

Table 24. Number of Patients Requiring Heparin Dosing After Administration of Study Medication Prior to Initiation of CPB and Prior to Heparin Reversal with Protamine (Intent-To-Treat Population)

* Does not include routine hepann primi ng of the CPB circuit * * The denominator is deriv ed from the number of patients in each treatment group that required an additional dose of heparin after administration of stud) medication up to the time of hepaπn rev ersal v. ith protamine

[00159] A total of 21 out of 27 placebo patients (78%) required at least one additional dose of heparin some time between administration of study medication and heparin reversal with protamine. However, only 12 out of 26 patients (46%) treated with rhAT required at least one additional heparin dose during the same period. The most important observation among these patients is the distribution of the number of doses that were required across each treatment group. Twelve placebo patients and eight rhAT patients required only one additional dose of heparin. However, 9 placebo patients and only 4 rhAT patients required between 2 and 4 additional doses of heparin. [00160] Among the 12 rhAT patients that required an additional heparin dose prior to heparin reversal with protamine only 3 (25%) of these patients required an additional dose of heparin before CPB, while 10 (48%) of the 21 placebo patients required additional heparin dosing prior to initiation of CPB. Additionally, the need for administration of more heparin doses among placebo patients may explain the cohort differences in mean time from the first dose of heparin to initiation of CPB. The mean time w as 60 minutes for the rhAT patients not receiving FFP, while the mean time for placebo patients requiring FFP was 69 minutes (p = 0 331) [00161 ] All of the heparin dosing discussed abov e w as administered by the attending physician in conjunction with the effort to maintain adequate anticoagulation and, therefore, qualify as an indicator of the impact of each of the study treatments The clinical relev ance of these findings is discussed further in Section 13 0 of this report

Patient Disposition and Characteristics Disposition and Accountability [00162] All patients randomized into the stud) are included in the summary of patient disposition and accountability No inferential statistical tests w ere performed Frequencies and percentages of patients enrolled into the study, discontinued from the study, and completing the study bv treatment group are summarized For accountability frequencies of patients (total and by study center) are provided at baseline, mtra-operativ ely, and at the 4-w eek follow -up

Demographics [00163] Summary statistics by treatment group are prov ided for the following • Age • Gender • Weight (in kg) • Ethnicity

[00164] Frequencies and percents are provided for gender and ethnicity categories, while descriptive statistics including N, mean, median, standard deviation, minimum, and maximum values are provided for age and weight

Physical Exam and Medical History [00165] Frequencies and percents by treatment group are provided for all 12 categoπes of the physical exam and all 16 categories of the medical history taken pre- operati ely Items are classified as 'normal,' 'abnormal,' or 'not examined ' In addition, pre-operativ e ital signs including blood pressure, heart rate, and w eight are summarized with descπpti e statistics including N, mean, median, standard deviation, minimum, and maximum v alues No inferential tests w ere performed

Efficacy Endpoints [00166] The primary efficacy analysis as based on the Intent-To-Treat population and included all randomized patients who received the blinded study drug (rhAT Patient 02-035 w as excluded from the ITT population because study drug administration w as stopped after only 1 mL because the surgeon decided to perform an "off pump^"' procedure)

Primary Efficacy Endpoint [00167] Treatment group differences in the proportion of patients requiring the administration of FFP w ere to be assessed using the Pearson Chi-square test w ith treatment group and FFP status as factors How ever, the assumptions of this test w ere not met, therefore, Barnard's Unconditional Exact Hypothesis Test for the null hypothesis, Ho p_tn ⁼ Piont- v> as employed, as allow ed for in the Statistical Analysis Plan Due to the relati ely large number of centers inv olv ed in this protocol, the small number of patients recruited at each center, and the standardized measuπng of ACTs among centers, center-to-center vaπation w as not assessed

Secondary Efficacy Endpoints [00168] Treatment group and cohort differences in changes from baseline (time 0) in plasma le els of Fibnn Monomer, D-Dimer, and plasma AT III activity level at 30 minutes post-CPB initiation and preprotamine were assessed using an Analysis of Vaπance (ANOVA) model when assumptions were met. When assumptions were not met, a Rank Sum test was used Since secondary endpoints w ere determined from assays performed by a single central lab, study center vaπation and center/treatment interaction were not assessed [00169] In addition to the exact value summaries for the fibnn monomer data, shift tables w ere also prov ided, due to the large number of tnm values reported as > or

< some value for the fibnn monomer lev els The percentage of patients in each treatment group shifting from normal to abnormal at both the 30 minutes on CPB and just pnor to protamine reversal time points w ere compared Results w ere commensurate with the exact v alue analyses (Section 14 2) Safety Endpoints [00170] All analy ses of safety parameters were conducted on the safety population The safety population w as comprised of all patients who met the inclusion/exclusion criteria, signed an informed consent, and ere randomized to a treatment arm

Adv erse Ev ents [00171 ] All adv erse expeπences are summanzed by body system and by preferred term based on the WHOART coding dictionary Frequencies and percents of treatment-emergent adv erse ev ents by treatment group are tabulated [00172] Tables are presented for treatment-emergent adverse events by intensity or seventy and by relationship to the treatment and/or concomitant medications The intensity has three categories mild, moderate, or sev ere The most sev ere intensity was assigned to a patient w hen more than one occurrence of the same .AE as reported Relationships of the AE to treatment and concomitant medications are categonzed as 'not related,' 'possible,' 'probable,' or 'definite ' The highest lev el of association was reported in patients w ith diffenng relationships for the same AE Listings of the abov e, as w ell as actions taken regarding treatment, episode type, and patient outcome, are provided for all patients Separate listings for patients who w ithdrew from the study due to AEs, as well as all patients reporting senous AEs, are also prov ided [00173] In addition to the adverse event tables and listings, it was decided, in consultation w ith Clinical and Medical Affairs and Pharmacov igilance, to compare the proportion of patients reporting specific ev ents in each treatment group for the most frequently reported adverse ev ents The incidences of the adverse ev ents that occurred in at least 10% of either or both treatment groups were compared

Tabulation of Indiv idual Response Data [00174] Indiv ldual patient response data for the single pπmary efficacy endpoint, and the three secondary efficacy endpoints, are pro ided in Appendix 16 2 6 These tabulations are for the Intent-To-Treat population and are displayed by treatment group Drug Dose, Drug Concentration, and Relationships to Response [00175] Direct measurement of rhAT was not performed because of the unavailability of an assay procedure that could distinguish between endogenous AT III and rhAT in blood plasma. Further, this study was not designed to evaluate dose response or to determine the pharmacokinetics of rhAT. However, the change in blood plasma AT III levels after the administration of the single dose of study medication was a secondary efficacy endpoint of the study.

Drug-Drug and Drug-Disease Interactions [00176] This study was not intended to evaluate drug-drug or drug-disease interactions. However, no apparent relationships between response and concomitant therapy, and between response and past and/or concurrent illness were observed.

Efficacy Conclusions [00177] This study established that treatment with rhAT significantly reduced the requirement for administration of fresh frozen plasma per protocol to heparin resistant CPB patients in order to achieve an ACT > 480 seconds. Hepaπn resistance in this study was defined as having failed to achieve an ACT > 480 seconds within 5 minutes after intravenous administration of a total of 400 U kg of heparin. Specifically, the proportion of rhAT treated patients in the Intent-To-Treat population who required administration of two units of FFP to achieve an activated clotting time > 480 seconds was significantly less than the proportion of placebo treated patients who required two units of FFP (pθ.001 ). Similar results for this primary efficacy endpoint were seen in the Per Protocol population (p<0.001 ). [00178] Further support of the efficacy of rhAT was provided by one of the three secondary study endpoints. Treatment with rhAT produced a significant increase in plasma AT III activity levels, while AT III activity levels among placebo treated patients continued to decline. The differences in AT III activity levels between the two treatment groups were statistically significant (p<0.001 ) at both time points (30 minutes on CPB and preprotamine administration) and for both the Intent-To-Treat and the Per Protocol populations. The same result was observed when the two treatment cohorts (rhAT without FFP and placebo with FFP) were compared at each time point and for each study population. [00179] For the Intent-To-Treat population, there were no statistically significant differences in mean change in D-Dimer levels or in fibrin monomer levels between the two treatment groups at 30 minutes after initiation of CPB or just prior to protamine administration. The same result was obtained when the Per Protocol population was analyzed. In addition, no statistically significant differences were observed for either parameter using either patient population when the rhAT without FFP and the placebo with FFP cohorts were compared. However, for D-Dimer, a trend toward smaller increases in levels was observed for the cohort analysis at the preprotamine time point for both the Intent-To-Treat (p=0.083) and the Per Protocol (p=0.067) populations. [00180] Several exploratory efficacy evaluations were performed. Among these evaluations was a comparison of the effect of placebo versus rhAT on activated clotting time (ACT). The mean change in ACT value from baseline (post heparinization) to 5 minutes after study drug administration was significantly greater (p<0.001 ) for the rhAT patients compared to the placebo patients. This result demonstrates that rhAT administration at 75 U kg has a more rapid and clinically relevant effect on the ACT in heparin resistant patients than administration of two units of FFP. A significant difference in activated clotting time between treatment groups was also observed at the 30 minutes after initiation of CPB time point (p<0.001) and the preprotamine time point (p=0.003). [00181 ] Another exploratory efficacy evaluation retrospectively examined two additional markers of thrombin activation: prothrombin fragment 1.2 and antithrombin- thrombin complex. Compared to placebo patients, patients who received rhAT showed significant inhibition of the generation of prothrombin fragment 1.2 (p = 0.020) and thrombin-antithrombin complex (p = 0.029) from baseline levels at the time just prior to reversal with protamine. [00182] The effect of study medication on subsequent heparin dosing was also assessed. Seventy-eight percent (78%) of placebo patients required at least one additional dose of heparin some time between administration of study medication and heparin reversal with protamine, while only 46% of patients treated with rhAT required at least one additional heparin dose during the same period. [00183] Based on having achieved the primary efficacy endpoint, rhAT at a dose of 75 U/kg effectively restores the anticoagulant activity of heparin in heparin resistant patients, thereby avoiding the use of FFP to achieve an ACT >480 seconds. This finding was further supported by the normalization of serum AT III levels among patients administered rhAT for the duration of CPB. In addition, although not an efficacy endpoint that could be prospectively defined, the statistically significant difference in activated clotting time (ACT) between rhAT patients and placebo patients is consistent with the primary endpoint result and circulating AT III levels. Finally, retrospective evaluations of two additional biochemical markers of thrombin activation, prothrombin fragment 1.2 and thrombin-antithrombin complex, support the prospectively-defined efficacy evaluations and provide a more reliable measure of thrombin production and activity than fibrin monomer levels, where no drug effect was observed.

Clinical Laboratory Evaluation

Listing of Individual Laboratory Measurements by Patient and Each Abnormal Laboratory Value [00184] Individual patient laboratory values by study center, including blood chemistry, hematology, urinalysis, and the normal ranges are provided herein.

Individual patient AT III antibody test results, measured as either positive (antibody present) or negative (antibody not present), are also provided herein. Because of the small amount of study drug (1 mL) administered to rhAT patient 02-035, laboratory tests were not performed for this patient. Post-operative laboratory specimens were not obtained from rhAT patient 13-197 prior to his death.

