AU7737401A - Composition and method for enhancing fibrinolysis using antibodies to alpha-2-antiplasmin - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A DIVISIONAL PATENT

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TO BE COMPLETED BY APPLICANT Name of Applicant: 99040: Actual Inventor: Address for Service: ******nvention Title: Jnvention Title: THE GENERAL HOSPITAL CORPORATION and THE PRESIDENT AND FELLOWS OF HARVARD COLLEGE Guy L Reed CALLINAN LAWRIE, 711 High Street, Kew, Victoria 3101, Australia COMPOSITION AND METHOD FOR ENHANCING FIBRINOLYSIS USING ANTIBODIES TO ALPHA-2-ANTIPLASMIN The following statement is a full description of this invention, including the best method of performing it known to me:- 03/10/01,ckl 2330oct3.front,l la- Field of the Invention The present invention relates to a composition and method of treatment for pulmonary embolism, myocardial infarction, thrombosis, and stroke in a patient, and more specifically to a therapy which enhances fibrinolysis comprising administering an alpha-2-antiplasmin-binding molecule. The invention also relates to a treatment for enhancing fibrinolysis comprising administering an alpha-2antiplasmin-binding molecule together with a thrombolytic agent.

This application is a divisional of patent no. 734997 (44134/97) filed by The General Hospital Corporation and The President and Fellows of Harvard College.

15. Description of Background Art c 0 Venous thrombosis and pulmonary embolism are major causes of morbidity and mortality in the United States, accounting for about 270,000 hospitalizations a year (Anderson, Jr. et Arch. Intern. Med. 151:933-938 (1991)). In addition, it is estimated that about 50,000-200,000 patients a year die from pulmonary emobolism (Lilienfeld, D.E. et al., Chest 98:1067-1072 (1990)). In surprising contrast with the mortality rate for myocardial infarction, the mortality rate for pulmonary embolism (estimated at 9.2% in treated patients) has not :improved in the last 30 years (Lilienfeld, D.E. et Chest 98:1067-1072 *2 (1990); Giuntini, C. et Chest 107:3S-9S (1995)). Moreover, survivors of venous thromboembolism are known to be at risk for recurrent thrombosis, 0 ck12330oct3.speci WO 98/12329 PCTIUS97/16122 -2postphlebitic syndrome, and pulmonary hypertension (Sutton, G.C. et al., Br.

HeartJ. 39:1135-1192 (1977); Salzman, E.W. and Hirsch, "The Epidemiology, Pathogenesis and Natural History of Venous Thrombosis," in Hemostasis and Thrombosis: Basic Principles and Clinical Practice, Coleman, R.W. et al., eds., 3rd ed. Philadelphia, PA (1994), pp. 1275-1296).

A. Mechanism of Clot Formation and Lysis Clots (or thrombi in a patient) are composed of fibrin and blood platelets in various ratios. The fundamental reaction in blood clotting involves the conversion of a soluble plasma protein (fibrinogen) into insoluble fibrin. The conversion offibrinogen into fibrin is catalyzed by the enzyme, thrombin, which is a serine protease.

Clot lysis is mediated by plasmin. Under natural conditions, plasminogen is converted to plasmin by plasminogen activators. Natural plasmin inhibitors include a 2 -antiplasmin, a2-macroglobulin and a-1-antitrypsin, all glycoproteins.

Alpha-2-antiplasmin has a much higher affinity for plasmin than a2-macroglobulin and binds specifically to plasmin in a 1:1 ratio. The larger pool of amacroglobulin acts as a reservoir inhibitor (Kane, Ann. Clin. Lab. Sci.

14:443-449 (1984)). Thus, clot lysis by the administration oft-PA is limited by the rapid and irreversible inactivation of plasmin by plasmin inhibitors. see* B. Treatmentfor Venous Thrombosis and Pulmonary Embolism Standard therapy for venous thromboembolism is heparin, which potentiates thrombin and factor Xa inhibition by antithrombin III (Goldhaber,

S.,

Chest 107:45S-51S (1995)). Although heparin decreases new thrombus (clot) formation, clinical studies suggest that there is little early endogenous lysis of the large thrombi that often exist at the time of diagnosis in patients with venous WO 98/12329 PCT/US97/16122 -3thromboembolism (Goldhaber, S.Z. et al., Lancet 2:886-889 (1986); "The Urokinase Pulmonary Embolism Trial," Circulation 47:1-108 (1973); Goldhaber, S.Z. et al., Am. J. Med. 88:235-240 (1990); Goldhaber, S.Z. et al., Lancet 341:507-511 (1993)). Since large thrombi are associated with an increase in morbidity and mortality, several studies have examined the effects of plasminogen activators in patients with venous thromboembolism (Goldhaber, S.Z. et al., Lancet 2:886-889 (1986); "The Urokinase Pulmonary Embolism Trial," Circulation 47:1-108 (1973); Goldhaber, S.Z. et al., Am. J. Med 88:235-240 (1990); Goldhaber, S.Z. et al., Lancet 341:507-511 (1993)).

Compared with heparin alone, plasminogen activators cause significant increases in the lysis of venous thromboemboli, but patients are frequently left i"with large amounts of residual thrombi in the lungs or deep veins immediately after therapy (Goldhaber, S.Z. et al., Lancet 2:886-889 (1986); "The Urokinase Pulmonary Embolism Trial," Circulation 47:1-108 (1973); Goldhaber, S.Z. et al., Am. J. Med 88:235-240 (1990); Goldhaber, S.Z. et al., Lancet 341:507-511 (1993)). None of the randomized, controlled trials of patients with pulmonary embolism have demonstrated a mortality benefit from plasminogen activators, although this may well be due to the small numbers of patients enrolled in these studies. Use ofplasminogen activators for myocardial infarctions has shown that 45-70% of patients with coronary thrombosis have failed to achieve full minutes reperfusion with these agents.

Why venous thromboemboli resist fibrinolysis is unknown. Physical i characteristics such as size, retraction, exposure to blood flow, and age may affect the lysis of these large fibrin-rich thrombi (Prewitt, Chest 99:157S-164S 235 (1991)). However, it is also likely that the fibrinolytic resistance of these thrombi is regulated by specific molecular factors such as factor XIII, plasminogen activator inhibitor 1 (PAI-1), and alpha-2-antiplasmin (a2AP) (Collen, Eur.

J. Biochem. 69:209-216 (1976); Moroi, M. and Aoki, J. Biol. Chem.

251:5956-5965 (1976); Mullertz, S. and Clemmensen, Biochem. J. 159:545- 553 (1976); Sakata, Y. and Aoki, J. Clin. Invest. 69:536-542 (1982); Robbie, WO 98/12329 PCTIUS97/16122 -4- L.A. etal., Thromb. Haemostas. 70:301-306 (1993); Francis, C.W. and Marder, J. Clin. Invest. 80:1459-1465 (1987); Jansen, J.W.C.M. et al., Thromb.

Haemostas. 57:171-175 (1987); Reed, G.L. et al., Trans. Assoc. Am. Phys.

104:21-28 (1991); Stringer, H.A. and Pannekoek, J. Biol. Chem. 270:11205- 11208 (1995); Carmeliet, P. et al., J. Clin. Invest. 92:2756-2760 (1993); Lang, I.M. et al., Circulation 89:2715-2721 (1994); Marsh, J.J. et al., Circulation 90:3091-3097 (1994)).

Because a2AP is an ultrafast covalent inhibitor of plasmin (the enzyme that degrades thrombi), a2AP is a particularly likely cause of thrombus resistance (Collen, Eur. J. Biochem. 69:209-216 (1976); Moroi, M. and Aoki, N., J. Biol. Chem. 251:5956-5965 (1976); Mullertz, S. and Clemmensen, Biochem.

J. 159:545-553 (1976)). Moreover, a2AP is the only fibrinolytic inhibitor that is covalently crosslinked to the fibrin surface (Sakata, Y. and Aoki, J. Clin.

Invest. 69:536-542 (1982)). This crosslinking (by activated factor XIII) 1concentrates a2AP on the fibrin surface, where it inhibits the initiation of fibrinolysis (Sakata, Y. and Aoki, J. Clin. Invest. 69:536-542 (1982)).

Previous in vitro studies have shown that clots from a2AP-deficient patients lyse spontaneously, suggesting that a2AP plays a critical role in thrombus resistance to endogenous plasminogen activators (Aoki, N. et al., Blood 62:1118-1122 *:Ui (1983); Miles, L.A. et al., Blood 59:1246-1251 (1982)). These observations led to the hypothesis that a2AP is a molecular mediator of the thrombus resistance S. seen in patients with pulmonary embolism. To test this hypothesis, we generated a specific inhibitor of c2AP and used it to determine the role played by a2AP in the regulation of lysis of experimental pulmonary emboli.

25 If an individual has formed a fibrin clot (thrombus) prior to the availability of medical assistance, the clot may be dissolved through the use of agents capable oflysing the fibrin thrombus, and thereby permitting blood to again flow through the affected blood vessel. Such agents include plasmin, anti-coagulants (such as, for example, heparin, hirudin and activated protein plasminogen activators (such as, for example, streptokinase, prourokinase, urokinase, tissue-type WO 98/12329 PCT/US97/16122 plasminogen activator, staphylokinase, and vampire bat plasminogen activator), and other such agents (Ganz, W. et al., J. Amer. Coll. Cardiol. 1:1247-1253 (1983); Rentrop, K.P. et al., Amer. J. Cardiol. 54:29E-31E (1984); Gold, H.K.

et al., Amer. J Cardiol. 53:122C-125C (1984)).

At present, treatment of pulmonary embolism, myocardial infarction, thrombosis, and stroke is partially achieved through the administration of thrombolytic agents. Use of such agents in therapy often results in incomplete lysis, and promotes the reformation of thrombi and reocclusion of the affected blood vessels. Hence, a need exists for an improvement in thrombolytic therapy which enhances fibrinolysis, while minimizing fibrinogen breakdown and preventing reformation of thrombi.

C Alpha-2 antiplasmin Antibodies

S

Alpha-2-antiplasmin (a2AP) has three functional domains: the reactive site for plasmin, the plasmin(ogen) or LBS-binding site [complementary to the LBS (lysine-binding site) ofplasmin(ogen)], and the crosslinking site for fibrin.

Mimuro, J. et al., Blood 69:446-453 (1987). Mimuro et al. discloses antibodies to a2AP, one of which (JPTI-1) was specific to the reactive site of a2AP and prevented formation of a2AP complexes, thereby inhibiting antiplasmin activity.

However, Mimuro et al. does not.teach administration of the JPTI-1 antibody to 20 enhance clot lysis. Other antibodies specific for a2AP are taught by Plow, E.F.

et al., Biol. Chem. 255:2902-2906 (1980); Wimen, B. et al., Scan. J. Clin. Lab.

Invest. 43:27-33 (1983); Hattey, E. et al., Thromb. Res. 45:485-495 (1987); Collen, U.S. Patent No. 4,346,029 (1980); and Collen, U.S. Patent No. 4,198,335 (1980).

WO 98/12329 PCT/US97/16122 -6- Summary of the Invention The present invention relates to an improved thrombolytic therapy for the treatment of pulmonary embolism, myocardial infarction, thrombosis and stroke in patients. The invention is directed to an immunologic molecule capable of binding to both human and nonhuman circulating a 2 -antiplasmins and (2) human and nonhuman fibrin crosslinked a 2 -antiplasmins. In preferred embodiments, the immunologic molecule is a chimeric antibody, a humanized antibody, or a single chain antibody. The invention is also directed to a method for treating pulmonary embolism, myocardial infarction, thrombosis and stroke in a patient comprising administering an a 2 -antiplasmin-binding molecule capable of binding to both human and nonhuman circulating a2-antiplasmins and (2) 0*94 human and nonhuman fibrin crosslinked a2-antiplasmins. The invention further provides a method of treatment for pulmonary embolism, myocardial infarction, thrombosis and stroke in a patient which comprises co-administrating to a patient 15 in need of such treatment: a therapeutically effective amount of an immunologic molecule capable of binding to both human and nonhuman circulating a 2 -antiplasmins .and human and nonhuman fibrin crosslinked a2-antiplasmins; and a therapeutically effective amount of a thrombolytic agent, wherein *26& the immunologic molecule is different from the thrombolytic agent thereby treating the patient.

The invention provides a monoclonal antibody or fragment thereof wherein the monoclonal antibody is capable of binding to both human and nonhuman circulating a 2 -antiplasmins and human and nonhuman fibrin crosslinked a2antiplasmins. In one embodiment, the invention is monoclonal antibody 77A3.

In another embodiment, the invention is monoclonal antibody 49C9. In another embodiment, the monoclonal antibody is 70B11.

The invention also provides a method of making the monoclonal antibody comprising: WO 98/12329 PCT/US97/16122 -7immunizing an animal with a2-antiplasmin or fragment thereof; fusing cells from the animal with tumor cells to make a hybridoma cell line; cloning the hybridoma cell line; selecting for the monoclonal antibody capable of binding to both human and nonhuman circulating a2-antiplasmins and human and nonhuman fibrin crosslinked a2-antiplasmins; and obtaining the monoclonal antibody.

The invention provides a hybridoma cell line which produces the monoclonal antibody capable of binding to both human and nonhuman circulating a 2 -antiplasmins and human and nonhuman fibrin crosslinked a2antiplasmins. In one embodiment, the invention is hybridoma cell line 77A3 (ATCC Accession No. HB-12192; Deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20862 on September 15 1996).

S. The invention is directed to a method of making the hybridoma cell line comprising: immunizing an animal with a2-antiplasmin or fragment thereof; fusing the cells from the animal with tumor cells to make the :20: hybridoma cell line; and obtaining the hybridoma cell line which produces the monoclonal.

antibody capable of binding to both human and nonhuman circulating a2sees antiplasmins and human and nonhuman fibrin crosslinked a2-antiplasmins.

The invention also provides a method for treating a number of diseases and 25 conditions, including pulmonary embolism, myocardial infarction, thrombosis and stroke in a patient comprising administering a therapeutically effective amount of an immunologic molecule which is capable of binding to both human and nonhuman circulating a 2 -antiplasmins and human and nonhuman fibrin crosslinked a 2 -antiplasmins, thereby treating the patient.

WO 98/12329 PCT/US97/16122 -8- The invention further provides a method of treatment for pulmonary embolism, myocardial infarction, thrombosis or stroke in a patient which comprises co-administering to a patient in need of such treatment: a therapeutically effective amount of an immunologic molecule which is capable of binding to both human and nonhuman circulating a2antiplasmins and human and nonhuman fibrin crosslinked a 2 -antiplasmins; and a therapeutically effective amount of a thrombolytic agent, wherein the immunologic molecule is different from the thrombolytic agent thereby treating the patient.

In preferred embodiments, the thrombolytic agent is plasmin, anticoagulant, or plasminogen activator. In one embodiment, the anti-coagulant is 0.$9 selected from the group consisting of heparin, hirudin and activated protein C. In ::09 another embodiment, the plasminogen activator is selected from the group consisting of staphylokinase, streptokinase, prourokinase, urokinase, tissue-type :.155 plasminogen activator, and vampire bat plasminogen activator.

SOther embodiments of the invention include, the immunologic molecule $00 -provided to the patient by an intravenous infusion, by an intravenously injected bolus, or with a first bolus containing the immunologic molecule and a subsequently administered second bolus containing the thrombolytic agent "0 Further embodiments include, the immunologic molecule provided to- the *goo** patient at* a dose of between 3 to 300 nmole per kg of patient weight; and the •0000. thrombolytic agent provided to the patient at a dose of between 0.01 to 3.0 mg per kg of patient weight.

The invention provides a kit useful for carrying out the method of 72 treatment for pulmonary embolism, myocardial infarction, thrombosis or stroke 0 V '"in a patient, being compartmentalized in close confinement to receive two or more container means therein, which comprises: a first container containing a therapeutically effective amount of the immunologic molecule and WO 98/12329 PCT/US97/16122 -9a second container containing a therapeutically effective amount of the thrombolytic agent wherein the immunologic molecule is different from the thrombolytic agent The invention also provides nucleic acid molecules encoding immunologic molecules capable of binding to both human and nonhuman circulating a2antiplasmins and human and nonhuman fibrin crosslinked a 2 -antiplasmins.

Also provided are molecules comprising an amino acid sequence of the binding region of an immunologic molecule described herein.

Brief Description of the Figures 1 Figure 1. Comparison of binding to 1 2 'I-a2-antiplasmin of monoclonal antibodies 49C9, 70B11, 77A3, RWR and anti-digoxin (control). Wells of a microtiter plate were coated with goat antimouse antibody. The wells were incubated in duplicate with 49C9, 70B11, 77A3, RWR or a control (antidigoxin) MAb (Mudgett-Hunter, M. et al., Mol. Immunol. 22:477-488 (1985)). After a wash, mI-a2AP (60,000 cpm) was added for an hour. The wells were rinsed and the amount of bound mI-a2AP was measured in a gamma counter.

Figure 2. Competition binding assays of monoclonal antibodies 49C9, 11, 77A3, RWR and anti-digoxin with immobilized 70B11. Competition radioimmunoassays were performed by coating wells of a microtiter plate with pl of purified MAb (70B11) in duplicate (10 ig/ml) for 1 hour. The wells were washed and blocked with 1% BSA for 1 hour. After washing, 25 p1 of a competitor MAb, same MAb or negative control MAb was added to different wells (50 pg/ml) followed by 25 pl of 2 sI-a2-antiplasmin (100,000 cpm). After 1 hour incubation, the wells were washed, cut and the radioactivity was measured in a gamma scintillation counter.

WO 98/12329 PCT/US97/16122 Figure 3. Comparison of amount of lysis by different monoclonal antibodies (or TBS alone) as a function of dose ofurokinase. See Example 1, below, for detailed description of the method. The amount of lysis was determined by gamma counting. The percent lysis was defined at 100 x (total supernatant cpm total clot cpm).

Figure 4. Dose response studies in the absence or presence of MAb 77A3. Lysis by urokinase is increased approximately 100-fold by 77A3.

Figure 5. Reduced SDS-polyacrylamide gel electrophoresis of 77A3 purification. Ascites containing 77A3 were harvested and purified. Lane 1, J protein standards with molecular mass in kDa (left); lane 2, supernatant after recipitation with 40% ammonium sulfate; lane 3, purified 77A3. The reduced 77A3 immunoglobulin consists of bands of-50 kDa, corresponding to the heavy chain, and -25 kDa, corresponding to the light chain.

Figure 6. Effect of.77A3 on the rate of lysis of ferret plasma clots in vitro. Ferret plasma clots formed with trace amounts of 12 I-labeled human fibrinogen were incubated with 100 pl of TBS (control) or purified MAb (25 gg, 77A3 or RWR). Clot lysis was initiated by adding 0.1 unit of rt-PA per tube. The clots were incubated at 37 0 C and the amount of lysis was determined by sampling for the release ofradiolabeled fibrin degradation products into the supernatant as described (Reed, G.L. m et al., Proc. Natl. Acad Sci. USA 87:1114-1118 (1990)).

Figure 7 Effect of in vivo administration of MAb 77A3 on functional a2AP levels in ferrets. In dose finding experiments, two anesthetized ferrets (A, B) were given 77A3 intravenously (22.5 mg/kg) and the amount of functional a2AP was measured in citrated plasma samples drawn before (time 0) and 1 and WO 98/12329 PCT/US97/16122 -11- 4 hours after infusion. The data represent the mean±SD inhibition of a2AP in plasma samples.

Figure 8. Effect of rt-PA and a2AP inhibition on the lysis of pulmonary emboli in vivo. Anesthetized ferrets were given a heparin bolus (100 U/kg) and "I-labeled fibrin clots were embolized into the lungs. After embolization, three groups of ferrets were given rt-PA 1, or 2 mg/kg) over 2 hours intravenously (plain bars). Two other groups of ferrets also received rt-PA (1 mg/kg) and a control MAb (antidigoxin, black bar, 22.5 mg/kg) or a MAb that inhibits a2AP (77A3, striped bar, same dose). The graph shows the amount of lysis (mean±SD) for each treatment group. The number of ferrets in each treatment group is shown, and the P values for differences between groups are indicated.

