Thrombus-Targeted Complexes of Plasminogen Activator and Fibrin Fragments

Europa,schesP_ MM M II II II M INI MMMMI Ml J European Patent Office ^ _ _ _ © Publication number: 0 473 689 B1 Office_„. europeen des brevets

© Date of publication of patent specification: 14.12.94 © Int. CI.5: A61 K 37/54, A61 K 37/547, A61K 37/02, C12N 9/50, © Application number: 90908772.8 C12N 9/70 C12N 9/72 C07K 15/06 ©r\ Daten * of,„■ filing: 21.05.90o, neon

(54) THROMBUS-TARGETED COMPLEXES OF PLASMINOGEN ACTIVATOR AND FIBRIN FRAGMENTS.

® Priority: 24.05.89 US 356978 © Proprietor: Temple University of the Common- wealth System of Higher Education @ Date of publication of application: 11.03.92 Bulletin 92/11 Philadelphia PA 19122 (US)

© Publication of the grant of the patent: @ Inventor: BUDZYNSKI, Andrei, Z. 14.12.94 Bulletin 94/50 2050 Wharton Road Glenslde, PA 19038 (US) © Designated Contracting States: Inventor: KNIGHT, Linda, C. AT BE CH DE DK ES FR GB IT LI LU NL SE 158 Oak Creek Road East Windsor, NJ 08520 (US) © References cited: Inventor: HASAN, Ahmed, Ak. US-A- 4 427 646 885 North Easton Road US-A- 4 536 391 Apt. 4A6 US-A- 4 673 573 Glenslde, PA 19038 (US)

CIRCULATION, vol. 80, no. 4, suppl. 2, Octo- ber 1989, page II-642; A. HASAN etal.: "Bind- © Representative: W.P. THOMPSON & CO. Ing of one-chain and two-chain t-PA to fibrin Eastcheap House fragments" Central Approach 00 Letchworth, Hertfordshire SG6 3DS (GB) o> TAKEDA ET AL, BIOLOGICAL ABSTRACTS, 00 volume 86, number 7, Issued 01 October CO 1988 00

Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid (Art. 99(1) European patent convention). Rank Xerox (UK) Business Services (3. 10/3.09/3.3.3) EP 0 473 689 B1

BODE ET AL, J. Mol. Cell Cardiol., Issued 1987, vol.19, pages 335-341

BODE ET AL, The Journal of Biological Chemistry, Issued 05 August 1987, volume 262, number 22, see pages 10819-10823 deMUNK ET AL, Biochemistry, Issued 1989, vol.28, pages 7318-7325

NIEUWENHUIZEN ET AL, Biochimica et Blophyslca Acta, Issued 1983, vol. 755, pages 531-533

2 1 EP 0 473 689 B1 2

Description solve thrombi if administered locally, but local administration requires catheterization, which may of- The invention relates to novel agents for throm- ten preclude administration promptly enough to bolytic therapy, more particularly to hybrids formed provide significant benefit to the patient. Systemic by joining plasminogen activators and fibrin frag- 5 administration of streptokinase in acute myocardial ments. infarction causes an undesirable side effect by A safe, effective method of dissolving vascular degrading fibrinogen and severely depleting plas- thrombi is urgently needed, owing to the life-threat- minogen in the circulation. Mentzer et al., Am. J. ening complications of thromboembolic disease. Cardiol. 57:1220-1226 (1986). The most common of such disorders is the forma- io Another widely used plasminogen activator, tion of thrombi, in a blood vessel or heart cavity urokinase, is available in three forms. So called that remain at the point of formation. Thrombi in "two-chain urokinase" (hereinafter "urokinase") can heart vessels, for example, can restrict blood flow, be extracted from human urine or prepared from resulting in myocardial infarction. Coronary ar- kidney cell cultures. Another form, "single-chain teriographic studies indicate that 87% of transmural 75 urokinase" or "pro-urokinase" (hereinafter "scu- myocardial infarctions are caused by occlusive PA"), a precursor of urokinase, has been isolated coronary artery thrombosis. DeWood et al., N. Eng. from human fluids and also obtained by recom- J. Med. 303:397-902 (1980). binant DNA technology. A low molecular weight In addition, parts of a thrombus may dislodge form of urokinase is obtained from long plasmin from its point of attachment and move through the 20 digestion of two-chain urokinase. blood vessels until it reaches a point where pas- Although urokinase is not antigenic, its mecha- sage is restricted. The resulting blockage is known nism of plasminogen activation parallels that of as a thromboembolism. streptokinase, resulting in a significant Plasminogen activators function mainly by con- fibrinogenolytic response in the circulating blood. verting the zymogen plasminogen into plasmin, 25 Single-chain urokinase has promising characteris- which is regarded as the end stage non-specific tics. However, it has not yet been adequately test- proteolytic enzyme in fibrinolysis. Plasmin lyses ed in man. In a small study of normal human the fibrin matrix and acts on other components of subjects, scu-PA displayed fibrinolytic activity com- the thrombus as well. The efficiency of the lytic parable to urokinase, and a lower systemic action activity of exogenous plasminogen activator de- 30 than urokinase. Trubestein et al., Hemostasis pends significantly on the amount of plasmin it 17:238-244 (1987). generates from endogenous plasminogen, which is Non-urokinase plasminogen activators, which present in free-form in circulating blood and bound include tissue plasminogen activator ("t-PA"), are form on the fibrin surfaces of thrombi and hemo- physiologic activators of plasminogen which may static plugs. In any thrombolytic therapy, it is desir- 35 be manufactured by cell culture and genetic en- able to generate the maximum amount of plasmin gineering. t-PA is capable of binding fibrin, and is on the fibrin surface i.e., plasmin immobilized on believed to convert plasminogen to plasmin at the the thrombus, with a minimum quantity of plasmin fibrin surface. Thus, it works more efficiently than (if possible, none at all) in the circulating blood. streptokinase or urokinase when administered sys- While immobilized plasmin efficiently carries out 40 temically. However, initial clinical trials indicate that the lysis of the thrombus and helps establish reper- lysis of fibrinogen is still significant during admin- fusion, circulating plasmin exhibits fibrinogenolytic istration of t-PA. Sherry, N. Engl. J. Med. activity, degrades platelet receptors, digests throm- 313:1014-1017 (1985). bospondin, and consequently decreases the plate- Although it has high specificity for fibrin, t-PA let aggregation capacity of the patient. The result is 45 has certain limitations. It has a very short biological the so called "lytic state", which is accompanied half-life time in the circulation of humans, on the by various undesired complications. order of five minutes. Cellular clearance of t-PA by A variety of plasminogen activators have been the liver is believed responsible for this limited utilized to promote dissolution of thrombi and res- biological half-life time. Rapid decrease of t-PA in toration of blood circulation to blocked vessels. 50 the blood after intravenous administration consti- However, the available plasminogen activators are tutes a major difficulty in drug therapy with respect far from ideal. Streptokinase, which is of bacterial to maintaining an adequate concentration of the origin, is antigenic. A loading dose is needed to activator. neutralize preexisting anti-streptokinase antibodies. Plasma contains plasminogen activator inhibi- Streptokinase treatment must be limited in duration 55 tors which can bind and inactivate plasminogen because of an increase in such antibody levels. activators. Continuous infusion of large doses of t- Conard et al., Semin. Throm. Haemostas. 13:212- PA are therefore necessary to overcome the effect 222 (1987). Streptokinase works efficiently to dis- of these inhibitors. At physiologic levels, free t-PA

3 3 EP 0 473 689 B1 4 is bound by the plasma inhibitor, PAI-1. Wun et al., Fibrinogen must be treated with thrombin, in Blood 69:1354-1362 (1987). The latter has been order to maximize targeting to thrombi. Thus, the shown to be present in elevated levels in the plas- fibrinogen-urokinase hybrid of Patent 4,564,596 re- ma of patients with myocardial infarction, Hamsten quires the presence of a thrombotic state for op- et al., N. Engl. J. Med. 313:1557-1563 (1985); 5 timal performance. Moreover, fibrinogen is an ad- recurrent deep venous thrombosis, deJong et al., hesive protein, since it contains the amino acid Thromb. Haemostas. 57:140-143 (1987); and sequence Arg-Gly-Asp. Fibrinogen binds to a vari- coronary artery disease, Paramo et al., Br. J. Med. ety of cells, providing a bridge between cells. Con- 291:573-574 (1985). When purified t-PA is added to sequently, much of the injected fibrinogen- plasma to create higher-than physiologic levels to io urokinase hybrid may bind to cells in the circulat- overcome the inhibitory effect of PAI-1, it com- ing blood, making less hybrid available to bind to plexes with other inhibitors present in plasma, such thrombi. as C1 -inhibitor, alpha2-antiplasmin and macro- Human fibrin fragment Ei binds specifically to globulin. Thorsen et al., Blochem. Blophys. Acta polymers of fibrin, Olexa et al., Proc. Natl. Acad. 802:111-118 (1984); Rijken et al., J. Lab. Clin. is Scl. USA 77:1374-1378 (1980), and has the ability Med. 101:285-294 (1983); Kruithoff et al., Blood to bind to aged as well as fresh thrombi. Knight et 64:907-913 (1984). al., J. Clin. Invest. 72:2007-2013 (1983). ^-label- Administration of urokinase, streptokinase and ed fragment Ei has been used to detect venous t-PA results in an increased tendency for bleeding, thrombi in patients. Knight et al., Radiology limiting the usefulness of these agents, particularly 20 156:509-514 (1985). In addition to fragment Ei , during surgical procedures. fragment E2 and peptides having an amino acid Streptokinase and urokinase do not have inher- sequence intermediate between fragments Ei and ent fibrin specificity. They indiscriminately convert E2 may be labeled to detect thrombi, as disclosed both circulating and fibrin-surface bound plas- in U.S. Patent 4,427,646. minogen into plasmin at presently-used therapeutic 25 CNBr-digest fragments of fibrinogen have been dosages. While t-PA and scu-PA have fibrin speci- shown to accelerate the activation of t-PA. ficity, they have short half-life times in the blood. A Nieuwenhuizen et al, Blochem. Blophys. Acta high dosage must be infused over a long period of 755:531-533 (1983); Lijnen et al, Eur. J. Blochem. time in order to maintain a therapeutic concentra- 144:541-544 (1984); Nieuwenhuizen et al, tion in the blood. Consequently, hemostatic imbal- 30 Blochem. Blophys. Acta 748:86-92 (1983). How- ances are encountered. Moreover, since the speci- ever the fibrinogen fragments afford no protection ficities of t-PA and scu-PA for fibrin are not ab- from inactivation of t-PA by plasminogen activator solute, they leave a small amount of activators in inhibitors present in plasma. the liquid phase, and some amount of plasmin is What is needed is a thrombus-dissolving agent gradually produced in the circulating blood. 35 having more potent lytic activity than the known Complexes have been formed from urokinase plasminogen activators, but without the disadvan- and antifibrin murine monoclonal antibodies. Bode tages of such known activators. Ideally, a throm- et al., Science 229:765-767 (1985); Bode et al., J. bolytic agent should have very high fibrin specific- Biol. Chem. 262:10819-10823 (1987) and J. Mol. ity, and should immobilize itself on or near the Cell Cardiol. 19:335-341 (1987). In an In vitro 40 fibrin surface of a thrombus. It should activate only model of a thrombus in a circulating plasma loop, fibrin surface-bound plasminogen without activating the antibody-urokinase complex displayed a great- circulating plasminogen. In addition, a lytic agent er rate of fibrinolysis than an equivalent amount of should have a very high potential for plasmin gen- pure urokinase. However, therapeutic use of such eration on a thrombus surface, and have a pro- complexes is unattractive due to the immunogenic- 45 longed half-life time in the circulation so that ity of murine monoclonal antibodies. lengthy, high-dose infusions are not necessary. U.S. Patent 4,564,596 discloses urokinase de- The thrombolytic agent which has the potential to rivatives comprising urokinase covalently bonded to produce more plasmin from a given amount of fibrinogen, either directly or through a specific al- plasminogen will perform as a better lytic agent, iphatic diamine. The derivatives are reported to 50 because the required amount of plasmin concentra- have an increased affinity for fibrin and prolonged tion on the thrombus surface can be achieved by fibrinolytic effect. However, fibrinogen has pro- using a smaller amount of activator units. coagulant action, and may promote further devel- By "plasminogen activator" is meant any agent opment of thrombi. Thrombus-bound fibrinogen capable of activating the zymogen plasminogen to which is carrying urokinase will promote extension 55 the fibrinolytic enzyme plasmin. of the thrombus via either platelet aggregation or By "fibrin fragment" is meant any fragment fibrin deposition, acting locally as a procoagulant resulting from the single or sequential plasmin agent. This may lead to reocclusion of the vessel. cleavage of intact cross-linked or non-cross-linked