Evaluation of Each Laborator Parameter

Laboratory Values Over Time [00185] Blood chemistry, hematology, and urinalysis parameters were monitored at baseline and at 24 to 48 hours post-operatively or prior to discharge from the hospital, whichever occuπed first. The mean changes in the individual laboratory parameters were not reviewed; however, shifts from normal at baseline to abnormal post-operatively were reviewed below. In addition, selected post-operative laboratory values that met the criteria for the Eastern Cooperative Oncology Group (ECOG) grade 3+ toxicity were identified retrospectively and evaluated. Individual Patient Changes

Patient Shifts from Normal to Abnormal [00186] Examination of the shift from normal values at baseline to postoperative abnormal values (either above the upper limit of normal or below the lower limit of normal) by treatment group for each laboratory parameter provides a reasonable estimation of any dramatic effects between the treatment groups. Tables Table 25, Table 26, and Table 27 below provide the percent of patients whose laboratory value shifted from normal at baseline to abnormal post-operatively for blood chemistry, hematology. and urinalysis parameters, respectively. [00187] Blood chemistry, hematology, and urinalysis results did not appear to reveal any safety concerns associated with rhAT treatment. The single-dose study design, the size of the study population in both treatment groups, the underlying patient disease status, and the impact of the surgical procedures that were performed make it difficult to differentiate treatment-related effects on these laboratory measurements. [00188] Because of the study conditions described above, statistical comparisons between the rhAT and placebo groups were not performed for any of the blood chemistry, hematology, or urinalysis parameters measured during the study. Treatment group shifts for blood chemistry parameters are displayed in Table 25.

Table 25. Blood Chemistry - Shifts from Normal at Baseline to Abnormal Post-operatively Safet Po ulation

Percentages arc based on the total number of patients in each treatment group. ALAT = Alanine Aminotransferase; ASAT = Aspartate Aminotransferase; CPK = Creatinine Phosphokinase; LDH = Lactate Dehydrogenase * Designates the group in which the percentage of patients that shifted to abnormal was greater.

[00189] Of the 19 blood chemistry parameters that were measured, the percentage shift toward abnormal was greater among the placebo patients for 12 parameters, while the greater percentage shift occurred in rhAT patients for 6 parameters. There was no difference between the treatment groups for the remaining parameter (inorganic phosphorus). The most pronounced treatment differences observed in the blood chemistry profile were greater percentage shifts (from normal baseline values to abnormal post-operative values) in the placebo group for glucose (56% vs. 33% in rhAT group) and urea nitrogen (30% vs. 15%), and a greater shift in the rhAT group for total serum protein (37 % vs. 22% in the placebo group). The shifts for glucose were to values above normal for all placebo and rhAT patients listed, while the shifts for total serum protein were to values below normal for all patients listed. The shifts for urea nitrogen were to values above normal for 7 placebo patients and all 4 rhAT patients, with one placebo patient having a shift to below normal. Treatment group shifts for hematology parameters are displayed in Table 26. Table 26. Hematology - Shifts from Normal at Baseline to Abnormal Post-operatively (Safety Population)

Percentages are based on the total number of patients in each treatment group, Ct = Count. Neutroph = Neutroph Seg, = neutrophils segmented, MCV = Mean Corpuscular Volume; MCH Mean Corpuscular Hemoglobin, MCHC = Mean Corpuscular Hemoglobin Concentration * Designates the group in w hich the percentage of patients that shifted to abnormal w as greater,

[00190] Among the 12 parameters that were measured as part of hematology, the percentage shift toward abnormal was greater among the rhAT patients for 5 parameters, while the greater percentage shift occurred in placebo patients for 2 parameters. There was no difference between the treatment groups for the remaining 5 parameters The greater shift tow ard abnormal among rhAT patients for hemoglobin and hematocrit inv olv ed too few patients in each treatment group to be considered clinically relev ant The larger shifts ( 18-48%) for both w hite blood cell count (all to above normal) and platelet count (all to below normal) in both treatment groups are not unexpected phy siologic responses to the patients' surgical procedures Likewise, the significant shifts (>48%) for both lymphocytes (all to below normal v alues) and segmented neutrophils (all to abov e normal v alues) in both treatment groups are not unexpected immunologic responses to the patients' surgeries The hematology data are difficult to interpret because of the many confounding factors associated w ith each patient's underlying disease, the complexity of the surgical procedures, and the frequent administration of v arious blood products to the majority of patients in each treatment group Treatment group shifts for urinalysis parameters are displayed in Table 27

Table 27. Urinalysis - Shifts from Normal at Baseline to Abnormal Post-operatively (Safety Population)

RBC ΗPF = Red Blood Cel ls H igh Pow ered Field. WBC/ HPF = White Blood Cells Ηigh Pow ered Field Percentages are based on the total number of patients in each treatment gτoup ^♦Designates the gτoup in w hich the percentage of patients that shifted to abnormal as greater

[00191 ] Among the 12 parameters that were measured as part of urinalysis, the percentage shift toward abnormal was greater among the placebo group for 6 parameters. There was no difference between the treatment groups for the other 6 parameters. None of the differences between the two treatment groups appeared to be clinically relevant.

ECOG Grade 3+ Laboratory Values [00192] In addition to the shifts in laboratory values discussed above, selected post-operative laboratory values that met the Eastern Cooperative Oncology Group (ECOG) grade 3+ toxicity criteria after administration of study drug were identified retrospectively. These criteria are displayed in Table 28. Table 28. Eastern Cooperati e Oncology Group (ECOG) Grade 3+ Toxicity Criteria for Selected Laboratorv Parameters

[00193] Abnormalities meeting the ECOG grade 3+ criteria provided in Table 28 were observed for ALAT, creatinine, total bilirubin, and platelet count. These abnoπnalities involved 2 placebo patients and 4 rhAT patients. Table 29 lists each of the abnormal values by patient by treatment group and includes the baseline value for each patient.

Table 29. Patients Having Selected Post-operative Laboratory Values Meeting ECOG Grade 3+ Toxicity Criteria (Safety Population)

ALAT = Alanine Aminotransferase

[00194] There were no statistically significant differences (p > 0.05) between the two treatment groups in the percentages of patients who demonstrated any postoperative laboratory value at or above the ECOG Grade 3 toxicity level. Among these patients, one placebo patient and three rhAT patients each had a single elevated liver enzyme. Therefore, these results are not suggestive of hepatotoxicity. The remaining findings — elevated creatinine in one placebo patient and decreased platelet count in one rhAT patient — are not considered indicative of a clinical trend and are unlikely related to the study medication that was administered.

AT III Antibody Testing and Retrospective Viral Testing Sample Collection [00195] Serum antibody testing (ELISA) and the collection of a serum sample for retrospective viral screening were performed at baseline (i.e., just prior to study drug administration) and again approximately 4 weeks post-operatively. ELISA testing was employed for initial screening for rhAT antibody formation (IgG seroconversion) for all patients in both the rhAT group and the placebo group A positive ELISA result required confirmation using a radio-immunoprecipitation (RIP) assay [00196] ELISA results w ere negativ e (1 e . w ithin normal range) for all patients in both treatment groups at both baseline and 4 w eeks post-operativ ely Seroconv ersion was not obseπ ed for any patient based on the ELISA results It should be noted that antibody testing at 4 w eeks post-operativ ely as not done for 5 placebo patients and 4 rhAT patients, so seroconv ersion status for these 9 patients is unknow n [00197] Serum samples for retrospectiv e v iral screening w ere collected in v lew of rhAT being denved from the milk of transgenic goats and the unknow n potential for transmission of v irus of animal (goat) origin No safety issue has prompted analysis of the samples The samples are being stored at the The Inv entors Corporation facility in Framingham, MA, w here they w ill be kept for a peπod of two years (until at least July 2001 )

Indiv idual Clinically Significant Changes [00198] One patient in each treatment group had abnormal laboratory values that ere reported as senous adverse e ents [00199] Patient 10- 145, w ho received placebo, had pre-existing anemia at the time of surgery (hemoglobin v alue of 8 5 g/dL), w hich w as reported in association w ith bleeding and was considered not related to study medication [00200] Patient 04-024, who received rhAT, experienced coagulopathy post- operativ ely, as well as acute renal failure demonstrated by worsening BUN and creatinine lev els These serious adv erse ev ents, w hich w ere considered not related to study drug, occurred concurrently with worsening cardiac failure, from w hich the patient ultimately died

Vital Signs, Ph sical Findings, and Other Observations Related to Safety Vital Signs [00201 ] Systolic and diastohc blood pressure and heart rate were measured at baseline (just prior to administration of study medication) and again at 1 , 5, and 10 minutes after study medication administration No other v ital sign measures w ere recorded dunng the study The collection of all v ital sign v alues occurred after anesthesia induction, surgical incision, and hepanmzation had been performed but pnor to the surgeon's decision regarding hether or not to administer FFP The single-dose study design, the size of the study population in both treatment groups, the underlying patient disease status, and the fact that the surgical procedures were in progress at the time of data collection make it difficult to differentiate treatment-related effects on v ital sign measurements The mean changes from baseline for vital signs at each time point are provided in Table 30

Table 30. Mean Changes from Baseline for Vital Signs Post-Dosing (Safety Population)

[00202] All mean changes from baseline in v ital signs post-dosing in both treatment groups at ev ery time point were small in magnitude No acute change in blood pressure or heart rate w as observed in conjunction with administration of rhAT (measurements taken w ithin 10 minutes of dosing) None of these mean changes was considered to be clinically relevant, and none of the differences between treatment groups was statistically significant (ANOVA, p>0 05) Physical Findings [00203] A physical examination was performed at the baseline v isit only Baseline physical examinations w ere similar for the t o treatment groups, and were consistent with and reflective of a patient population with pre-existing cardio ascular disease Three patients randomized to receiv e rhAT and five patients randomized to receiv e placebo had normal baseline physical exams All remaining patients in both treatment groups had one or more abnormalities None of these abnormalities was considered by the respective investigators to be exclusionary for study participation [00204] Electrocardiograms (ECG) were performed pre- and post-operatively Only 2 of the 27 placebo patients and 6 of the 27 rhAT patients had a normal ECG pre- operatively. Post-operatively, only 2 of the 27 placebo patients and 6 of the 27 rhAT patients had normal ECG results. Only one patient, in the placebo group, had a normal ECG pre-operatively and an abnormal ECG post-operatively. Patient 04-023 developed a lateral injury but had normal sinus rhythm. There were no statistically significant shifts in ECG status within either treatment group. The general ECG findings are not unexpected based on the patient population, patient age, and medical history.