Figure 9. Residual fibrinogen levels in animals treated with heparin, Vo. rt-PA, and an a2AP inhibitor. Blood samples were collected (on EDTA with aprotinin) from ferrets before pulmonary embolization and at the end of the experiment. Residual fibrinogen levels were measured as described (Rampling, M.W. and Gaffney, Clin. Chim. Acta.67:43-52 (1976)). The graph shows the mean±SD percentage residual fibrinogen level for animals receiving rt-PA S alone 1, or 2 mg/kg; plain bars) and those receiving rt-PA and the a2AP inhibitor (striped bar).

3. Figure 10. The peptide sequences of the amino terminus of purified light chains from 49C9 (SEQ ID NO:) 70B11 (SEQ ID NO:2) and 77A3 (SEQ ID NO:3) are shown.

Figure 11. The cDNA sequence (SEQ ID NO:4) and corresponding deduced amino acid sequence of the signal peptide (amino acids -20 to -1 of SEQ ID NO:5) and light chain variable regions (amino acids 1 to 107 of SEQ ID of 49C9 are shown.

WO 98/12329 PCT/US97/16122 -12- Figure 12. The cDNA sequence (SEQ ID NO:6) and corresponding deduced amino acid sequence of the signal peptide (amino acids -20 to -1 of SEQ ID NO:7) and light chain variable regions (amino acids 1 to 107 of SEQ ID NO:7) of 70B 11 are shown.

Figure 13. The cDNA sequence (SEQ ID NO:8) and corresponding deduced amino acid sequence of the signal peptide (amino acids -20 to -1 of SEQ ID NO:9) and light chain variable regions (amino acids 1 to 107 of SEQ ID NO:9) of 77A3 are shown.

Figure 14. The cDNA sequence (SEQ ID NO:10) and corresponding deduced amino acid sequence of the signal peptide (amino acids -19 to -1 of SEQ ID NO:11) and heavy chain variable regions (amino acids 1-119 of SEQ ID NO: 11) of49C9 are shown.

NO:13) of 70B 1 are shown.

Figure 16. The cDNA sequence (SEQ ID NO:12) and corresponding deduced amino acid sequence of the signal peptide (amino acids -19 to -1 of SEQ ID NO:13) and heavy chain variable regions (amino acids 1-119 of SEQ ID 20 NO:13) of 77A3 are shown.

Figure 17. The cDNA sequence (SEQ ID NO:14) and corresponding "deduced amino acid sequence of the signal peptide (amino acids -19 to -1 of SEQ ID NO:15) and heavy chain variable regions (amino acids 1-119 of SEQ ID NO: 15) of77A3 are shown.

00* Figure 17. The cDNA sequence (SEQ ID NO:16) and corresponding amino acid sequence (SEQ ID NO:17) of humanized77A3-1 and humanized 77A3-2 light chain. Positions falling within the CDR loops are shown enclosed within the boxes with solid borders.

WO 98/12329 PCT/US97/16122 -13- Figure 18. The cDNA sequence (SEQ ID NO: 18) and corresponding amino acid sequence (SEQ ID NO:19) of humanized 77A3-1 heavy chain.

Positions falling within the CDR loops are shown enclosed within the boxes with solid borders.

Figure 19. The cDNA sequence (SEQ ID NO:20) and corresponding amino acid sequence (SEQ ID NO:21) of humanized 77A3-2 heavy chain.

Positions falling within the CDR loops are shown enclosed within the boxes with solid borders.

Figure 20. Results of murine 77A3 chimeric 77A3 and .6 humanized 77A3-1 in the plasmin assay with chromogenic substrate are shown.

Figure 21. The amino acid sequences of the light chains are shown: h77A3-1 and h77A3-2 (SEQ ID NO:17); m77A3 (SEQ ID NO:9); m49C9 (SEQ ID NO:5); m70B11 (SEQ ID NO:7); murine consensus (SEQ ID NO:75), which .*15 shows the consensus between m77A3, m49C9, and m70Bll; 77A3/49C9 consensus (SEQ ID NO:76), which shows the consensus between 77A3 and 49C9; and all (SEQ ID NO:77), which shows the consensus between h77A3-1, h77A3-2, m77A3, m49C9, and m70B 11. Positions falling withing the CDR loops are shown enclosed within the boxes.

D Figure 22. The amino acid sequences of the heavy chains are shown.

h77A3-1 (SEQ ID NO:19); h77A3-2 (SEQ ID NO:21); m77A3 (SEQ ID m49C9 (SEQ ID NO:11); m70B 11 (SEQ ID NO: 13); humanized consensus

(SEQ

ID NO:78), which is the consensus between h77A3-1 and h77A3-2; murine consensus (SEQ ID NO:79), which is the consensus between m77A3, m49C9, and m70Bll; 77A3/49C9 consensus (SEQ ID NO:80), which is the consensus between 77A3 and 49C9; and all (SEQ ID NO:81), which is the consensus WO 98/12329 PCT/US97/16122 -14between h77A3-1, h77A3-2, m77A3, m49C9, and m70Bll. Positions falling withing the CDR loops are shown enclosed within the boxes.

Detailed Description of the Preferred Embodiments Alpha-2-antiplasmin (a2AP) is a molecular mediator of the thrombus resistance in patients with pulmonary embolism. A specific inhibitor of a2AP is described which is used to determine the role played by a2AP in the regulation of fibrinolysis.

A. Immunologic Molecules e..

*S

In the following description, reference will be made to various methodologies well-known to those skilled in the art of immunology. Standard reference works setting forth the general principles of immunology include Klein, Immunology: The Science of Cell-Noncell Discrimination, John Wiley Sons, New York (1982); Kennett, R. et al.,Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, New York (1980); Campbell, "Monoclonal Antibody Technology," in Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Burdon, et al., eds., Elsevier, Amsterdam (1984); and Eisen, Microbiology, 3rded, Davis, et al., Harper Row, Philadelphia (1980).

As used herein, a2AP-binding molecule includes antibodies (polyclonal or *0 monoclonal), as well as ligands. As used herein, an "immunologic molecule" refers to polypeptides comprising the binding region of a monoclonal antibody.

Thus, monoclonal antibodies, antibody fragments, chimeric antibodies, humanized antibodies, and fusion proteins comprising antibody binding regions are "immunologic molecules". The term "antibody" (Ab) or "monoclonal antibody" (MAb) is meant to include intact molecules as well as antibody fragments (such as, for example, Fv, Fab and F(ab') 2 fragments), single chain antigen-binding WO 98/12329 PCT/US97/16122 proteins, "humanized" antibodies, and chimeric antibodies which are capable of specifically binding to a2AP. Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less nonspecific tissue binding of an intact antibody (Wahl et al, J Nucl. Med 24:316-325 (1983)). Thus, these fragments are preferred.

An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. As used herein, the term "hapten" is intended to refer to any molecule capable of being bound by an antibody. The term "epitope" is meant to refer to 10 that portion of a hapten which can be recognized and bound by an antibody. A hapten or antigen may have one, or more than one epitope. An "antigen" or se •"immunogen" is a hapten which is additionally capable of inducing an animal to 0 produce antibody capable of binding to an epitope of that antigen. The specific reaction referred to above is meant to indicate that the hapten will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

The antibodies of the present invention may be prepared by any of a variety of methods. For example, cells expressing a2AP (or fractions, lysates, etc.

thereof) can be administered to an animal in order to induce the production of sera containing polyclonal antibodies that are capable of binding a2AP. In a preferred method, a preparation of x2AP of the present invention is prepared and purified •@005•5 to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater 6 specific activity.

The antibodies of the present invention may also be prepared using phage display technology. Methods of preparing antibodies using phage display are known in the art. See, for example, U.S. Patent No. 5,565,332; Clarkson et al., Nature 352:624-628 (1991); Huse, Science 246:1275-1281 (1989); Kang, Proc.

Nat. Acad Sc! USA 88:11120-11123 (1993); Marks, J. Mol. Biol. 222:581-597 (1991); and McCafferty et al., Nature 348:5 52-554 (1990).

WO 98/12329 PCT/US97/16122 -16- In one preferred method, the immunogenic molecules of the present invention are monoclonal antibodies (or a2AP binding molecules). Such monoclonal antibodies can be prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J Immunol 6:292 (1976); Hammerling et al., in MonoclonalAntibodies and T-CellHybridomas, Elsevier, pp. 563-681 (1981)). In general, such procedures involve immunizing an animal (preferably a mouse) with the antigen or with a cell which expresses the antigen. A preferred antigen is purified a2AP.

The most preferred antigen is a2AP fragment (fibrin binding region) obtained by trypsin digest of a plasma clot, then affinity purified with a SEPHAROSE-coupled monoclonal antibody, RWR (Reed, G.L. III et al., Trans. Assoc. Am. Phys.

101:250-256 (1988); U.S. Patent No. 5,372,812, issued December 13, 1994).

Suitable cells can be recognized by their capacity to secrete anti-a2AP antibody.

Such cells may be cultured in any suitable tissue culture medium; however, it is *1 preferable to culture cells in Earle's modified Eagle's medium supplemented with fetal bovine semm (inactivated at about 56"C), and supplemented with about ag/1 of nonessential amino.acids, about 1,000 U/ml of penicillin, and about 100 i g/ml of streptomycin. The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. The method of somatic cell fusion is described in Galfre, G. and Milstein, Meth. Enzymol. 73:3-46 (1981). After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al., Gastroenterology 80:225- 232 (1981). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding a2AP.

Alternatively, additional antibodies capable of binding to the a2AP antigen may be produced in a two-step procedure through the use of anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and that, therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, a2AP-specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an WO 98/12329 PCT/US97/16122 -17animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the a2AP-specific antibody can be blocked by the a2AP antigen. Such antibodies comprise anti-idiotypic antibodies to the a2AP-specific antibody and can be used to immunize an animal to induce formation of further a2AP-specific antibodies.

It will be appreciated that Fab and F(ab') 2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments). Alternatively, a2AP-binding fragments can be produced through the application of recombinant DNA technology, through synthetic chemistry, or biotinylation.

Also intended within the scope of the present invention are humanized or chimeric antibodies, produced using genetic constructs derived from hybridoma S. 15 cells producing the MAbs described above. Humanized antibodies are antibodies in which the framework or other regions of the murine Ab is replaced with the homologous regions of a nonmurine antibody. Chimeric antibodies are antibodies in which the murine constant region has been replaced with a non-murine constant S" region. Methods for production of chimeric antibodies are known in the art. See, for review: Morrison, Science, 229:1202-1207 (1985); Oi et al., BioTechniques 0 ~4:214 (1986); see also, Cabilly et al., U.S. Patent 4,816,567 (3/28/89); Taniguchi et al., EP171496 (2/19/86); Morrison et al., EP173494 Neuberger et al., W08601533 (3/13/86); Robinson et al., WO 8702671 Boulianne et al., Nature 312:643-646 (1984); and Neuberger et al., Nature 314:268-270 (1985).

Methods for production of humanized antibodies are known in the art. See, for example, U.S. Patent 5,585,089; Jones et al., Nature 321:522-525 (1986); and Kettleborough et al., Protein Engineering 4:773-783 (1991).

Also provided in the present invention are single-chain antibodies capable of binding to both human and nonhuman circulating a2-antiplasmins and (2) human and nonhuman fibrin crosslinked a 2 -antiplasmins. Methods of making -18single chain antibodies are well known in the art. See, for example, U.S. Patent No. 4,946,778; U.S. Patent No. 5,260,203; U.S. Patent No. 5,091,513; and U.S.

Patent No. 5,455,030, all of which are herein incorporated by reference.

Also intended within the scope of the present invention are variants of the monoclonal antibodies described above.

The present inventors have determined the nucleotide and amino acid sequence of several immunologic molecules capable of binding to both human and nonhuman circulating a2-antiplasmins and human and nonhuman fibrin crosslinked a2-antiplasmins. Accordingly, the present invention provides for .1Q. nucleic acid molecules comprising a nucleotide sequence encoding for an immunologic molecule of the present invention or fragment thereof.

*00 Due to the degeneracy of the genetic code, and to the fact that the genetic o. code is known, all other nucleotide sequences which encode the same amino acid sequence as the nucleotides of the present invention can be determined and used 5' in the practice of the present invention.

DNA clones containing nucleotide sequences encoding the following antibody chains were deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20862 on September 19, 1997: light chain of 77A3 (77A3 LC), ATCC Accession No. 209290; light chain of 49C9 (49C9 LC), ATCC Accession No. 209291; light chain of 70B 1 (70B 11 LC), ATCC Accession No. 209292; heavy chain of 77A3 (77A3 HC), ATCC Accession No.

209287; heavy chain of 49C9 (49C9 HC), ATCC Accession No. 209289; and heavy chain of70B1 I (70B1 1 HC), ATCC Accession No. 209288.

The nucleic acid molecules of the present invention include: nucleic acid molecules containing a nucleotide sequence encoding the mature light chain of 77A3 as shown in SEQ ID NO:9 or as encoded by the clone contained in the ATCC Accession No. 209290; nucleic acid molecules containing a nucleotide sequence encoding the mature light chain of 49C9 as shown in SEQ [D NO:5 or as encoded by the clone contained in ATCC Accession No. 209291; and nucleic acid molecules containing a nucleotide sequence encoding the mature light chain AMENDED SHEET -19of 70B 11 as shown in SEQ ID NO:7 or as encoded by the clone contained in ATCC Accession No. 209292.

Also included in the present invention are nucleic acid molecules containing a nucleotide sequence encoding an antibody heavy chain, including: nucleic acid molecules containing a nucleotide sequence encoding the mature heavy chain of 77A3 as shown in SEQ ID NO: 15 or as encoded by the clone contained in ATCC Accession No. 209287; nucleic acid molecules containing a nucleotide sequence encoding the mature heavy chain of 49C9 as shown in SEQ ID NO: 11 or as encoded by the clone contained in ATCC Accession No. 209289; 0 and nucleic acid molecules containing a nucleotide sequence encoding the mature heavy chain of 70B11 as shown in SEQ ID NO: 13 or as encoded by the clone contained in ATCC Accession No. 209288.

Also included are nucleic acid molecules encoding humanized antibodies including: nucleic acid molecules comprising a nucleotide sequence encoding for gee amino acid residues 1 to 107 of SEQ ID NO:17; nucleic acid molecules comprising a nucleotide sequence encoding for amino acid residues 1 to 119 of SEQ ID NO: 19; and nucleic acid molecules comprising a nucleotide sequence encoding for amino acid residues 1 to 119 of SEQ ID NO:21.

Also intended within the scope of the invention are nucleic acid molecules encoding "consensus" amino acid sequences of heavy and light chain of antibodies, including: nucleic acid molecules comprising a nucleotide sequence encoding for amino acid residues 1 to 107 of SEQ ID NO:75; nucleic acid molecules comprising a nucleotide sequence encoding for amino acid residues 1 to 107 to of SEQ ID NO:76; nucleic acid molecules comprising a nucleotide sequence encoding for amino acid residues 1 to 107 of SEQ ID NO:77; nucleic acid molecules comprising a nucleotide sequence encoding for amino acid residues 1 to 119 of SEQ ID NO:78; nucleic acid molecules comprising a nucleotide sequence encoding for amino acid residues 1 to 119 of SEQ ID NO:79; nucleic acid molecules comprising a nucleotide sequence encoding for amino acid residues AMENDED

SHEET

WO 98/12329 PCT/US97/16122 1 to 119 of SEQ ID NO:80; and nucleic acid molecules comprising a nucleotide sequence encoding for amino acid residues I to 119 of SEQ ID NO:81.

Nucleic acid molecules encoding an immunologic molecule of the present invention can be used to express recombinant proteins. A nucleic acid molecule encoding an immunologic molecule of the present invention can be inserted into a vector in accordance with conventional techniques. A "vector" should be understood as a nucleic acid vehicle used for cloning or expressing a desired sequence in a host.

In one embodiment, the recombinant vector is capable of expressing the immunologic molecule of the present invention. A vector is said to be "capable of expressing" a polypeptide if it contains a nucleotide sequence that encodes for the polypeptide, as well as transcriptional and translational regulator information operably linked to the nucleotide sequence that encodes the polypeptide. Two nucleotide sequences are said to be "operably linked" if the nature of the linkage 5. between the two nucleotide sequences does not: result in the introduction of a frame-shift mutation; interfere with the ability of the promoter region sequence to direct the transcription of the desired sequence; or interfere with the ability of the desired sequence to be transcribed by the promoter region sequence. Thus, a promoter region would be operably linked to a desired nucleotide sequence if the promoter were capable of effecting transcription of that nucleotide sequence.

""Once the recombinant vector is constructed, it can be introduced into a S* host cell, either prokaryotic or eukaryotic, by a variety of conventional techniques including transfection, transduction, electroporation, calcium-phosphate precipitation, and microinjection. Prokaryotic hosts include bacteria such as E.

coli, Bacillus, Streptomyces, and Salmonella. The most preferred prokaryotic host is E. coli. Eukaryotic hosts include yeast cells, insect cells, and mammalian cells, such as COS cells, CHO cells, and myeloma cells. In one embodiment of the invention, CHO cells are preferred.

In one embodiment of the invention, a nucleic acid molecule comprising a nucleotide sequence encoding for the light chain of an antibody is introduced WO 98/12329 PCT/US97/16122 -21into a vector, and a nucleic acid molecule comprising a nucleotide sequence encoding for the heavy chain of an antibody is introducing into another vector.

Both vectors are introduced into the same host cell. Alternatively, both chains could be introduced into the same vector.

Following expression in an appropriate host, the polypeptide can be readily isolated using standard techniques, including affinity chromatography.

Also intended within the scope of the present invention are molecules comprising an amino acid sequence of the binding region of an immunologic molecule described herein. Molecules comprising an amino acid sequence of the binding region of an immunologic molecule described herein include, but are not limited to, monoclonal antibodies, humanized antibodies, chimeric antibodies, fragments of any such antibodies, single chain antibodies, fusion proteins, and the like. Such molecules can be used in the assays and methods of treatment of the present invention described below.

*5 The amino acid sequence of the binding region of the immunologic molecules of the present invention are shown in Figure 21 for the light chains and Figure 22 for the heavy chains. In Figure 21, the amino acid sequence of the binding regions of the light chains ofh77A3-1 and h77A3-2 (amino acid residues 24 to 34, 50 to 56 and 89 to 97 ofSEQ ID NO:17), m77A3 (amino acid residues 24 to 34, 50 to 56 and 89 to 97 ofSEQ ID NO:9), m44C9 (amino acid residues 24 to 34, 50 to 56 and 89 to 97 of SEQ ID NO:5), m70B11 (amino acid residues 24 to 34, 50 to 56 and 89 to 97 of SEQ ID NO:7), the murine consensus (amino i acid residues 24 to 34, 50 to 56 and 89 to 97 of SEQ ID NO:75), the 77A3/49C9 consensus (amino acid residues 24 to 34, 50 to 56 and 89 to 97 of SEQ ID NO:76) and the consensus of all light chains (amino acid residues 24 to 34, 50 to 56 and 89 to 97 of SEQ ID NO:77) are shown in the larger boxes.

In Figure 22, the amino acid sequence of the binding regions of the heavy chains of h77A3-1 (amino acid residues 26 to 35, 50 to 66 and 99 to 108 of SEQ ID NO:19), h77A3-2 (amino acid residues 26 to 35, 50 to 66 and 99 to 108 of SEQ ID NO:21), m77A3 (amino acid residues 26 to 35, 50 to 66 and 99 to 108 WO 98/12329 PCT/US97/16122 -22of SEQ ID NO:15), m49C9 (amino acid residues 26 to 35, 50 to 66 and 99 to 108 of SEQ ID NO:11), m70B 11 (amino acid residues 26 to 35, 50 to 66 and 99 to 108 of SEQ ID NO:13), the humanized consensus (amino acid residues 26 to to 66 and 99 to 108 of SEQ ID NO:78), the murine consensus (amino acid residues 26 to 35, 50 to 66 and 99 to 108 of SEQ ID NO:79), the 77A3/49C9 consensus (amino acid residues 26 to 35, 50 to 66 and 99 to 108 of SEQ ID and the consensus of all the heavy chains (amino acid residues 26 to to 66 and 99 to 108 of SEQ ID NO:81) are shown in the overlapping boxes.