4 5 EP 0 473 689 B1 6 fibrin ("native fibrin fragment"), and any such frag- PA activity as a function of incubation time in ment which is further cleaved, derivatized or modi- plasma is expressed as a percentage of initial fied in any manner, while retaining a substantial activity. portion of the native fragment amino acid sequence Figure 3 is a plot of the fibrinolytic activity of t- ("non-native fibrin fragment"). 5 PA as a function of the molar ratio of fibrin frag- By the term "linked", as used herein in de- ment to t-PA. The t-PA was incubated for 5 hours scribing the union between a plasminogen activator in normal human plasma either alone or complexed and a fibrin fragment, is meant any form of chemi- with various ratios of fragment DD, (DD)E complex cal association or bond, including, but not limited to or fragment E3. The fibrinolytic activity remaining non-covalent complex formation, covalent bonding 70 at t = 5 hours as a function of the fibrin fragmentt- (including but not limited to covalent bonding by PA molar ratio (FDP:t-PA) is shown. means of one or more cross-linking agents), and Figure 4 is a plot of the duration of the the like. Also included in the scope of such associ- fibrinolytic activity of t-PA in plasma in the pres- ations is the formation of a unitary protein by ence of fragment DD, (DD)E complex, cyanogen genetic engineering, resulting from the co-expres- 75 bromide cleavage fragments of fibrinogen (CNBr), sion of genetic information for all or part of the or buffer (control). The experimental conditions of fibrin fragment and plasminogen activator as a sin- complex formation and incubation are the same as gle protein. those in Figure 2. A purified thrombolytic agent is provided com- Figure 5 is a plot of the duration of urokinase prising a fibrin fragment linked to a plasminogen 20 fibrinolytic activity in plasma in the presence of activator. The fibrin fragment may be a native frag- fragment DD, (DD)E complex, cyanogen bromide ment of fibrin, or a non-native fragment. Native cleavage fragments of fibrinogen (CNBr), or buffer fragments of fibrin include, for example, fragments (control). The experimental conditions of complex Ei, E2, E3, D and DD, and the (DD)E complex, formation and incubation are the same as those in which are obtained by plasmin degradation of 25 Figure 2. cross-linked and/or non-cross-linked fibrin. Non-na- Figure 6 is a plot of plasmin generation by t-PA tive fibrin fragments include, for example, smaller in the presence of fragment DD, (DD)E complex, fragments of the native fragments, such as pep- cyanogen bromide cleavage fragments of tides having amino acid sequences intermediate fibrinogen (CNBr), or buffer (control). Plasmin activ- between fragments Ei and E2. Non-native fibrin 30 ity was determined at various intervals from hy- fragments further include modified, synthetic or de- drolysis of the chromogenic substrate S-2251 . rivatized native fragments. Covalent or non-covalent Figure 7 is a plot of fibrin clot lysis by either t- complexes of such native or non-native fibrin frag- PA alone, or t-PA complexed with fragment DD or ments are formed with a plasminogen activator (DD)E complex in a 1:100 molar ratio. The lytic such as t-PA, streptokinase, urokinase or scu-PA. 35 agents were incubated in human plasma at 37 °C A thrombolytic composition is provided com- for 1 hour. The mixture was then supplemented prising a thrombolytic agent of the invention in an with 125 l-fibrinogen and clotted with thrombin. Clot amount sufficient for thrombolysis, and a phar- lysis, measured at various time intervals, is plotted maceutical^ acceptable carrier suitable for intra- as a percentage of initial values. The top curve venous administration. 40 (solid circles) was obtained using t-PA with no plasma preincubation. Brief Description of the Figures Detailed Description of the Invention Figure 1 is a plot of the binding of various concentration of one-chain t-PA to fibrin monomer 45 We have found that when a plasminogen ac- (FM), (DD)E complex, fragment DD or fragment Ei . tivator is linked to a fibrin fragment, the resulting The amount of t-PA bound to microtiter plates hybrid retains the ability of the parent fibrin frag- coated with fibrin monomer or fibrin fragments was ment to bind fibrin clots (thrombi), and also retains determined by enzyme-linked immunosorbent as- the ability of the parent plasminogen activator mol- say ("ELISA") measuring absorbance at 405 nm. 50 ecule to convert plasminogen to its active enzyme The plates were coated with bovine serum albumin form, plasmin. The fibrin fragment effectively ("BSA") as a negative control. serves as a vehicle for transporting the plas- Figure 2 is a plot of the duration of fibrinolytic minogen activator directly to the thrombus surface. activity of t-PA in plasma in the presence of frag- As a result, larger amounts of plasmin are gen- ment DD, (DD)E complex, or buffer (control). T-PA 55 erated In situ at the thrombus surface from the was complexed with a 500-fold molar excess of thus surface-bound plasminogen activator than either fragment DD or (DD)E complex and incu- from equivalent dosages of free plasminogen ac- bated in normal human plasma for up to 5 hours, t- tivator. Administration of the thrombolytic agents of

5 7 EP 0 473 689 B1 8 the invention thus mimics local administration of invention are not procoagulant. Moreover, unlike plasminogen activator, which has been previously the aforementioned prior art fibrinogen-containing shown to be about five times more effective than hybrids, the hybrids of the invention do not bind systemically-infused plasminogen activator. avidly to platelets, and do not promote platelet Because of the thrombus-targeting effect of the 5 aggregation. Thus, the instant thrombolytic hybrids fibrin fragment component, the hybrids of the in- do not promote reocclusion, which is primarily trig- vention are more efficient thrombolytic agents than gered by platelet aggregation and deposition. plasminogen activator alone. They produce more The hybrids of the invention, being formed plasmin per amount of endogenous plasminogen from fibrin fragments as opposed to fibrinogen, are than the corresponding uncomplexed plasminogen io smaller than the prior art fibrinogen-urokinase hy- activator molecules. They also generate more plas- brids, and therefore are believed to exhibit better min from fibrin-surface bound plasminogen than thrombus penetration. They do not require activa- the plasminogen activator molecule alone. Con- tion in the body by a thrombotic (or other) state for sequently, the dosage of plasminogen activator ad- targeting to thrombi. Since the fibrin fragments ministered as a complex according to the invention is either do not contain the Arg-Gly-Asp sequence, or may be reduced from the high levels of free plas- contain a greatly reduced amount in comparison to minogen activator presently being administered in fibrinogen, the hybrids of the invention should not thrombolytic therapy. The hybrids generate bind appreciably to cell surface receptors. They therapeutically useful levels of plasmin at the may be administered at lower doses than that thrombus site without significant systemic plas- 20 required for the fibrinogen-urokinase prior hybrids. minogen activation. The frequency and duration of Plasma constitutes a hostile environment for infusion of lytic agents of the invention is less than exogenous plasminogen activator. Cellular compo- that required for free plasminogen activator. nents of the blood and the hepatocytes of the liver Systemic activation of plasminogen results in rapidly take up exogenous plasminogen activator excess plasmin generated in circulating blood. This 25 from the circulation. In contrast to the short half-life causes degradation of fibrinogen, which results in of plasminogen activators in present use, the lytic lowering of platelet adhesion and aggregation. Ex- hybrids of the invention are protected, by virtue of cess circulating plasmin also degrades throm- the fibrin fragment component, from the inhibitors bospondin and other components of the hemostatic normally present in plasma, and from rapid uptake plug. These developments are indicative of a lytic 30 by the liver. Consequently, the thrombolytic agents state, which may give rise to bleeding complica- of the invention enjoy an increased half-life in the tions when vascular lesions and pre-existing im- circulation in comparison to naked plasminogen paired hemostatic plugs are present. Pre-existing activators. impaired hemostatic plugs are easily dissolved, The thrombolytic agents of the invention are which may lead to bleeding. Pre-existing vascular 35 formed by linking a plasminogen activator to a or epithelial defects may also become very vulner- fibrin fragment. Plasminogen activators may be pu- able to bleeding. Moreover, heparin therapy in rified from natural materials, may be produced by combination with conventional thrombolytic therapy cells grown in culture, or may be obtained by facilitates the development of bleeding complica- recombinant DNA technology. Plasminogen activa- tions in dose and time-dependent manner. 40 tors include, but are not limited to, streptokinase, The thrombolytic hybrids of the invention, be- urokinase, scu-PA, t-PA, and the like. cause of the thrombus-targeting property of the fibrin fragment component, minimize excess cir- Plasminogen Activators culating plasmin and the aforementioned complications. In addition, it is believed that the fibrin frag- 45 One class of plasminogen activators which may ment component of the complex may exert some be linked to fibrin fragments according to the inhibitory effect on fresh fibrin formation or propa- present invention includes products of bacterial ori- gation of the formed thrombus. Use of the throm- gin, such as streptokinase. Also known as strep- bolytic hybrids allows clinicians to substantially re- tococcal fibrinolysin, streptokinase is a protein produce the dosage of heparin, and significantly lower 50 duced by hemolytic streptococci. The purification the risk of bleeding complications during throm- of streptokinase is described in one or more of the bolytic therapy. It is believed that the thrombolytic following U.S. Patents, the complete disclosures of hybrids of the invention are unlikely to cause sig- which are incorporated herein by reference: nificant bleeding episodes, based upon the known 3,063,913; 3,063,914; 3,138,452; 3,276,304; mechanism of hemorrhagic complications following 55 3,016,337; 3,042,586; and 3,107,203. thrombolytic therapy. Another class of plasminogen activators which In contrast to the fibrinogen-urokinase hybrids may be linked to fibrin fragments according to the of Patent 4,564,596, the hybrids of the present invention includes any of the various plasminogen