Other Observations Related to Safety [00205] Several prospectively defined parameters relating to safety were recorded. These included the length of stay in the SICU, the length of hospitahzation, and post-operative chest tube drainage volume (a potential measure of increased postoperative bleeding). These findings are displayed in Table 31. Table 31. Patient Outcome Measures Related to Safety - Quantitative Measures

* Patient 04-024 died in SICU, and Patient 13- 197 died post-operativ ely Note The completion time of placebo patient 10- 145 ^'s surgery is unknown [00206] Small differences between treatment groups were obser ed for the mean SICU stay and the total length of hospitahzation In both cases. rhAT patients stayed longer than placebo patients The mean SICU stay in the placebo group w as 2 5 days and in the rhAT group w as 3 8 days Mean total hospitahzation time for the placebo group as 10 6 day s, w hile total hospitahzation time in the rhAT group w as 1 1 8 days Howev er, there was no statistically significant difference betw een treatment groups in the length of SICU stay (Log-Rank test, p = 0 973) or in the length of hospitahzation (Log-Rank test, p = 0 713) [00207] Differences betw een the treatment groups w ere observ ed for the mean v olume of chest tube drainage for both time penods (12 and 24 hours post-operativ ely) In both cases, rhAT patients had larger mean volumes of chest tube drainage than placebo patients The mean chest tube drainage volume dunng the period 12 hours post-operativ ely w as 71 mL/hr for the placebo cohort and 124 mL/hr for the rhAT cohort Dunng the period 24 hours post-operativ ely . mean chest tube drainage v olume w as 52 mL hr for the placebo cohort and 86 mL hr for the rhAT cohort This difference w as statistically significant bet een treatment groups for both the penod 12 hours post-operativ ely (Rank Sum test, p = 0 045) and dunng the penod 24 hours post- operati ely (Rank Sum test, p = 0 030) [00208] With regard to safety, rhAT treatment appeared to result in a slight increase in SICU stay and length of hospitahzation, and a moderate increase in the amount of chest tube drainage, compared with placebo treatment How ev er, the treatment differences observed were not statistically significant (p>0 05) Furthermore, these results need to be interpreted with caution, as no attempt was made to standardize patient care across the 1 1 study sites [00209] Also included among these safety observ ations ere the incidences of significant post-operative events (cardiac or neurological, invasiv e therapeutic cardiac procedures, rehospita zations, or operations), administration of additional blood products, and administration of protamine These findings are displayed in Table 32 Table 32. Incidence of Other Patient Outcome Measures Related to Safety-Specific Events Safet Po ulation

Means for total protamine administered are for the rhAT without FFP and the Placebo with FFP cohorts only. All other parameters include all cohorts. N = 25 for rhAT group; status of protamine administration is unknown for 2 rhAT patients.

[00210] There was no difference between treatment groups in any of these parameters that would suggest a safety concern for treatment with rhAT. No patient in either group underwent an invasive therapeutic cardiac procedure post-operatively. Four rehospitahzations occurred in the rhAT group and one in the placebo group. In all cases, these rehospitahzations involved cardiac events or symptoms of cardiovascular disease (congestive heart failure, edema, atrial fibrillation, excess fluids, dyspnea). The treatment differences in the occuπence of cardiac/neurological events and rehospitahzations were not statistically significant (Barnard's Unconditional Exact test, p = 0.360 and 0.235, respectively). [0021 1 ] During the period from pre-bypass initiation to within 24 hours post surgery, significantly more placebo patients (85%) than rhAT patients (48%) received fresh frozen plasma (Barnard's Unconditional Exact test, p < 0.001 ). Also during this time period, significantly more placebo patients (100%) than rhAT patients (85%) received transfusions of packed red blood cells (Barnard's Unconditional Exact test, p = 0.041 ). No statistically significant differences were noted between treatment groups in the numbers of patients who received platelets or other blood products after the initiation of CPB (Barnard's Unconditional Exact test, p = 1.000 and 0.406, respectively). The rhAT without FFP cohort had a larger mean total dose of protamine administered than the placebo with FFP cohort (434 mg vs. 368 mg), but the difference was not statistically significant (ANOVA, p = 0.135). [00212] The data displayed in Table 32 should be interpreted cautiously, because no attempt was made to standardize hospital procedures relating to patient care, and no consideration was given to the potential variability in physician skill.

Safety Conclusions [00213] Adverse events, laboratory abnormalities, and other safety-related events observed across both the rhAT and placebo treatment groups were considered in the context of the study population's serious underlying disease(s). In addition, consideration was given to the fact that the events reported are known to be associated with the surgical procedures performed. These procedures, as well as the many concurrent medications that patients were receiving prior to, during, and subsequent to surgery, made it difficult to interpret the severity and relationship of safety-related events to the administration of rhAT. [00214] Differences in the incidence of adverse events between treatment groups are likely due to normal and potentially wide variations that would be expected to occur in the patient population studied. Indeed, a statistically significant difference between the rhAT and placebo treatment groups was not observed for any of the most frequently occurring (> 10% incidence) adverse events. No adverse event was considered by the study investigators to be definitely or probably related to rhAT administration. Possible relationship to study medication was reported in association with adverse events experienced by only 6 patients in the rhAT group and 1 patient in the placebo group (all events were hemorrhage NOS). The distribution of all adverse events by severity (mild, moderate, or severe) was similar in each treatment group, except for a tendency for more reports of severe unrelated adverse events in the rhAT group. [00215] Two deaths occurred during the study, both in the rhAT group. Neither death was considered related to treatment with rhAT. Ten rhAT patients and seven placebo patients reported one or more serious adverse events each. One SAE (hemorrhage NOS) was considered possibly related to rhAT administration; this event of "persistent bleeding" could also have been reasonably associated with the surgical procedure. [00216] Among the adv erse events associated w ith bleeding, hemorrhage NOS w as the most frequently reported ev ent in both treatment groups The ev ent occurred more frequently among rhAT patients than placebo patients, but this difference was not statistically significant In addition, thrombocytopenia occuπed only in one placebo patient, w hile coagulation disorder and hematoma occurred only in one rhAT patient each Adv erse ev ents associated w ith bleeding w ere reported as SAEs for four rhAT patients (3 hemorrhage NOS, 1 coagulation disorder) and for three placebo patients (3 hemorrhage NOS) Bleeding-related adv erse events w ere not associated w ith supra- normal plasma AT III lev els [00217] Blood chemistry , hematology, and urinalysis results did not appear to rev eal any safety concerns associated w ith rhAT treatment No statistically significant differences (p > 0 05) w ere observ ed betw een the two treatment groups in the percentages of patients w ho had laboratory abnormalities meeting the ECOG grade 3+ cnteπa Only one patient in each treatment group had abnormal laboratory v alues that w ere reported as serious adv erse ev ents, these ev ents w ere considered not related to administration of the study medication Ov erall, many factors likely confounded interpretation of the blood chemistry, hematology, and unnalysis results The most relevant of these may ha e been the fact that the majonty of blood and unne samples were collected shortly after surgery dunng the early stages of patient recov ery, a period when effects on laboratory values w ould hav e been most significant [00218] No significant treatment effect on v ital signs or ECG results was observed within either treatment group ELISA testing for rhAT antibody formation after administration of study medication was negativ e for all patients who received rhAT Among the vanous other safety-related parameters monitored dunng the study, rhAT treatment appeared to result in a slight increase in SICU stay and length of hospitahzation Compared w ith placebo treatment, this difference w as not statistically significant and did not suggest a safety concern for treatment with rhAT Furthermore, from pre-bypass initiation to w ithin 24 hours post surgery, significantly more placebo patients than rhAT patients received transfusions of fresh frozen plasma and packed red blood cells How ev er mean chest tube drainage v olume was significantly greater among patients who received rhAT compared to patients who received placebo dunng both the 12-hour postoperative penod and the 24-hour postoperative penod No statistically significant or clinically relevant differences w ere noted betw een treatment groups for the remaining safety-related parameters (l e , post-operative cardiac or neurological events, inv asiv e therapeutic cardiac procedures, rehospitahzations or operations, or administration of platelets or other blood products, or protamine administration) [00219] Safety results of Phase I clinical studies of rhAT, as w ell as the biochemical and functional similanty of rhAT to human AT III, indicate that the rhAT of the inv ention can be safely administered intravenously in AT Ill-depleted patients Overall, in this study, intrav enous administration of rhAT at a dose of 75 U kg in patients undergoing electiv e cardiac surgery requiring sternotomy and cardiopulmonary bypass (CPB) appeared to be safe, when compared to administration of a placebo control and to reliev e and or lessen negativ e neurocognitiv e outcomes [00220] Although not prospectiv ely proposed in this study, the Inv entors inv estigated other biochemical markers of hemostatic activ ation, including prothrombin fragment 1 2 and antithrombin-thrombin complex These inv estigations w ere carried out at the same time points using the same plasma samples from w hich D-Dimer. fibrin monomer, and AT III activity w ere determined Prothrombin fragment 1 2 is generated when prothrombin is converted to thrombin, and thus serv es as a marker of thrombin generation Thrombin-antithrombin complex, a stable complex formed when AT III inhibits activ ated thrombin, serves as a marker for the dynamic between thrombin generation and thrombin inhibition Compared to placebo patients, patients w ho received rhAT showed significant inhibition of the generation of these markers at the preprotamine time point in patients undergoing CPB This is an indication of decreased thrombin generation, and therefore indicates a reduced nsk of clot formation in the CPB circuit, systemic thrombosis, and bleeding post-operatively [00221 ] While gross clot formation in the extracoφoreal circuit rarely occurs in current clinical practice, inhibition of the activation of the hemostatic system has important clinical implications Activ ation of the hemostatic system dunng CPB is implicated in the generation of cerebral microemboli, one of the proposed causes of stroke and neurologic deficit often observ ed after CPB Similarly, coronary, pulmonary, and lntracardiac thrombosis has also been reported during cardiac surgery

Post-operativ e bleeding may result from consumption of labile coagulation factors secondary to thrombin activ ation during CPB Although this Phase 3 study was not designed to demonstrate these clinical outcome endpoints due to the large number of patients required for differential determinations, rhAT administration clearly suppressed activ ation of the hemostatic system of patients on CPB [00222] Additional benefits of improv ed heparin anticoagulation management were observ ed among patients w ho receiv ed rhAT Only 3 of 26 ( 12%) rhAT treated patients compared to 10 of 27 (37%) placebo treated patients required additional heparin to achiev e adequate anticoagulation before the initiation of CPB Accordingly, rhAT administration according to the current inv ention sav es operating room time, since clinical practice dictates taking the time to draw a blood sample and monitor the ACT after each hepaπn administration Patients receiv ing rhAT also required less total hepaπn dunng CPB Low er overall hepann administration during CPB requires less protamine for rev ersal and has been associated w ith a decreased incidence of hepaπn rebound, improving patient clinical outcomes, improving efficiency and low enng hospitahzation costs [00223] Administration of rhAT at a dose of 75 U/kg w as well tolerated by hepaπn resistant patients undergoing CPB There w ere no statistically significant differences in the incidence of adv erse experiences or the incidence of clinically significant abnormal laboratory parameters noted betw een the t o treatment groups Furthermore, no rhAT treated patients developed antibodies to rhAT The interpretation of hemorrhage-related adv erse ev ents w as confounded by the multiple etiologies of bleeding in the setting of heart surgery and CPB While it is conceivable that supra-normal AT III activ ity after rhAT administration might induce bleeding, AT III activity lev el data and FFP administration data do not support this hypothesis On the other hand, mean chest tube drainage was significantly greater among patients treated with rhAT This obser ation is not totally unexpected in view of provision having not been made to monitor and reduce hepann administration in the presence of normalized AT III activ ity or to closely monitor the effectiv eness of hepann reversal with protamine during the postoperative period Had these measures been taken, it is likely that there ould have been no difference between treatment groups in mean chest tube drainage [00224] In summary, dunng cardiopulmonary bypass, blood comes into contact with the artificial surface of the bypass circuit, w hich results in nonspecific activation of the hemostatic system Without anticoagulation, clots would be formed in the bypass circuit and could result in circuit occlusion In addition, activation products would be released into the systemic circulation, and the hemostatis system w ould be depleted of factors and platelets [00225] To prevent these e ents, the pnor art has utilized hepann alone to accelerate the action of antithrombin in the blood, reducing thrombin generation, thrombin activity, and fibnn generation in the bypass circuit and systemically Inadequate anticoagulation during CPB potentially leads to an increased πsk of clotting in the bypass circuit, thrombosis in the patient, and post-operativ e bleeding due to the consumption of hemostatic factors How ev er, hepann resistance may lead to inadequate anticoagulation during CPB associated w ith the risks described above [00226] The administration of rhAT administration suppressed increases in D- Dimer, prothrombin fragment 1 2, and antithrombin-thrombin complex normally associated w ith CPB, prov iding better control of hemostatic activ ation The current inv ention w as the first to utilize rhAT in patients w ith heparin resistance undergoing cardiopulmonary bypass Heparin resistance in this setting w as caused by AT III deficiency in the v ast majority of cases Administration of rhAT, 75 U/kg. restored AT III activ ity to normal range, hich led to improv ed heparin sensitiv ity. adequate anticoagulation for CPB, and no need for treatment ith FFP in the majonty of cases In contrast, AT III activity declined further from baseline in placebo treated patients, requiring treatment w ith FFP and other measures to obtain adequate anticoagulation before initiation of CPB The administration of rhAT resulted in significant inhibition of coagulation markers of thrombin generation compared to placebo treated patients Clinically, these findings translate into a reduced nsk of clotting in the bypass circuit, thrombosis in the patient, and post-operative bleeding due to the consumption of hemostatic factors Finally, rhAT at a dose of 75 U/kg was well tolerated in the setting of hepaπn resistant patients undergoing cardiopulmonary bypass MATERIALS AND METHODS