B. Assays 10. Methods for immunoblotting are known in the art (see, for example, Reed, G.L. et al., J. Immunol. 150:4407-4415 (1993)). In a preferred method, the a2AP is electrophoresed on a slab minigel under reducing and non-reducing conditions. The gel is electroblotted to polyvinylidene difluoride membrane. The blot is exposed to different hybridoma supernatants in the channels of a miniblotter apparatus. After washing, the bound antibody is detected by incubation with goat antimouse antibody. After additional washing, the membrane is exposed in Sa phosphorimager (Molecular Devices, Sunnyvale, CA).

Methods.for radioimmunoassays are also known. For example, the wells 0" of a microtiter plate are coated with goat antimouse antibody. The wells are washed and blocked with BSA. The hybridoma supernatants are added to the empty wells. After incubation, the wells are washed and 25 I-a2AP is added.

After washing, the wells are cut and the bound antibody is measured by gamma scintillation counting. For competition assays, the wells of a microtiter plate are coated with a competing MAb. In a preferred embodiment, the binding of MAbs to 2 I-a2AP (preferably, the fibrin binding region fragment of a2AP, obtained by binding to RWR) is assayed by reverse solid-phase radioimmunoassay.

Methods for clot assays are also known (see, for example, Reed, G.L. III et al., Proc. Nail. Acad. Sci. USA 87:1114-1118 (1990). In a preferred WO 98/12329 PCT/US97/16122 -23embodiment, plasma is mixed with 25 I-fibrinogen, then clotted by mixing with CaCl 2 and thrombin. Clots are compressed and washed with Tris-buffered saline to remove unbound proteins. The supernatant is removed and the clots counted in a gamma counter. To each set of duplicate clots is added, various amounts of plasminogen activator, anti-coagulant, and Tris-buffered saline containing the MAb or no MAb. The clots are incubated and at various intervals, a portion of the solution is temporarily removed and gamma-counted to determine the amount of lysis. The percent lysis may be defined at 100X (total supernatant cpm/total clot cpm).

Fibrinogen assays are known. Blood samples and platelet-poor plasma are assayed for fibrinogen by, for example, the sodium sulfite method (Rampling, 0* M.W. and Gaffney, Clin. Chim. Acta. 67:43-52 (1976)).

Alpha-2-antiplasmin levels in plasma are assayed, for example, with a chromogenic substrate assay for plasmin inhibition (Stachrom kit) as described in S'ai Reed, II etal., Proc. Natl. Acad Sci. USA 87:1114-1118 (1990).

Statistical tests may be analyzed by, for example, a one way analysis of variance followed by a Bonferroni-Dunn procedure for multiple comparison testing.

In vivo pulmonary embolism experiments are described in Example 2, 20 below.

SC Methods of Treatment By "patient" is intended, human or nonhuman. Nonhumans include, for example, baboon, green monkey, dog, cynamologus, marmoset, ferret, guinea pig, and gerbil.

By "clot" is intended, an in vitro blood or fibrin clot, or "thrombi" in a patient. Diseases treated according to the methods of his invention include, but are not limited to pulmonary thromboembolism; acute coronary syndrome, including unstable angina pectoris and non-Q-wave myocardial infarction; various WO 98/12329 PCT/US97/16122 -24forms of thrombosis, including venous thrombosis deep venous thrombosis), and arterial thrombosis renal, mesenteric, and limb thrombosis); and cerebral and thrombosis embolism; renal vein and peripheral arterial thrombosis, myocardial infarction, stroke, and other thromboses. This method may also be used to treat thrombotic conditions secondary or concomitant to surgical procedures, including percutaneous transluminal coronary angioplasty, peripheral arterial angioplasty, bypass graft, and stent. The "treating" or "treatment" is by, for example, inhibiting the formation of a thrombus, dissolving a thrombus, or by enhancing fibrinolysis.

By the term "co-administration" is intended that each of the hapten-binding molecule and thrombolytic agent will be administered during a time frame wherein the respective periods of pharmacological activity overlap. The two agents may 00" be administered simultaneously or sequentially.

The a2AP-binding molecules of the present invention may be monoclonal 15 antibodies or fragments thereof. It is preferable to employ the fragment of such an antibody for this purpose, in order to minimize any immunological reaction caused by the Fc portion of the immunoglobulin. Also preferred are single-chain antibodies, such as sFv. Procedures for preparing monoclonal antibodies are disclosed by Kaprowski, H. et al., United States Patent No.

4,172,124, and Kohler et al., Nature 256:495-497 (1975). The preparation of monoclonal antibodies capable of preventing the inhibition of plasmin are taught by Mimuro, J. et al., Blood 69:446-453 (1987), and described in the examples section of the present application.

As used herein, an "antigen" is a molecule capable of being bound by an .25. antibody such as, for example, a2AP. In order to be used in accordance with the 0 present invention, the "antigen-binding molecule" must be capable of binding to a plasmin inhibitor and thereby prevent such an inhibitor from forming inhibitorplasmin complexes. Any such antigen-binding molecule may be employed in accordance with the present invention. A preferred embodiment is a2AP-binding molecule which is capable of binding to a2AP or fragment thereof. An especially WO 98/12329 PCT/US97/16122 preferred a2AP-binding molecule for this purpose is a monoclonal antibody.

Preferred embodiments of the monoclonal antibody is 77A3, 70B 11 or 49C9, described more fully below. The hybridoma producing MAb 77A3 has been deposited under the terms of the Budapest Treaty with the International Depository Authority American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, on September 20, 1996; the ATCC Accession No.

is HB-12192.

Also preferred are chimeric an humanized antibodies. An especially preferred chimeric antibody for this purpose is c77A3, described more fully below.

Especially preferred humanized antibodies for this purpose are h77A3-1 and h77A3-2, described more fully below. Also preferred are antibody fragments and single-chain antibodies, including sFv77A3-1 and sFv77A3-2, described below.

The terms "thrombolytic agent" are meant to refer to any agent capable of either dissolving a fibrin and/or platelet clot (or thrombus), or inhibiting the S 1'i5 formation of such a clot. Examples of thrombolytic agents include fibrinolytic S molecules, such as plasmin, plasminogen activator (for example, staphylokinase, S0- streptokinase, prourokinase, urokinase, tissue-type plasminogen activator, and vampire bat plasminogen activator); anti-coagulants (for example, inhibitors of fibrin formation, such as heparin, hirudin and activated protein C; and anti-platelet 2 agents, such as ticlopidine, aspirin, and clopidigrel and inhibitors of glycoprotein IIb/lla function). Use of t-PA for these purposes is especially preferred.

Although natural t-PA may be employed, it is preferable to employ recombinant t-PA (rt-PA). The invention may additionally employ hybrids, physiologically active fragments or mutant forms of the above thrombolytic agents. For example, S5 the term "tissue-type plasminogen activator" as used herein is intended to include such hybrids, fragments and mutants, as well as both naturally derived and recombinantly derived tissue-type plasminogen activator.

As stated, the methods of the invention comprise the administration of the a2AP-binding molecule alone or in combination with a thrombolytic agent. When administered alone the molecule enhances endogenous fibrinolysis or thrombolysis WO 98/12329 PCT/US97/16122 -26by significantly augmenting clot lysis by endogenous plasminogen activators.

Further, administration of the a2AP-binding molecule does not increase fibrinogen consumption over that obtained with equivalent doses oft-PA alone. Thus, the present method of clot-specific inhibition of a2AP enhances the potency of the plasminogen activator and preserves its fibrin selectivity.

Alternatively, the a2AP-binding molecule is administered with a thrombolytic agent. In this embodiment, the a2AP-binding molecule and the thrombolytic agent of the present invention are intended to be co-administered to the recipient. It is preferable to provide the a2AP-binding molecule to the patient prior to the administration of the thrombolytic agent.

The a2AP-binding molecule of the present invention is provided for the purpose of preventing the inhibition ofplasmin by a plasmin inhibitor. It has been discovered that coadministration of the a2AP-binding molecule together with a thrombolytic agent causes a synergistic effect, and thereby enhances clot lysis (thrombolysis) to a greater extent than would be expected if the effects of a2APbinding molecule administration and thrombolytic agent administration was merely additive.

The a2AP-binding molecule of the present invention encompasses clotspecific inhibitors of a2AP. It is demonstrated that the combination oft-PA and 2.E the specific inhibitors, particularly monoclonal antibodies to a2AP, does not increase fibrinogen consumption over that obtained with equipotent doses of plasminogen activator alone. Further, clot-specific inhibition of a2AP enhances the potency ofplasminogen activators and preserves fibrin selectivity. For agents such as urokinase, which is not selective for fibrin, inhibition of clot bound a2AP would cause a similar, or more pronounced, enhancement in potency and lead to less fibrinogen consumption as well.

Thus, the inhibition of clot-bound a2AP enhances clot lysis by endogenous plasminogen activators. Further, when administered in combination with a thrombolytic agent, thrombolysis is significantly increased compared with the lysis achieved by equivalent doses of the thrombolytic agent alone. This increased lysis WO 98/12329 PCT/US97/16122 -27by the combination of the thrombolytic agent and a2AP inhibitor occurs without decreasing circulating fibrinogen or a2AP levels. The net result is a synergistic interaction between the two agents.

When used alone, an amount of a2AP-binding molecule capable of preventing inhibition of plasmin and thereby enhancing clot lysis when provided to a patient is a "therapeutically effective" amount. In order to enhance clot lysis and prevent clot reformation, it is desirable to provide between 3 to 300 nmole of a2AP-binding molecule per kilogram of patient weight. This dosage may be administered, in one embodiment, over a period of between 60 to 480 minutes, by continual intravenous infusion at a rate of 0.006 to 5 nmole/kg/min. Alternatively, it is possible to provide the a2AP-binding molecule in an intravenously injectable bolus at a dose of between 3 to 600 nmole/kg, and most preferably between 30 to 60 nmole (of a2AP-binding molecule) per kilogram of patient weight. If the a2AP-binding molecule is provided in this manner, a single bolus is sufficient to prevent potential clot reformation. The a2AP-binding molecule of the present invention may be dissolved in any physiologically tolerated liquid in order to prepare an injectable bolus. It is preferable to prepare such a bolus by dissolving the a2AP-binding molecule in normal saline.

When the a2AP-binding molecule capable of preventing inhibition of plasmin is co-administered with a thrombolytic agent, it is desirable to provide 3 to 300 nmole of a2AP-binding molecule per kilogram of patient weight. This dosage may be administered, in one embodiment, over a period of 60 to 480 minutes, by continuous intravenous infusion. Alternatively, it is possible to provide the a2AP-binding molecule in an intravenously injectable bolus at a dose of between 3 to 600 nmole/kg, and most preferably between 30 to 60 nmole/kg S of patient weight. An amount ofthrombolytic agent capable of causing such lysis is a "therapeutically effective" amount. It is desirable to provide between 0.01 to mg per kilogram of patient weight. In one embodiment, the thrombolytic agent is provided over a prolonged period from about 180 to about 1440 minutes). In a preferred embodiment, the thrombolytic agent of the present WO 98/12329 PCT/US97/16122 -28invention is provided as an intravenously injected bolus containing between 0.5 to mg/kg, and most preferably between 0.5 to 0.75 mg/kg. For example, for pulmonary embolism, the dosage oft-PA by continuous infusion is -100 mg for 2 hours (Goldhaber, S.C. et al., Lancet 341:507 (1993)). The dosage to be used ofthrombolytic agent of the present invention is generally known in the art (see, Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd ed. Philadelphia, PA (1994)).

The thrombolytic agent of the present invention may be dissolved in any physiologically tolerated liquid in order to prepare an injectable bolus. It is, however, preferable to prepare such a bolus by dissolving the thrombolytic agent in normal saline.

A patient treated according to the preferred embodiment will, therefore, receive an intravenously injected bolus of the a2AP-binding molecule in combination with an intravenously injected bolus of the thrombolytic agent. This 5 preferred treatment minimizes the amount oft-PA required for thrombolysis, thus reducing the extent of fibrinogen breakdown and lessening any tendency for general hemorrhage. Importantly, the use of the preferred treatment results in the dissolution of the occluding thrombus at a rate which greatly exceeds the rate of thrombus dissolution when either the a2AP-binding molecule or the thrombolytic agent is provided by infusion alone. Additionally, the risk of reocclusion is substantially reduced.

In previous models offibrinolysis the chief role assigned to a2AP has been to inactivate circulating plasmin and prevent a systemic lytic state. Thus, it may be surprising that an a2AP inhibitor can increase clot lysis without increasing fibrinogenolysis. This marked amplification of thrombolysis by a2AP inhibitor underscores the importance of fibrin bound a2AP in regulating fibrinolysis. Since the subject antibodies augment clot lysis by a fibrin-selective agent such as t-PA as well as that by the nonselective activators urokinase and streptokinase, it appears that fibrin-bound a2AP plays a critical role in determining the rate oflysis by any exogenous plasminogen activator.

WO 98/12329 PCT/US97/16122 -29- These unexpected findings are important because it had previously not been possible to accelerate the rate of clot lysis without increasing the tendency to hemorrhage. The preferred embodiment, therefore, provides a method of treatment in which the administration of a bolus of a a2AP-binding molecule in combination with the administration of a bolus of a thrombolytic agent are capable of dissolving an occluding thrombus at a faster rate than can be obtained when either compound is administered alone. Moreover, the preferred embodiment accomplishes this goal while minimizing both fibrinogen breakdown and the risk of reocclusion. Thus, the combination of agents can significantly increase the potency and specificity ofthrombolytic therapy.

As would be apparent to one of ordinary skill in the art, the required dosage of the anti-a2AP binding molecule or thrombolytic agent will depend upon the severity of the condition of the patient, and upon such criteria as the patient's height, weight, sex, age, and medical history.

S 15 The a2AP-binding molecule or thrombolytic agent of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, such as by admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation are described, for example, in Remington's Pharmaceutical Sciences, 16th Ed., Osol, ed., Mack, Easton PA 9 "20 (1980). In order to form a pharmaceutically acceptable composition suitable for *0 effective administration, such compositions will contain an effective amount of the S" a2AP-binding molecule or thrombolytic agent, either alone, or with a suitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control the .25 duration of action. Controlled release preparations may be achieved by the use of polymers to complex or absorb the a2AP-binding molecule or thrombolytic agents of the present invention. The controlled delivery may be exercised by selecting appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate). The rate of drug release may also be controlled by altering the WO 98/12329 PCT/US97/16122 concentration of such macromolecules. Another possible method for controlling the duration of action comprises incorporating the therapeutic agents into particles of a polymeric substance such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, it is possible to entrap the therapeutic agents in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, by the use ofhydroxymethylcellulose or gelatin-microcapsules or poly(methylmethacrylate) microcapsules, respectively, or in a colloid drug delivery system, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules, or in macroemulsions. Such teachings are disclosed in Remington's Pharmaceutical Sciences, 16th Ed., Osol, ed., Mack, Easton PA (1980).

The thrombolytic agent or a2AP-binding molecule may be provided to a patient by means well known in the art. Such means of introduction include oral means, intranasal means, subcutaneous means, intramuscular means, intravenous 15 means, intra-arterial means, or parenteral means. In one preferred method of treatment for pulmonary embolism, myocardial infarction, thrombosis or stroke, a patient is provided with a bolus (intravenously injected) containing between to 1.0 mg/kg of a thrombolytic agent.

Generally, the results reported herein demonstrate that an inhibitor, 0:0 2 particularly a monoclonal antibody, can be used to augment the catalytic function of an enzyme by neutralizing an inhibitor of that enzyme. This approach can be applied to biological processes which are tightly governed by inhibitors. Because coagulation is a finely balanced system in which the effects of enzymes (generally serine proteases) are pitted against the effects of inhibitors, frequently serpins S ,(serine protease inhibitors) pathological alterations in clotting can be treated by augmenting enzyme activity or by neutralizing an inhibitor.

Having now generally described this invention, the same will be better understood by reference to certain specific examples which are included herein for purposes of illustration and are not intended as limiting.

WO 98/12329 PCT/US97/16122 -31- Example 1 Preparation of an Antibody Directed to Alpha-2-antiplasmin A. Monoclonal Antibody Production, Purification and Characterization Two Balb/C mice were immunized subcutaneously with 25 zg of purified human a2AP fragments derived from the trypsin digest of a human plasma clot.

The a2AP fragments were affinity purified with a SEPHAROSE-coupled monoclonal antibody, RWR (Reed, G.L. Ill et al., Trans. Assoc. Am. Phys.

101:250-256 (1988); U.S. Patent No. 5,372,812, issued December 13, 1994), against human a2AP. Mice were initially immunized with complete Freund's adjuvant and boosted 90 days later with 50 yg of a2AP fragment in incomplete Freund's adjuvant. The antisera titer was tested in a solid-phase radioimmunoassay (Reed, G.L. II et al., Proc. Natl. Acad. Sci. USA 87:1114- 1118 (1990)) with a2AP immobilized in the wells of a microtiter plate. Four days before fusion, the mouse with the highest titer of a2AP antibody was hyperimmunized with 100 zg a2AP intraperitoneally. Somatic cell fusion was performed as described (Galfre, G. and Milstein, Meth. Enzymol. 73:3-46 (1981)).

Hybridomas were tested for the production of antibodies to the a2AP "20 fragment and for their ability to inhibit a2AP as described in Reed, G.L. III et al., Proc. Natl. Acad. Sci. USA 87:1114-1118 (1990). The binding of monoclonal antibodies (MAbs) to 25 I-a2AP was tested in a solid-phase radioimmunoassay.

Wells of a microtiter plate were coated with goat antimouse antibody (25 il, for 2 hours. The wells were rinsed and nonspecific protein binding sites were blocked with 1% bovine serum albumin in Tris-buffered saline, pH 7.4, for 1 hour. After a wash, 25 il ofhybridoma supernatant was added to the wells and incubated for 1 hour. The wells were rinsed and 125I-a2AP was added (25 1l, WO 98/12329 PCT/US97/16122 -32- 60,000 cpm) for 1 hour. The 12sI-a2AP was then removed and the wells were rinsed and gamma-counted.

Cloned hybridomas (limiting dilution) were expanded into ascites in pristane-primed Balb/C mice. Antibodies were purified from filtered ascites by precipitation with 40% ammonium sulfate, dialysis into 10 mM KH 2

PO

4 pH 7.2, and ion-exchange chromatography on DEAE-AFFIGEL BLUE SEPHAROSE (BioRad, Hercules, CA) with a linear gradient from 0 to 100 mM NaCI.

B. Immunoblotting These were performed largely as described in Reed, G.L. et al., J.

.P1 Immunol. 150:4407-4415 (1993). Purified human a2AP (5 pg, American Diagnostica, Greenwich, CT) was electrophoresed in a large single sample lane on a 12% slab minigel (BioRad, Hercules, CA) under reducing and non-reducing conditions. The sample was electroblotted (Kyhse-Anderson, 1084) to S. polyvinylidene difluoride membranes (Millipore, Bedford, MA) and nonspecific protein binding sites were blocked with 5% dry milk. The blots were exposed to different hybridomas supernatants for 1 hour in the channels of a miniblotter apparatus (Immunetics, Cambridge, MA). After washing, the bound antibody was detected by incubation with 25 1-goat antimouse antibody (1.5 million cpm/membrane). After additional washing, the membranes were exposed in a o20 phosphorimager (Molecular Devices, Sunnyvale, CA).

C Radioimnunoassays Wells of a microtiter plate were coated with goat antimouse antibody pl, 5 pl/ml) for 2 hours at 21 0 C. They were washed and blocked with 1% BSA (bovine albumin serum) for 1 hour. To the empty wells in duplicate were added 25 pl of hybridoma supernatants. After incubation for 1 hour the wells were washed and 25 pl of 12-a2AP was added to the wells for another hour.

WO 98/12329 PCT/US97/16122 -33- After washing the wells were cut and the bound antibody measured by gamma scintillation counting.

Competition radioimmunoassays were performed by coating wells of a microtiter plate with 25 pl of purified MAb (70B11) in duplicate (10 gg/ml) for 1 hour. The wells were washed and blocked with 1% BSA for 1 hour. After washing, 25 pl of a competitor MAb, same MAb or negative control MAb was added to different wells (50 ug/ml) followed by 25 pl of 25 1-a2-antiplasmin (100,000 cpm). After 1 hour incubation, the wells were washed, cut and the radioactivity was measured in a gamma scintillation counter.