6 9 EP 0 473 689 B1 10 activators distributed throughout mammalian urine, lecular weight 200-20,000) as described in U.S. blood or tissue. The two major groups include Patent 4,640,835, incorporated herein by reference. urokinase-type plasminogen activators, and non- Other known urokinase derivatives suitable for urokinase-type plasminogen activators. therapeutic purposes include, for example, com- The prototype urokinase-type plasminogen ac- 5 plexes of urokinase with heparin or dextran sul- tivator, urokinase, is formed in kidney tissue and phate, and urokinase derivatives wherein the car- excreted into urine. Several tissues and tumors in bohydrate structure of the native urokinase mol- tissue culture produce urokinase or a urokinase- ecule is modified to produce a longer-acting throm- type plasminogen activator. Urokinase is typically bolytic agent. Such carbohydrate-modified isolated from e.g., human urine or from tissue io urokinases are disclosed, for example, in U.S. Pat- culture of kidney cells, or is obtained by genetic ent 4,326,033, which is incorporated herein by ref- engineering. It may be isolated from human male erence. urine according to one or more of the following Non-urokinase plasminogen activators are pro- U.S. Patents, the entire disclosures of which are duced by human uterine tissue, as well as by incorporated herein by reference: 2,961,382; is certain tumor cells in culture. A subclass of non- 2,989,440; 2,983,647; and 3,081,236. Also see urokinase plasminogen activators known as "vascu- White et al., Biochemistry 5:2160 (1966). More lar" plasminogen activators (hereinafter collectively recent processes for isolation of urokinase are dis- "tissue plasminogen activator" or "t-PA"), appear closed in the following U.S. Patents, the entire to be produced by the human blood vessel wall. disclosures of which are incorporated herein by 20 These molecules activate plasminogen upon a reference: Re. 29,980, directed to a method for fibrin surface, whereas the urokinase-type plas- obtaining urokinase directly from human urine com- minogen activators do not require a fibrin clot net- prising contacting urine with an acrylonitrile poly- work to activate plasminogen to plasmin. mer of specific porous structure; Patent 4,259,447, Tissue plasminogen activator ("t-PA") exists in utilizing absorption with a porous solid matrix of 25 a form including a single protein chain ("one-chain specific surface area on which a protein is immo- t-PA"), and a two protein chain form ("two-chain t- bilized; 4,537,852; 4,259,448; 4,190,708; 3,957,582; PA"). The latter is proteolytically derived from the 3,930,945; 3,930,944; and 3,884,760. one-chain molecule. It is theorized that the two- There are three forms of native urokinase, all chain protein is associated with produced fibrin and having catalytic activity to convert plasminogen to 30 that proteolytic conversion from the one-chain to plasmin. The most intact is single-chain urokinase the two-chain material occurs at the locus of the (scu-PA) which is also called "prourokinase" (54 conversion of plasminogen to plasmin. Both forms kDa molecular weight). Plasmin cleaves this mol- may be utilized in the practice of the invention. ecule without a change in total molecular weight to Tissue plasminogen activator may be obtained form "two-chain urokinase", (typically referred to 35 from culture fluid of human or mammalian tissues simply as "urokinase"). The latter comprises an such as uterus, kidney, lung, and small intestine. about 22 kDa light chain linked to an about 32 kDa Normal or tumor cells are employed. Preparation of heavy chain by a disulfide bond. The heavy chain t-PA from various cultured normal human cells is contains the enzyme active site. Long plasmin di- disclosed in the following U.S. Patents, which are gestion of this 54 kDa high molecular weight 40 incorporated herein by reference: 4,335,215; urokinase form produces a low molecular weight 4,505,893; 4,537,860; and 4,550,080. Production of form of urokinase. The latter contains two chains, t-PA from normal human embryonic lung cells is one chain being approximately 32 kDa and the described by Rijken et al., J. Biol. Chem. 256(13)- other being approximately 2 kDa in molecular :7035-7041 (1981), incorporated herein by refer- weight. The 32 kDa chain corresponds to the heavy 45 ence. Pohl et al., FEBS Lett. 168(1 ):29-32 (1984), chain of high molecular weight urokinase. The two incorporated herein by reference, discloses produc- urokinase types do not differ significantly in in tion of t-PA from human uterine tissue. vitro enzymatic activity. Tissue plasminogen activator may also be ob- The present invention contemplates the linkage tained from tumor cells. For example, t-PA has of any of the various urokinase-type plasminogen 50 been prepared from Bowes melanoma cells in cul- activators to fibrin fragments to produce thrombus- ture according to Rijken et al., supra, and published targeted fibrinolytic hybrids. European Patent Application 41,766 (1981), incor- In addition to native urokinase-type plas- porated herein by reference. The Bowes cell t-PA minogen activators, the present invention contem- is a glycoprotein of about 68-70 kDa molecular plates the linkage of any modified urokinase-type 55 weight and 527 amino acids. It contains an A-chain plasminogen activator to a fibrin fragment. One and a B-chain. Two variants, known as types I and such modified urokinase-type molecule is obtained II, differ in the A-chain by about 2-3 kDa. Ranby et by coupling urokinase to polyalkylene glycol (mo- al., FEBS Lett. 146(2): 289-292 (1982); Wallen et

7 11 EP 0 473 689 B1 12 al., Eur. J. Biochem. 132:681-686 (1983). The of thrombin. Fibrin forms the clot matrix which is protein moiety of the melanoma tissue plas- cross-lined by Factor Xllla to form a stabilized clot. minogen activator has been cloned and expressed Human cross-linked fibrin is degraded by plasmin, in E. coll. See published British Patent Application thereby releasing characteristic degradation pro- 2,119,804; Pennica et al., Nature 301:214-221 5 ducts: (DD)E complex, fragment DD, fragment E (1983). Recombinant t-PA produced by expressing and alpha polymer remnants. The (DD)E complex Bowes melanoma genes in cultured mammalian contains fragment Ei and fragment DD. The (DD)E cells has been used in therapy. U.S. Patent complex is susceptible to further degradation by 4,661,453, incorporated herein by reference, dis- plasmin resulting in cleavage of fragment Ei to closes t-PA obtained from prostate adenocarcino- 10 fragment E2 without loss of the ability to bind ma cells. fragment DD. Digestion of fragment E2 to E3 re- Recombinant t-PA is disclosed in U.S. Patent sults in dissociation of the complex. The terminal 4,766,075, incorporated herein by reference. plasmin degradation products of cross-linked fibrin While recombinant t-PA derived from E. coll is are therefore fragments DD and E3. The complete non-glycosylated, and contains only the protein 15 reaction scheme is thus: fibrin --> (DD)Ei --> (DD)- moiety of t-PA, the present invention contemplates E2 --> DD + E3. the use of either glycosylated or non-glycosylated The fibrin fragments which may be linked to a t-PA to form the thrombus-targeted hybrids of the plasminogen activator to form the thrombus-tar- invention. Glycosylated t-PA may be obtained from geted complexes of the invention may be obtained non-human sources such as Chinese hamster ova- 20 by cleavage of cross-linked or non-cross-linked ry cells according to published European Patent fibrin. Such fragments have affinity for fibrin and/or Application 117,059 (1984) and Kaufman et al., protect the plasminogen activator to which they are Mol. Cell. Biol. 5:1750-1759 (1985), and from bound from inactivation in plasma. The fibrin frag- mouse L cells according to Browne et al., Gene ments also enhance the activity of the plasminogen 33:279-284 (1985), all incorporated herein by refer- 25 activator molecule. They may also exert an inhibi- ence. Glycosylated t-PA may be produced by re- tory effect on fresh fibrin formation or propagation combinant DNA techniques according to Zamarron of the formed thrombus. Fragments Ei , E2 and DD, et al., J. Biol. Chem. 259(4):2080-2803 (1984) and and (DD)E complex are particularly useful for bind- Collen et al., J. Pharmacol. Expertl. Therap. 231- ing with t-PA or scu-PA to form thrombolytic com- (1 ):1 46-1 52 (1984), incorporated herein by refer- 30 plexes. ence. Published European Patent Applications Native fibrin fragments useful in the practice of 143,081 (1985) and 174,835 (1986) disclose the the present invention include, for example, frag- expression of t-PA in yeast by recombinant tech- ments Ei , E2, E3, D and DD, and the (DD)E com- niques, while published European Patent Applica- plex. The various plasmin degradation products of tion 178,105 (1986) discloses a glycosylated uter- 35 fibrin, and their preparation, is reported in Olexa et ine t-PA expressed in mouse or yeast cells. The al., Biochemistry 18:991 (1979) and J. Biol. aforesaid European patent applications are incor- Chem. 254:4925 (1979), both of which are incorporated herein by reference. U.S. Patent 4,751,084 porated herein by reference. discloses glycosylated t-PA from normal human The E fragments comprise the plasmic colon fibroblast cell line CCD-18Co, ATCC CRL- 40 cleavage products of human cross-linked fibrin. 1459. They contain the NH2 -terminal regions of all six These and other native and modified t-PA mol- polypeptide chains of fibrin. The amino acid se- ecules known to those skilled in the art have utility quences of the various fragments E have been in the practice of the present invention. established. Olexa et al., Biochemistry 21:6139- Also useful in the practice of the invention is 45 6145 (1981). At least three species of fragment E the plasminogen activator known as "angiokinase", have been isolated and characterized, namely frag- the purification of which from highly vascularized ments Ei , E2 and E3, having molecular weights of connective tissue of mammals is disclosed in U.S. about 60 kDa, 55 kDa and 50 kDa, respectively. Patent 4,532,129, incorporated herein by reference. These species are sequential degradation products. While any plasminogen activator may be em- 50 Micro-heterogeneity of each species has been ployed in the practice of the invention t-PA and noted. Fragments Ei and E2, and peptides having scu-PA are preferred, owing to their superior an amino acid sequence intermediate between thrombus specificity. fragments Ei and E2, have affinity for preformed clots as disclosed in U.S. Patent 4,427,646. Each Native Fibrin Fragments 55 such peptide is useful in forming thrombus-targeting fibrinolytic hybrids. Fibrin is formed by cleavage of its precursor, Fragment Ei is particularly useful as a throm- the soluble plasma protein fibrinogen, by the action bus-targeting vehicle for plasminogen activators.

8 13 EP 0 473 689 B1 14

Fragment Ei specifically binds to polymers of the fibrin deposit in a thrombus will be higher than fibrin, Olexa et al., Proc. Natl. Acad. Scl. USA the affinity of the plasminogen activator for any of 77:1374-1378 (1980). It has the advantage of lower these fragments, so that the activator will dissociate likelihood of antigenicity than the murine mon- from the carrier fragment in the vicinity of the oclonal antibodies used in the prior art, because it 5 thrombus and bind to the fibrin to activate plas- is a fragment of a native human protein. Moreover, minogen on the surface of the thrombus. Moreover, fragment Ei has been shown to have the ability to while (DD)E complex does not bind fibrin, it has a bind to aged as well as fresh thrombi. Knight et al., potentiating effect on the lytic activity of the plas- J. Clin. Invest. 72:2007-2013 (1983). The binding minogen activators. Thus, the (DD)E complex may of fragment Ei to thrombi in patients with docu- io be efficiently used as a shield for the protection of mented venous thrombosis, Knight et al., Radi- the plasminogen activator molecule and therefore ology 156:509-514 (1985), demonstrates the use- enhance the activity of the plasminogen activator fulness of this fragment as a molecular marker for molecule. specifically targeting thrombi In vivo. It should be noted that while the native fibrin The basic process for the preparation of the is fragments Ei , E2, E3, DD and (DD)E are obtained various native plasmin degradation products of through degradation of cross-linked fibrin, fragment fibrin, as described in U.S. Patent 4,427,646, is as D is obtained from non-cross-linked fibrin. Frag- follows. A fibrin clot from fibrinogen enriched with ment D may also be obtained as a monomeric Factor XIII is formed. The clot is hydrolyzed with plasmic degradation product of fibrinogen. plasmin and the resulting digest is centrifuged to 20 The preparation of fibrin for the generation of remove large clot particles. The supernatant con- fibrin fragments, and for the preparation of a fibrin- tains soluble degradation products, which are sepa- diatomaceous earth affinity matrix utilized in the rated according to molecular weight, preferably on purification of fibrin fragments and/or fibrinolytic an agarose gel bead column, to give the (DD)E complexes, is described as follows. complex. Fragments Ei and E2 may be obtained 25 from the purified (DD)E complex by incubation in a Preparation of Fibrin concentrated salt solution to cause dissociation of fragment DD and fragments Ei and E2, followed by Sonicated human cross-linked fibrin may be separation according to molecular weight, prefer- prepared from plasminogen- and thrombin-free hu- ably by means of an agarose gel bead column. 30 man fibrinogen according to Lukas et al, J. Biol. Fragment DD, a dimeric degradation product of Chem. 258:4249-4256 (1983), incorporated herein fibrin, is capable of binding to fibrin polymers and by reference. Briefly, purified fibrinogen is clotted fibrin monomers but does not bind fibrinogen. It is with thrombin at a ratio of one unit per mg of more resistent to plasmic degradation than frag- fibrinogen in the presence of calcium ions. The ment Ei, Olexa et al, J. Biol. Chem. 254:4925- 35 fibrin clot is then exposed to sonication in an 4932 (1979), and because of its larger size may appropriate cell disrupter (e.g. from Heat Systems- offer greater protection of the plasminogen activa- Ultrasonics, Plainview, NY) with ice cooling. tor and a longer residence time in the blood than Fragment Ei . Preparation of Non-Cross-Linked And Cross- The fragments generally known as "fragment 40 Linked Flbrln-Dlatomaceous Earth D" comprise a group of fibrinogen or fibrin derivatives characterized by the same immunologic reac- Fibrin-diatomaceous earth comprises a fibrin tion and similar three-chain structure. They are clot immobilized on diatomaceous earth particles, monomeric plasmic degradation products of which serve as nuclei for small fibrin clot assem- fibrinogen, which are also obtained by plasmin 45 blies. The non-cross-linked and cross-linked fibrin- cleavage of non-cross-linked fibrin. They bind to a diatomaceous earth utilized as an affinity matrix site available on fibrin monomer, but not available material in the following procedure may be pre- on fibrinogen. Fragment Di has the highest molec- pared as follows: ular weight (100 kDa) of the various D fragments. Non-cross-linked fibrin-diatomaceous earth is As used herein, unless otherwise noted, "fragment 50 prepared essentially as described by Husain et al., D" means any of the various D fragments, frag- Proc. Natl. Acad. Scl. USA 78:4265-4269 (1981), ments such as Di , D2 and D3. with the following modifications. After removing fine While the (DD)E complex does not bind to particles from the starting diatomaceous earth fibrin, it is believed that this complex nevertheless (Hyflo Super Cel, Fluka Chemical Corp., Ronkon- offers a protective effect to the plasminogen activa- 55 koma, NY) by repeated suspension in water, the tor such that the latter may be transported safely to diatomaceous earth material is washed twice, each a thrombus. It is further believed that the affinity of time by gently stirring with one liter of 1 M NaCI for certain plasminogen activators, such as t-PA, for two hours. This treatment removes material present