Transgenic Goats & Cattle [00227] The herds of pure- and mixed- breed scrapie-free Alpine, Saanen and Toggenburg dairy goats used as cell and cell line donors for this study were maintained under Good Agncultural Practice (GAP) guidelines Similarly, cattle used should be maintained under Good Agncultural Practice (GAP) guidelines and be certified to oπginate from a scrapie and bovine encephalitis free herd

Pregnancy and Perinatal Care. [00228] For goats, pregnancy was determined by ultrasonography starting on day 25 after the first day of standing estrus. Does were evaluated weekly until day 75 of gestation, and once a month thereafter to assess fetal viability. For the pregnancy that continued beyond 152 days, parturition was induced with 5 mg of PGF2α (Lutalyse, Upjohn). Parturition occuπed within 24 hours after treatment. Kids were removed from the dam immediately after birth, and received heat-treated colostrum within 1 hour after delivery. Time frames appropriate for other ungulates with regard to pregnancy and perinatal care (e.g., bovines) are known in the art.

Cloned Animals. [00229] The present invention also includes a method of cloning a genetically engineered or transgenic mammal, by which a desired gene is inserted, removed or modified in the differentiated mammalian cell or cell nucleus prior to insertion of the differentiated mammalian cell or cell nucleus into the enucleated oocyte. [00230] Also provided by the present invention are mammals obtained according to the above method, and the offspring of those mammals. The present invention is preferably used for cloning caprines or bovines but could be used with any mammalian species. The present invention further provides for the use of nuclear transfer fetuses and nuclear transfer and chimeric offspring in the area of cell, tissue and organ transplantation. [00231 ] Suitable mammalian sources for oocytes include goats, sheep, co s, pigs, rabbits, guinea pigs, mice, hamsters, rats, primates, etc. Preferably, the oocytes will be obtained from ungulates, and most preferably goats or cattle. Methods for isolation of oocytes are well known in the art. Essentially, this will comprise isolating oocytes from the ovaries or reproductive tract of a mammal, e.g., a goat. A readily available source of ungulate oocytes is from hormonally induced female animals. [00232] For the successful use of techniques such as genetic engineering, nuclear transfer and cloning, oocytes may preferably be matured in vivo before these cells may be used as recipient cells for nuclear transfer, and before they can be fertilized by the sperm cell to develop into an embryo. Metaphase II stage oocytes, which have been matured in vivo, have been successfully used in nuclear transfer techniques. Essentially, mature metaphase II oocytes are collected surgically from either non-super ovulated or super ovulated animals sev eral hours past the onset of estrus or past the injection of human choπonic gonadotropin (hCG) or similar hormone [00233] Moreov er, it should be noted that the ability to modify animal genomes through transgenic technology offers new alternativ es for the manufacture of recombinant proteins The production of human recombinant pharmaceuticals in the milk of transgenic farm animals solv es many of the problems associated w ith microbial bioreactors (e g . lack of post-translational modifications, improper protein folding, high purification costs) or animal cell bioreactors (e g , high capital costs, expensiv e culture media, low yields) The current invention enables the use of transgenic production of biopharmaceuticals. transgenic proteins, plasma proteins, and other molecules of interest in the milk or other bodily fluid (1 e , unne or blood) of transgenic animals homozygous for a desired gene [00234] According to an embodiment of the current inv ention when multiple or successive rounds of transgenic selection are utilized to generate a cell or cell line homozygous for more than one trait such a cell or cell line can be treated w ith compositions to lengthen the number of passes a given cell line can w ithstand in in vitro culture Telomerase w ould be among such compounds that could be so utilized [00235] The use of v ing organisms as the production process means that all of the matenal produced w ill be chemically identical to the natural product In terms of basic amino acid structures this means that only L-optical isomers, having the natural configuration, will be present in the product Also the number of w rong sequences w ill be negligible because of the high fidelity of biological synthesis compared to chemical routes, in w hich the relative inefficiency of coupling reactions ill alw ays produce failed sequences The absence of side reactions is also an important consideration w ith further modification reactions such as carboxy-terminal amidation Again, the enzymes operating in

giv e a high degree of fidelity and stereospecificity which cannot be matched by chemical methods Finally the production of a transgenic protein of interest in a biological fluid means that low-lev el contaminants remaining in the final product are likely to be far less toxic than those onginating from a chemical reactor [00236] As previously mentioned, expression lev els of three grams per liter of ov ine milk are well within the reach of existing transgenic animal technology Such levels should also be achievable for the recombinant proteins contemplated by the cuπent inv ention [00237] In the practice of the present inv ention, obesity related transgenic proteins are produced in the milk of transgenic animals The human recombinant protein of interest coding sequences can be obtained by screening libraries of genomic material or rev erse-translated messenger RNA deriv ed from the animal of choice (such as cattle or mice), or through appropnate sequence databases such as NCBI, genbank, etc These sequences along w ith the desired polypeptide sequence of the transgenic partner protein are then cloned into an appropnate plasmid v ector and amplified in a suitable host organism, usually £ coli The DNA sequence encoding the peptide of choice can then be constructed, for example, by polymerase chain reaction amplification of a mixture of overlapping annealed oligonucleotides [00238] After amplification of the v ector, the DNA construct w ould be excised w ith the appropnate 5' and 3' control sequences, purified aw ay from the remains of the v ector and used to produce transgenic animals that hav e integrated into their genome the desired obesity related transgenic protein Conv ersely. with some v ectors, such as yeast artificial chromosomes (YACs), it is not necessary to remov e the assembled construct from the ector, in such cases the amplified v ector may be used directly to make transgenic animals In this case obesity related refers to the presence of a first polypeptide encoded by enough of a protein sequence nucleic acid sequence to retain its biological activity, this first polypeptide is then joined to a the coding sequence for a second polypeptide also containing enough of a polypeptide sequence of a protein to retain its physiological activity The coding sequence being operatively linked to a control sequence which enables the coding sequence to be expressed in the milk of a transgenic non-human placental mammal [00239] A DNA sequence which is suitable for directing production to the milk of transgenic animals carries a 5'-promoter region deπved from a naturally- deπv ed milk protein and is consequently under the control of hormonal and tissue- specific factors Such a promoter should therefore be most active in lactating mammary tissue According to the current inv ention the promoter so utilized can be followed by a DNA sequence directing the production of a protein leader sequence which would direct the secretion of the transgenic protein across the mammary epithelium into the milk At the other end of the transgenic protein construct a suitable 3'-sequence, preferably also denved from a naturally secreted milk protein, and may be added to improve stability of mRNA An example of suitable control sequences for the production of proteins in the milk of transgenic animals are those from the caprine beta casein promoter. [00240] The production of transgenic animals can now be performed using a variety of methods. The method preferred by the current invention is nuclear transfer.

Milk Specific Promoters. [00241 ] The transcriptional promoters useful in practicing the present invention are those promoters that are preferentially activated in mammary epithelial cells, including promoters that control the genes encoding milk proteins such as caseins, beta-lacto globulin (Clark et al., ( 1989) BIO/TECHNOLOGY 7: 487-492), whey acid protein (Gorton et al. (1987) BIO/TECHNOLOGY 5: 1 183- 1 187), and lactalbumin (Soulier et al., (1992) FEBS LETTS. 297: 13). Casein promoters may be derived from the alpha, beta, gamma or kappa casein genes of any mammalian species; a preferred promoter is derived from the goat beta casein gene (DiTullio, (1992) BIO/TECHNOLOGY 10:74-77). The milk-specific protein promoter or the promoters that are specifically activated in mammary tissue may be derived from either cDNA or genomic sequences. Preferably, they are genomic in origin. [00242] DNA sequence information is available for all of the mammary gland specific genes listed above, in at least one, and often several organisms. See, e.g., Richards et al., J. BiOL. CHEM. 256, 526-532 (1981 ) (α-lactalbumin rat); Campbell et al., NUCLEIC ACIDS RES. 12, 8685-8697 (1984) (rat WAP); Jones et al., J. BIOL. CHEM. 260, 7042-7050 (1985) (rat β-casein); Yu-Lee & Rosen, J. BIOL. CHEM. 258, 10794- 10804 (1983) (rat γ-casein); Hall, BIOCHEM. J. 242, 735-742 (1987) (α-lactalbumin human); Stewart, NUCLEIC ACIDS RES. 12, 389 (1984) (bovine ocsl and K casein cDNAs); Gorodetsky et al., GENE 66, 87-96 (1988) (bovine β casein); Alexander et al., EUR. J. BIOCHEM. 178, 395-401 (1988) (bovine K casein); Brignon et al., FEBS LETT. 188, 48-55 (1977) (bovine αS2 casein); Jamieson et al., GENE 61 , 85-90 (1987), Ivanov et al., BIOL. CHEM. Hoppe-Seyler 369, 425-429 (1988), Alexander et al., NUCLEIC ACIDS RES. 17, 6739 ( 1989) (bovine β lactoglobulin); Vilotte et al., BIOCHIMIE 69, 609- 620 (1987) (bovine α-lactalbumin). The structure and function of the various milk protein genes are reviewed by Mercier & Vilotte, J. DAIRY SCI. 76, 3079-3098 (1993) (incorporated by reference in its entirety for all puφoses). To the extent that additional sequence data might be required, sequences flanking the regions already obtained could be readily cloned using the existing sequences as probes Mammary-gland specific regulatory sequences from different organisms are likew ise obtained by screening branes from such organisms using known cognate nucleotide sequences, or antibodies to cognate proteins as probes