D. Plasma Clot Lysis Assays These were performed largely as described in Reed, G.L. III et al., Proc.

Natl. Acad Sci. USA 87:1114-1118 (1990). Pooled fresh frozen plasma was Sobtained from 5 random donors to the Massachusetts General Hospital Blood Bank. The plasma was mixed with '"I-fibrinogen to achieve -1,000 cpm/pl. The t plasma was clotted for 1 hour at 37 0 C in a 12 x 65 mm test tube by mixing 50 pl with 50 pl ofCaC1 2 (5 mM final) and thrombin (1 U/ml). Clots were compressed and washed in 1 ml Tris-buffered saline (pH 7.4) to remove unbound proteins.

The supernatant was removed and the clots were counted in a gamma counter.

To each set of duplicate clots was added 100 pl containing various amounts of 20 urokinase, 100 pl of pooled plasma containing 1 u/ml of hirudin and 100 pl of Tris-buffered saline containing 7 pg (Figure 4) or 21 pg (Figure 5) of MAb or no MAb. The clots were placed in a 37°C water bath and at various intervals 100 pl of solution was temporarily removed and gamma-counted to determine the WO 98/12329 PCTIUS97/16122 -34amount of lysis. The percent lysis was defined at 100 x (total supernatant cpm total clot cpm).

E. Results Three hybridomas were selected that appeared to inhibit a2AP function in screening assays. The serotypes of these MAbs were: 49C9 (Igy2aK), 70B 11 (Igy 1K), and 77A3 (Igy2aK). Figure 1 compares the binding of these MAbs to s' 2 -a2AP in a reverse solid-phase assay. When compared to the original a2AP inhibitor RWR, these MAbs bound with greater avidity. To determine if the MAbs bound to the same epitopes, competition assays is shown for 70B 11 in Figure 2. Compared to the negative control, anti-digoxin MAb, RWR had no significant inhibitory effects on the binding of 2 5 1l-a2AP to immobilized 70B 11.

In contrast, when 70B 11 was used as a competitor, it completely inhibited the binding of 2 I-a2AP to immobilized 70B 11, as expected. However, 49C9 and 77A3 were also excellent competitors as well. The results of these assays are 6. shown in tabular form in Table 1, below. MAbs 49C9, 70B 11, 77A3 also fully inhibited the binding of each other to 1 25 I-a2AP, but they had no inhibitory effects on the binding of RWR. The converse was also true, RWR as a competitor had no effect on the binding of 12sI-a2AP to the other MAbs. This indicated that MAbs 49C9, 70B 11 and 77A3 competed for binding to the same epitope, while RWR appeared to bind to a separate region of a2AP.

To determine if the MAbs recognized a continuous or discontinuous epitope in a2AP, immunoblotting experiments were performed with denatured and reduced a2AP. In these studies RWR bound well to denatured and reduced a 2 AP, indicating that it recognized an epitope which was not affected by boiling with SDS, nor reduction of disulfide bonds. In contrast, MAbs 49C9, 70B11 and 77A3 did not bind to denatured a2AP, suggesting that they recognize a conformation-dependent epitope.

Il (I WO 98/12329 PCT/US97/16122 Clot lysis assays were performed to examine the relative potency of these MAbs in amplifying the fibrinolysis by urokinase. Figure 3 compares the amount of lysis achieved by 7 ug of different purified MAbs (or TBS alone) as a function of dose of urokinase. Compared to urokinase alone (TBS) or urokinase with the control antidigoxin MAb, RWR, 49C9, 70B 11 and 77A3 all accelerate clot lysis.

However, 49C9, 70B11 and 77A3 appear to be significantly more potent than RWR in these assays. To examine the increase in fibrinolytic potency of urokinase achieved by one of these antibodies, dose response studies were performed in the absence or presence of MAb 77A3. Figure 4 shows that MAb 77A3 markedly increases the potency of lysis of urokinase by roughly 100-fold.

As a means of further discriminating among the functional and epitope binding specificities of these MAbs, their ability to inhibit the a2AP from different animal species in plasma clot lysis assays was examined. The results of these assays are summarized in Table 2, below. In the different species of animal f5 plasmas tested, RWR appeared to inhibit only human a2AP. In contrast, the other MAbs showed a broader species cross-reactivity and ability to inhibit nearly all primate and some non-primate a2APs.

i* 0* WO 98/12329 PCTIUS97/16122 -36- 105 Seio*:

C

00 0@000 2 5 cc c 250

C

eec.

C C

C

Table 1 Effect of Different MAb Inhibitors on the binding of 25 -a2AP to Immobilized MAbs.

Immobilized MAb Inhibitor RWR 49C9 70B 11 77A3 RWR 49C9 I 77A3 anti-digoxin To wells of a microtiter plate containing an immobilized MAbs was added "I-a2AP and a different competitor MAbs. indicates that the competitor inhibited the binding of 2 51-a2AP to the plate, whereas a indicates that there was no inhibition.

Table 2 The cross reactivity of MAbs with different ca2-antiplasmins.

Species RWR 49C9 70B ll 77A3 HUMAN 4 Baboon Grn Monkey Dog cynamologus

II+

marmoset ferret guinea pig gerbil The crossreactivity of each MAb was determined by its ability to accelerate the lysis of that species' plasma clots. A indicated that the MAb did not accelerate plasma clot lysis, a indicated modest effects, and indicates significant acceleration of plasma clot lysis significant functional crossreactivity).

WO 98/12329 PCT/US97/16122 -37- Example 2 In Vivo Study of Pulmonary Embolism A. Materials Materials were obtained from the following suppliers: rt-PA with a specific activity of 580,000 IU/mg, Genentech (South San Francisco, California); ketamine (100 mg/ml), Fort Dodge Laboratories (Fort Dodge, Iowa); acepromazine maleate, Fermenta Animal Health Co: (Kansas City, MO); heparin (1000 U/ml), Elkins-Sinn Inc. (Cherry Hill, NJ); sodium iodide, Aldrich Chemical Co.

(Milwaukee, WI); calcium chloride, Mallinckrodt (Paris, Kentucky); normal saline for intravenous use, Travenol Laboratories (Deerfield, IL); a2AP assay kit, e e Stachrom (Asnieres, France); purified a2AP and fibrinogen, American Diagnostica v (Greenwich, CT); goat antimouse antibody, Cappel Organon Technika (Durham, 0* NC); human plasma pooled from random donors, Massachusetts General Hospital (Boston); bovine thrombin, Parke-Davis (Morris Plains, NJ); Na 25 I, Dupont-NEN (Cambridge, MA); Bard Parker surgical blade, Becton Dickinson (Franklin Lake, NJ); 4.0 silk sutures, American Cyanamid Co. (Danbury, CT); SURFLO IV catheter and 20 gauge 1-1/4-inch VENOJECT tubes with K 3 EDTA, Terumo e Medical Corp. (Elkton, MD); sterile three-way stopcock, Mallinckrodt Critical S. Care (Glens Falls, NY); auto syringe infusion pump, Baxter Health Care Corp.

(Hooksett, NH); infusion pump tubing and microbore 60-inch extension set, e McGaw of Puerto Rico (Sabana Grand, Puerto Rico); surgical instruments,

VWR

(Boston); tubing, Namic (Glens Falls, NY); ferrets 1 kg), Marshall Farms J. (New York, NY); aprotinin, Sigma (St. Louis, MO); and microcentrifuge tubes, National Scientific Supply Co. (San Rafael, CA).

n WO 98/12329 PCTIUS97/16122 -38- B. In Vitro Clot Lysis Assays Sees

,SJ

IS 6 00 :61 0 6S e:G 0~ 0 1*C* 0 Pooled, fresh-frozen, citrated ferret plasma (1100 was mixed with Al of LI-labeled human fibrinogen (-40,000 cpm/clot). Ferret plasma (35 /l) was mixed with 35 /l ofTris-buffered saline (TBS) containing 10 mM CaCI 2 and thrombin (1 U/ml) in twelve 65-mm plastic tubes and clotted for 1 hour at 37 0

C.

The clots were washed in TBS, the supematant was removed, and then 100 pl of TBS or 25 jg of purified MAb (RWR or 77A3) was added to tubes in duplicate.

Clot lysis was initiated by adding 0.1 .U of rt-PA per tube. The clots were incubated at 37 0 C for 5 hours and the amount of lysis was determined by sampling for the release ofradiolabeled fibrin degradation products into the supernatant, as described (Reed, G.L. m et al., Proc. Natl. Acad. Sci. USA 87:1114-1118 (1990)).

C Pulmonary Embolism Experiments Male ferrets were anesthetized by intramuscular injection (0.4 ml) of a mixture of ketamine and acepromazine (two parts acepromazine [10 mg/ml] to one part ketamine [100 mg/ml]). Intraperitoneal injections were repeated as necessary to keep the animals anesthetized. After an anterior midline incision had been made in the neck, the jugular vein and the carotid artery were exposed by blunt dissection and cannulated with 20G catheters that were secured at the proximal and distal ends with 4-0 silk sutures. The catheters were capped with three-way stopcocks.

Pooled, citrated human plasma was mixed with 'I 2 -fibrinogen to achieve -1,000,000 cpm/ml. Individual clots were formed by mixing '2sI-fibrinogenlabeled plasma (45 with 2.5 p1 of bovine thrombin (100 U/ml) and 2.5 /l of calcium chloride (0.4 These clots were incubated at 37 0 C for 90 minutes, compressed, and washed thoroughly with saline three times to remove unbound proteins. The radioactive content of the clots was measured in a gamma counter WO 98/12329 PCT/US97/16122 -39immediately before clot injection. Blood samples were drawn at base line and at the end of the experiment. Sodium iodide (10 mg) was injected to block thyroid uptake. Clots were embolized into the lungs by injection through the internal jugular vein. Ferrets weighing less than 1 kg received three clots; those weighing 1 kg or more received four clots. Successful embolization was evidenced by the accumulation of radioactivity in the thorax. After the clots had been injected, the ferrets were turned on their sides to ease breathing.

All animals received weight-adjusted heparin at 100 U/kg (bolus), a dose sufficient to keep the activated partial thromboplastin time (aPTT) above 150 seconds throughout the procedure. The a2AP inhibitor (sterile-filtered, 14 mg/ml in saline) or a purified control MAb (antidigoxin) was given intravenously as a single dose (22.5 mg/kg). The rt-PA was given as a continuous infusion over 2 hours (1 or 2 mg/kg in 5 ml normal saline). Animals were observed for a total of four hours after pulmonary embolization and then killed by lethal injection of 15 anesthesia or by CO 2 inhalation. The thorax was dissected and all intrathoracic structures were removed for gamma counting to detect residual thrombi. The percentage of clot lysis was.determined for each ferret by dividing the total residual radioactivity in the thorax (cpm) by that in the initial thrombi.

This experimental protocol was approved by the Harvard Medical Area Standing Committee on Animals. The Harvard Medical School animal management program is accredited by the American Association of Laboratory Animal Care, and the procedures were conducted in accordance with National Institutes of Health standards, as set forth in the Guide for the Care and Use of Laboratory Animals (DHHS Publication No. [NIH] 85-23, revised 1985), the 26 Public Health Service Policy on the Humane Care and Use of Laboratory Animals by Awardee Institutions, and the NIH Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training.

WO 98/12329 PCT/US97/16122 D. Statistical Tests The data were analyzed by a one way analysis of variance followed by a Bonferroni-Dunn procedure for multiple comparison testing.

E. Fibrinogen Assays Blood samples were collected on K 3 EDTA (0.15% solution final) with aprotinin (50 kallikrein U/ml). Platelet-poor plasma was obtained by centrifugation of whole blood (Mustard, J.F. el al., Meth. Enzymol. 169:3-11 (1989)) and assayed for fibrinogen by the sodium sulfite method (Rampling,

M.W.

and Gaffney, Clin. Chim. Acta.67:43-52 (1976)).

::06 F. a2-Antiplasmin Assays To measure c2AP levels, we collected ferret blood on sodium citrate (1/10 volume) and centrifuged it to obtain plasma (Mustard, J.F. et al., Meth. Enzymol.

169:3-11 (1989)). The plasma was tested for functional a2AP with a chromogenic substrate assay for plasmin inhibition (Stachrom kit) as described (Reed, G.L. III et al., Proc. Natl. Acad Sci. USA 87:1114-1118 (1990)).

Results *see From a panel ofhybridomas we selected 77A3, a MAb that bound tightly to human a2AP. MAb 77A3 was purified from mouse ascites by ion exchange 0 chromatography, and its purity was confirmed by SDS-polyacrylamide gel analysis (Figure To study the role of a2AP in experimental pulmonary embolism in vivo, we tested purified 77A3 in several different animal plasma clot lysis assays to determine whether it could bind and inhibit a non-human a2AP. Of various small animal plasmas tested hamster, gerbil, guinea pig, rat, etc.), 77A3 WO 98/12329 PCT/US97/16122 -41significantly crossreacted with ferret plasma. Figure 6 compares the lytic effects of 77A3 with those of another MAb inhibitor of human a2AP, RWR (Reed, G.L.

III et al., Trans. Assoc. Am. Phys. 101:250-256 (1988); U.S. Patent No.

5,372,812, issued December 13, 1994), and with buffer alone. Figure 6 shows that in comparison with the control (buffer alone), 77A3 accelerated the lysis of ferret plasma clots induced by a low dose of rt-PA (0.1 unit). In contrast, RWR, which inhibits human a2AP (Reed, G.L. III et al., Trans. Assoc. Am. Phys.

101:250-256 (1988); U.S. Patent No. 5,372,812, issued December 13, 1994) but does not crossreact with nonhuman a2AP, had no detectable effect. This experiment indicated that 77A3 inhibited ferret a2AP and amplified ferret clot lysis in vitro.

0 The cross-reactivity of 77A3 allowed us to investigate the role of a2AP in a ferret model of pulmonary embolism. In humans, pulmonary embolism is usually treated with heparin (Goldhaber, Chest 107:45S-51S (1995)).

15 Consequently, ferrets were treated with a weight-adjusted bolus dose of heparin at 100 U/kg. This dose was sufficient to keep the aPTT above 150 seconds throughout the experiment To investigate the effects of intravenous MAb 77A3 on the activity of a2AP in the blood, we selected a dose, 22.5 mg/kg, that was in molar excess to the level of ferret a2AP. Our ex vivo measurements of ferret a2AP activity, I and 4 hours after intravenous dosing, showed that of ferret a2AP activity was inhibited at this dose (Figure 7, n=2).

Using heparin at 100 U/kg and 77A3 at 22.5 mg/kg, we then investigated the effect of these agents and rt-PA on the lysis of pulmonary emboli (Figure 8).

All animals received heparin. Control animals which received no rt-PA, '25 showed 15.6±10.5% (mean±SD) lysis of their pulmonary emboli. Animals 0: receiving rt-PA at 1 mg/kg over 2 hours showed 38.5±6.3% lysis, which was significantly greater than lysis obtained in those receiving heparin alone Similarly, animals receiving rt-PA at 1 mg/kg and a control (antidigoxin) MAb showed 35.2-4.6% lysis. Ferrets treated with rt-PA at 2 mg/kg (n=4) showed a minimal increase in lysis over those treated at 1 mg/kg (45.0±6.5% vs I I WO 98/12329 PCT/US97/16i22 -42- 38.5±6.3%, However, animals receiving rt-PA at 1 mg/kg together with the a2AP inhibitor showed greater lysis than those receiving an equivalent dose of rt-PA alone with or without the control (antidigoxin) MAb or those receiving twice the dose of rt-PA alone In addition to inhibiting plasmin on the thrombus surface, a2AP and other inhibitors inactivate plasmin in the blood (Collen, Eur. J. Biochem. 69:209-216 (1976); Moroi, M. and Aoki, J Biol. Chem. 251:5956-5965 (1976); Mullertz, S. and Clemmensen, Biochem J. 159:545-553 (1976)). We measured fibrinogen levels in the blood to determine if inhibition of a2AP led to nonspecific plasminolysis of a circulating clotting factor. Figure 9 shows residual fibrinogen i levels expressed as a function of their initial values in four treatment groups. In animals that received no rt-PA, fibrinogen levels varied moderately but did not diminish during the experiment. Ferrets receiving 1 mg/kg and 2 mg/kg of rt-PA 1 alone showed no significant change in fibrinogen level. Similarly, animals receiving the combination of rt-PA and the a2AP inhibitor showed no detectable change in circulating fibrinogen levels.

H. Discussion Clinical and experimental studies suggest that pulmonary emboli and venous thrombi resist endogenous fibrinolysis and lysis induced by plasminogen activators (Goldhaber, Chest 107:45S-51S (1995); Goldhaber, S.Z. et al., Lancet 2:886-889 (1986); The Urokinase Pulmonary Embolism Trial, Circulation S 47:1-108 (1973); Goldhaber, S.Z. et al., Am. J. Med 88:235-240 (1990); Goldhaber, S.Z. etal., Lancet 341:507-511 (1993)). This resistance to lysis is due in part to specific molecular factors in the thrombus that act to oppose fibrinolysis.

During thrombus formation, a2AP is covalently crosslinked to fibrin by activated factor XIII (Sakata, Y. and Aoki, J. Clin. Invest. 69:536-542 (1982)). Studies in vitro indicate that when a2AP in the clot is absent or inhibited by MAbs, clots WO 98/12329 PCT/US97/16122 -43undergo spontaneous lysis (Aoki, N. et al., Blood 62:1118-1122 (1983); Miles, L.A. et al., Blood 59:1246-1251 (1982); Reed, G.L. Ill et al., Trans. Assoc. Am.

Phys. 101:250-256 (1988); Reed, G.L. III et al., Proc. Natl. Acad Sci. USA 87:1114-1118 (1990)). Conversely, when levels of a2AP in clots are increased by supplementation in vitro, fibrinolysis is inhibited (Sakata, Y. and Aoki,

J.

Clin. Invest. 69:536-542 (1982)). In the present study we investigated the hypothesis that a2AP plays a major regulatory role in fibrinolysis and that it contributes to the thrombus resistance obtained in pulmonary embolism.

We measured the effect ofrt-PA, with and without a2AP inhibition, on the net lysis of pulmonary emboli in ferrets. Because heparin is the established therapy for humans with pulmonary embolism, we considered animals treated with heparin alone as the control group. The weight-adjusted bolus dose of heparin given to the ferrets was sufficient to maintain a high level of anticoagulation throughout the experiment. In animals treated with rt-PA, at a dose comparable .1 to that used in humans (1 mg/kg), lysis of pulmonary emboli was enhanced significantly in comparison with lysis in animals treated with heparin alone.

Increasing the dose of rt-PA to 2 mg/kg, a dose higher than is safe in humans, led to a minimal increase in lysis. A similar plateau in the dose response for t-PA-induced lysis has been noted in experimental studies of pulmonary embolism in dogs (Werier, J. et al., Chest. 100:464-469 (1991)). However, specific inhibition of a2AP markedly potentiated the lysis of experimental pulmonary emboli by rt-PA (1 mg/kg), causing significantly more lysis than was seen in ferrets treated with the same dose ofrt-PA: alone or with a control MAb, the lysis achieved with a2AP inhibition was also greater than that achieved in ferrets 2 treated with high-dose rt-PA (2 mg/kg). At the same time, despite the higher total lysis obtained in animals treated with the a2AP inhibitor, there was no significant consumption of circulating fibrinogen. In these studies of experimental pulmonary embolism, a2AP played an important role in thrombus resistance to lysis induced by rt-PA. Further studies will be necessary to establish the relative quantitative roles of circulating and thrombus bound a2AP in this process.