9 15 EP 0 473 689 B1 16 in some batches of diatomaceous earth that would dibromopropanol.) Approximately 200-250 mg of otherwise retard clotting of the fibrinogen. To ob- protein in a volume of 10-15 ml is applied in each tain fibrin-diatomaceous earth, plasminogen-free run and the flow rate is adjusted to about 40-45 fibrinogen is clotted by thrombin in the presence of ml/hour. Fractions of 8 ml within main peaks are diatomaceous earth and then washed on a Buchner 5 combined and concentrated and subsequently ana- funnel with 0.05 M Tris-HCI buffer, containing 0.15 lyzed on SDS-PAGE and alkaline-PAGE. The main M NaCI, pH 7.4. Fibrin clots which form become (second) peak fractions contain pure homogeneous incorporated into the particles. Residual thrombin is (DD)E complex. The preparation is concentrated by inhibited by D-Phe-Pro-Arg-chloromethylketone (al- ultrafiltration and pooled. biochem-Behring Corp. San Diego, CA). Cross- 10 linked fibrin-diatomaceous earth is prepared by Preparation Of Fragment DD suspending washed, non-cross-linked fibrin-diatomaceous earth in Tris buffer, containing 5 mM Fragment DD is prepared in a similar manner CaCb and 1 M mercaptoethanol, and incubating but with a different proportion of plasmin to dry the suspension at 20 °C for 15 hours. The cross- is fibrin. To achieve a maximum yield of fragment linked fibrin-diatomaceous earth is subsequently DD, 1 g of dry cross-linked fibrin is digested by 10 washed in a column. Sodium dodecylsulfate CTA units of plasmin (10 CTA units/1 g fibrin) for polyacrylamide gel electrophoresis ("SDS-PAGE") strictly 21.5 hrs. Then digestion is terminated by 10 may be used to show complete cross-linking of the ml (100 KIU/1 CTA unit of plasmin) of aprotinin and alpha and gamma fibrin chains. 20 the reaction mixture is stored frozen until control The preparation and characterization of certain SDS-PAGE is performed. The digest is then cen- pure, homogeneous soluble fibrin fragments is il- trifuged at 3000 g for 15 minutes at 4°C and the lustrated as follows. supernate is then chromatographed on the same type of column as used in the above-identified Preparation Of (DD)E Complex 25 (DD)E complex preparation, equilibrated and run by the same buffer system as above, but with a higher (DD)E complex may be prepared from purified molar concentration of sodium chloride (1 M NaCI), human cross-linked fibrin as described in Olexa et pH 7.4. The fractions (8 ml) within the main peak al, Biochemistry 18:991-995 (1979). Accordingly, are combined and analyzed on SDS-PAGE. The 1 g of dry cross-linked fibrin and 1 CTA unit of 30 main peak fractions contain relatively pure and plasmin (1 CTA unit/1 g fibrin; Kabi, Stockholm, homogeneous fragment DD. The sample is con- Sweden) are suspended together as quickly as centrated and pooled. It is observed that the tail of possible in 20 ml of prewarmed 0.15 M Tris-HCI the main peak also contains a significant amount of buffer containing 5 mM CaCb and 0.02% sodium fragment DD with traces of impurities of fragments azide, pH 7.4. (A "CTA unit" is the Committee on 35 Di and E3. These fractions are purified on an anti- Thrombolytic Agents standard unit which defines E affinity column (Anti-E Sepharose®) and run the proteolytic activity of plasmin by its action on a through a fibrin-diatomaceous earth affinity column casein substrate.) The digestion is carried out un- to pool the fragment with total preservation of bind- der constant gentle stirring at 37 ° C for strictly 21 .5 ing affinity for fibrin. hours for maximum yield of (DD)E complex. The 40 digestion is then stopped by addition of 0.01 ml of Preparation of Fragments Ei 1 1 and E222 aprotinin (Trasylol®, Bayer & Co.) to a final concentration of 100 KIU/1 CTA unit of plasmin. (The About 50 mg of (DD)E complex is dissociated potency of aprotinin as an enzyme inhibitor is with 3 M urea, pH 5.5 for 1 hr at 37 °C. The calibrated according to the amount of kallikrein it 45 resulting species comprise denatured fragment DD can inhibit. Hence its activity is expressed in units and native and functionally active fragment Ei . The of kallikrein inhibitor units per ml or "KIU/ml".) The sample is then chromatographed on a Sepharose® digest is stored frozen and control SDS-PAGE and CL-6B (0.9 x 90 cm) column, equilibrated and alkaline-PAGE are performed. The small amount of eluted with the same buffer system as employed in particulate material is removed by centrifugation at 50 the fragment DD separation. Denatured fragment 3,000 g for 15 minute at 4°C. The digest super- DD and fragments E (Ei and/or E2) elute in two natant is then chromatographed on a Sepharose® markedly separated peaks. Control SDS-PAGE is CL-6B column (Pharmacia, Piscataway, NJ; 2.5 x performed. Fragments E are concentrated and 190 cm) equilibrated and eluted with 0.05 M Tris- pooled. The preparation contains about 25% frag- HCI buffer containing 0.1 M sodium chloride, 0.028 55 ment E2 and 75% fragment Ei . The E species are M sodium citrate, 25 KIU/ml of aprotinin and 0.02% separated by fast liquid protein chromatography sodium azide, pH 7.4. (Sepharose® CL is highly ("FPLC") on a Mono-Q® an HR5/5 ion exchange purified agarose which has been treated with column. (5mm x 5cm glass column packed with 1

10 17 EP 0 473 689 B1 18 ml of Mono Q® anion exchange resin; ion capacity Synthetic peptides containing a portion of the = 0.27-0.37C1-. Mono Q® anion exchange resin native fibrin fragment are also useful in the practice comprises the relatively strong anionic exchanges of the invention. One such fragment is the function, -CH2N + (CH3)3, on a base of mon- gamma39i -gamma405 fragment described by Cier- odisperse hydrophilic polymer particles. Negatively 5 niewski et al, J. Biol. Chem. 262:13896-13901 charges species are bound through the quaternary (1987), incorporated herein by reference. Other amine groups, which remain negatively charged synthetic peptides comprising portions of native over the pH range 2-12. To isolate functionally fibrin degradation fragments are known to those intact fragment Ei , affinity chromatography on skilled in the art. fibrin-diatomaceous earth is performed. io Derivatized fragments may be formed by reac- ting a fibrin fragment with, e.g. methyl iodide, ac- Preparation of Fragment E ylating agents, carbohydrates, carbodiimides, etc. which may be used to facilitate alteration of one or Fragment E3 is obtained from a terminal plas- more properties, such as prolongation of biological mic digest of cross-linked fibrin by column gel is half-life. filtration according to Olexa et al, Biochemistry Derivatization may also include blockage of the 18:911-995 (1979). Accordingly, cross-linked fibrin plasminogen binding site on the fibrin fragment is digested by 100 CTA units plasmin/1 g fibrin for with an appropriate blocking agent. Some fibrin 24 hours and then gel-filtered on a Sepharose® degradation products retain intact binding sites for CL-6B column as for fragment DD. Fragment E3, 20 plasminogen. Hybrids formed from such fragments the second peak, is separated from fragment DD could theoretically bind plasminogen from the sys- and alpha polymer remnant present in the first and temic circulation and generate plasmin in the cir- third peaks, respectively. The purity and homo- culating blood before immobilizing on a clot sur- geneity of the fragment are tested on SDS-PAGE. face. This may lead to undesirable fibrinogenolytic The fractions are pooled, then concentrated by 25 activity in the circulation. To prevent this, the plas- ultrafiltration and subsequently purified on an anti-E minogen binding site on the fibrin fragment is bloc- affinity column. ked. Blockage of the plasminogen binding site may Preparation of Fragment D be accomplished in several ways. Inactive plasmin 30 derivatives may be prepared by treating plasmin Fragment D is prepared by a prolonged plas- with, e.g., diisopropyl fluorophosphate, or other mic digestion of non-cross-linked fibrin in the pres- plasmin-inactivating agent. Such inactivated plas- ence of 25 mM CaCb and purified by Pevikon min derivatives may be non-covalently bound to a block preparative electrophoresis according to Mar- fibrin fragment to block the fragment's plasminogen der et al, Throms. Dlath. Haemorrh. 22:234-239 35 binding site. According to one method, plasmin (1969), incorporated herein by reference. A mixture molecules are inactivated by inhibitors such as of fragments D2 and D3 is obtained from this phenylmethylsulfonyl fluoride ("PMSF") and procedure in the absence of 25 mM CaCb. isopropyl-fluorophosphate ("DFP"). The inhibitors inactivate the plasmin molecule's enzyme active Non-Native Fibrin Fragments 40 site. These inactivated plasmin molecules are then used to saturate the fibrin fragment. The deacti- As an alternative to native fibrin fragments, vated plasmin molecules, which bind to the fibrin non-native fibrin fragments may be linked to an fragment via their fibrin-binding function, may thus appropriate plasminogen activator to form throm- block all the plasminogen-binding sites on the fibrin bus-targeting fibrinolytic hybrids. Such non-native 45 fragment. The plasmin molecules so bound will not fibrin fragments include modified plasmin degrada- express their enzyme activity due to their prior tion fibrin fragments, such as heat-denatured, inactivation with inhibitor. CNBr-modified or acid-denatured fragments; syn- Alternatively, the fibrin fragment's plasminogen thetic fragments, i.e., synthetic peptides having the binding site may be blocked by covalently attach- same sequence as a portion of or all of the amino so ing inactivated plasminogen derivatives to the fibrin acid sequence of the corresponding naturally-oc- fragment by specific cross-linking reagents. Inac- curring fibrin fragment, or synthetic peptides with tivated plasmin is coupled through its active en- one or more amino acids replaced, such synthetic zyme site to the fibrin fragment's plasminogen peptides comprising single chains or chains linked binding site by means of the cross-linking agent. together by disulfide or other chemical bonds; and 55 Examples of suitable heterobifunctional cross-link- derivatized fragments, that is, native fibrin frag- ing reagents for this purpose are those reagents ments which have been reacted with or treated with which carry a PMSF moiety as one functional one or more chemical agents. group and an aromatic azido residue as the other