Signal Sequences. [00243] Among the signal sequences that are useful in accordance w ith this invention are milk-specific signal sequences or other signal sequences w hich result in the secretion of eukaryotic or prokaryotic proteins Preferably, the signal sequence is selected from milk-specific signal sequences. 1 e , it is from a gene which encodes a product secreted into milk Most preferably, the milk-specific signal sequence is related to the milk-specific promoter used in the expression system of this inv ention The size of the signal sequence is not cπtical for this invention All that is required is that the sequence be of a sufficient size to effect secretion of the desired recombinant protein, e g , in the mammary tissue For example, signal sequences from genes coding for caseins, e g , alpha, beta, gamma or kappa caseins, beta lactoglobuhn, whey acid protein, and lactalbumin are useful in the present inv ention The preferred signal sequence is the goat β-casein signal sequence [00244] Signal sequences from other secreted proteins, e g , proteins secreted by liver cells, kidney cell, or pancreatic cells can also be used

Amino-Terminal Regions of Secreted Proteins. [00245] The efficacy w ith which a non-secreted protein is secreted can be enhanced by inclusion in the protein to be secreted all or part of the coding sequence of a protein which is normally secreted Preferably the entire sequence of the protein which is normally secreted is not included in the sequence of the protein but rather only a portion of the ammo terminal end of the protein which is normally secreted For example, a protein which is not normally secreted is fused (usually at its amino terminal end) to an amino terminal portion of a protein which is normally secreted [00246] Preferably, the protein w hich is normally secreted is a protein which is normally secreted in milk Such proteins include proteins secreted by mammary epithelial cells, milk proteins such as caseins, beta lacto globulin, whey acid protein, and lactalbumin Casein proteins include alpha, beta, gamma or kappa casein genes of any mammalian species A prefeπed protein is beta casein, e g , a goat beta casein The sequences which encode the secreted protein can be deπv ed from either cDNA or genomic sequences Preferably, they are genomic in origin, and include one or more introns

DNA Constructs. [00247] The expression system or construct, described herein, can also include a 3' untranslated region downstream of the DNA sequence coding for the non-secreted protein This region apparently stabilizes the RNA transcπpt of the expression system and thus increases the yield of desired protein from the expression system Among the 3' untranslated regions useful in the constructs of this inv ention are sequences that provide a poly A signal Such sequences may be deriv ed, e g , from the SV40 small t antigen, the casein 3' untranslated region or other 3' untranslated sequences well know n in the art Preferably, the 3' untranslated region is derived from a milk specific protein The length of the 3' untranslated region is not critical but the stabilizing effect of its poly A transcnpt appears important in stabilizing the RNA of the expression sequence [00248] Optionally, the expression system or construct includes a 5' untranslated region between the promoter and the DNA sequence encoding the signal sequence Such untranslated regions can be from the same control region from which promoter is taken or can be from a different gene, e g , they may be den ed from other synthetic, semi-synthetic or natural sources Again their specific length is not critical, how ev er, they appear to be useful in improving the level of expression [00249] The construct can also include about 10%, 20%, 30%, or more of the N-terminal coding region of the gene preferentially expressed in mammary epithelial cells For example, the N-terminal coding region can coπespond to the promoter used, e g , a goat β-casein N-terminal coding region [00250] The above-descπbed expression systems may be prepared using methods well known in the art For example, various ligation techniques employing conventional linkers, restnction sites etc may be used to good effect Preferably, the expression systems of this invention are prepared as part of larger plasmids Such preparation allows the cloning and selection of the correct constructions in an efficient manner as is well known in the art Most preferably, the expression systems of this inv ention are located betw een conv enient restriction sites on the plasmid so that they can be easily isolated from the remaining plasmid sequences for incoφoration into the desired mammal [00251] Pnor art methods often include making a construct and testing it for the ability to produce a product in cultured cells prior to placing the construct in a transgenic animal Suφnsingly, the inv entors have found that such a protocol may not be of predictiv e v alue in determining if a normally non-secreted protein can be secreted, e g , in the milk of a transgenic animal Therefore, it may be desirable to test constructs directly in transgenic animals, e g , transgenic mice, as some constructs which fail to be secreted in CHO cells are secreted into the milk of transgenic animals

Sequence Production and Modification [00252] The inv ention encompasses the use of the descnbed nucleic acid sequences and the peptides expressed therefrom in v arious transgenic animals The sequences of specific molecules can be manipulated to generate proteins that retain most of their tertiary structure but are physiologically non-functional [00253] PCR technology may also be utilized to isolate full length cDNA sequences For example, RNA may be isolated, follow ing standard procedures, from an appropriate cellular or tissue source (l e , one kno n, or suspected, to express a target receptor gene, such as, for example from , skin, testis, or brain tissue) A reverse transcnption (RT) reaction may be performed on the RNA using an oligonucleotide pπmer specific for the most 5' end of the amplified fragment for the pnming of first strand synthesis The resulting RNA/DNA hybrid may then be "tailed" using a standard terminal transferase reaction, the hybrid may be digested with RNase H, and second strand synthesis may then be pnmed w ith a complementary primer Thus. cDNA sequences upstream of the amplified fragment may easily be isolated For a review of cloning strategies which may be used, see e g , Sambrook et al , 1989 [00254] A cDNA of a mutant target gene may be isolated, for example, by using PCR In this case, the first cDNA strand may be synthesized by hybndizing an ohgo-dT oligonucleotide to mRNA isolated from tissue know n or suspected to be expressed in an indiv idual putatively carrying a mutant target allele, and by extending the new strand with reverse transcnptase The second strand of the cDNA is then synthesized using an oligonucleotide that hybπdizes specifically to the 5' end of the normal gene Using these two pnmers, the product is then amplified via PCR, optionally cloned into a suitable v ector, and subjected to DNA sequence analy sis through methods well known to those of skill in the art. By comparing the DNA sequence of the mutant target allele to that of the normal target allele, the mutation(s) responsible for the loss or alteration of function of the mutant target gene product can be ascertained. [00255] Alternatively, a genomic library can be constructed using DNA obtained from an individual suspected of or known to carry the mutant target allele, or a cDNA library can be constructed using RNA from a tissue known, or suspected, to express the mutant target allele. A normal target gene, or any suitable fragment thereof, can then be labeled and used as a probe to identify the coπesponding mutant target allele in such libraries. Clones containing the mutant target gene sequences may then be purified and subjected to sequence analysis according to methods well known to those of skill in the art. [00256] Additionally, an expression library can be constructed utilizing cDNA synthesized from, for example, RNA isolated from a tissue known, or suspected, to express a mutant target allele in an individual suspected of or known to carry such a mutant allele. In this manner, gene products made by the putatively mutant tissue may be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against the normal target product. [00257] The invention also encompasses nucleotide sequences that encode mutant target receptor protein sequences, peptide fragments of the target receptor proteins, truncated target receptor proteins, and target receptor protein fusion proteins. These include, but are not limited to nucleotide sequences encoding mutant target receptor proteins described herein; polypeptides or peptides corresponding to one or more domains of the target receptor protein or portions of these domains; truncated target receptor protein in which one or more of the domains is puφosefully deleted, or a truncated non-functional target receptor protein so as to generate a puφosefully dysfunctional receptor protein. [00258] Puφosefully dysfunctional receptor proteins can be made and expressed in a transgenic system to provide a composition that can bind to physiological agents that would maintain obesity or work to increase weight gain.

Nucleotides encoding fusion proteins may include, but are not limited to, full length target receptor protein sequences, truncated target receptor proteins, or nucleotides encoding peptide fragments of a target receptor protein fused to an unrelated protein or peptide that will facilitate expression in a transgenic mammal or other transgenic animal expression model, such as for example, a target receptor protein domain fused to an Ig Fc domain which increases the stability and half-life of the resulting fusion protein in the bloodstream such that retains its ability to ameliorate obesity or related pathologies. [00259] The target receptor protein amino acid sequences of the invention include the amino acid sequences presented in the sequence listings herein as well as analogues and derivatives thereof. Further, corresponding target receptor protein homologues from other species are encompassed by the invention. The degenerate nature of the genetic code is well known, and, accordingly, each amino acid presented in the sequence listings, is generically representative of the well known nucleic acid "triplet" codon, or in many cases codons, that can encode the amino acid. As such, as contemplated herein, the amino acid sequences presented in the sequence listing, when taken together with the genetic code (see, pp 109, Table 4-1 of MOLECULAR CELL BIOLOGY, (1986), J. Darnell et al. eds., incoφorated by reference) are generically representative of all the various permutations and combinations of nucleic acid sequences that can encode such amino acid sequences. [00260] According to a prefeπed embodiment of the invention random mutations can be made to target gene DNA through the use of random mutagenesis techniques well known to those skilled in the art with the resulting mutant target receptor proteins tested for activity, site-directed mutations of the target receptor protein coding sequence can be engineered to generate mutant target receptor proteins with the same structure but with limited physiological function, e.g., alternate function, and/or with increased half-life. This can be accomplished using site-directed mutagenesis techniques well known to those skilled in the art. [00261 ] One starting point for such activities is to align the disclosed human sequences with coπesponding gene/protein sequences from, for example, other mammals in order to identify specific amino acid sequence motifs within the target gene that are conserved between different species. Changes to conserved sequences can be engineered to alter function, signal transduction capability, or both. Alternatively, where the alteration of function is desired, deletion or non-conservative alterations of the conserved regions can also be engineered. [00262] Other mutations to the target protein coding sequence can be made to generate target proteins that are better suited for expression, scale-up, etc. in the host cells chosen For example, cysteine residues can be deleted or substituted w ith another amino acid in order to eliminate disulfide bridges [00263] While the target proteins and peptides can be chemically synthesized, large sequences deriv ed from a target protein and full length gene sequences can be advantageously produced by recombinant DNA technology using techniques w ell know n in the art for expressing nucleic acid containing target protein gene sequences and/or nucleic acid coding sequences Such methods can be used to construct expression vectors containing appropnate transcnptional and translational control signals These methods include, for example, in \ ιtro recombinant DNA techniques, synthetic techniques, and in vι\ o genetic recombination Transgenic Mammals. [00264] Preferably, the DNA constructs of the inv ention are introduced into the germ-line of a mammal For example, one or several copies of the construct may be incoφorated into the genome of a mammalian embryo by standard transgenic techniques known in the art [00265] Any non-human mammal can be usefully employed in this inv ention Mammals are defined herein as all animals, excluding humans, w hich have mammary glands and produce milk Preferably, mammals that produce large v olumes of milk and have long lactating penods are prefeπed Preferred mammals are cows, sheep, goats, mice, oxen, camels and pigs Of course, each of these mammals may not be as effective as the others with respect to any given expression sequence of this invention. For example, a particular milk-specific promoter or signal sequence may be more effectiv e in one mammal than in others However, one of skill in the art may easily make such choices by following the teachings of this invention [00266] In an exemplary embodiment of the current invention, a transgenic non-human animal is produced by introducing a transgene into the germline of the non- human animal. Transgenes can be introduced into embryonal target cells at vanous developmental stages Different methods are used depending on the stage of development of the embryonal target cell The specific hne(s) of any animal used should, if possible, be selected for general good health, good embryo yields, good pronuclear v isibility in the embryo, and good reproductive fitness [00267] The litters of transgenic mammals may be assayed after birth for the incoφoration of the construct into the genome of the offspring Preferably, this assay is accomplished by hybndizing a probe corresponding to the DNA sequence coding for the desired recombinant protein product or a segment thereof onto chromosomal material from the progeny. Those mammalian progeny found to contain at least one copy of the construct in their genome are grown to maturity, The female species of these progeny will produce the desired protein in or along with their milk. Alternatively, the transgenic mammals may be bred to produce other transgenic progeny useful in producing the desired proteins in their milk. [00268] In accordance with the methods of the cunent invention for transgenic animals a transgenic primary cell line (from either caprine, bovine, ovine, porcine or any other non-human vertebrate origin) suitable for somatic cell nuclear transfer is created by transfection of the transgenic protein nucleic acid construct of interest (for example, a mammary gland-specific transgene(s) targeting expression of a transgenic protein to the mammary gland). The transgene construct can either contain a selection marker (such as neomycin, kanamycin, tetracycline, puromycin, zeocin, hygromycin or any other selectable marker) or be co-transfected with a cassette able to express the selection marker in cell culture. [00269] Transgenic females may be tested for protein secretion into milk, using any of the assay techniques that are standard in the art (e.g., Western blots or enzymatic assays). [00270] The invention provides expression vectors containing a nucleic acid sequence described herein, operably linked to at least one regulatory sequence. Many such vectors are commercially available, and other suitable vectors can be readily prepared by the skilled artisan. "Operably linked" or "operatively linked" is intended to mean that the nucleic acid molecule is linked to a regulatory sequence in a manner which allows expression of the nucleic acid sequence by a host organism. Regulatory sequences are art recognized and are selected to produce the encoded polypeptide or protein. Accordingly, the term "regulatory sequence" includes promoters, enhancers, and other expression control elements which are described in Goeddel, GENE EXPRESSION TECHNOLOGY : METHODS IN ENZYMOLOGY 185, (Academic Press, San Diego, Calif. (1990)). For example, the native regulatory sequences or regulatory sequences native to the transformed host cell can be employed. [00271 ] It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. For instance, the polypeptides of the present invention can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells or both (A L ABORATORY MANUAL, 2nd Ed , ed Sambrook et al (Cold Spnng Harbor Laboratory Press, 1989) Chapters 16 and 17)) [00272] Follo ing selection of colonies recombinant for the desired nucleic acid construct, cells are isolated and expanded, with aliquots frozen for long-term preser ation according to procedures know n in the field The selected transgenic cell- lines can be characterized using standard molecular biology methods (PCR. Southern blotting, FISH) Cell lines caπying nucleic acid constructs of the obesity related transgenic protein of interest, of the appropnate copy number, generally w ith a single integration site (although the same technique could be used w ith multiple integration sites) can then be used as karyoplast donors in a somatic cell nuclear transfer protocol know n in the art Follo ing nuclear transfer, and embryo transfer to a recipient animal, and gestation, liv e transgenic offspnng are obtained [00273] Typically this transgenic offspring carnes only one transgene integration on a specific chromosome, the other homologous chromosome not carrying an integration in the same site Hence the transgenic offspnng is heterozygous for the transgene, maintaining the cuπent need for at least tw o successiv e breeding cycles to generate a homozygous transgenic animal