WO 98/12329 PCT/US97/16122 -44- Besides a2AP, other molecular factors may regulate the thrombus resistance of pulmonary emboli. A leading candidate is PA-1, a serine protease inhibitor of t-PA and urinary-type plasminogen activator (u-PA or urokinase) (Stringer, H.A. and Pannekoek, J. Biol. Chem. 270:11205-11208 (1995); Carmeliet, P. et al., J. Clin. Invest. 92:2756-2760 (1993); Lang, I.M. et al., Circulation 89:2715-2721 (1994); Marsh, J.J. et al., Circulation 90:3091-3097 (1994)). Unlike a2AP, PAI-1 is not specifically crosslinked to fibrin in the thrombus, although it has been shown to bind to fibrin in vitro (Stringer, H.A. and Pannekoek, J Biol. Chem. 270:11205-11208 (1995)). By adding recombinant PAI-1 to developing thrombi, Marsh et al. (Marsh, J.J. et al., Circulation 90:3091-3097 (1994)) have shown that PAI-l-enriched clots can suppress the spontaneous lysis of pulmonary emboli in a canine model; however, the role of PA-1 in the lysis ofautologous thrombi was not investigated. Pathologic studies of pulmonary emboli extracted by thrombectomy have suggested that PAI-I 15 expression increases in the endothelial cells at the margins of fresh thrombi but is not detectable in the thrombi themselves (Lang, I.M. et al., Circulation 89:2715- 2721 (1994)). Since PAl-1-deficient mice (by gene deletion) are less likely than regular mice to develop venous thrombosis induced by endotoxin (Carmeliet,

P.

et al., J Clin. Invest. 92:2756-2760 (1993)), the expression of PA-1 in endothelial cells at the margin of the developing thrombus may be functionally important. Nonetheless, the role of PA-1 in thrombus resistance to S. pharmacologic plasminogen activators is less clear: in patients given t-PA, the inhibitory capacity ofPAI-1 is overwhelmed completely (Lucore, C.L. and Sobel, Circulation 77:660-669 (1988)), and thrombus resistance is also observed 25 in patients given streptokinase, against which PAI-1 has no effect.

Another potential cause of thrombus resistance in pulmonary embolism is activated factor XIII. Several studies in vitro suggest that this coagulation enzyme renders the fibrin in clots more resistant to degradation by plasmin by crosslinking fibrin chains together and by crosslinking a2AP to fibrin. (Sakata, Y.

and Aoki, J. Clin. Invest. 69:536-542 (1982); Robbie, L.A. et al., Thromb.

WO 98/12329 PCTIUS97/16122 Haemostas. 70:301-306 (1993); Francis, C.W. and Marder, J. Clin. Invest.

80:1459-1465 (1987); Jansen, J.W.C.M. et al., Thromb. Haemostas. 57:171-175 (1987); Reed, G.L. etal., Trans. Assoc. Am. Phys. 104:21-28 (1991)) However, little is known about activated factor XIII and thrombus resistance in vivo. This is probably due to the fact that a potent inhibitor of factor XIII function has only recently become available (Reed, G.L. and Lukacova, Thromb. Haemostas.

74:680-685 (1995)). One study has suggested that when factor XIII is partially inhibited, coronary thrombi lyse at accelerated rates in response to t-PA (Shebuski, R.J. et al., Blood 75:1455-1459 (1990)). This observation argues that factor XIII, through its effects on fibrin-fibrin and a2AP-fibrin crosslinking, also contributes to thrombus resistance.

Improving the lysis of thrombi in patients with pulmonary embolism and deep venous thrombosis remains a challenge. Unfortunately, increasing the dose ofplasminogen activators is not a promising approach. High dose t-PA has been 15 associated with an unacceptable increase in the risk of cerebral bleeding (Passamani, E. et al., J. Am. Coll. Cardiol. 10:51B-64B (1987)). In addition, in the present study and others (Werier, J. et al., Chest. 100:464-469 (1991)), high-dose t-PA (22 mg/kg) produced only minimal increases in net lysis. The current FDA-approved doses ofurokinase and streptokinase cause plasminogen "depletion"; thus, increasing the doses of these agents is also not likely to have an effect on net lysis (Onundarson, P.T. et al, J. Lab. Clin. Med 120:120-128 (1992)). Several potent inhibitors of thrombin generation and activity are under development. Although these agents may further reduce the formation of new thrombi, they will not directly improve lysis of the large thrombi that typically 25 exist in patients at the time they are diagnosed. These considerations suggest that fundamental insights into the molecular factors that oppose physiologic or pharmacologic lysis in thrombi will be necessary to spark improved treatments for venous thromboembolism. The results of the present study indicate that a2AP is a major contributor to thrombus resistance in experimental pulmonary embolism, WO 98/12329 PCT/US97/16122 -46and they suggest that inhibiting a2AP might improve lysis in patients with thrombotic disease.

Example 3 Cloning and Sequencing ofAntibody cDNA A. Amino Terminal Sequences ofAntibodies Monoclonal antibodies (49C9, 70B11 and 77A3) were expanded into ascites and purified by ion exchange chromatography on DEAE Affigel Blue or by protein A agarose as described in Lukacova, D. et al., Biochemistry 30:10164-10170 (1991). The purified MAbs (15 pg) were subjected to .J0 SDS-PAGE on 10% minigels (BioRad, Hercules, CA). The protein samples were electroblotted to PVDF membranes (Millipore, Bedford, MA) using semi-dry technique (Kyhse-Anderson, J. Biochem. Biophys. Meth. 10:203-209 (1984)) at 4"C for 2 hrs at 75 milliamps (Millipore electroblotter). The bands were stained with Ponceau Dye (Sigma, St. Louis) and excised. The amino terminal sequences of the light chain of the antibodies are shown in Figure 10 (SEQ ID NOS: 1-3).

B. Molecular Cloning ofAntibody cDNA Cloned hybridoma cell lines 49C9, 70B11 and 77A3 were grown in 150 S mm tissue culture plates in 20% fetal bovine serum in Dulbecco's modified Eagle's 020 medium with 4.5 g/1 of glucose and penicillin and streptomycin. The cells were harvested and centrifuged at 1200 rpm for 7 min. The cell pellet was resuspended in sterile phosphate buffered saline (pH 7.4) and re-centrifuged. Then 5 ml of RNAzol (Teltest, Friendswood, TX) was added and the pellet was homogenized for 2 min. Chloroform (500 pl) was added and the mixture was vortexed and left to incubate on ice for 15 min. The samples were centrifuged at 12,000 rpm for WO 98/12329 PCT/US97/16122 -47min. The aqueous layer was mixed with 4.5 ml of isopropanol and vortexed. The mixture was precipitated at -700C for 90 min. and recentrifuged at 12,000 rpm for min. The pellet was washed in 2 ml of 70% ethanol in DEPC-treated water.

After repeat centrifugation, the supernatant was removed and the pellet air-dried.

The pellet was dissolved in 200 pl ofdiethyl-pyrocarbonate (DEPC)-treated water and 20 pl of 3 M NaCI and 800 pl of ethanol were added. The mRNA was precipitated overnight at -70*C and the pellet resuspended in DEPC-water.

The cDNA corresponding to the light and heavy chain sequences were isolated by primer guided reverse transcription followed by polymerase chain reaction as described (Gene Amp Thermostable rTth Reverse Transcriptase

RNA

PCR kit (Perkin-Elmer Cetus, San Francisco, CA). The light chain mRNA was primed for reverse transcription with a 3' primer N6GAATTCACTGGATGG TGGGAAGATGGA 3' (SEQ ID NO:22)) corresponding to the constant region ofthe light chain Coloma, et al., Biotechniques 11:152-154, 156 (1991)) 15 and the heavy chain was primed with a 3' primer N6GAATTCA(TC) CTCCACACACAGG(AG)(AG)CCAGTGGATAGAC 3' (SEQ ID NO:23)) corresponding to the constant region of the heavy chain (Coloma, et al., Biotechniques 11:152-154, 156 (1991)). Because the light chain amino terminal sequences were known, a specific primer corresponding to the likely 5' sense sequence was used ACTAGTCGACATGAGTGTGCTCACTCAGGTCCTGG (GC)GTTG 3' (SEQ ID NO:24); Jones, and Bendig, Bio/Technology 9:88-89 (Erratum) (1991)) for cDNA amplification. For cloning of the heavy chain, mouse heavy chain variable primers 1-12 were used as described (Jones, and Bendig, Bio/Technology 9:88-89 (Erratum) (1991)). All heavy chains amplified best with primer 9; though lesser amplification was also seen with Sprimers 12, 10 and 6. The PCR products were isolated by low melt agarose fractionation and ligated into a vector. The light chain PCR product was ligated into PCR II vector (Invitrogen, San Diego, CA) The heavy chain PCR product from primer 9 was ligated into PCR II.1 vector (Invitrogen, San Diego, CA).

After transformation, the plasmid DNA was isolated and subjected to restriction WO 98/12329 PCT/US97/16122 -48digestion with EcoR1. Two clones from each heavy and light chain were expanded and the DNA harvested. Both strands of the cDNA clones were sequenced using T7 and M13 primers with an ABI Prism automated sequencing apparatus. The cDNA sequences and deduced amino acid sequences are shown in Figures 11-16 (SEQ ID NOS:4-15).

Example 4 Preparation and Characterization of Chimeric and Humanized Antibodies In designing the sequence for a chimeric or humanized antibody, there are many parameters to consider. In the constant regions, a whole antibody may be made, or an antibody fragment (Fab and Fab'2) can be made. The constant regions may be murine or human. It is an accepted practice to replace murine constant regions with human constant regions, thus forming a "chimeric" antibody.

Chimeric antibodies are less immunogenic than murine antibodies and are thus more acceptable in the clinic.

The subclass of the antibody must also be considered. It is most common S* 0* to express recombinant antibodies as IgGs, but within this class, one must choose amongst recombinant chimeric human IgGI, IgG2, IgG3, and IgG4. These subclasses have different biological properties. The present inventors took a conservative approach of using IgG2 because 1) the strong complement activating properties of IgGI and IgG3 were not needed for this antibody and 2) IgG2 may be more straightforward to manufacture than IgG4. Any of the other subclasses could be made with the same specificity following similar strategies.

There are also parameters to consider in designing the variable region. The antibodies could be constructed to be chimeric or humanized. The chimeric antibody (murine V region, human constant region) is a more conservative approach, and virtually guarantees very similar antigen-binding activity to the murine antibody. With humanization, there is the risk of reducing the affinity WO 98/12329 WO 9812329PCTIUS97/16122 -49and/or biological activity of the antibody, but it can be presumed that the antibody will be less immunogenic. The present inventors have produced chimeric antibody as well as three forms of the humanized antibody.

Depending upon the strategy taken, humanization of any particular antibody can result in many different variable regions. At the simplest level, humanization consists of choosing a human variable region to serve as a template, and then deciding which residues should be "human" and which "murine". Thus, the choice of both the human template and which residues to maintain as human will affect the final sequence.

In general the strategy the present inventors have taken is to choose from among the human germline variable region genes for the templates. Alternatively, 0000 one can choose from rearranged variable region genes, both those which have and have not undergone somatic mutation. The rationale for the first strategy is that :0 *.:somatic mutations can introduce immunogenic, epitopes, while germline genes would have less potential for doing so. The selection was further limited to germline genes which are known to be rearranged and expressed as functional proteins in humans.

The choice of which germline gene to use as template is governed by the overall sequence similarity between the murine sequence and the human s equence; 0 200 the structural similarities between the two sequences (Chothia and Lesk, J Mol.

too* Biod. 196:901.(1987)); the anticipated ability of the chosen heavy chain template 00 to pair with the chosen light chain template; and the presence of the germline gene in the majority of humans. The choice of which residues should be murine is governed by which residues are thought to come in contact with antigen and 25 which are necessary to maintain the positioning and orientation of those residues which might contact antigen.

Variable regions were assembled from oligonucleotides and inserted into expression vectors containing the human gamma 2 constant region (for the VH region) and human kappa constant region (for the VL region). Heavy and light chain vectors were verified by nucleotide sequence and ability to direct the WO 98/12329 PCT/US97/16122 synthesis of antigen binding immunoglobulin (Ig) in COS cells (transient expression). Selected heavy and light chain vectors were then cotransfected into CHO cells to produce stable cell lines expressing the chimeric and humanized antibodies. Antibody was purified and tested for activity by antigen binding ELISA, ability to block the inhibitory activity of a2-AP in a plasmin assay, and ability to facilitate lysis of human clots by urokinase.

A. Construction of Chimeric and Humanized Antibody Vectors A functional light chain variable region is formed by the rearrangement and juxtaposition of a V gene segment and J gene segment. Therefore, it was necessary to find the best match for each of these segments and combine them to •00 form a human template. A FASTA search (using the Wisconsin Package Interface) of amino acids 1-95 (Kabat numbering system; V gene proper) of *I murine 77A3 (m77A3) light chain against a database of human Vk germline genes showed that m77A3 is clearly most similar to the human VkI subgroup (69.2% 71.6% identity vs less than 60% identity to sequences outside this subgroup).

From among the Vk I sequences, the sequence with GenBank accession X59312 (also known as the 02/012 gene) was chosen as a likely candidate because of the S0* match with structurally important positions and because of its prevalent expression 0 in humans. The human template for the light chain was completed by the addition 0 of the human Jk2 sequence. This J region was chosen because of its high degree of similarity with the murine J region of 77A3.

A functional heavy chain variable region is formed by the rearrangement S and juxtaposition of a V gene segment, a D gene segment, and a J gene segment.

0* Therefore, it was necessary to find the best match for each of these segments and combine them to form a human template. A FASTA search (using the Wisconsin Package Interface) of amino acids 1-94 (Kabat numbering system; V gene proper) of murine 77A3 heavy chain against a database of human VH germline genes showed that m77A3 is clearly most similar to the human VH7 family (77 WO 98/12329 PCT/US97/16122 -51identity) with the human VH1 family having the next best match (about 60 identity). The human VH7 family is mostly composed ofpseudogenes; the only active gene (7-04.1, Accession X62110) is polymorphic in the human population not all people have it) and therefore, in some people, this V gene could be more immunogenic than others. As an alternative human template for the heavy chain, the V gene with accession number Z12316 (1-18 gene) was chosen. This sequence is very similar to 7-04.1 except for the H2 loop and FR3 region. A human template for the D region was not considered because this region lies entirely within the H3 loop, the sequence of which is generally pivotal for antigen binding and therefore likely to entirely follow the murine sequence in a humanized antibody. The human template for the heavy chain was completed by the addition S" of the human JH5 sequence. This J region was chosen because of its high degree of similarity with the murine J region of 77A3.

Following the selection of human templates for the heavy and light chain variable regions, it was necessary to determine which positions should follow the murine sequence vs which positions should follow the human sequence. The following criteria were used in, selecting positions to follow the murine sequence: all positions falling within the CDR loops; all positions known to influence the conformation and/or spatial position of CDR loops (so called structural determinants; Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Lesk and Tramontano, in: Antibody Engineering, W.H. Freeman and Co., pp.7-38 (1992)); residues which were close enough to interact with residues in the CDR loops; and residues at or proximal to the VH-VL domain interface. All other residues followed the human sequence. These items are discussed in greater detail below.

Positions falling within the CDR loops are shown enclosed within the 6** boxes with solid borders and structural determinants are marked with an in the row below the position number in Figures 17-19.

In order to determine which residues were close enough to interact with the CDR loops, it was necessary to generate an approximate molecular model of the Fv region of murine 77A3. The molecular model was built based on the WO 98/12329 PCT/US97/16122 -52combined variable light chain of an anti-lysozyme mAb (D1.3) and the variable heavy chain of an anti-neuraminidase mAb (Incca) as structural template. CDR loop sequences were assigned to canonical loop conformations and a possible conformation for CDR H3 was extracted form the Protein Data Bank. The modeling building protocol followed procedures described by Bajorath Novotny (Therapeutic Immunol. 2:95-105 (1995)). Likewise, residues at or proximal to the VH-VL domain interface were identified and the murine residues were used for the humanized antibody. In all, for h77A3-1 heavy chain, h77A3-2 heavy chain, and for the common light chain there were 7, 18, and 11 murine residues, respectively, used outside of the CDR loops.

In order to prepare vectors encoding these chains, the amino acid sequence must be back translated into nucleotide sequence. For the most part, this was done simply by using the nucleotide sequence from the human template in cases where the amino acid residue is derived specifically from the human 15 template; otherwise, the nucleotides from the murine sequence were used. At a few positions, silent substitutions were made in order to eliminate restriction sites.

Finally, signal peptides must be added to the sequence. For both the chimeric and humanized light chains, signal peptides corresponding to that of the Smurine 77A3 light chain were used. For the chimeric and humanized heavy chains, the same signal peptide as for the light chains was used. Alternatively, signal peptides corresponding to that of murine 77A3 VH or any other signal peptide can be used in the chimeric and humanized heavy chains.

Two humanized antibodies were created: h77A3-1 and h77A3-2. A third version of the humanized heavy chain was created by including an oligonucleotide designed for h77A3-1 in the construction of h77A3-2. This resulted in a hybrid 0 molecule that was identical to h77A3-2 except for amino acids Ser and Leu at positions 9 and 11 of the heavy chain. One chimeric antibody, c77A3, was generated.

Amino acid and nucleotide sequences of h77A3-1 and h77A3-2 heavy and light chains are shown in Figures 17-19 (SEQ ID NOS:16-21). The common light WO 98/12329 PCT/US97/16122 -53chain is shown in Figure 17 (mature protein is amino acid residues 1 to 107 of SEQ ID NO:17). The heavy chain ofh77A3-1 is shown in Figure 18 (mature protein is amino acid residues I to 119 of SEQ ID NO:19). The heavy chain of h77A3-2 is shown in Figure 19 (mature protein is amino acid residues 1 to 123 of SEQ ID NO:21).

Expression vectors for chimeric and humanized 77A3 light and heavy chains were prepared in three stages: construction of cassettes containing human light or heavy chain constant region genes (pD16-hCka and pD20-hy2a, respectively); preparation of a PCR product containing the light or heavy chain variable region; and insertion of the variable region into the appropriate expression cassette.

Plasmid pD13 was constructed and derived from the pcDNA3 plasmid t* (Invitrogen) in two steps. The SV40 promoter/enhancer and neomycin resistance genes were removed from pcDNA3 by digestion with NaeI and isolation of the *1 5 3.82 kb fragment. These genes were replaced by the SV40 promoter/enhancer and dhfr gene from pSV2-dhfr. The DNA containing the pSV2-dhfr sequence was isolated as.a 1.93 kb fragment after digestion with PvuII and BamHI. The 3.82 and 1.93 kb fragments were ligated together and used to transform MC1061 bacteria following filling in the protruding ends of the 1.93 kb fragment from pSV2-dhfr. The correct product (designated pD12) was confirmed by the release of an 890 bp fragment following Hindm digestion.

g The polylinker was replaced with alternative restriction sites by digesting the resultant vector above with Asp718 and Bspl20I. The following oligonucleotides were annealed to the vector and cloned by ExoIII cloning (K.

*25 Hsiao, Nucl. Acid Res. 21:5528-5529 (1993)) to complete the plasmid pD13: 5' TAGGGAGACCCAAGCTTGGTACCAATTTAAATTGATATCTCCTT

AG

GTCTCGAGTCTCTAGATAACCGGTCAATCGATTGGGATTCTT 3' (SEQ ID NO:25) and

GACACTATAGAATAGGGCCCTTCCGCGGTTGGATCCAACAGT

GAAGCTAGCAAGCGGCCGCAAGAATTCCAATCGATTGACCGGTTA 3' WO 98/12329 PCT/US97/16122 -54- (SEQ ID NO:26). The resulting plasmid was used to transform competent E. coli and the correct product was confirmed by sequencing the polylinker region.

Plasmid pD16 was derived from the pcDNA3 plasmid (Invitrogen) in a series of steps which: add a polylinker sequence upstream of the CMV promoter for linearization; delete the SV40 promoter/enhancer and neomycin resistance gene and replace them with the histone H3 transcription termination sequence, the promoter (enhancer deleted) and DHFR gene; and insert the gastrin transcription termination sequence upstream of the CMV promoter.

pcDNA3 (Invitrogen) was digested with BglII and annealed to the following oligonucleotides: 5' primer: GCGCC-3' (SEQ ID NO:27);and 3' primer: 3

'-ACGATCGGGCCCACTGGACGCCGCGCGGAAACCGCGG

CTAG-5' (SEQ ID NO:28).

The plasmid was then ligated. After ligation, the resulting plasmid (pcDNA3-LSI) was used to transform competent E. coli DH5a and the correct construct was confirmed by release of a 230 bp fragment following restriction enzyme digestion with NheI and NruI.

.20 Plasmid pcDNA3-LSI was then digested with NgoMI, PvuI and BsmI.