11 19 EP 0 473 689 B1 20 functional group. The PMSF moiety binds covalen- plexes may be prepared provided the fibrin frag- tly with the active site of the plasmin molecule, ment and plasminogen activator molecule have an while the azido group will couple the plasmin mol- inherent binding affinity between them. ecule to the plasminogen binding site of the fibrin The non-covalent complexes of the present fragment portion of the fibrinolytic hybrid. One 5 invention are prepared by incubating a fibrin frag- such heterobifunctional agent believed suitable for ment with a plasminogen activator molecule in a this purpose is: molar ratio favoring complex formation, under conditions favoring stabilization of the resulting complex. Generally, a reaction vessel is pre-coated with N=N io serum albumin. An amount of plasminogen activator molecule, depending upon the required activity, is incubated with about a 1 to about 1000-fold, preferably about 10 to about 1000-fold, molar excess of fibrin fragment at, e.g., 37 °C for thirty is minutes. Following incubation, human serum al- CH2 CH2 CH2 CH2 bumin and/or other stabilizer and 1-2 mM CaCb are added to the mixture. Such other stabilizers include, for example, non-ionic detergents, 0.01%- Another approach to prevent plasminogen 0.1% concentration. Such detergents include, for binding to the fibrin fragment portion of the 20 example, polysorbate 80 (Tween® 80) and polysor- fibrinolytic hybrid utilizes isolated plasminogen bate 20 (Tween® 20). Formation of complexes is kringles as a blocking agent to saturate the fibrin verified by non-dissociating PAGE. The complexes fragment's plasminogen binding site. Kringles com- are then purified by FPLC. They may then be prise a class of disulfide-bridged, triple loop struc- tested for intactness of fibrin binding affinity and tures existing as independent, autonomous folding 25 plasminogen activating activity. The functional ac- domains in a number of proteins. Kringles have tivity of the complexes may then be calibrated by been found to exist in, for example, scu-PA (kringle standard chromogenic substrate assay or other 1), t-PA (kringle 2) and plasminogen (kringles 4 and suitable functional assay. The chromogenic plasmin 5). Plasminogen may be cleaved between kringle 4 substrate H-D-Val-Leu-Lys-p-nitroaniline (S-2251) and kringle 5 by 100 mM elastase. Machovich et 30 may be used for this purpose. al., Circulation 78(4), Supplement 11, Abstract No. Thrombolytic hybrids may also be formed by 2039 of "Abstracts of the 61st Scientific Session of covalently coupling the fibrin fragment to a plas- the American Heart Association. Synthesis of iso- minogen activator. The components may be lated plasminogen kringles is possible by recom- covalently cross-linked by appropriate homobifunc- binant DNA technology. The kringles may be com- 35 tional or heterobifunctional cross-linking agents. bined with the fibrinolytic hybrids of the invention The covalently cross-linked fibrinolytic hybrids and irreversibly cross-linked so as to be indifferent of the present invention may be prepared by utiliz- to circulating plasminogen molecules. ing homobifunctional cross-linking reagents, e.g. The various native and non-native fibrin frag- disuccinimidyl tartrate, disuccinimidyl suberate, ments which find utility in the present invention 40 ethylene glycolbis(succinimidyl succinate), 1,5- may be prepared by appropriate enzyme digest of difluoro-2,4-dinitrobenzene ("DFDNB"), 4,4'- the parent molecule fibrin. It is also contemplated diisothiocyano-2,2'-disulfonic acid stilbene that fibrin fragments, which may be linked to plas- ("DIDS"), and bismaleimidohexane ("BMH"). The minogen activator molecules to form fibrinolytic cross-linking reaction occurs randomly between the hybrids according to the invention, may be pre- 45 fibrin fragment and plasminogen activator. pared by genetic engineering techniques known to Alternatively, heterobifunctional cross-linking those skilled in the art. reagents may be employed. Such agents include, for example, N-succinimidyl-3-(2-pyridyldithio)- Preparation of Plasminogen Activator-Fibrin propionate ("SPDP"), sulfosuccinimidyl-2-(p- Fragment Fibrinolytic Complexes 50 azidosalicylamido)ethyl-1 -3'-dithiopropionate ("SASD", Pierce Chemical Company, Rockford, IL), Non-covalent fibrinolytic complexes may be N-maleimidobenzoyl-N-hydroxy-succinimidyl ester prepared between fibrin fragments and plas- ("MBS"), m-maleimidobenzoylsulfosuccinimide es- minogen activator molecules which have inherent ter ("sulfo-MBS"), N-succinimidyl(4-iodoacetyl)- binding affinity between them. Exemplary complex- 55 aminobenzoate ("SIAB"), succinimidyl 4-(N-mal- es include, but are not limited to, complexes of eimidomethyl)-cyclohexane-1-carboxylate fragments Ei , E2, D and DD, and (DD)E complex ("SMCC"), succinimidyl-4-(p-maleimidophenyl) linked to t-PA or scu-PA. Other non-covalent com- butyrate ("SMPB"), sulfosuccinimidyl(4-iodoacetyl)-

12 21 EP 0 473 689 B1 22 aminobenzoate ("sulfo-SlAB"), sulfosuccinimidyl 4- The purified, functionalized fibrin fragment is (N-maleimidomethyl)cyclohexane-l-carboxylate collected by affinity chromatography using a matrix ("sulfo-SMCC"), sulfosuccinimidyl 4-(p-mal- having affinity for fibrin fragments, e.g., a fibrin- eimidophenyl)-butyrate ("sulfo-SMPB"), diatomaceous earth (1.5 ml) affinity column. Immo- bromoacetyl-p-aminobenzoyl-N-hydroxy- 5 bilized fibrin with the functionalized fibrin fragment succinimidyl ester, iodoacetyl-N-hydroxysuc- bound thereto is then removed from the column cinimidyl ester, and the like. and suspended in a solution of an appropriate For heterobifunctional cross-linking, the fibrin plasminogen activator. An ultra-violet light source fragment is derivatized with, e.g., the N-hydrox- (e.g. Mineralight UVSL-25, Ultra Violet Products, ysuccinimidyl portion of the bifunctional reagent, io Inc., San Gabriel, CA) is positioned 1 cm from the and the derivatized fibrin fragment is purified by gently stirred suspension and irradiated in a long- gel filtration. Next, plasminogen activator is reacted wavelength range for about 10 minutes. The sus- with the second functional group of the bifunctional pension is put back into the fibrin-diatomaceous reagent, assuring a directed sequence of binding earth column and washed with a buffer containing between components of the fibrinolytic hybrid. 15 0.15 M NaCI, 0.1% bovine serum albumin, 0.01% Typical heterobifunctional cross-linking agents polysorbate 80 and 25 KIU/ml of aprotinin to re- for forming protein-protein conjugates have an ami- move reaction byproducts. The covalently cross- no reactive N-hydroxy-succinimide ester as one linked fibrinolytic hybrid is eluted with the same functional group and a sulfhydryl reactive group as buffer system containing 0.5 M arginine. The com- the other functional group. First, epsilon-amino 20 plex is then separated from arginine by BioGel™ groups of surface lysine residues of the fibrin frag- P-2 column chromatography. ment are acylated with the NHS-ester group of the Alternatively, conjugation of the fibrin fragment cross-linking agent. The plasminogen activator mol- to the plasminogen activator may be accomplished ecule, possessing free sulfhydryl groups, is reacted by first derivatizing the plasminogen activator mol- with the sulfhydryl reactive group of the cross- 25 ecule. The plasminogen activator molecule is con- linking agent to form a covalently cross-linked jugated with, e.g. SASD as above, and the func- fibrinolytic hybrid. Common thiol reactive groups tionalized plasminogen activator molecule is puri- include maleimides, pyridyl disulfides, and active fied as above by fibrin-diatomaceous earth affinity halogens. For example, MBS contains a N- chromatography. Benzamidine-agarose chromatog- hydroxy-succinimide ester as the amino reactive 30 raphy may be substituted for fibrin-diatomaceous group, and a maleimide moiety as the sulfhydryl earth chromatography. reactive group. Benzamidine binds specifically to certain plas- Photoactive heterobifunctional cross-linking minogen activators such as t-PA and urokinase, reagents, e.g. photoreactive phenyl azides, may which contain a benzamidine binding site. An affin- also be employed. One such reagent, SASD, may 35 ity column for purifying plasminogen activators be linked to the fibrin fragment via its N-hydrox- may thus be prepared by immobilizing ben- ysuccinimidyl ester group. The conjugation reaction zamidine on a suitable matrix, e.g., highly purified is carried out at pH 7 at room temperature for agarose (Sepharose®, Pharmacia Fine Chemicals, about 10 minutes. Molar ratios between about 1 Uppsala, Sweden), particularly, agarose which has and about 20 of the cross-linking agent to fibrin 40 been treated with dibromopropanol (Sepharose® fragment are used. Low ratios are favored in order CL-6B). Benzamidine-agarose is available from to minimize chemical modification of binding sites Pharmacia Fine Chemicals (Uppsala, Sweden) as a on the fibrin fragments. The fibrin fragment to preswollen affinity gel in 0.9 M NaCI, containing which cross-linking agent has been bound is then 0.01% merthiolate. purified in the dark from reaction by-products by 45 Following reaction with fibrin fragment under column gel filtration, e.g. polyacrylamide column ultraviolet irradiation, and chromatography of the filtration. Polyacryamide gel materials for gel filtra- reaction mixture as above, the covalently cross- tion chromatography, consisting of porous linked fibrinolytic hybrid is eluted with either 0.5 M polyacrylamide beads are available, for example, arginine in the case of fibrin-diatomaceous earth as BioGel™ beads from Bio-Rad Corp., Richmond, 50 chromatography or 0.1 M acetate, 0.4 M NaCI, pH CA. The gels are available with a range of pore 4.0, in the case of benzamidine-agarose sizes, indicated by a suffix (P-2, P-4, P-6, etc.). The chromatography. When fibrin-diatomaceous earth is suffix corresponds to the smallest molecule which employed as the affinity matrix, the covalent com- is completely excluded from the pores. For purify- plexes are further purified by BioGel™ P-2 ing fibrin fragments, a gel with pore sizes excluding 55 chromatography, whereas if benzamidine-agarose 2 kDa and larger molecules is utilized, e.g., chromatography is employed, the eluate is simply BioGel™ P-2 (1 x 10 cm column). quickly neutralized.