Animal Promoters [00274] Useful promoters for the expression of obesity related in mammary tissue include promoters that naturally dnve the expression of mammary-specific polypeptides, such as milk proteins, although any promoter that permits secretion of obesity related into milk can be used These include, e g , promoters that naturally direct expression of w hey acidic protein (WAP), alpha S I -casein, alpha S2_:caseιn, beta-casein, kappa-casein, beta-lactoglobuhn, alpha-lactalbumin (see, e g , Drohan et al . U S Patent No 5,589,604, Meade et al , U S Patent No 4, 873,316, and Karatzas et al , U S Patent No 5,780,009), and others described in U S Patent No 5,750,172

Whey acidic protein (WAP, Genbank Accession No XOl 153), the major whey protein in rodents, is expressed at high lev els exclusiv ely in the mammary gland dunng late pregnancy and lactation (Hobbs et al , J BIOL CHEM 257 3598-3605, 1982) For additional information on desired mammary gland-specific promoters, see, e g ,

Richards et al , J BIOL CHEM 256 526-532, 1981 (α-lactalbumin rat), Campbell et al ,

NUCLEIC ACIDS RES 12 8685-8697, 1984 (rat WAP), Jones et al , J BIOL CHEM 260:7042-7050, 1985 (rat /3-casein); Yu-Lee & Rosen, J. BIOL. CHEM. 258: 10794- 10804, 1983 (rat γ-casein); Hall, BIOCHEM. J. 242:735-742, 1987 (human α- lactalbumin); Stewart, NUCLEIC ACIDS RES. 12:3895-3907, 1984 (bovine α-sl and K- casein cDNAs); Gorodetsky et al., GENE 66:87-96, 1988 (bovine /3-casein); Alexander et al., EUR. J. BIOCHEM. 178:395-401 , 1988 (bovine K -casein); Brignon et al., FEBS LETT. 188:48-55, 1977 (bovine α-S2 casein); Jamieson et al., GENE 61 :85-90, 1987, Ivanov et al., BiOL. CHEM. Hoppe-Seyler 369:425-429, 1988, and Alexander et al., NUCLEIC ACIDS RES. 17:6739, 1989 (bovine /3-lactoglobulin); and Vilotte et al., BIOCHIMIE 69:609-620, 1987 (bovine α-lactalbumin). The structure and function of the various milk protein genes are reviewed by Mercier & Vilotte, J. DAIRY SCI. 76:3079- 3098, 1993. [00275] If additional flanking sequences are useful in optimizing expression, such sequences can be cloned using the existing sequences as probes. Mammary-gland specific regulatory sequences from different organisms can be obtained by screening libraries from such organisms using known cognate nucleotide sequences, or antibodies to cognate proteins as probes. [00276] Useful signal sequences-for expression and secretion of obesity related into milk are milk-specific signal sequences. Desirably, the signal sequence is selected from milk-specific signal sequences, i.e., from a gene which encodes a product secreted into milk. Most desirably, the milk-specific signal sequence is related to a milk- specific promoter described above. The size of the signal sequence is not critical for this invention. All that is required is that the sequence be of a sufficient size to effect secretion of a target transgenic protein of use in the treatment of obesity, e.g., in the mammary tissue. For example, signal sequences from genes coding for caseins, e.g., alpha, beta, gamma, or kappa caseins, beta lactoglobulin, whey acidic protein, and lactalbumin are useful in the present invention. Signal sequences from other secreted proteins, e.g., proteins secreted by liver cells, kidney cell, or pancreatic cells can also be used. [00277] Useful promoters for the expression of a recombinant polypeptide transgene in urinary tissue are the uroplakin and uromodulin promoters (Ken et al., NAT. BIOTECHNOL. 16:75-79, 1998; Zbikowska, et al., BIOCHEM. J. 365 :7- 1 1 , 2002; and Zbikovvski et al., TRANSGENIC RES. 1 1 :425-435, 2002), although any promoter that permits secretion of the transgene product into urine may be used. [00278] A useful promoter for the expression and secretion of obesity related into blood by blood-producing or serum-producing cells (e g , liv er epithelial cells) is the albumin promoter (see, e g , Shen et al , DNA 8 101 - 108, 1989, Tan et al , DEV BIOL 146 24-37, 1991 , McGrane et al . TIBS 17 40-44, 1992, Jones et al , J BIOL CHEM 265 14684-14690, 1990, and Shimada et al . FEBS LETTERS 279 198-200, 1991). although any promoter that permits secretion of the transgene product into blood may be used The nativ e alpha-fetoprotein promoter can also be used (see, e g , Genbank Accession Nos AB053574, AB053573, AB053572, AB053571 , AB053570, and AB053569) Useful promoters for the expression of obesity related in semen are descnbed in U S Patent No 6,201 ,167 Useful av ian-specific promoters are the ov albumin promoter and the apo-B promoter [00279] Another three grams is produced in the liv er (serum hpoproteins) and deposited in the egg yolk In addition, since birds do not typically recognize mammalian proteins lmmunologically because of their ev olutionary distance from mammals, the expression of obesity related in birds is less likely to have any deleterious effect on the iability and health of the bird [00280] Other promoters that are useful in the methods of the inv ention include inducible promoters Generally, recombinant proteins are expressed in a constitutive manner in most eukaryotic expression systems The addition of inducible promoters or enhancer elements prov ides temporal or spatial control over expression of the transgenic proteins of interest, and provides an alternative mechanism of expression Inducible promoters include heat shock protein, metallothionien, and MMTV-LTR, while inducible enhancer elements include those for ecdysone, munsterone A, and tetracycline/ doxycyclme

Therapeutic Uses [00281 ] The combination herein is preferably employed for in

use in treating these tissue cultures The combination, however, is also be effectiv e for in \ι\o applications Depending on the intended mode of administration in

the compositions used may be in the dosage form of solid, semi-solid or liquid such as, e g , tablets, pills, powders, capsules, gels, ointments, liquids, suspensions, or the like

Preferably the compositions are administered in unit dosage forms suitable for single administration of precise dosage amounts The compositions may also include, depending on the formulation desired, pharmaceutically acceptable earners or diluents, which are defined as aqueous-based v ehicles commonly used to formulate pharmaceutical compositions for animal or human administration The diluent is selected so as not to affect the biological activity of the human recombinant protein of interest Examples of such diluents are distilled w ater, physiological saline, Ringer's solution, dextrose solution, and Hank's solution The same diluents may be used to reconstitute lyophilized a human recombinant protein of interest In addition, the pharmaceutical composition may also include other medicinal agents, pharmaceutical agents, earners, adjuvants, nontoxic, non-therapeutic, non-immunogenic stabilizers, etc Effective amounts of such diluent or earner will be amounts w hich are effectiv e to obtain a pharmaceutically acceptable formulation in terms of solubility of components, biological activ ity, etc [00282] The compositions herein may be administered to human patients via oral, parenteral or topical administrations and otherwise systemic forms for anti- melanoma and anti-breast cancer treatment

Bacterial Expression [00283] Useful expression v ectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter The vector will comprise one or more phenotypic selectable markers and an ongin of replication to ensure maintenance of the v ector and, if desirable, to provide amplification w ithin the host Suitable prokaryotic hosts for transformation include E coli , Bacillus subtihs, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomy ces, and Staphylococcus, although others may, also be employed as a matter of choice In a prefeπed embodiment, the prokaryotic host is £

[00284] Bactenal vectors may be, for example, bactenophage-, plasmid- or cosmid-based These vectors can compnse a selectable marker and bactenal ongin of replication denved from commercially available plasmids typically containing elements of the w ell known cloning v ector pBR322 (ATCC 37017) Such commercial vectors include, for example, GEM 1 (Promega Biotec, Madison, Wis , USA), pBs, phagescπpt, PsιX174, pBluescnpt SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene), pTrc99A, pKK223-3, pKK233-3, pKK232-8, pDR540, and pRIT5 (Pharmacia) A preferred v ector according to the inv ention is THE Pt7I expression v ector [00285] These "backbone" sections are combined w ith an appropnate promoter and the structural sequence to be expressed Bactenal promoters include lac, T3, T7. lambda PR or PL. tφ. and ara T7 is a prefeπed bacterial promoter [00286] Follow ing transformation of a suitable host strain and growth of the host strain to an appropnate cell density , the selected promoter is de-repressed/mduced by appropriate means (e g , temperature shift or chemical induction) and cells are cultured for an additional penod Cells are typically harv ested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification

Eukary otic Expression Vectors [00287] Vaπous mammalian cell culture systems can also be employed to express recombinant proteins Examples of mammalian expression systems include selected mouse L cells, such as thymidine kmase-negativ e (TK) and adenine phosphonbosul transferase-negativ e ( APRT) cells Other examples include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, CELL 23 175 ( 1981 ), and other cell lines capable of expressing a compatible v ector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines In particular, as regards yeasts, there may be mentioned yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula Among the fungi capable of being used in the present inv ention, there may be mentioned more particularly Aspergillus ssp, or Trichoderma ssp [00288] Mammalian expression vectors will compnse an ongin of replication, a suitable promoter and enhancer, and also any necessary πbosome binding sites, polyadenylation site, splice donor and acceptor sites, transcπptional termination sequences, and 5' flanking non-transcnbed sequences DNA sequences denv ed from the SV40 viral genome, for example, SV40 ongin, early promoter, enhancer, splice, and polyadenylation sites may be used to prov ide the required non-transcnbed genetic elements [00289] Mammalian promoters include beta-casein, beta-lactoglobu n, w hey acid promoter others include HSV thymidine kinase, early and late SV40, LTRs from retrov irus, and mouse metallothιoneιn-1 Exemplary mammalian vectors include pWLneo, pSV2cat, pOG44, pXTl , pSG (Stratagene) pSVK3. pBPV, pMSG. and pSVL (Pharmacia) In a prefeπed embodiment, the mammalian expression v ector is pUCIG- MET Selectable markers include CAT (chloramphenicol transferase) [00290] The nucleotide sequences w hich can be used ithin the framework of the present inv ention can be prepared in various w ays Generally, they are obtained by assembling, in reading phase, the sequences encoding each of the functional parts of the polypeptide The latter may be isolated by the techniques of persons skilled in the art, and for example directly from cellular messenger RNAs (mRNAs). or by recloning from a complementary DNA (cDNA) library, or alternatively they may be completely synthetic nucleotide sequences It is understood, furthermore, that the nucleotide sequences may also be subsequently modified, for example by the techniques of genetic engineenng, in order to obtain denvativ es or v aπants of the said sequences

Fluorescence In Situ Hy bridization (FISH) Analy sis. [00291 ] Standard culture and preparation procedures are used to obtain metaphase and inteφhase nuclei from cultured cells denv ed from animals carrying the desirable transgene Nuclei are deposited onto slides and ere hybndized w ith a digoxigenin-labeled probe denved from a construct containing 8kb of the genomic sequence for the obesity related protein of interest Bound probe w as amplified using a horseradish peroxidase-conjugated antibody and detected w ith tyramide-conjugated fluorescein isothiocyanate (FITC, green fluorochrome) Nuclei w ere counterstained with 4', 6-dιamιdιno-2-phenyhndole (DAPI, blue dye) FISH images were obtained using MetaMoφh soft are

Therapeutic Compositions. [00292] The proteins of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, w hereby the inventive molecules, or their functional denv atives, are combined in admixture with a pharmaceutically acceptable earner vehicle Suitable vehicles and their formulation, inclusive of other human proteins, e g , human serum albumin, are described, for example, in order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of one or more of the proteins of the present invention, together w ith a suitable amount of earner vehicle [00293] Pharmaceutical compositions for use in accordance with the present inv ention may be formulated in conv entional manner using one or more physiologically acceptable earners or excipients Thus, the obesity related molecules and their physiologically acceptable salts and solv ate may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration [00294] For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e g , pregelatinised maize starch, polyv iny lpyrrohdone or hydroxypropyl methylcellulose), fillers (e g , lactose, microcrystalhne cellulose or calcium hydrogen phosphate), lubncants (e g , magnesium stearate, talc or silica), disintegrants (e g , potato starch or sodium starch glycolate), or w etting agents (e g , sodium lauryl sulphate) The tablets may be coated by methods w ell know n in the art Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they maybe presented as a dry product for constitution w ith w ater or other suitable vehicle before use Such liquid preparations may be prepared by conv entional means with pharmaceutically acceptable additiv es such as suspending agents (e g , sorbitol syrup, cellulose denv atives or hydrogenated edible fats), emulsifying agents (e g , lecithin or acacia), non-aqueous vehicles (e g , almond oil, oily esters, ethyl alcohol or fractionated v egetable oils), and preservativ es (e g , methyl or propyl-p-hydroxybenzoates or sorbic acid) The preparations may also contain buffer salts, flavoπng, colonng and sweetening agents as appropnate [00295] Preparations for oral administration may be suitably formulated to give controlled release of the active compound For buccal administration the composition may take the form of tablets or lozenges formulated in conventional manner [00296] For administration by inhalation, the obesity related molecules for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressunzed packs or a nebulizer, with the use of a suitable propellant, e g , dichlorodifluoromethane, tnchlorofluoromethane, dichlorotetrafluoroethan- e, carbon dioxide or other suitable gas In the case of a pressunzed aerosol the dosage unit may be determined by prov iding a v alv e to dehv er a metered amount Capsules and cartndges of, e g gelatin for use in an inhaler or insufflator may be formulated containing a pow der mix of the compound and a suitable pow der base such as lactose or starch [00297] The obesity related transgenic proteins of the inv ention may be formulated for parenteral administration by injection, e g , by bolus injection or continuous lntransgenic Formulations for injection may be presented in unit dosage form, e g . in ampules or in multi-dose containers, w ith an added preser ativ e The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous v ehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents Alternativ ely, the activ e ingredient may be in povv der form for constitution with a suitable vehicle, e g , steπle pyrogen-free water, before use [00298] The compounds may also be formulated in rectal compositions such as suppositoπes or retention enemas, e g , containing conventional suppository bases such as cocoa butter or other glycendes [00299] In addition to the formulations described prev lously, the obesity related molecules may also be formulated as a depot preparation Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection Thus, for example, the compounds may be formulated with suitable polymenc or hydrophobic mateπals (for example as an emulsion in an acceptable oil) or ion exchange resins, or as spanngly soluble denv atives, for example, as a spanngly soluble salt [00300] The compositions may, if desired, be presented in a pack or dispenser device w hich may contain one or more unit dosage forms containing the active ingredient The pack may for example comprise metal or plastic foil, such as a blister pack The pack or dispenser device may be accompanied by instructions for administration

Treatment Methods. [00301 ] The inventive therapeutic methods according to the invention generally utilize the obesity related proteins identified above The domains of the transgenic proteins share the ability to specifically target a specific tissue and/or augment an immune response to targeted tissue A typical method, accordingly, involv es binding a receptor of a targeted cell to the receptor-antagonizing domain of the transgenic protein and/or stimulating a T-cell dependent immune response [00302] Therapeutic methods involve administering to a subject in need of treatment a therapeutically effective amount of a transgenic protein. "Therapeutically effective" is employed here to denote the amount of transgenic proteins that are of sufficient quantity to inhibit or reverse a disease condition (e.g., reduce or inhibit cancer growth). Some methods contemplate combination therapy with known cancer medicaments or therapies, for example, chemotherapy (preferably using compounds of the sort listed above) or radiation. The patient may be a human or non-human animal. A patient typically will be in need of treatment when suffering from a cancer characterized by increased levels of receptors that promote cancer maintenance or proliferation. [00303] Administration during in vivo treatment may be by any number of routes, including parenteral and oral, but preferably parenteral. Intracapsular, intravenous, intrathecal, and intraperitoneal routes of administration may be employed, generally intravenous is prefeπed. The skilled artisan will recognize that the route of administration will vary depending on the disorder to be treated. [00304] Determining a therapeutically effective amount specifically will depend on such factors as toxicity and efficacy of the medicament. Toxicity may be determined using methods well known in the art and found in the foregoing references. Efficacy may be determined utilizing the same guidance in conjunction with the methods described below in the Examples. A pharmaceutically effective amount, therefore, is an amount that is deemed by the clinician to be toxicologically tolerable, yet efficacious. Efficacy, for example, can be measured by the induction or substantial induction of T lymphocyte cytotoxicity at the targeted tissue or a decrease in mass of the targeted tissue. Suitable dosages can be from about 1 mg/kg to 10 mg/kg. [00305] The foregoing is not intended to have identified all of the aspects or embodiments of the invention nor in any way to limit the invention. The accompanying drawings, which are incoφorated and constitute part of the specification, illustrate embodiments of the invention, and together with the description, serve to explain the principles of the invention. [00306] All publications and patent applications mentioned in this specification are herein incoφorated by reference to the same extent as if each independent publication or patent application is specifically indicated to be incoφorated by reference. [00307] While the inv ention has been descnbed in connection with specific embodiments thereof, it w ill be understood that it is capable of further modifications and this application is intended to cov er any v aπations, uses, or adaptations of the inv ention follow mg, in general, the principles of the invention and including such departures from the present disclosure that come ithin know n or customary practice w ithin the art to w hich the inv ention pertains and may be applied to the essential features hereinbefore set forth

Literature Cited and Incorporated bv Reference:

1. Alberio R, et al , (2001 ). Mammalian Oocy te Activation Lessons from the Sperm and Implications for Nuclear Transfer, INT J DEV BIOL 2001 ; 45 : 797-809.

2. Andrew M.J. et al., Neuropsychological Dysfunction After Minimally Invasive Direct Coronary Artery Bypass Grafting, A NALS OF THORACIC SURGERY,

3. Arroyo. A. et al., (2000). In Vivo Roles oflntegrins During Leukocyte Development and Traffic Insights From the Analysis of Mice Chimeric For s,

and a<ι Integrins. THE JI 165 : 4667-4675.

4 Arrowsmith J.E. et al., Central Nervous System Complications of Cardiac Surgery , BRITISH JOURNAL OF ANAESTHESIA. 84(3)-378-93, (2000).

5. Baguisi A, (1999) et al., Production of Goats by Somatic Cell Nuclear Transfer. NATURE BIOTECH; 17: 456-461.

6. Buss M.I. et al., Cardiopulmonary Bypass, Rewarming, and Central Nervous System Dysfunction, ANNALS OF THORACIC SURGERY. 61 (5): 1423-7, (1996)

7. Carrascal Y. et al., Neurological Complications After Cardiopulmonary Bypass An Update, EUROPE ΛN NEUROLOGY, 41 (3): 128-34, ( 1999).

8. Despotis, G.J., et al., The Impact Of Heparin Concentration And Activated Clotting Time Monitoring On Blood Conservation, J.THOR. CARD. SURG., 1 10:46-54, (1995).

9. Despotis, G.J., et al., Antithrombin III During Cardiac Surgery: Effect On Response Of Activated Clotting Time To Heparin And Relationship To Markers Of Hemostatic Activation, ANESTHESIA AND ANALGESIA, (1997).

10. Despotis GJ, et al., Anticoagulation Monitoring During Cardiac Surgery - A Review Of Current And Emerging Techniques, AN'ESTHESIOLOGY, Vol. 91 , (1999).

1 1. Edmunds, T. et al., Transgenically Produced Human Antithrombin. Structural and Functional Comparison to Human Plasma-Derived Antithrombin, BLOOD, 12(June 15): 4561 -71 (1998).