Following digestion, a 2.0 kb NgoMI-PvuI fragment was isolated. Plasmid pD12 (described above) was digested with PvuI and SphI to remove the SV40 enhancer and a 3.6 kb fragment was isolated. The following oligonucleotides, encoding the histone H3 transcription termination sequence were annealed and then ligated with 25 the 2.0 kb NgoMI-PvuI fragment and 3.6 kb PvuI-SphI fragment: primer: 5'-CCGGGCCTCTCAAAAAAGGGAAAAAAAGCATG-3' (SEQ ID NO:29); and 3' primer: 3 '-CGGAGAGTTTTTCCCTTTTT C-5' (SEQ ID 11 WO 98/12329 PCT/US97/16122 The resulting plasmid pcTwD-LSI was confirmed by the production of 3.3, 0.95, 0.82 and 0.63 kb fragments after digestion with NheI plus NciI and the production of 4.2, 1.0, 0.26 and 0.23 kb fragments after digestion with SphI plus BstEII.

Insertion of the gastrin transcription termination sequence to form plasmid pD16 was accomplished by digesting pcTwD-LS with BssHII and Narl and isolating the 5.7 kb fragment and ligating with the following annealed oligonucleotides: primer: TTTTATTTTATTTTATTTTTGAGATGGAGTTTGG-3' (SEQ ID NO:31); and 3' primer: 3'-GGCCGAAGCTTATCGGTCTCATTGGAAAAAAAAATTAAAAT AAAAT AAAATAAAAACTCTACCTCAAACCGC-5' (SEQ ID NO:32).

After ligation, the product was used to transform competent E. coli MC1061 and the correct construction was confirmed by the production of 4.8, 0.66 and 0.31 kb fragments after digestion with NgoMI plus Spel and the production of 3.3, 1.0, 0.82 and 0.67kb fragments following digestion with NgoMI plus Ncol.

Plasmid pD17 was derived from pD16 by the removal of the Nhel site from the linearization polylinker. This was accomplished by digestion of pD16 with BstlI and NheI and filling the protruding ends using Klenow polymerase. The reaction mixture was self-ligated and used to transform competent E. coli 20, pD17 was digested with Asp7181 and Bspl20I to remove a polylinker which was replaced by the 113 bp Asp718I/Bsp 1201 polylinker from pD 13. After ligation, .the resulting intermediate plasmid pD20 had the Nhel site required for inserting heavy chain V genes. pD20 was distinguished from pD17 by linearization with Nhel, and distinguished from pD13 by linearization with BssH II which cuts only 2 once within the linearization site polylinkers of pD16, pD17 and pD20. Finally, DNA sequencing was used to confirm the polylinker in A 2.9 kb EcoRI fragment was isolated from pGk.11 (Walls et al., Nucl.

Acid Res. 21:2921-2929 (1993)) and this was ligated into the plasmid pD13 (described above) previously digested with EcoRI. This construct (pD13-hCka) containing the human CK exon and flanking intron sequences was used to transform it r lI WO 98/12329 PCT/US97/16122 -56- E. coli DH5a and the correct product was confirmed by restriction digestion.

Digestion with EcoRI resulted in fragments of 5.7, 2.8 and 0.3 kb and digestion with SacI resulted in fragments of 7.1, 1.1 and 0.5 kb.

Construction of the light chain expression cassette was completed by removing the CK fragment along with the flanking polylinker sequences from pD 13 and inserting it into pD16. Plasmid pD13-hCka was digested with Asp718I and to release the CK fragment and polylinker sequences. The same enzymes were used to linearize pD16 and the CK containing fragment was ligated into pD16 to form pD16-hCka. Following transformation ofDH5a E. coli and amplification, the correct construct was confirmed by the release of 2.9 kb fragment following digestion with Asp7181 and Bspl201 and linearization following digestion with a restriction enzyme present in pD16, but not pD13. The nucleotide sequence was also confirmed by sequencing various regions of the construct.

A genomic DNA fragment encoding the human y2 gene was preassembled S in pIC, and then transferred into pD20 as follows. Phage clone Phage 5A (Ellison and Hood, Proc. Natl. Acad Sci, 79:1984-1988 (1982)), containing the human y2 gene was digested with HindIII and cloned into the HindII site ofpUC18 to form the vector py2. In py2, the 5' end of the y2 gene is adjacent to the polylinker region.

pG was derived from pSV2-gpt by digestion with Hind III and Bgl II, Klenow fill in, and religation. This served to remove a 121 bp Hind II-Bgl II fragment. py2 was then digested with BamH I and inserted into the BamH I site of pG to form pGy2.2. pGy2.2 contains a BglII site 3' of the coding region that would interfere with later cloning steps. To remove this restriction site, pGy2.2 was first digested with Bgl II, the sticky ends filled in by Klenow DNA polymerase I, then the plasmid religated. The resulting intermediate plasmid, pGy2.3 was screened for lack of digestibility with Bgl II.

For purposes of later cloning in variable region genes, it was important to provide a restriction site in the y2 containing cassette. This is conveniently done by mutating the nucleotides encoding the first two amino acids of the CH1 exon to (1 WO 98/12329 PCT/US97/16122 -57encode an Nhe I site (Coloma M.J. et al, J. Immunological Methods 152:89-104(1992)). Previously, an Nhe I to Bst E II fragment from the human y4 gene was cloned. In this region, human y2 and human y4 genes encode identical amino acids. Thus, the y4 containing vector (pIChy4.1) could serve as a source for the 5' end of the y2 gene. This vector was obtained as follows: The 8.6 kb BamH I fragment from Phage 5D (Ellison, J. et alDNA 1:11-18 (1981)), containing the human y4 gene, was subcloned into pUC, resulting in the plasmid pUChy4.

pUChy4 served as the template for a PCR reaction involving the following primers: sense primer: 5'-ATCGATGCTA% CACCAAGGGCCCA-3' (SEQ ID NO:33); and antisense primer: 5'-CTCGAGGG~TCACCACGCTGCTGA-3' (SEQ ID NO:34).

The sense primer contained a Clal site for subcloning the PCR product into pIC20R (Marsh et al, Gene 32: 481-485 (1984)) adjacent to a synthetic Nhel site (underlined). Note that the bases for the Nhel site can encode the first two amino acids (Alanine and Serine) for the human y 1, y2, y3 or y4 CHI exon. The antisense primer has an Xho I site for subcloning into pIC20R, next to a BstE II site (underlined) which is in the CH1 exon of the human y4 and y2 gene. The PCR product formed was restricted with Cla I Xho I then ligated into pIC20R which had been digested by the same enzymes, to generate the intermediate pIChy4.1.

pGy2.3 was digested with BamH I and HinD III and a 6.1 Kb fragment Q .2Q including the human y2 gene locus was isolated from a 1.4% agarose gel for purification by the Qiaex' gel extraction kit (Qiagen, Chatsworth, CA). The 2.9 Kb pIChy4.1 plasmid was treated in a similar manner, and the two fragments were S* ligated together to form the intermediate vector pIChy2.1. To screen, an EcoR I digest yielded appropriate fragment sizes of 6.3 Kb and 2.6 Kb.

pIChy2.1 contained a duplication of the 5' portion of the human y2/y4 CHI exon. In order to remove the duplicated region, it was digested with BstE II giving fragment sizes of 4.0 Kb, 1.8 Kb, 1.6 Kb, 1.1 Kb, and 0.4 Kb. The 4.0 Kb fragment was isolated from a 1.4% agarose gel, while the 1.6 Kb fragment was separated and isolated away from the 1.8 Kb fragment in 4% NuSieve' M GTG (FMC Bioproducts, Rockland, ME) agarose. Both fragments were purified by Qiagen gel extraction WO 98/12329 PCT/US97/16122 -58prior to ligating them together to prepare pIChy2.2. In order to confirm the proper orientation of the two fragments the following primers were used to determine that the 3' portion of the human y4 CH1 exon's BstE II sticky end had joined with the end of the human y2 CHI exon (thus forming a contiguous human y2 locus in sense primer: 5'-AACAGCTATGACCATGATTAC-3' (SEQ ID NO:35); and antisense primer: 5'-CACCCAGCCTGTGCCTGCCTG-3' (SEQ ID NO:36).

The sense primer is homologous to sequence 5' of the pIC20R EcoR I site that is adjacent to the Cla I site. The antisense primer was chosen to be 500 bp downstream of the sense strand primer, and is homologous to sequence within the human y2 CHI to CH2 intron. Thus, visualization of a 500 bp PCR product in a 1.4% agarose gel confirmed that the hybrid human y4- y2 CH1 exon formed and was oriented in a contiguous manner to the remainder of the locus. pIChy2.2 was digested with EcoR I to give the expected 2.6 Kb and 1.9 Kb fragments. The entire .I human y2 CH1 exon was confirmed by DNA sequencing.

0 The 1.8 Kb Nhe I HinD II fragment containing the human y2 gene locus was removed from pIChy2.2 for ligation into plasmid pD20 opened by Nhe I HinD m. The resulting vector is the expression cassette pD20-hy2a.

The variable region genes for both chimeric and humanized antibodies 20 were synthesized by a modification of the non template specific PCR protocol (Prodromou and Pearl L. Protein Eng. 5: 827 -829 (1992)). The PCR products included DNA encoding both the signal peptide and variable region proper O as well as flanking sequences to facilitate insertion into the vector as well as correct splicing (light chain only).

The following primers were used: LH1, sense chimeric 77A3 VH outer primer (30mer), CGGCCGCTTGCTAGC-3' (SEQ ID NO:37); WO 98/12329 PCTIUS97/16122 -59- LH2, sense chimeric 77A3 VH primer 1 (80 mer),

GCATGGATTGGGTGTGGACTTGCTATTCCTGATGCAGCTCCA

AGTATCCAAGCACAGA-.31 (SEQ IOD NO:38); LH3, anti-sense chimeric 77A3 VH primer 2 (80 mer),

TCCAGGCTTCTTCAGCTCAGGTCCAGACTCACCACTGATCTGTGC

TTGGATACTTTG3(CAY2TG.3' (SEQ DD NO:39); LH4, sense chimeric 77A3 VII primer 3 (80 mer),

GGAGAAACAGTCAAGATCTCCTGCAGGCTTCTGGTATACCTUCAC

AAACTATGGAATGAACTGGGtT.3- (SEQ IID LH5, anti-sense chimeric 77A3 VII primer 4 (80 mer),

CCAGCCCATCCACTTTACCCTTUCCTGGAGCTGCTTCACCCAGTT

CATTCCATAGTTTGTGPAG-3- (SEQ ED NO:41); LH6, sense chimeric 77A3 VII primer 5 (80 mer), .1.5,GGGACGGTTTrGCCTTCTCTTTG3- (SEQ IID NO:42); LH7, anti-sense chimeric 77A3 VII primer 6 (80 mer), AAGGCAAACCGTCCCT-TGAA..3 (SEQ ID NO:43); 00060:LH8, sense chimeric 77A3 VII primer 7 (80 mer), 0**qo

TCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGATGGGTACCT

GGGACCTATGCCATG(JACT.Y .(SEQ ID NO:44); LII9, anti-sense chimeric 77A3 VII primer 8 (80 mer),

TAGCTGAGGAGACGGTGACTGAGG-TTCCTTGACCCCAGTAGTCCATG

:0 GCATAGGTCCCAGGTACCC.31(SEQ ED LI1, anti-sense murine 77A3 VII outer primer (29 mer), GGCCCTTGGTGCTAGC-3' (SEQ ID NO:46); LII 11, sense chimeric 77A3 VL outer primer (30Omer), ATCTCCTTAGGTCTCGAG.3- (SEQ ED NO:47); WO 98/12329 PCTIUS97/16122 LH12, sense chimeric 77A3 VL primer 1(79 mer),

TTAGGTCTCGAGATGAGTGTGCTCACTCAGGTCCTGGCGTTCTG

GCTGTGGCTTACAG.3' (SEQ ID NO:48); LHI 3, anti-sense chimeric 77A3 VL primer 2 (78 mer),

GAGGCTGGAGACTGAGTCATCTGATGTCACATCTGACCTGTA

CCACAGCAGCAGCAACGY-3- (SEQ ED NO:49); LH14, sense chimeric 77A3 VL primer 3 (78 mer),

TCTGCATCTGTGGGAGACTGTCACCATCACATGTCGACAAGTG

GAATATTCACAPJTA..3' (SEQ ID LHI5, anti-sense chimeric 77A3 VL primer 4 (78 mer),

GAGCTGAGGAGATTTTCCCTGTTTCTCTGATACCATGCTATJJTT

GTGAATATTCCCACTT(Y2TC-31(SEQ ED NO:5 1); LH16, sense chimeric 77A3 VL primer 5 (78 mer), VOODe

CCTGGTCTATAATGCAAACCTTAGCAGATGGTGTGCCATCAGG

TCAGTGGCAGTGj(ATCA.3- (SEQ ID NO: 52); LH17, anti-sense chimeric 77A3 VL primer 6 (78 mer);

TCAGGCTGCAGGCTGTTGATCCTGAGAGAAATGTGTCCTGATCC

ACTGCCACTGAALCCTTGAT..3 (SEQ ED NO:53); LH18, sense chimeric 77A3 VL primer 7 (78 mer), .:o.2b

'-GCCTGCAGCCTGAGATTTTGGAGTCATTACTGTCAACATTTTT

GACCCGGGCTCGGGGCCA3(SEQ ID NO:54); 0000 LH19, anti-sense chimeric 77A3 VL primer 8 (81 mer), 0

CCGGTTATCTAGAGACTCGAGACTTACGTGATCCACTTGG

S000 CTCCACCGAACGTCCACG 3 I (SEQ ID) NO:5 0 b125 LH2O, anti-sense chimeric 77A3 VL outer primer (3Omer), S CCGGTTATCTAGAGACTCGAGA-3- (SEQ ED NO:5 6); LH2 1, anti-sense humanized 77A3 VL primer 2 (78 mer),

GGGAGGATGGAGACTGAGTCATCTGGATGTCACATCTGGCCTA

AGCCACAGCAGCACC-

3 (SEQ In NO:69) WO 98/12329 PCTIUS97/16122 -61- LH22, sense humanized 77A3 VL primer 3 (78 mer),

CTATCTGCATCTGTGGGAGACAGAGTCACCATCACATGTCGAAG

TGGGAATATTCACAATTA (SEQ DD LH23, sense humanized 77A3 VL primer 5 (78 mer),

CTCGTTTAGAAACTGAGGTTCACA

GTTCAGTGGCAGTGGATCA (SEQ ED NO:71) LH24, anit-sense humanized 77A3 VL primer 6 (78 mer),

TTCAGGCTGCAGGCTGCTGATGGTGAGAGTAATCTGTCCTGATC

CACTGCCACTGAACCUTGAT (SEQ ID NO: 72) LH25, sense humanized 77A3 VH -I primer 1 (80 mer),

CTAGCATGAGTGTGCTCACTCAGGTCCTGGCGTGCTGCTGCTGTGG

0:60CTTACAGGTGCCAGATGTC (SEQ ED NO:57); L1126, anti-sense humanized 77A3 VH -I primer 2 (80 mer); *a*

CCAGGCTTCTTCAGCTCAGATCCAGACTGCACCACTGATCTGACA

TCTGGCACCTGTAAJCACAGY2A (SEQ ID NO: 58); LI{27, sense humanized 77A3 VII -1 primer 3 (80 mer),

CTGGAGCCTCAGTCAAGATCTCCTAGGTTCTGGTATACCTTCA

CAAACTATGGAATGAACTG (SEQ ID NO:59); goes&: LH28, anti-sense humanized 77A3 VII -1 primer 4 (80 mer) ATTCCATAGTTTGTGAGGTA (SEQ ID LH29, sense humanized 77A3 VH -1 primer 5 (80 mer), :000*0AGGGACG{3TTTGTCTTCTCT (SEQ ED NO:6 1); 45 LEBO, anti-sense humanized 77A3 VII -1 primer 6 (80 mer), GACA AACCGTCCCTTGAACTC (SEQ ID NO: 62); LH3 1, sense humanized 77A3 VI AI primer 7 (80 mer),

CAGCCTCAAGCTGAGGACACGTGTGTATTTCTGTAGATGG

TACCTGGGACCTATGCCATLI( (SEQ ED NO:63); WO 98/12329 PCTIUS97/16122 -62- *0 15 L1132, anti-sense humanized 77A3 VII -1 primer 8 (80 mer),

CTAGCTGAGGAGACGGTGACCGTGGTTCCTGACCCCAGTAGTCA

GGAAGCCGTCCT (SEQ ID NO:64); LH33, anti-sense humanized 77A3 VII -2 primer 2 (80 mer),

TACAGGTGCCAGATGTCAGATCCAGTTGGTGCAGTCTGACTGG

TGAAGAAGCCT(YJA(YCTCAGTC (SEQ ID LH34, sense humanized 77A3 VII -2 primer 5 (80 mer),

TGAAAACAATGGGCAAAGTAGGTA

GOGACGGTTTACCTTCACC -3 (SEQ ED NO:66); LH35, anti-sense humanized 77A3 VII -2 primer 6 (80 mer), GCTCCTGATCTCCA4TAGGCAGTGCTCGTAGAGGTGTCCAAGGA AGGTAAACCGTCCCTTGA&ACTC (SEQ ID NO:67); and LH36, sense humanized 77A3 VH -2 primer 7 (80 mer,

AGGAGCTCAGATCTGACGACACTGTGTACTGTCAGAT

GGACGGCTTCAG 3 (SEQ ID NO:68).

Table 3 summarizes how the above primers were used in the non-template PCR protocol.

0 *OSbOO 0**S 0 0 0505

S

555500 S S SS SO 5 0 0

SOS

S

WO 98/12329 PCTIUS97/16 122 -63- 3. Use of primers in non-template

PCR

chimeric Heavy chains humanized-ij humanized-21 outer primer (sense) VI (sense) LH1 (SEQ ID NO:37) LH I (SEQ ID NO:37) Light chains chimeric humanized LHI (SQ LHI I (SEQ ID NO:47) ID NO:47) LH I (SEQ ID NO:37) LH2 (SEQ ID NO:38) LH25 (SEQ LH25 (SEQ LH12 (SEQ LH12 (SEQ IDNO:57) ID NO:57) IDNQ:48'~ Tfl WA.A~ T~1 t 61a.

:0 *0: 0 V2 (antis V3 (s V4 (antiS4 (s V6 (antise V7 (se ense) LH3 (SEQ ID NO:39) LH26 (SEQ ID NO:58) LH33 (SEQ LH13 (SEQ LH{21 (SEQ ense) LH4 (SEQ LH27 (SEQ LH27 (SEQ LH14 (SEQ LH22 (SEQ NO:40) ID NO:59) ID NO:59) ID NO:50) ID NO:7o) LH5 (SEQ ILH128 (SEQ LH28 (SEQ LH15 (SEQ LH15 (SEQ ense) ID NQ:41) ID NO:60) ID NO:60) ID NO:5I1) ID NO:5 1) ense) LH6 (SEQ LH29 (SEQ LH34 (SEQ LH16 (SEQ LH23 (SEQ NO:42) ID NO:6 1) ID NO:66) ID NO:52) ID NO:7 1) LH7 (SEQ LH30 (SEQ. LH35 (SEQ LH17 (SEQ LH24 (SEQ ~nse) ID NO:43) ID NO:62) ID NO:67) ID NO:53) ID NO:72) ~nse) LH8 (SEQ LH31 (SEQ LH36 (SEQ LHl18 (SEQ LH18 (SEQ NO:44) ID NQ:63) ID NO:68) ID NO:54) ID NO:54) V8 H (E (antisense) ID NO:45) outer primer LHIO (SEQ (antisense) ID NO:46) JLH32 (SEQ ID NO:64) LH32 (SEQ LH19 (SEQ LH19 (SEQ ID NO:6I LH1O (SEQ ID NO:46) LHI10 (SEQ ID NO:46) LH20 (SEQI LH20 (SEQ ID NO:56) ID NQ:56) Briefly, 8 adjacent oligonucleotides which represent a synthetic light or heavy chain V gene are synthesized (VI-V8 in Table Four sense strand oligonucleotides alternate with 4 overlapping antisense strand oligonucleotides of 78-8 1 nt in length. These PCR primers overlap each other by 24-27 nt. Note that for oligonucleotide primers VI and V8 (Table their 5' end is designed to overlap with 15 -30 nt of the vector sequence, while their 3' end overlaps 48-65 nt of the signal peptide (VI) or the V gene sequence The 8 oligonucleotide primers I i, I WO 98/12329 PCT/US97/16122 -64are all included in the same first round PCR. Reaction conditions for this 1st round PCR were 0.125 picomoles of each primer, 10 Jl of 10X Pfu buffer (Stratagene Inc., San Diego, CA), 10 nanomoles dNTP's (Boehringer Mannheim, Indianapolis, IN), 10% dimethylsulfoxide (DMSO), and 2.5 units cloned Pfu DNA polymerase I (Stratagene Inc., San Diego, CA) in a 100 /l reaction volume. Reactants were first denatured at 95C for 5 min, annealed at 45 *C for 5 min, and extended at 72 *C for 1 min, followed by 25 cycles of denaturation at 94 *C for 30 sec, annealing at 55 °C for 30 sec, and extension at 72 *C for 30 sec. The 25 cycles were followed by a final extension at 72 *C for 7 min in a Perkin-Elmer DNA Thermal Cycler (Norwalk,

CT).