13 23 EP 0 473 689 B1 24

While the above-described procedure utilizes sodium phosphate, 0.3 M NaCI and 0.05% polysor- SASD, a cleavable cross-linker, non-cleavable bate 20, pH 7.4. t-PA molecules not bound by the cross-linking reagents may be utilized which con- benzamidine-agarose affinity matrix, non-functional tain, e.g. alpha-hexanoate, rather than beta-ethyl- fibrinolytic hybrids having impaired plasminogen 1 ,3-dithiopropopionate moieties. MSB is one exam- 5 activator portions, and free fibrin fragments are ple of a non-cleavable cross-linking reagent. washed from the column with 8 column washes of The ability of fibrin fragments to form non- equilibration buffer. The affinity matrix-bound covalent complexes with plasminogen activators is fibrinolytic hybrids and uncomplexed t-PA are then demonstrated as follows. Fibrin monomer (FM), eluted from the column with 0.1 M acetate, 0.4 M (DD)E complex, and fragments DD and Ei were io NaCI, pH 4.0. The purified complexes are assayed absorbed to the wells of Immulon C microtiter for functional activity by S-2251 chromogenic as- plates (Dynatech Laboratories, Chantilly, VA). Un- say. Briefly, the complexes are incubated with plas- occupied binding surfaces were blocked by BSA. minogen (0.04 U/ml) and the synthetic One-chain t-PA purified from a human melanoma chromogenic substrate SS-2251 (3 mM) at 37 °C cell line (American Diagnostica, NY, NY) was ad- is for 15 minutes. The reaction is stopped with 50% ded to the plates in incremental amounts from 0.1 acetic acid. The absorbance of the reaction mixture to 15 nM concentration range and incubated for 3 is read at 405 nm. The activity of the complex may hours. The amount of t-PA bound to the fibrin be derived from a curve created from the same fragments on the plate was quantified by ELISA. assay using known amounts of t-PA. Complex formation was evident throughout the en- 20 While gel filtration HPLC may be substituted tire range of t-PA concentration tested and was for FPLC, the latter is preferred because of the similar for fibrin monomer (FM), (DD)E complex, higher quality and purity of the product, better fragment DD and fragment Ei (Figure 1). Specific- retentional of biological activity, greater ease of ity of binding was confirmed by the lack of t-PA separation, greater speed, and minimal contamina- binding to BSA. These results demonstrate that t- 25 tion by closely related fragments and molecules. PA forms non-covalent complexes with fibrin frag- The uniform size (dp = 9.8 urn) and distribution of ments. the anion exchanger material in the Mono Q® col- Non-covalent complexes of plasminogen ac- umn, and its small-ion capacity (0.28-0.36 tivators and fibrin fragments may be prepared ac- mmol/ml), ensures its suitability for the herein-de- cording to the following non-limiting example: 30 scribed purification process. In addition, low pres- sure chromatographic techniques are utilized in Example 1 FPLC. The following non-limiting example illustrates Fragment DD/t-PA Non-Covalent Complex the preparation of a fragment DD/t-PA covalent 35 hybrid according to the present invention, using a A reaction vessel is pre-coated with 10 mg/ml heterobifunctional cross linking reagent. solution of serum albumin and dried. Fragment DD (640 nmoles) is incubated with one-chain t-PA (3.2 Example 2 nmoles, American Diagnostica, NY, NY) in the albumin pre-coated vessel for 1/2 hour at 37 °C in 40 Fragment DD/t-PA Covalent Complex the presence of 2 mM Ca++. Ten milligrams of serum albumin and ten microliters of 10% solution One-chain t-PA (American Diagnostica, 0.5 mg of polysorbate 20 are added as a stabilizer. Phos- containing 250,000 units of activity) is dissolved in phate buffered saline ("PBS"), pH 7.4, is added to 0.5 ml of PBS, pH 7.4. Immediately thereafter, adjust the volume and standardize the activity 45 SPDP in a 20-fold molar excess is added dropwise units. The reaction mixture is fractionated by FPLC to the t-PA solution. This mixture is stirred gently at utilizing a Mono Q® anion exchange column to room temperature for 30 minutes and immediately isolate the fibrinolytic hybrid. Accordingly, 0.15 mg dialyzed against a buffer (0.14 M NaCI, 3.7 mM of the reaction mixture comprising the fibrin frag- NaH2PO+, 1 mM KCI, pH 7.4) for 3 changes over ment and plasminogen activator is introduced into 50 12 hours at 4°C. Fragment DD (10 mg/ml, 0.5 ml) a Mono Q® HR 5/5 column at a flow rate of 1 is mixed in equal amounts (v/v) with a 1000-fold ml/min, gradient = 2.5 mM/min NaCI. The mobile molar excess of 2-iminothiolane in 25 mm sodium phase consists of 20 mM Tris-HCI, pH 7.4. The borate, pH 9.3. The reaction is allowed to continue fibrinolytic hybrid is detected at 280 nm. The hy- for 25 minutes at room temperature with gentle brid is subsequently purified on a benzamidine- 55 stirring. Excess 2-iminothiolane is immediately re- agarose affinity matrix column. Thus the FPLC moved by gel filtration on Sephadex® G-25 pre- eluate is applied to a 1.5 ml benzamidine-agarose equilibrated with PBS containing 0.02% sodium column (0.8 x 3.5 ml) and eluted with 0.05 M azide, pH 6.6. The SPDP-modified t-PA is then

14 25 EP 0 473 689 B1 26 mixed with the 2-iminothiolated fragment DD, with Plasminogen Actlvator-Flbrln Fragment Com- gentle stirring at room temperature for 7 hours. At plex Activity this time, a 100-fold molar excess of iodoacetamide to protein is added in 0.1 M NaH2PO+, pH 8.0, in The ability of fibrin fragments to protect plas- order to complete the reaction. 5 minogen activators against inactivation in plasma Covalent complexes which contain intact fibrin was demonstrated by the following series of experi- binding sites of the parent fibrin fragment compo- ments. According to a unique radiolabeled plasma nent, and intact catalytic sites of the parent plas- clot lysis assay for plasminogen activator activity, minogen activator component, are purified from the activator sample (fibrinolytic hybrid or uncomplex- reaction mixture in two steps. First, the mixture is io ed plasminogen activator) is collected and incu- subjected to benzamidine-agarose affinity bated with radiolabeled clot for 10 minutes. chromatography as in Example 1. Second, imme- Solubilized radiolabeled fibrin is counted and ex- diately following elution from the benzamidine- pressed in terms of net percent of radio-activity agarose column and neutralization of the eluent, released by the plasma clot into the supernatant. the fractions are applied to a fibrin-diatomaceous is The amount of protein solubilized is calculated. earth affinity column, which retains only conjugates The activator activity in units/ml of the sample is containing fibrin fragments with fibrin-binding affin- derived from a standard curve constructed utilizing ity, as well as uncoupled plasminogen activator. t-PA or urokinase of known activity. The percent Species without fibrin affinity are not retained. initial activity is calculated and plotted from the Cross-linked fibrin-diatomaceous earth (1.5 ml) 20 initial and residual activities. is packed in a column (0.8 x 3.5 cm) and equili- Fragment DD or the (DD)E complex was mixed brated with 0.05 M sodium phosphate buffer, con- with one-chain t-PA in a 500:1 molar ratio. The taining 0.3 M NaCI and 0.05% polysorbate 80, pH mixture was incubated in plasma for various 7.4. The eluted, neutralized fractions from the ben- periods of time. The remaining t-PA activity was zamidine-agarose chromatography are then applied 25 measured. As shown in Figure 2, t-PA in plasma to the fibrin-diatomaceous earth column. lost its activity very rapidly, whereas t-PA stored in Fibrinolytic hybrids with impaired fibrin-binding buffer retained its activity during 5 hours of incuba- function do not bind to the fibrin-diatomaceous tion. Fragment DD and (DD)E complex conferred a earth affinity matrix. The matrix retains only hybrids protective effect on t-PA, such that the latter did containing intact fibrin fragments having fibrin-bind- 30 not lose activity as rapidly as t-PA incubated in ing affinity, as well as uncoupled plasminogen ac- plasma alone. The half-life ("T1/2") of t-PA in plas- tivator. Species without fibrin affinity are not re- ma (i.e., the time when t-PA activity is one-half its tained. The impaired, non-binding hybrids are initial value), when protected with fragment DD or washed from the column with 8 column volumes of (DD)E complex, was 2.08 and 2.03 hours, respec- the equilibration buffer. The bound proteins, that is, 35 tively, as compared with 1 .3 hours for unprotected free plasminogen activator and hybrid molecules t-PA. Thus, complexation with fibrin fragment re- with intact fibrin binding function, are then eluted sulted in about a 57% improvement in the half life with equilibration buffer containing 0.5 M arginine, of t-PA activity in plasma. 0.1 mg/ml human serum albumin, 0.01% polysor- The protective effect afforded by the fibrin frag- bate 80, and 25 KIU/ml aprotinin, pH 7.4. Arginine 40 ment may be influenced by the molar ratio be- is removed from the eluate on a prepacked desal- tween the fragment and the plasminogen activator, ting column (Econo-Pac 10DG Column, Bio-Rad as demonstrated by the following experiment. t-PA Corp., which is packed with Bio-Gel P-6 desalting was incubated in normal human plasma for 5 hours gel, a hydrophilic polyanlamide having a molecular alone or in the presence of different molar ex- weight exclusion limit of about 6KDa and a hy- 45 cesses of the following fibrin fragments: (DD)E drated particle size of about 90-1 80m..) The eluate complex, fragment DD and fragment E3. After the is then dialyzed against PBS with 0.02% sodium incubation time, residual t-PA activity was assayed. azide, pH 7.4. and stored in this buffer at 4°C. It was found that uncomplexed t-PA retained only Where the plasminogen activator portion of the 22% of its initial activity in plasma (data not shown) hybrid is t-PA or scu-PA uncoupled plasminogen 50 whereas near total original activity (92-100%) was activator may co-elute with the fibrinolytic hybrid preserved by complexation with fibrin fragments upon fibrin-diatomaceous earth chromatography. (FDP:t-PA = 100:1 molar). See Figure 3. Thus, co-eluted t-PA molecules are then separated It has been reported that CNBr-cleavage frag- from the hybrid molecules by an additional gel ments of human fibrinogen potentiate the rate of filtration step. The resulting fragment DD/t-PA cova- 55 plasmin generation from plasminogen by t-PA, lent complex is then assayed for functional activity Neuwenhuizen et al. Blochem. Blophys. Acta. by S-2251 chromogenic assay, as in Example 1 . 755:531-533 (1983); Lijnen et al., Eur. J. Biochem. 144:541-544 (1984), and that the potentiating effect