12. Engelman R.M., et al., Influence of Cardiopulmonary Bypass Perfusion Temperature on Neurologic and Hematologic Function After Coronary Artery Bypass Grafting, ANNALS OF THORACIC SURGERY, 67(6): 1547- 1555, (1999).

13. Georgiadis D. et al., Predictive Value ofS-100 beta and Neuron-Specific Enolase Serum Levels for Adverse Neurologic Outcome after Cardiac Surgery, JOURNAL OF THORACIC & CARDIOVASCULAR SURGERY, 1 19( 1 ): 138-47, (2000). 14. Harrison M.J., Neurologic Complications of Coronary Artery Bypass Grafting: Diffuse or Focal Ischemia '*, ANNALS OF THORACIC SURGERY, 59(5): 1356-8, (1995).

15. Herczeg L. et al., Morphological Damage to the Central Nervous System (CNS) Follow ing Open Heart Surgery , FORENSIC SCIENCE INTERNATIONAL, 79(2): 103-1 1 , (1996).

16. Herrmann M. et al., Neurobehavioral Outcome Prediction after Cardiac Surgery : Role of Neurobiochemical Markers of Damage to Neuronal and Glial Brain Tissue, STROKE, 31 (3):645-50, (2000).

17. Inoue K. et al., Incidence and Risk Factors ofPenoperatne Cerebral Complications. Heart Transplantation Compared to Coronary Artery Bypass Grafting and Valve Surger . JOURNAL OF C ARDIOVASCULAR SURGERY, 39(2):201 -8, (1998).

18. Janssen D.P., et al., Predictors of Neurological Morbidity After Coronary Artery Bypass Surgery , EUR. J ADOLESC HEALTH, 16(3):397-8 ( 1999).

19. Johnsson P., Markers of Cerebral Ischemia After Cardiac Surgery, JOURNAL OF CΛRDIOTHORACIC & VASCULAR AN ESTHESIA, 10(1): 120-6, (1996).

20. Johnsson P. et al.. Cerebral Complications After Cardiac Surgery Assessed by S- 100 and NSE Levels in Blood, JOURNAL OF C.ARDIOTHORACIC & VASCULAR ANESTHESIA, 9(6):694-9, (1995).

21 . Kasinathan P,(2001 ) et al, Effect of Fibroblast Donor Cell Age and Cell Cycle on Development of Bovine Nuclear Transfer Embryos In Vitro, BlOL REPROD.; 64(5): 1487-1493.

22. Levy J.H. et al., Recombinant Antithrombin: Production and Role in Cardiovascular Disorder, SEMINARS IN THROMBOSIS AND HEMOSTASIS, 27(4): 405-16, (2001 ).

23. Meng L, et al., (1997) Rhesus Monkeys Produced by Nuclear Transfer, BlOL REPROD. Aug; 57(2):454-9.

24. Mickleborough L.L. et al., Risk Factors For Stroke In Patients Undergoing Coronary Artery Bypass Grafting, JOURNAL OF THORACIC & CARDIOVASCULAR SURGERY, 1 12(5): 1250-58, (1996).

25. Mortensen, R, et al., (1992) Production of Homozygous Mutant ES Cells with a Single Targeting Construct, MOL.CELL. BIOL. 12, 2391-2395.

26. Murkin JM., The Role of CPB Management In Neurobehavioral Outcomes After Cardiac Surgery, ANNA LS OF THORACIC SURGERY, 59(5): 1308- 1 1 , (1995).

27. Murkin JM., Etiology snd Incidence of Brain Dysfunction After Cardiac Surgery, JOURNAL OF C.ARDiOTHORACic & VASCULAR ANESTHESIA. 13(4 Suppl 1 ): 12-7; discussion 36-37, (1999). 28. Nagy, A, et al., (1996) Targeted Mutagenesis Analysis of Phenotype Without Germ-Line Transmission. J. CLIN. INV EST. 97. 1360-1365.

29. Newman M.F et al , Cardiopulmonary Bypass And The Centra! Nerv ous System - Potential For Cerebral Protection, JOURNAL OF CLINICAL ANESTHESIA. 8(3 Suppl).53S-60S, (1996).

30. New man M.F., et al , Cerebral Physiologic Effects Of Burst Suppression Doses Of Propofol During Nonpulsatile Cardiopulmonary Bypass, ANESTHESIA & ANALGESIA. 81 (3):452-57, (1995).

31. Newman M.F. et al.. (2001 ) Longitudinal Assessment of Neurocognitive Function After Coronary -Artery Bypass Surgery, NEW ENG. J. MED. 344(6), 395-402.

32. Ongen EM, et al., (2001 ) Development of Goat Embryos After In VitroFertilization and Parthenogenetic Activation by Different Methods, THERIOGENOLOGY Jun l ;55(9): 1933-45.

33. Rasmussen L.S. et al., Do Blood Levels of Neuron-Specific Enolase and S-100 Protein Reflect Cognitive Dysfunction After Coronary Artery Bypass? ACTA AN AESTHESIOLOGIC A SCANDINAV ICA, 43(5).495-500, ( 1999).

34. Roach G.W , et al., Ineffectiveness of Burst Suppression Therapy in Mitigating Perwperatn e Cerebrovascular Dysfunction, ANESTHESIOLOGY, 90(5): 1255-64, (1999).

35. REMINGTON'S PH ARM ΛCEUTICΛL SCIENCES (16^th ed., Osol, A., editor; Mack, Easton Press. (1980)).

36. Sambrook et al., MOLECULAR CLONING--A LABORATORY MAN UAL, Cold Spring Harbor Laboratory Press, (2nd Edition) (1989).

37. Seines O.A., et al., Neurobehavioural Sequelae of Cardiopulmonary Bypass, LANCET. 353(9164): 1601 -06, (1999).

38. Soloway. H.B , Chπstiansen, T.W., Heparin Anticoagulation During Cardiac Surgery in an A T III Deficient Patient, AM. J CLIN . PATHOL. 73: 723-5, 1980.

39. Stump, DA, et al. Cerebral Emboli and Cognitive Outcome After Cardiac Surgery, JOURNAL OF CARDIOTHORACIC AND VASCULAR ANESTHESIA, 10 (1 ): 1 13-19, ( 1996).

40. Subramanian VA., Less Invasive Arterial CABG On A Beating Heart, ANNALS OF THORACIC SURGERY, 63(6 Suppl):S68-71 , (1997).

41. Taylor KM., Central Nervous System Effects Of Cardiopulmonary Bypass, ANNALS OF THORACIC Surgery. 66(5 Suppl):S20-4; discussion S25-8, (1998).

42. Utley, J.R., Pathophysiology of Cardiopulmonary Bypass: Current Issues. JOURNAL OF CARDIAC SURGERY 5.3 : 177-89, 1990. 43. Videm V. et al., Hepann-Coated Cardiopulmonary_' Bypass Equipment I. Biocompatibility Markers And Development Of Complications In A High-Risk Population, JOURNAL OF THORACIC & CARDIOVASCULAR SURGERY. 1 17(4):794- 802, (1999).

44. Wilmut I, et al., (2002) Somatic Cell Nuclear Transfer, NATURE 10,419(6907):583- 6.

45. Wilmut I, et al., (1997) Viable Offspring Derived From Fetal and Adult Mammalian Cells, NATURE Feb 27;385(6619):810-3.

46. Zou X, et al. ,(2002) Generation of Cloned Goats (Capra Hircus) from Transfected Foetal Fibroblast Cells, The Effect of Donor Cell Cycle. MOL REPROD DEV. ; 61 : 164- 172.

Claims

What is claimed is

1 A method for the treatment of prophylaxis of a neurocogniti e disorder expenenced by a surgical patient comprising administeπng an effective amount of antithrombin

2 The method of claim 1 , w herein said antithrombin is human recombinant antithrombin

3 The method of claim 2, wherein said antithrombin is produced by a transgenic non-human animal or a plant

4 The method of claim 3, w herein said antithrombin is produced by a transgenic non-human mammal

5 The method of claim 3, wherein said antithrombin is produced by an ungulate

6 The method of either claims 3 or 5, wherein said transgenic animal is an ungulate selected from the group consisting of bovine, ovine, porcine, equine, caprine and buffalo

7 The method of claim 4, wherein said transgenic non-human mammal provides a donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus and that such donor cell nucleus is from an adult non-human mammalian somatic cell

8 The method of claim 3, wherein said non-human animal is a rodent

9 The method of claim 4, wherein said transgenic non-human mammal provides a donor differentiated mammalian cell to be used as a source of donor nuclei or donor cell nucleus is a non-quiescent somatic cell or a nucleus isolated from said non-quiescent somatic cell

10. The resultant offspring of the methods of claims 3 or 6.

1 1 . The method of claim 1 further comprising using a second pharmaceutical agent.

12. The method of claim 1 1 wherein said second pharmaceutical agent is a compound selected from the group consisting of: urokinase, PF4, alpha- fetoprotein, C-l esterase inhibitor, tPa, decorin, interferon, transferrin conjugates with biologically active peptides or fragments thereof, human serum albumin, thiopental sodium, thrombin, heparin, blood Factor X, blood Factor VIII, as well as monoclonal antibodies.

13. The method of claim 2 wherein said antithrombin is rhAT and is produced in milk.

14. The resultant milk derived from the offspring of the methods of claim 13.

15. The method of claim 1 wherein said surgical patient has undergone or is undergoing a surgical procedure selected from the group consisting of: heart transplantation, CABG, valve surgery and CABG simultaneously with valve surgery.

16. The method of claim 1 wherein said surgical patient has undergone a surgical procedure that has caused a central nervous system injury.

17. A method of treating a neurocognitive disorder or other disease condition medically related to said neurocognitive disorder comprising administering to a mammal in need of such treatment a therapeutically effective amount of a human recombinant antithrombin (rhAT) or a prodrug thereof or a pharmaceutically acceptable salt of said compound or of said prodrug.

18. A method as recited in claim 17 wherein the amount of said rhAT is about 0.01 mg/kg/day to about 50 mg/kg/day.

I l l

19. A recombinant protein as recited in claim 17 wherein the mammal is a human.

20. A recombinant DNA vector comprising the nucleic acid sequence of the transgenic protein of claim 17.

21. A host cell transformed with said recombinant DNA vector of claim 20.

22. The method of claim 17 wherein said disease condition is a neurocognitive disorder.

23. The method of claim 17 wherein said disease condition is an embolism.

24. The method of claim 17 wherein said disease condition is an parieto-occipital watershed area injury.

25. The method of claim 17 wherein said disease condition is a visuoconstruction related deficiency.

26. A method as recited in claim 17 wherein the amount of said rhAT is about 0.01 mg/kg day to about 50 mg/kg/day in conjunction with the administration an effective amount of a second pharmaceutical compound.

27. The method as recited in claim 26 wherein said second pharmaceutical compound is sodium pental.

28. The method as recited in claim 27 wherein the amount of said sodium pental is about 0.01 mg/kg/day to about 50 mg/kg/day.

29. The method of claim 2 wherein said rhAT administered to a surgical patient has a faster plasma clearance time and an increased affinity for heparin both compared to plasma derived human antithrombin.

30. The method of claim 2 wherein the activity of said rhAT is measured by a kit comprising neuron-specific enolase (NSE) and the S- 100 protein.

1. The method of claim 4 or 17, wherein said transgenic non-human mammal is generated through a nuclear microinjection procedure.