The amplified PCR product was electrophoresed through a 1.4% agarose gel and the smear ofDNA running between approximately 350 bp 500 bp was cut out prior to purification by the Qiaex' II gel extraction kit (Qiagen, Chatsworth, CA). This purified non template specific PCR product served as the template for a 2nd round PCR. To complete the 2nd round PCR, two additional outer primers are utilized. These outer primers are homologous to 29 30 nt of the vector sequence Sthat is either 5' (sense primer) or 3' (antisense primer) of the linearized cloning site within the mammalian expression cassette vector. This allowed for the amplified PCR product to be subcloned into the vector by bacterial homologous "0 recombination (Jones, D.H. and Howard, B. BioTechniques 10: 62-66 (1991)).

Thus, the reaction conditions for the 2nd round PCR were 0.125 picomoles each of outer sense and antisense primers, 10 pl of 10X Pfu buffer, 10 nanomoles dNTP's, i 10% DMSO, 2.5 units Pfu DNA polymerase I, and approximately 100 ng of 1st round PCR template DNA. The reactants underwent the same thermocycle program 5 described above. Subsequently, the amplicand from this reaction was removed from a 1.4% agarose gel and purified with the Qiagen' T II gel extraction kit.

2 00ng -1000ng of PCR product was mixed with an equal weight of linearized vector, and this mixture was used to transform 200 ml of competent

E.

coli DH5a cells (GIBCO BRL/ Life Technologies, Gaithersburg,

MD).

Transformed cells were selected by 100 gg/ml ampicillin in LB agarose. Typically, WO 98/12329 PCT/US97/16122 pD16-hCka digested with Xho I was used for subcloning light chain V genes.

pD20-hg2a digested with Nhe I served as the vehicle for heavy chain V gene constructs.

In order to confirm that the V gene of interest had been inserted into the expression vector, two screens were performed. The primary screen was by PCR, while the secondary screen was by restriction digest. Each individual colony of bacteria was picked into 5 ml of T broth (GIBCO BRL/ Life Technologies, Gaithersburg, MD) containing 100 /2g/ml ampicillin and grown 8 16 hr at 37 "C with shaking. The conditions for the PCR screen were 0.125 picomoles of both outer primers (Table 2 ml 10X Mg 2 buffer (Boehringer Mannheim, Indianapolis, IN), 10 nanomoles dNTP's (Boehringer Mannheim, Indianapolis, IN), 1 unit Taq DNA polymerase I (Boehringer Mannheim, Indianapolis, IN), and 1 pl of the liquid culture growth (which served as the source of DNA template since the cells lysed at high temperature) in a 20 /l volume. Reactants first underwent denaturation at 1.5 94 °C for 5 min, followed by 25 cycles of denaturation at 94 °C for 25 sec, annealing at 45 °C for 25 sec, and extension at 72 "C for 12 sec. The cycles were followed by a final extension at 72 "C for 7 min. Positives were determined by size comparison relative to a DNA standard marker after electrophoresis through a 1.4% agarose gel.

For the secondary screen, midi DNA preparations (Qiagen, Chatsworth, CA) were made from bacterial pellets and a portion was digested with either Xho I (VL genes) or Nhe I (VH genes). Again, after electrophoresis through a 1.4% *00: agarose gel, size comparison of the fragment released due to enzyme digestion served to identify potentially positive clones.

"0 The above procedures were used to confirm the presence of a potentially correct insert. However, they were not specific enough to detect small errors in the sequence (insertions, deletions and substitutions). To determine which clones contained DNA encoding complete Ig genes, each potentially positive heavy chain clone was cotransfected into COS cells with each potentially positive light chain 1. 1.

WO 98/12329 PCT/US97/16122 -66clone. Culture supematants were screened by ELISA for the presence of human IgG, and then for the presence of IgG binding to a 2 -antiplasmin (see below).

DNA for COS transfections was derived from midi DNA preparations described above. COS tranfections were performed in 60 mm dishes. Complete details of the DEAE- dextran technique employed have been described (Linsley P.S. et al, J. Exp. Med 173: 721-730 (1991)). Typically, 1.5 /g 6 gg of whole antibody is derived from small scale COS transfections As a final confirmation, the V region inserts from the above clones were sequenced by the dideoxy nucleotide procedure.

B Production of Humanized and Chimeric Antibodies Once heavy and light chain vectors encoding each of the desired antibodies were qualified, sufficient quantities of chimeric and humanized antibody for testing in functional assays were needed. This was first done as a scale-up of the COS transfections using the selected vectors. Finally, stable cell lines were prepared by high copy number electropQration. The electroporation protocol of Barsoum (Barsoum, DNA and Cell Biology 9:293-300 (1990)) was followed with the exception that 100 g each of the heavy and light chain vector were used (following restriction with BssHII) and the electroporation was performed in PFCHO media (PX-CELL PFCHO media, JRH Biosciences, Lenexa, Kansas).

Transfected cells were selected in media containing either 20 nM or 100 nM methotrexate (MTX). Culture supernatants were assayed for the presence of whole antibody using the non-specific IgG ELISA. Cells from master wells containing the most antibody in the supernatant were expanded into larger volumes. In some cases, the methotrexate concentration was also increased in order to amplify the vector in the cell lines. The vector pairs in Table 4 were electroporated into DG44 CHO cells.

I

WO 98/12329 PCT/US97/16122 -67- Table 4. Vector pairs for production of antibody Product Heavy Chain Light Chain Vector Vector c77A3 (chimeric 77A3) pD20-cR1.HI pD16-cR1.L1 h77A3-1 (humanized 77A3) pD20-hR1.H1 pD16-hR1.L1 h77A3-2 (humanized 77A3) pD20-hR2.H1 pD16-hRl.Ll h77A3-3 (humanized 77A3) pD20-hR3.H1 pD16-hRl.L1 0 0 0*@O

S.

400 0 0 00 00 *0 0 .:0 aO 6 *0 *q 1.) *000 So C Purification of Humanized and Chimeric Antibodies The purification of the antibody was first performed using protein-A affinity chromatography. A Pharmacia column, sized so that 5 mg of antibody to be loaded per 1 ml of resin, was packed with Perseptive Biosystems Poros 50 A protein-A resin. The column was then sanitized according to the methods recommended by the resin supplier. The column was equilibrated with pyrogen free 10 mM sodium phosphate, 150 mM sodium chloride pH 7.0 (PBS). The cell culture supernatant was adjusted to pH of 7.0-7.5 and loaded on the column at a flow rate equal to 2 3 column volume/min (CV/min). The column was then washed with 15 CV pyrogen free PBS or until a stable base line has been achieved. The antibody was eluted with 20 mM glycine/HCI pH 3.0 elution buffer. The eluted peak was collected in a pyrogen free vessel that contained 1/20 CV of 1 M Tris base solution.

The pH of the eluted antibody solution was adjusted to pH 8.0 with 1M Tris base immediately. The column was then cleaned with 5 CV 12 mM HCI solution. The column was stored in 20% ethanol/water at The antibody was next purified using anion exchange chromatography.

A

Pharmacia column, sized so that 5 10 mg of antibody to be loaded per 1 ml of resin, was packed with Perseptive Biosystems Poros HQ 50 anion exchange resin.

The column was then sanitized according to the methods recommended by the resin WO 98/12329 PCT/US97/16122 -68supplier. The column was equilibrated with pyrogen firee 50 mM Tris/HCI, 50 mM NaCI, pH 8.0. The protein-A purified antibody adjusted to pH of 8.0 was loaded on the column with flow rate equal to 1 CV/min. The column was then washed with 5 CV pyrogen free 50 mM Tris/HCI, 1M NaCI pH 8.0. The antibody does not bind to this column under the running conditions and was present in the flowthrough fraction. The column was stored in 20% ethanol/water at 4.0*C. The antibody was then concentrated and diafiltered against PBS using a 30K cut off membrane.

D. Non-specific IgG ELISA to detect presence of antibody This ELISA detects whole antibody (containing both heavy and light chain) *IV and relies on a capture antibody specific for human IgG Fc region and a conjugate specific for human kappa chains. In this assay, Immunlon II flat bottom plates (Dynatech) were coated with goat anti-human IgG (Fc specific, adsorbed on mouse IgG) (Caltag, Inc. catalog #H10000) at 0.5 iig/ml in carb/bicarb buffer pH 9.6 and then blocked with PTB (PBS containing 0.05% Tween 20 and 1.0% BSA). Sample was added (either undiluted or diluted in PTB or Genetic Systems specimen diluent), the plates were incubated o/n at 4"C or for a few hours at room i temperature. After washing, conjugate (goat anti-human kappa conjugated with horseradish peroxidase from Southern Biotech) was added at 1:10000 in PTB.

After approximately 1 hour incubation at room temperature, plates were washed and 100 /l chromagen/substrate was added (Genetic Systems chromagen diluted 1:100 into Genetic Systems substrate). After sufficient color development (usually 5 to 15 minutes) 100 p1 1 N H 2

SO

4 was added to stop the reaction. Optical densities were determined using a Biotek plate reader set at 450 and 630 nm wavelengths.

In the occasional case that none of the samples from small COS transfections showed the presence of whole antibody, similar ELISAs were performed to determine whether any light chain was being secreted. In this case, WO 98/12329 PCT/US97/16122 -69the plates were coated with a goat anti-human kappa chain at 1 /g/ml. The rest of the assay was done exactly as above.

The assay was used for three purposes. First, to screen small COS transfections that were set up to qualify various heavy and light chain vectors. In this case, the presence or absence of a signal was sufficient and it was not necessary to quantify the amount of antibody present. Second, to determine which of many master wells from CHO transfections were producing the most antibody. In this case, culture supernatants were diluted so that relative signals could be compared and the master wells containing the most antibody could be distinguished and thus selected for cloning and expansion. Thirdly, to determine amounts of antibody, either in culture supernatants or following purification. In this case, a standard consisting of either a chimeric or human IgG1 or a human myeloma IgG2 were used. Both standard and sample were serially diluted (2x) across a plate and sample concentration relative to standard was determined by comparing position of the curves. The concentrations thus determined were used for following antibody production during the cloning and amplification process and for determining specific Sactivity in the antigen binding ELISA and any of the functional assays.

E. ELISAs to show that antibody is capable of binding to antigen This ELISA relies on an antigen capture and a human kappa chain specific conjugate. It was used for two purposes. Initially, to qualify a vector, supernatants from COS transfections were screened for the ability of antibody to bind to antigen.

Vectors passing this test were then submitted to DNA sequencing. Secondly, to S determine relative antigen binding ability of the various chimeric and humanized antibodies. This ELISA is very similar to the non-specific IgG ELISA described above except that the plates were coated with a2-antiplasmin (obtained from American Diagnostica) at 1 pg/ml in PBS.

To determine relative antigen binding ability of various antibodies, scatter plots were used with log antibody concentration along the X axis and optical density WO 98/12329 PCT/US97/16122 along the Y axis. Antigen concentration was determined either from the non-specific ELISA or based on optical density of purified preparations. All three forms of humanized antibody (h77A3-1, and show antigen binding similar to that of the chimeric antibody. Comparisons were not made with the murine antibody (m77A3) because the m77A3 cannot be detected in the assay as described (the antibody-conjugate used in the second step recognizes only human constant regions).

F. Functional Assays Two functional assays were performed. The first, known as the "plasmin 0'assay with chromogenic substrate" is based on the ability of plasmin to convert Spectrozyme PL, H-D-Nle-HHT-Lys-pNA.2AcOH into pNA, which absorbs light

*D)

c at 405 nm. Ifunblocked a2-antiplasmin is present, little or no conversion occurs.

Active antibody is capable of blocking the inhibitory activity of a2-antiplasmin. The second assay, the clot lysis assay, is a measure of the ability of antibody along with urokinase to lyse preformed clots.

The plasmin assay with chromogenic substrate is designed based on the action of plasmin on its chromogenic substrate according to the reaction: Plasmin H-D-Nle-HHT-Lys-pNA.2AcOH H-D-Nle-HHT-Lys-OH pNA Plasmin Chromogenic Substrate *eec The generation ofpNA was monitored by the increase in absorption at 405 nm using a SpectraMax 250 spectrophotometer. The addition of a2-antiplasmin inhibits the plasmin activity and no increase in absorption at 405 nm will be observed. Premixing of a2-antiplasmin with functional antibody blocks the ability of a2-antiplasmin to inhibit the plasmin activity. Plasmin activity was measured as the initial rate of color development.

Assays are performed in 96 well microtiter plates. The chromogenic substrate Spectrozyme PL, H-D-Nle-HHT-Lys-pNA.2AcOH, human plasmin, and human a 2 -antiplasmin were purchased from American Diagnostica. Stock and WO 98/12329 PCT/US97/16122 -71working solutions are prepared as follows: Spectrozyme PL stock solution mM in H20; Spectrozyme PL working solution 1:12.5 dilution of stock solution in H 2 0; human plasmin stock solution 0.2 mg/ml in 50% glycerol, 50% 2 mM HCI; human plasmin working solution 1 12.5 dilution of stock solution in 0.11 mM HCI, which must be prepared immediately before use; human a2-antiplasmin stock solution 0.2 mg/ml in PBS; and human a2-antiplasmin working solution 1:15 dilution of stock solution in PBS. Stock solutions were stored at -70 and should not be refrozen after thawing.

Reagents are added in the following order, with mixing after each addition: 80 ul antibody or PBS, 40 ul a2-antiplasmin working solution, 40 ul plasmin working solution, and 40 ul Spectrazyme PL working solution. R is the rate of color development. Rp, which represents maximum plasmin activity, is determined in wells lacking both antibody and a2-antiplasmin. Ro, which represents minimal plasmin activity, is determined in wells lacking antibody. Rs is the rate of color 15 development in the sample. Antibody activity is calculated as (Rs Ro)/(Rp Ro) 100. Values should range between 0% and 100%. Antibody activity was plotted vs. amount antibody (on a log scale). Curves generated by test antibody and standard (usually murine 77A3) were compared.

The data for murine 77A3, c77A3, and h77A3-1 are shown in Figure The curves for murine and chimeric 77A3 were superimposable. The curve for h77A3-2 indicates a potential small loss in activity (20-30%).

The clot lysis assays were performed as follows. Test clots were formed in 96-well Corning #25805 microtiter plates by mixing 25 uL 16 mM CaCI 2 50 uL of pooled human plasma, and 25 uL of 4 NIH unit/ml of human alpha-thrombin i* (Sigma) in 30mM Hepes buffer, pH 7.40. Plates were incubated overnight at room temperature to allow clots to achieve maximum clot turbidities. Clot lysis was initiated by adding 10 uL of antibody to give 5 or 10 ug/well and 100 uL of urokinase to give 1, 3 or 5 units ofurokinase/well (Abbott Labs) at pH 7.40. Plates were mixed on a table top microplate vortexer for 30 sec before the initial reading at 405 nm to get values corresponding to 0% lysis. Plates were sealed with Coring 4. WO 98/12329 PCT/US97/16122 -72sealing tape #430454 and incubated at 37"C. During the course of 24 hrs, the decrease of turbidity was measured at 405 nm to quantify the progress of clot lysis.

The results of a clot lysis experiment of humanized 77A3-1 indicate that h77A3-1 enhances clot lysis dramatically in comparison to buffer controls in each of the conditions tested. There was significant separation between the humanized and murine 77A3 in clots containing 5 ug antibody in the presence of 1 or 3 units ofurokinase indicating that humanized 77A3 was somewhat less active than murine 77A3, even though the lysis profiles were similar at the remaining four conditions tested. It should be noted that murine RWR, a monoclonal antibody with a lower affinity than murine 77A3, causes no lysis at 10 ug per clot in the presence of 1 unit ofurokinase and would give a lysis profile like buffer control.

0* Example Preparation and Characterization of Single Chain Fv Fragments A. Deign and expression of sFvform of 77A3 The sFv fragment of an antibody is most commonly obtained by the tandem expression of the variable region of the antibody heavy chain along with the variable region of the antibody light chain spaced by a linker of 15-20 amino acids. sFv fragments are expected to have superior clot penetration to parent antibodies. Two S constructs, p53-6 and p52-12, were prepared using murine variable regions with a VH-(linker)-VL polarity using YPRSIYIRRRHPSPSLTT (SEQ ID NO:73) as linker 1 for sFv77A3-1 and GGSGSGGSGSGGSGS (SEQ ID NO:74) as linker 2 0 for sFv77A3-2. Both constructs were cloned into the pET-22b vector from Novagen and transformed into the BL21 (DE3) strain ofE. coli grown in minimal M9 media. Though the majority of the His-tagged product was found in inclusion bodies, supematants of cell lysate contained sufficient quantities of soluble sFv fragments for nickel-column purification.

1* It WO 98/12329 PCT/US97/16122 -73sFv77A3-2 present in fractions 7-11 collected from a nickel-column gave a single Coomasie staining band with a MW about 30,000 agreeing well with the calculated MW of 29,986. A similar but more weakly staining gel was obtained for sFv77A3-1.

B. Activity of sFv77A3-1 and sFv77A3-2 Preparations of both sFv77A3-1 and sFv77A3-2 were tested for alpha2antiplasmin binding activity in a competition binding assay. Microplate wells coated with 77A3 were treated with mixtures of biotinylated-human alpha2antiplasmin and either sFv77A3-1 or sFv77A3-2 along with positive control 77A3 and negative control, mAb-59D8. Increasing quantities of 77A3 prevented binding S.of biotinylated-human alpha2-antiplasmin whereas negative control 59D8 had little 0 effect as an competitive inhibitor. With concentrations of test samples estimated by intensity of Coomasie stained bands, both sFv77A3-1 and sFv77A3-2 completely 00 inhibited the binding of biotinylated-human a2-antiplasmin with a profile of 15* inhibition nearly superimposible to the parental 77A3 reference.

Idiotypic markers present on 77A3 were probed with a sandwich

ELISA

using a biotinylated polyclonal reagent rendered specific by multiple 000 immunoadsorbtion steps through columns bearing immunoglobulins from man, mouse, baboon and cynomologous monkey (P Stenzel-Johnson D Yelton, Seattle). Microplate wells were coated with 77A3 and 59D8 as controls along with 00, sFv77A3-1 and sFv77A3-2. It is evident that 77A3 control and both sFv fragments bear idiotypic markers at each dose tested indicating that the sFv fragments "look" like the parental 77A3.

It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples.

WO 98/12329 PCT/US97/161 2 2 -74- Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

The disclosure of all references, patent application, and patents referred to herein are hereby incorporated by reference.

Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof.