15 27 EP 0 473 689 B1 28 is attributable to fragment alpha 148-197 of the results are shown in Figure 6. While CNBr- fibrinogen alpha chain. Neuwenhuizen et al. cleavage fragments of fibrinogen were more potent Biochem. Biophys. Acta. 748:86-92 (1983). Not- than fibrin fragments in accelerating the rate of withstanding the presence of the same alpha chain plasmin generation over brief incubation times (up segment in fragment D, in fragment DD, and in the 5 to 60 minutes), the reverse was observed at longer (DD)E complex, CNBr-cleavage fragments of incubation times. fibrinogen offer no protective effect on plasminogen In clinical applications, the thrombolytic hybrids activators in normal plasma, as determined by the of the present invention will generate more plasmin following experiment. on fibrin surfaces from a given amount of plas- One-chain t-PA was mixed with a 100-fold mo- io minogen than the activator molecule may generate lar excess of (DD)E complex, fragment DD, or in the absence of the fibrin fragment. This permits CNBr-cleavage fragments of fibrinogen and incu- the use of a lower amount of activator than pres- bated at 37 °C for 45 minutes to allow complex ently used in clinical practice. formation. Normal human plasma was added and The hybrids accelerate lysis of plasma clots, as the mixture was incubated for various time inter- is observed from the following experiment. Ten units vals. The plasminogen activator activity as a func- of one-chain t-PA were added to 1 ml of normal tion of time was measured by adding aliquots taken human citrated plasma containing 125 l-fibrinogen from the incubation mixtures at various times to and incubated at 37 °C for 1 hour. Lysis of the human plasma supplemented with 125 l-fibrinogen, plasma clot was measured by release of radioactiv- immediately followed by the addition of thrombin 20 ity counts into the supernatant. As shown in Figure and incubation at 37 °C for 10 minutes. The half- 7, only 2.9% of the clot was lysed. When the same lives for fragment DD-protected t-PA, (DD)E com- amount of plasma was clotted without preincuba- plex-protected t-PA and unprotected t-PA were tion with t-PA, the same amount of t-PA achieved 4.87, 4.56 and 3.37 hours, respectively. While the dissolution of 24.3% of the clot. Thus, there was a two fibrin fragments exerted a slight protective ef- 25 significant inactivation of t-PA by the plasma milieu. fect on t-PA inactivation in plasma, the CNBr- Fibrinolytic complexes of one-chain t-PA were cleavage fragments of fibrinogen were devoid of then formed with a 10-fold excess of either DD or any protective effect. See Figure 4. (DD)E, and were added to plasma. Following in- Fibrin fragments have a similar protective ef- cubation at 37 ° C for 1 hour, the plasma was clot- fect on urokinase activity in plasma. Fragment DD, 30 ted by addition of thrombin. As shown in Figure 7, (DD)E complex or CNBr-cleavage fragments of the fibrinolytic complexes of the invention caused a fibrinogen were mixed in a 100-fold molar excess significantly greater plasma clot lysis than plas- with urokinase and incubated at 37 °C for 45 min- minogen activator alone. While the lytic activity of utes. The mixture was then added to 6 ml of naked plasminogen activator molecules is rapidly plasma, and the incubation was continued. Figure 5 35 suppressed in plasma, complexation with fibrin shows that fragment DD and (DD)E complex pro- fragments has a substantial protective effect from vided a protective effect, while the CNBr-cleavage inhibitors found in plasma. fibrinogen fragments provided no protective effect. The thrombolytic hybrids of the invention, due The half-lives for DD- and (DD)E-protected to their fibrin specificity, readily localize at the fibrin urokinase were 4.6 and 1 .6, respectively, while the 40 surface of thrombi. As a result, only clot-surface half-lives for unprotected urokinase was 1.2 hours. bound plasminogen is activated, without significant Complexation with fragment DD extends the half- systemic plasminogen activation and possible in- life of urokinase almost four-fold. ducement of a lytic state. The risk of hemorrhagic The fibrinolytic hybrids of the invention accel- complication is thus reduced. Moreover, the throm- erate plasmin generation at a rate almost two-fold 45 bus-targeting property of the hybrids will provide a higher than CNBr-cleavage fragments of fibrinogen. thrombolytic effect of comparable degree to locally The latter are known accelerators of plasminogen infused thrombolytic agent. activation. One-chain t-PA was incubated with a The hybrids are strong potentiators of plas- 100-fold molar excess of fragment DD, (DD)E com- minogen activation, providing more plasmin from plex or CNBr-cleavage fragments of fibrinogen to 50 the same amount of plasminogen than naked plas- form non-covalent complexes. Plasminogen and minogen activator. Moreover, the fibrin component the chromogenic substrate S2251 were added to of the hybrid affords considerable protection each mixture and incubated at 37 °C for different against inhibition of the plasminogen activator com- time intervals, according to the method of Friberger ponent by inhibitors present in the blood, and pro- et al., Haemostasis 7:138-145 (1978). The amount 55 tection against rapid uptake by the liver, contribut- of plasmin generated was recorded from absor- ing to an increased half-life of activity compared to bance at 405 nm, and the activity in milliunits per uncomplexed plasminogen activator. Substitution of ml was determined from a standard curve. The the thrombolytic hybrids of the invention for the

16 29 EP 0 473 689 B1 30 presently available plasminogen activators will per- tide having the properties of the fibrinolytic hybrids mit clinicians to decrease the frequency, duration described herein, which polypeptides are prepared and dosage of administration of therapeutic agent by genetic engineering techniques. Thus, it is con- without sacrificing efficacy. Treatment with the hy- templated that genetic information coding for a brids will permit the clinician to lower the dose and 5 fibrin fragment and information coding for a plas- delay the initiation of heparin therapy and other minogen activator may, by DNA recombinant tech- anticoagulant therapies for the prevention of re- niques, be co-expressed as a single unitary mol- stenosis and fresh clot formation (reocclusion). ecule having the properties of the fibrinolytic hy- The thrombolytic hybrids of the invention may brids described herein. be administered intravascularly or intramuscularly io The present invention may be embodied in in the form of a composition containing the hybrid other specific forms without departing from the and a pharmaceutically acceptable carrier suitable essential attributes thereof and, accordingly, refer- for intravascular or intramuscular administration. ence should be made to the appended claims, The composition may be in the form of a solution rather than to the foregoing specifications, as in- in a suitable carrier, for example isotonic saline and 15 dicating the scope of the invention. sterile water. The composition may further include any of the additives typically utilized in in- Claims travascular or intramuscular administration, e.g. buffer salts, L-arginine, glucose, etc. The composi- 1. A purified thrombolytic agent comprising a tion may contain stabilizers such as, for example, 20 fibrin fragment linked to a plasminogen activa- human serum albumin or non-ionic detergents such tor. as polysorbate. The dosage, expressed in terms of units of 2. A thrombolytic agent according to claim 1, plasminogen activator, is generally less than the wherein the fibrin fragment comprises a native dosages presently used for corresponding uncom- 25 plasmin degradation fragment of fibrin. plexed plasminogen activator. It is contemplated that the hybrids of the invention may be utilized in 3. A thrombolytic agent comprising a non-native dosages as much as an order of magnitude lower fragment of fibrin linked to a plasminogen ac- than the corresponding uncomplexed plasminogen tivator. activator. 30 Presently, the typical dosage practices for ad- 4. A thrombolytic agent according to claim 2, ministering the principal plasminogen activators are wherein the fibrin fragment is selected from as follows: fragments Ei , E2, E3, D and DD, and (DD)E t-PA or scu-PA: complex. One hundred mg (60 mg bolus, 20 mg infusion 35 in first hour, then 20 mg infusion in next hour); 5. A thrombolytic agent according to claim 4, streptokinase or urokinase: wherein the plasminogen activator is t-PA. 1.5 million units (bolus, infusion over three hours) 6. A thrombolytic agent according to claim 4, It is contemplated that the same affect may be 40 wherein the plasminogen activator is urokinase. obtained by administering only one tenth the standard dosage, when the plasminogen activator is 7. A thrombolytic agent according to claim 4, coupled to a fibrin fragment according to the wherein the plasminogen activator is strep- present invention. Thus, it is contemplated that 10 tokinase. mg of t-PA or scu-PA, or 150,000 units of strep- 45 tokinase or urokinase, is an effective dosage when 8. A thrombolytic agent according to claim 4, administered in the form of a fibrinolytic hybrid. wherein the plasminogen activator is scu-PA. The hybrids of the invention may be utilized in any situation where the corresponding uncomplex- 9. A thrombolytic agent according to claim 5, ed plasminogen activator is utilized, e.g., in the 50 wherein the fibrin fragment is fragment DD or treatment of myocardial infarctions, pulmonary em- (DD)E complex. boli, deep venous thrombosis, etc. where vascular thrombi are to be dissolved. 10. A thrombolytic agent according to claim 1, The invention has been illustrated by the cova- which is a non-covalent complex of a fibrin lent or non-covalent linkage of fibrin fragments with 55 fragment and a plasminogen activator. appropriate plasminogen activators to form fibrinolytic hybrids. Also within the scope of the 11. A thrombolytic agent according to claim 10, invention is preparation of a single unitary polypep- formed by combining one molar amount of

17 31 EP 0 473 689 B1 32

plasminogen activator with about one molar 22. A composition according to claim 20, wherein amount to about a one thousand molar amount the fibrin fragment comprises a non-native of fibrin fragment. fragment of fibrin.

12. A thrombolytic agent comprising a fibrin frag- 5 23. A composition according to claim 21, wherein ment covalently coupled to a plasminogen ac- the fibrin fragment is selected from fragments tivator. Ei , E2, E3, DD, and (DD)E complex.

13. A thrombolytic agent according to claim 12, 24. A composition according to claim 23, wherein wherein the plasminogen activator and the io the plasminogen activator is t-PA. fibrin fragment are coupled by a homobifunctional cross-linking agent. 25. A composition according to claim 23, wherein the plasminogen activator is urokinase. 14. A thrombolytic agent according to claim 12, wherein the plasminogen activator and the 75 26. A composition according to claim 23, wherein fibrin fragment are coupled by a heterobifunc- the plasminogen activator is streptokinase. tional cross-linking agent. 27. A composition according to claim 23, wherein 15. A thrombolytic agent according to claim 14, the plasminogen activator is scu-PA. wherein the heterobifunctional cross-linking 20 agent is selected from N-succinimidyl-3-(2- 28. A composition according to claim 24, wherein pyridyldithio)propionate and sulfosuccinimidyl- the fibrin fragment is fragment DD or (DD)E 2-(p-azidosalicylamido)ethyl-1-3'- complex. dithiopropionate. 25 29. A thrombolytic agent according to claim 3 12 16. A thrombolytic agent comprising a fibrin frag- or 16, wherein the plasminogen activator is ment linked to a plasminogen activator, selected from t-PA, urokinase, streptokinase, wherein the fibrin fragment has a plasminogen scu-PA, and combinations thereof. binding site which has been blocked by a blocking agent. 30 30. A thrombolytic agent according to claim 29, wherein the plasminogen activator comprises t- 17. A thrombolytic agent according to claim 16, PA. wherein the blocking agent comprises inactivated plasmin. 31. A thrombolytic agent according to claim 29, 35 wherein the plasminogen activator comprises 18. A thrombolytic agent according to claim 17, scu-PA. wherein inactivated plasmin has been non- covalently bound to the fibrin fragment. 32. A thrombolytic agent according to claim 29, wherein the plasminogen activator comprises 19. A thrombolytic agent according to claim 17, 40 urokinase. wherein inactivated plasmin has been coupled through its active enzyme site by a cross- 33. A thrombolytic agent according to claim 29, linking agent to the plasminogen binding site wherein the plasminogen activator comprises of the fibrin fragment. streptokinase. 45 20. A thrombolytic composition comprising: 34. A composition comprising a fibrin fragment a purified thrombolytic agent comprising a linked to a plasminogen activator, for use as a fibrin fragment linked to a plasminogen activa- medicament. tor, and a pharmaceutically acceptable carrier suit- 50 35. Use of a composition comprising a fibrin frag- able for intravascular or intramuscular admin- ment linked to a plasminogen activator in the istration. preparation of a medicament for use as a thrombolytic agent. 21. A composition according to claim 20, wherein the fibrin fragment comprises a native plasmin 55 36. A composition according to claim 34, wherein degradation fragment of fibrin. the fibrin fragment is as specified in claim 2, 3, 4, 9, 16 or 17.

18 33 EP 0 473 689 B1 34

37. A composition according to claim 34, wherein Fibrinfragment gebildet wird. the plasminogen activator is as specified in any of claims 5 to 8. 12. Thrombolytisches Mittel umfassend ein Fibrin- fragment, das kovalent an einen Plasminogen- 38. A composition according to claim 34, wherein 5 aktivator gebunden ist. the fibrin fragment and plasminogen activator are linked as specified in any of claims 10 to 13. Thrombolytisches Mittel gemaB Anspruch 12, 15 and 19. worin der Plasminogenaktivator und das Fibrin- fragment durch ein homobifunktionelles Ver- 39. Use according to claim 35, wherein the com- io netzungsmittel miteinander verbunden sind. position is in accordance with any of claims 36 to 38. 14. Thrombolytisches Mittel gemaB Anspruch 12, worin der Plasminogenaktivator und das Fibrin- Patentanspruche fragment durch ein heterobifunktionelles Ver- 15 netzungsmittel miteinander verbunden sind. 1. Gereinigtes thrombolytisches Mittel umfassend ein mit einem Plasminogenaktivator verbunde- 15. Thrombolytisches Mittel gemaB Anspruch 14, nes Fibrinfragment. worin das heterobifunktionelle Vernetzungsmit- tel aus N-Succinimidyl-3-(2-pyridylthio)- 2. Thrombolytisches Mittel gemaB Anspruch 1, 20 propionat und Sulfosuccinimidyl-2-(p-azidosali- Worin das Fibrinfragment ein natives Plasmin- cylamido)ethyl-1-3'-dithiopropionatausgewahlt abbaufragment von Fibrin umfaBt. ist.

3. Thrombolytisches Mittel umfassend ein nicht- 16. Thrombolytisches Mittel umfassend ein mit ei- natives, mit einem Plasminaktivator verbunde- 25 nem Plasminogenaktivator verbundenes Fibrin- nes Fibrinfragment. fragment, worin das Fibrinfragment eine Plas- minogenbindungsstelle besitzt, die durch ein 4. Thrombolytisches Mittel gemaB Anspruch 2, Blockierungsmittel blockiert worden ist. wobei das Fibrinfragment aus den Fragmenten Ei , E2, E3 und DD und dem (DD)E-Komplex 30 17. Thrombolytisches Mittel gemaB Anspruch 16, ausgewahlt ist. worin das Blokkierungsmittel inaktiviertes Plas- min umfaBt. 5. Thrombolytisches Mittel gemaB Anspruch 4, worin der Plasminogenaktivator t-PA ist. 18. Thrombolytisches Mittel gemaB Anspruch 17, 35 worin das inaktivierte Plasmin nicht-kovalent an 6. Thrombolytisches Mittel gemaB Anspruch 4, das Fibrinfragment gebunden worden ist. worin der Plasminogenaktivator Urokinase ist. 19. Thrombolytisches Mittel gemaB Anspruch 17, 7. Thrombolytisches Mittel gemaB Anspruch 4, worin das inaktivierte Plasmin durch ein Ver- worin der Plasminogenaktivator Streptokinase 40 netzungsmittel uber seine aktive Enzymstelle ist. mit der Plasminogenbindungsstelle des Fibrin- fragments verbunden worden ist. 8. Thrombolytisches Mittel gemaB Anspruch 4, worin der Plasminogenaktivator scu-PA ist. 20. Thrombolytische Zusammensetzung umfas- 45 send: 9. Thrombolytisches Mittel gemaB Anspruch 5, ein gereinigtes thrombolytisches Mittel, das ein worin das Fibrinfragment das Fragment DD mit einem Plasminogenaktivator verbundenes oder der (DD)E-Komplex ist. Fibrinfragment umfaBt, und einen pharmazeutisch annehmbaren, zur intra- 10. Thrombolytisches Mittel gemaB Anspruch 1, 50 vaskularen oder intramuskularen Verabrei- welches ein nicht-kovalenter Komplex eines Fi- chung geeigneten Trager. brinfragments und eines Plasminogenaktivators ist. 21. Zusammensetzung gemaB Anspruch 20, worin das Fibrinfragment ein natives Plasminabbau- 11. Thrombolytisches Mittel gemaB Anspruch 10, 55 fragment von Fibrin enthalt. das durch Kombinieren einer molaren Menge Plasminogenaktivator mit etwa einer molaren 22. Zusammensetzung gemaB Anspruch 20, worin Menge bis etwa einer tausendmolaren Menge das Fibrinfragment ein nicht-natives Fibrinfrag-

19 35 EP 0 473 689 B1 36

ment enthalt. spruche 5 bis 8 bezeichnet ist.

23. Zusammensetzung gemaB Anspruch 21, worin 38. Zusammensetzung gemaB Anspruch 34, worin das Fibrinfragment aus den Komplexen Ei , E2, das Fibrinfragment und der Plasminaktivator E3 und DD und dem (DD)E-Komplex ausge- 5 wie in einem der Anspruche 10 bis 15 und 19 wahlt ist. bezeichnet verbunden sind.

24. Zusammensetzung gemaB Anspruch 23, worin 39. Verwendung gemaB Anspruch 35, wobei die der Plasminogenaktivator t-PA ist. Zusammensetzung einem der Anspruche 36 10 bis 38 entspricht. 25. Zusammensetzung gemaB Anspruch 23, worin der Plasminogenaktivator Urokinase ist. Revendicatlons

26. Zusammensetzung gemaB Anspruch 23, worin 1. Agent thrombolytique purifie qui comprend un der Plasminogenaktivator Streptokinase ist. is fragment de fibrine lie a un activateur de plasminogene. 27. Zusammensetzung gemaB Anspruch 23, worin der Plasminogenaktivator scu-PA ist. 2. Agent thrombolytique selon la revendication 1 , caracterise en ce que le fragment de fibrine 28. Zusammensetzung gemaB Anspruch 24, worin 20 comprend un fragment de fibrine natif de de- das Fibrinfragment DD oder der (DD)E-Kom- gradation par la plasmine. plex ist. 3. Agent thrombolytique qui comprend un frag- 29. Thrombolytisches Mittel gemaB Anspruch 3, ment de fibrine non natif lie a un activateur de 12 oder 16, worin der Plasminogenaktivator 25 plasminogene. aus t-PA, Urokinase, Streptokinase, scu-PA und deren Kombinationen ausgewahlt ist. 4. Agent thrombolytique selon la revendication 2, caracterise en ce que le fragment de fibrine 30. Thrombolytisches Mittel gemaB Anspruch 29, est choisi parmi les fragments Ei , E2, E3, D et worin der Plasminogenaktivator t-PA umfaBt. 30 DD et le complexe (DD)E.

31. Thrombolytisches Mittel gemaB Anspruch 29, 5. Agent thrombolytique selon la revendication 4, worin der Plasminogenaktivator scu-PA umfaBt. caracterise en ce que I'activateur de plasminogene est t-PA. 32. Thrombolytisches Mittel gemaB Anspruch 29, 35 worin der Plasminogenaktivator Urokinase um- 6. Agent thrombolytique selon la revendication 4, faBt. caracterise en ce que I'activateur de plasminogene est I'urokinase. 33. Thrombolytisches Mittel gemaB Anspruch 29, worin der Plasminogenaktivator Streptokinase 40 7. Agent thrombolytique selon la revendication 4, umfaBt. caracterise en ce que I'activateur de plasminogene est la streptokinase. 34. Zusammensetzung umfassend ein mit einem Plasminogenaktivator verbundenes Fibrinfrag- 8. Agent thrombolytique selon la revendication 4, ment zur Verwendung als Arzneimittel. 45 caracterise en ce que I'activateur de plasminogene est scu-PA. 35. Verwendung einer Zusammensetzung, die ein mit einem Plasminogenaktivator verbundenes 9. Agent thrombolytique selon la revendication 5, Fibrinfragment umfaBt, bei der Herstellung ei- caracterise en ce que le fragment de fibrine nes Arzneimittels zur Verwendung als throm- 50 est le fragment DD ou le complexe (DD)E. bolytisches Mittel. 10. Agent thrombolytique selon la revendication 1, 36. Zusammensetzung gemaB Anspruch 34, worin caracterise en ce qu'il est un complexe non das Fibrinfragment wie in Anspruch 2, 3, 4, 9, covalent d'un fragment de fibrine et d'un acti- 16 oder 17 bezeichnet ist. 55 vateur de plasminogene.

37. Zusammensetzung gemaB Anspruch 34, worin 11. Agent thrombolytique selon la revendication der Plasminogenaktivator wie in einem der An- 10, caracterise en ce qu'il est forme en combi-

20 37 EP 0 473 689 B1 38

nant une quantite molaire d'activateur de pla- 21. Composition selon la revendication 20, carac- sminogene avec environ une quantite molaire terisee en ce que le fragment de fibrine pour environ mille fois la quantite molaire de contient un fragment de fibrine natif de degra- fragment de fibrine. dation par la plasmine. 5 12. Agent thrombolytique caracterise en ce qu'il 22. Composition selon la revendication 20, carac- comprend un fragment de fibrine couple d'une terisee en ce que le fragment de fibrine maniere covalente a un activateur de plasmi- contient un fragment non natif de fibrine. nogene. io 23. Composition selon la revendication 21, carac- 13. Agent thrombolytique selon la revendication terisee en ce que le fragment de fibrine est 12, caracterise en ce que I'activateur de pla- choisi parmi les fragments Ei , E2, E3, DD et le sminogene et le fragment de fibrine sont cou- complexe (DD)E. ples par un agent de reticulation homobifonc- tionnel. is 24. Composition selon la revendication 23, caracterisee en ce que I'activateur de plasminogene 14. Agent thrombolytique selon la revendication est t-PA. 12, caracterise en ce que I'activateur de plasminogene et le fragment de fibrine sont cou- 25. Composition selon la revendication 23, carac- ples par un agent de reticulation heterobifonc- 20 terise en ce que I'activateur de plasminogene tionnel. est I'urokinase.

15. Agent thromoolytique selon la revendications 26. Composition selon la revendication 23, carac- 14, caracterise en ce que I'agent de reticula- terisee en ce que I'activateur de plasminogene tion heterobifonctionnel est choisi parmi le N- 25 est la streptokinase. succinimidyl-3-(2-piridyldithio)-propionate et le sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl- 27. Composition selon la revendication 23, carac- 1 -3'-dithiopropionate. terisee en ce que I'activateur de plasminogene est scu-PA. 16. Agent thrombolytique comprenant un fragment 30 de fibrine lie a un activateur de plasminogene, 28. Composition selon la revendication 24, carac- caracterise en ce que le fragment de fibrine a terisee en ce que le fragment de fibrine est le un site de liaison au plasminogene qui a ete fragment DD ou le complexe (DD)E. bloque par un agent de blocage. 35 29. Agent thrombolytique selon les revendications 17. Agent thrombolytique selon la revendication 3, 12 ou 16, caracterise en ce que I'activateur 16, caracterise en ce que I'agent de blocage de plasminogene est choisi parmi le t-PA, comprend de la plasmine inactivee. I'urokinase, la streptokinase, le scu-PA et les combinaisons de ceux-ci. 18. Agent thrombolytique selon la revendication 40 17, caracterise en ce que la plasmine inactivee 30. Agent thrombolytique selon la revendication a ete liee d'une maniere non-covalente au frag- 29, caracterise en ce que I'activateur de pla- ment de fibrine. sminogene comprend du t-PA.

19. Agent thrombolytique selon la revendication 45 31. Agent thrombolytique selon la revendication 17, caracterise en ce que la plasmine inactivee 29, caracterise en ce que I'activateur de pla- a ete couplee par son site d'enzyme actif par sminogene comprend du scu-PA. un agent de reticulation au site de liaison au plasminogene du fragment de fibrine. 32. Agent thrombolytique selon la revendication 50 29, caracterise en ce que I'activateur de pla- 20. Composition thrombolytique caracterisee en ce sminogene comprend de I'urokinase. qu'elle comprend : - un agent thrombolytique purifie conte- 33. Agent thrombolytique selon la revendication nant un fragment de fibrine lie a un acti- 29, caracterise en ce que I'activateur de pla- vateur de plasminogene et 55 sminogene comprend de la streptokinase - un support pharmaceutiquement acceptable approprie pour I'administration par 34. Composition contenant un fragment de fibrine voie intravasculaire ou intramusculaire. lie a un activateur de plasminogene pour I'utili-

21 39 EP 0 473 689 B1 40

sation comme medicament.

35. Utilisation d'une composition caracterisee en ce qu'elle contient un fragment de fibrine lie a un activateur de plasminogene pour la prepa- 5 ration d'un medicament destine a I'emploi comme agent thrombolytique.

36. Composition selon la revendication 34, caracterisee en ce que le fragment de fibrine est 10 comme specifie dans les revendications 2, 3, 4, 9, 16 ou 17.

37. Composition selon la revendication 34, caracterisee en ce que I'activateur de plasminogene is est comme specifie dans I'une des revendications 5 a 8.

38. Composition selon la revendication 34, caracterisee en ce que le fragment de fibrine et 20 I'activateur de plasminogene sont lies comme specifie dans I'une des revendications 10 a 15 et 19.

39. Utilisation selon la revendication 35, caracteri- 25 see en ce que la composition est en conformi- te avec I'une des revendications 36 a 38.

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