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INDICATIONS RELATING TO A DEPOSITED

MICROORGANISM

(PCT Rule l3bis) A. The indications made belo0w relate to the microorganismi referred to in the description on page 7 ,line 12 D3. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet J Name of depositary institution AMERICAN TYPE CUL'IURE

COLLECION

Address of depositary institution (includ ing Postal code and country) 12301 Parkiawn Drive ROckville, Marylan~d 20852 United States of America

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0 0 Date of deposit 20 September 1996 Accession Number tm I I) I rl') C. ADDITIONAL INDICATIONS (leave blank ifot applicabic) This information is continued on an additional sheet 3 Hybridzta cell line, 77a3 D. DESIGNATED STATES FOR WHIICHI INDICATIONS ARE MADE (ifth indications are"no for all desi ptated States) SEPARATE FURNISIUNG OF INDICATIONS (leavye blank if not applicable) Th indimucations listed below will be submitted to the Interna tionalI Burea u la ter (specify the general noture of thi.ndicons eg., 'Accession Number of Dcposit*) For receiving office use only For international Bureau use only Thnis sheet was received with the international application I'i he a eevdb teitrainlBra n A u l b 'I z~A t h o r d o f f i c e r u f c r z e i -f c r Fo'rm: 11717/1(0/11-4 (July 199~2) WO 98/12329 PCTIUS97/16122 Applicants or agent's (tic 09. 432PC01 -7.2 nternaitional ppiickliod' reference number TEA INDICATIONS RELATING TO A DEPOSITE-D MICROORGANISM (PCT Rule I3bis) A. The indications made below relate to the microorganism referred to in the description on page .18 line BI. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet E Name of depositary instituhion AM'ERICAN~ TYPE CULTURE COLLECTION Address of depositary institution (incduding postal code and country) 12301 Parkiawn Drive Rockville, M'aryland 20852 United States of America Date of deposit Accession Number 19 September 1997 TEA C. ADDITIONAL INDICATIONS (leavie blank if not applicable) This information is continued on an additional sheet E] 77A3-HC agee 0 000@ 0* 0 *00 S. es @6 9 5 0

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For receiving Office use only SThis sheet was rceivcd with the international application Authorizcd officer Foirnm ICAM1.- ll(ll 4Julv y i2 For International Bureau use only [IIThis sheet was received by the International Bureau on: Auilioriizvd officcr WO 98/12329 PCTIUS97/16122 -74.3- A I: Iu ppcn ao gent'& file Lreference number jUl. 432PCOI Inte"mmtional pplication I rrqqh Liz I International appliuadon INDICATIONS RELATING TO A DEPOSITED

MICROORGANISM

(PCT Rule 13Lns) A. The indications made below relate to the microorganism referred to in the description on page 18 line 19 H. IDENTIFICATION OF DEPOSIT Name of depositary institution Further deposits are identified on an additional sheet jLI] AMERICAN T1YPE CUTURE COLLECTION Addres of depositary institution (including postal code and country) 12301 Parkiawn Drive Rockville, Maryland 20852 United States of Anerica 0

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C. ADDITIONAL INDICATIONS (leave. blank if no applicable) This information is continued on an additional sheet 0 77A3-iC D_ DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (ifth eindications are nofo all deintdStates) E. SEPARATE FURNIStING OF INDICATIONS (leavye blank if not applicable) T'he indlications litdblwwill bec subimitted to the international B~ureau later (spccify the general nature of theindicalions 'Accession Number of Deposig Accession number will be furnished at a later date.

I-or recciving Office use only Thskecaa rcic with the international application Authorized officer K2+31 'For International Bureau use only IJThis sheet was received by the International Bureau on: Aullirized telficcr WO 98/12329 PCTIUS97/16122 -74.4- Applicant's or agent's file 09.4 32PC0 1 Interrnational application INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT' Rule l3bis) A. Thbe indicationsmrgde below relate to the microorganism rffrred to in the description on page line U. IDENTIFICATION OF DEPOSIT Further deposits arc identified on an additional sheet Name of depositary institution AM~ERICAN~ TYPE CM=1UR COLLECIION Address of depositary institution (including postal code and country) 12301 Parkiawn Drive Rockville, Maryland 20852 United States, of America S* Date of deposit Accession Number :0 19 Sepitenber 1997 B ADDITIONAL INDICATIONS (leave blank if"no applicable) This information is continued on an additional sheet jJ3 49C9-HC D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indicationsr are nat/or all designated Stalejr) E.SPRT*URIHN FINIAIN lav ln fno plcbe neidctoslse eo i e umte oteItra oaIBreultr(pcf h eea.aur fhidctoseg,'ceso be*f*eost Aceso nme il efrise talae ae *o reevn fieueol -r nentoa ueuueol F hssetwsrcie ih...nentoa plcto iF 7 he a eevdbyteItrainlBra n Authorized Officer 1-or 1 4 7 It,1vI w Autllliirzicd ofificer WO 98/12329 PCT/US97/16122 -74.5- Applicant's or agent's file 09. 432PCO1 International applicatii I reference number TBA INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13bis) A. The indications made below relate to the microorganism referred to in the description on page 1]8 line 19 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet Name of depositary institution AMERICAN TYPE CULTURE COLLECTION Address of depositary institution (including postal code and country) 12301 Parklawn Drive Pockville, Maryland 20852 United States of America 6•09 •Date of deposit Accession Number -19 September 1997 TBA C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet L 49C9-LC *0 S D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE fifthe indications are not for all designated States) E. SEPARATE FURNISH-ING OF INDICATIONS (leave blank if not appicable) 0 The ind ications listed below will be submitted to the nicerat 0•a I Bureau later (specify the gneral nature of theindications 'Accession a Number of Deposit*) Accession number will be furnished at a later date.

For receiving Office use only For International Bureau use only This sheet was rceived with the international application b" •hsseet was received by the international Bureau on:

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0 *0 E. SEPARATE FURNISHING OF INDICATIONS (leave blank ifnot applicable) The indications listed below will be submitied to the International Bureau later (specify the general nature of the indications *Accession S* Number ofJDeposit') Accession number will be furnished at a later date.

For receiving Offtce use only For International Bureau use only P|This sheet was received with tli international application This sheet was received by the International Bureau on: Authorrized officer l irnm July Authorinzcd fficer WO 98/12329 PCT/US97/16122 -74.6- Applicant's or agent's file -09.432PC01 Internationalapplication' reference number TBA INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13bis) A. The indications made below relate to the microorganism referred to in the description on page 18 line 23 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet Name of depositary institution AMERICAN TYPE CULTURE COLLECTION Address of depositary institution (including postal code and country) 12301 Parklawn Drive Rockville, Maryland 20852 United States of America Date of deposit Accession Number 19 September 1997 TBA C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet 0- 70B11-HC

C.,

000 gem

COO

ft S f D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (ifthe indications are not for all designated States)

C

CCC CCC

S

C

003@ 0 0 005600 0 S. CO *6 0 6

C

C..

0 E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the International Bureau later (specify thegeneral nature ofthe indications "Accession Number ofDeposit') Accession number will be furnished at a later date.

For receiving Office use only rM This sheet was received with the international application Authorized officer l:urn 34 July I 1 4'2) For International Bureau use only E1 This sheet was received by the International Bureau on: Aulthrizecd officcr WO098/12329 -74.7- PCTIUS97/16122 Applicantsor agent,. file 0609. 432PC01 Ineratonljjj]ator" INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule I3bis) A. The indications Made below relate to the microorganism referred to in the description on page 18 *line D. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet Name of depositary institution AMEP.ICiAN TYPE CULTURE CDLLE=ION Address of depositary institution (including postal code and country) 12301 Parkiawn Drive Rockville, Maryland 20852 United States. of America Date of deposit 19jg Accession Number 1 Setember 1997

ITEA

000 :0 0 00 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet Eli 70B11-LC D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (iffheindaionsatforall eigalState) 0 000000 g 0000 0 ~0*0 0 0000 6 6000 0 000000 0 00 00 0 B 0 0 000 0 E. SEPARATE FURNISIHNG OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the Internat iona I Burea u later (specify the general nature of the andicationzs 'Ace~son Number oflDcposuf) Accession numtber will be furnished at a later date.

For receiving Office use only jIJ This sheet was reccived with the international application Aulitrizcd olffi.cC For International Bureau use only M This sheet was received by the International Bureau on: Authorized 4ifificrr -74.8- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT/INVENTOR: REED, GUY L.

(ii) TITLE OF INVENTION: COMPOSITION AND METHOD FOR ENHANCING FIBRINOLYSIS USING ANTIBODIES TO ALPHA-2 ANTIPLASMIN (iii) NUMBER OF SEQUENCES: 81 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: STERNE, KESSLER, GOLDSTEIN FOX P.L.L.C.

STREET: 1100 NEW YORK AVENUE, N.W. SUITE 600 CITY: WASHINGTON STATE: D.C.

COUNTRY: USA ZIP: 20005 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30

C

(vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT/US97/16122 FILING DATE: 19-SEP-1997

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 60/026,356 FILING DATE: 20-SEP-1996

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: GOLDSTEIN, JORGE A REGISTRATION NUMBER: 29,021 REFERENCE/DOCKET NUMBER: 0609.432PC01 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (202) 371-2600 TELEFAX: (202) 371-2540 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids 0: TYPE: amino acid STRANDEDNESS: single TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Xaa Ile Gin Met Thr Gin Ser Pro Ala Ser Leu Ser Ala Ser Val AUEIOt0 SHE;; f 1 1 n~ -74.9- INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Asp Ile Gin Met Thr 1 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 0SSO 0 0S a 0*e *0 0e *0* 0 0

S

00 00 0

S

Xaa Ile Gin Met Thr Gin Ser Pro Ala 1 5 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 381 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear Ser Leu Ser Ala Ser Val 10 (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..381 (ix) FEATURE:

NAME/KEY:

LOCATION:

sig peptide 1..60 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ATG AGT GTG CTC ACT CAG GTC CTG GSG TTG CTG CTG CTG TGG CTT ACA Met Ser Val Leu Thr Gin Val Leu Xaa Leu Leu Leu Leu Trp Leu Thr V, GH EET 3rLL $9

GGT

G1 y

GCA

Al a

ATT

lie

GAG

Gin

AGG

Arg

AGC

Ser

ACC

Thr

GCC

Al a

TCT

Ser

GAG

H is

CTC

Leu

TTC

Phe

CTG

Le u

ACT

Thr

AGA

Arg

GTG

Val.

AAT

Asnr

CTG

Le u

AGT

Ser

GAG

Gin

CCG

Pro 95

TGT

Cys -1

GGA

Gi y

TAT

Tyr

GTC

Vai

GGC

Gly

CCT

Pro 80

TGG

T rp

GAG

Asp

GAA

Giu

TTA

Le u

TAT

Tyr

AGT

Ser

GAA

Gi u

AG

Thr -i5

ATC

Ile

ACT

Thr

GGA

Al a

PAT

As n 50

GGA

Gi y

GAT

Asp

TTC

Phe GAG ATG Gin Met GTC ACC Vai Thr 20 TGG TAT Trp Tyr 35 GCA AAA Ala Lys TCA GGA Ser Gly TTT GGG Phe Gly GGT GGA Giy Giy 100

AC

Th.

AT(

Ii(

CA(

GIr

AC(

Thi

AC;

Thi

AGI

Sei

GGC

Gi -74.10- T GAG TGT CCA GCC TGC CTA r Gin Ser Pro Ala Ser Leu 5 CACA TGT CGA GCA AGT GGG e Thr Cys Arg Ala Ser Gly G AG AAA GAG GGA AAA TCT i Gin Lys Gin Giy Lys Sez -TTA GGA GAT GGT GTG CGA *Leu Ala Asp Gly Val Pro 55 ~CPA TTT TCT CTC AGG ATC -Gin Phe Ser Leu Arg Ile 70 CAT TAC TGT CAA GAT TTT *His Tyr Cys Gin His Phe *ACC PAG CTG GAA ATC PAA Thr Lys Leu Giu Ile Lys 105 o ii,, .9 ~9

TCT

Ser

AAT

As n

CCT

TCA

Ser

AAC

As n

TGG

T rp 96 144 192 240 336 *000 0 *000 *0 0 *0.

@0 @0 00 0 0

S.

0 000 es 00 000 INFORMATION FOR SEQ ID Ci) SEQUENCE CHARACTERISTICS: LENGTH: 127 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: CA) NAME/KEY: Modified-site 0000 LOCATION: 9 0 CD) OTHER INFORMATION: /note= "Can be Cxi) SEQUENCE DESCRIPTION: SEQ ID .Met Ser Vai Leu Thr Gin Vai Leu Xaa Leu Leu Leu 20 -15 Giy Ala Arg Cys Asp Ile Gin Met Thr Gin Ser Pro -1 Ala Ser Val Giy Giu Thr Val Thr Ile Thr Cys Arg 20 Ile His Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Gin 35 Gin Leu Leu Val. Tyr Asn Aia Lys Thr Leu Ala Asp 50 "tiMENDED SHEET either Gly or Ala" Leu Al a Al a Gly Gly Leu Leu Gi y Ser Pro I. I' I I .4 6661 66 6 66 4 0 6 66 66 4 06 6 4 *661 a *61 664 @66 606 *666

S

66 6S 6 6 66 6 -74.11- Arg Phe Ser Gly Ser Gly Ser Gly Thr Gin Phe Ser Leu Arg Ile Asn 65 70 Ser Leu Gin Pro Giu Asp Phe Giy Ser His Tyr Cys Gin His Phe Trp 80 85 Thr Thr Pro Trp Thr ?he Gly Giy Gly Thr Lys Leu Glu Ile Lys 100 105 INFORMATION FOR SEQ ID NO:6: SEQUENCE

CHARACTER.ISTICS:

LENGTH: 381 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix)

FEATURE:

NAME/KEY:

CDS

LOCATION: l. .381 (ix)

FEATURE:

NAME/KEY: sig_peptide 6* LOCATION: 1. (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ATG AGT GTG CTC ACT CAG GTC CTG GGG TTG CTG CTG CTG TGG CTT

A

**Met Ser Val Leu Thr Gin Val Leu Gly Leu Leu Leu Leu Trp Leu

T

-15 -10 GGT GCC AGA TGT GAC ATC CAG ATG ACT CAG TCT CCA GCC TCC CTA T Gly Ala Arg Cys Asp Ile Gin Met Thr Gin Ser Pro Ala Ser Leu

S

66-1 5 10 GCA TCT GTG GGA GAA ACT GTC ACC GTC ACA TGT CGA GCA AGT

GGG

Ala Ser Val Gly Giu Thr Val Thr Val Thr Cys Arg Ala Ser Gly 20 25 ,*ATT CAC AAT TAT TTA GCA TGG TAT CAG CAG AAA CAG GGA AAA TCT Sle His Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Gin Gly Lys Ser 30 35 40 CAG OTO CTG GTC TAT AAT GCA AGA ACC TTA GCA GAT GGT GTG

CCA

Gin Leu Leu Val Tyr Asn Ala Arq Thr Leu Ala Asp Gly Val Prc 45 50 55 AGG TTC AGT GGC AGT GGA TCA GGA ACA CAA TAT TCT CTC AAG

ATC

Arg Phe Ser Gly Ser Gly Ser Gly Thr Gin Tyr Ser Leu Lys Ile 65 70 75 AGC CTG CAG CCT GAA GAT TTT GGG AGT TAT TAC TGT CAA CAT TTT Ser Leu Gin Pro Giu Asp Phe Gly Ser Tyr Tyr Cys Gin His Phe 80 85 90 AGT AAT CCG TGG ACG TTC GGT GGA GGC ACC AAG CTG GAA ATC

AAA

Ser Asn Pro Trp Thr Phe Gly Gly Giy Thr Lys Leu Giu Ile Lys 100 105

CA

hr

CT

e r

AT

ks n Prc T CA Ser

AAC

As n

TGG

T rp 48 96 144 192 240 288 336 38 1 1 AiDDSHEET *I r *1 -74.12- INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 127 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met Gly Ala Ile Gin Arg Ser Ser (xi) SEQUENCE Ser Val Leu Thr Ala Arg Cys Asp -1 Ser Val Gly Glu His Asn Tyr Leu Leu Leu Val Tyr Phe Ser Gly Ser 65 Leu Gin Pro Glu 80 Asn Pro Trp Thr DESCRIPTION: SEQ ID Gin Val Leu Gly Leu -15 Ile Gin Met Thr Gin Thr Val Thr Val Thr 20 Ala Trp Tyr Gin Gin 35 Asn Ala Arg Thr Leu 50 Gly Ser Gly Thr Gin 70 Asp Phe Gly Ser Tyr Phe Gly Gly Gly Thr 100 NO:7: Leu Leu -10 Ser Pro Cys Arg Lys Gin Ala Asp 55 Tyr Ser Tyr Cys Lys Leu Leu Ala Ala Gly Gly Leu Gin Glu 105 Leu Gly Ser Pro Ile Phe Lys Ser Asn Pro Ser Asn Trp e *b* INFORMATION FOR SEQ ID NO:8: *0 00 SEQUENCE CHARACTERISTICS: LENGTH: 381 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..381 (ix) FEATURE: NAME/KEY: sig peptide LOCATION: 1..60 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: AGT GTG CTC ACT CAG GTC CTG GCG TTG CTG CTG CTG TGG CTT ACA Ser Val Leu Thr Gin Val Leu Ala Leu Leu Leu Leu Trp Leu Thr -15 -10 GCC AGA TGT GAC ATC CAG ATG ACT CAG TCT CCA GCC TCC CTA TCT Ala Arg Cys Asp Ile Gin Met Thr Gin Ser Pro Ala Ser Leu Ser :.;iIDEO SHEET

ATG

Met

GGT

Gly 7 7

C

-74.13- GCA TCT Ala Ser ATT CAC Ile His CA. CTC Gin Leu AGG TTC Arg Phe AGC CTG Ser Leu ACC ACT Thr Thr

GTG

Va1

AAT

Asn

CTG

Leu

AGT

Ser

CAG

Gin

CCG

Pro 95 -1

GGA

Gly

TAT

Tyr

GTC

Va1

GGC

Gly

CCT

Pro

TGG

Trp

GAA

Glu

TTA

Leu Tyr

AGT

Ser

GAA

Glu

ACG

Thr

ACT

Thr

GCA

Ala AAikT Asn 50

GGA

Gly

GAT

Asp

TTC

Phe

GTC

Va1

TGG

Trp 35

GCA

Ala

TCA

Ser

TTT

Phe

GGT

Gly

ACC

Thr 20

TAT

Tvr Lys

GGA

Giy

GGG

Glv

GGA

Gly 100 5

ATC

Ile

CAG

Gin

ACC

Thr

ACA

Thr

AGT

Ser 85

GGC

Gly

ACA

Thr

GAG

Gin

TTA

Leu

CAA

Gin 70

CAT

His

ACC

Thr

TGT

Cys

AAA

Lys

GCAN

Ala 55

TTT

Phe

TAC

Tyr

AAG

Lys

CGA

Arg

GAG

Gin

CAT

Asp

TCT

Ser

TGT

Cys

CTG

Leu

GCA

Ala

GGA

Gly

GGT

Gly

CTC

Leu

CA

Gin

GAA

Glu 105

AGT

Ser

AAA

Lys

GTG

Va1

AAG

Lys

CAT

His

ATC

Ile

GGG

Gly

TCT

Ser Pro

ATC

Ile

TTT

Phe

AAA

Lys

AAT

Asn

CCT

Pro

-CA

Ser

AAC

Asn

TGG

Trp 0)00 a Come 0 00 0e 0 0 C 0* 0O 0*0 00 *0

S

oem.

0 0000 *000 0 0 *0 06 00 0 0 INFORMATION FOR SEQ ID NO:9: Met -20 Gly Ala Ile Gin Arg Ser Thr SEQUENCE CHARACTERISTICS: LENGTH: 127 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Ser Vai Leu Thr Gin Val Leu Ala Leu Leu Leu -15 Ala Arg Cys Asp Ile Gin Met Thr Gin Ser Pro -1 5 Ser Vai Gly Glu Thr Val Thr Ile Thr Cys Arg 20 His Asn Tyr Leu Aia Trp Tyr Gin Gin Lys Gin 30 35 Leu Leu Vai Tyr Asn Ala Lys Thr Leu Ala Asp 50 Phe Ser Gly Ser Giy Ser Gly Thr Gin Phe Ser 70 Leu Gin Pro Giu Asp Phe Gly Ser His Tyr Cys 85 Thr Pro Trp Thr Phe Cly Gly Gly Thr Lys Leu 100 Leu Al a Ala Gly Gly Leu Gin Glu 105 Trp Ser Ser Lys Va1 Lys His Ile Leu Leu Gly Ser Pro Ile Phe Lys A%.heIfkF SHEET fl,~ S S 0 C -71~ -74.14- INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 414 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .414 (ix) FEATURE: NAME/KEY: sig_peptide_ LOCATION: 1. .57 (xi) SEQUENCE DESCRIPTION: SEQ ID ATG GMT TGG GTG TGG AMC TTG CTA TTC CTG ATG GCA GCT GCC CAA AC-T 000S 0 0000

S.

0 @00 @0 00 0 S 0 0* e 000 0S 0 000 0 0000SS 0 0 0 0 0 *00550 0 00 @0 So S 0

S

000 0 Met Xaa Trp Val. Trp Xaa Leu Leu Phe Leu Met Ala

CTC

Leu

CCT

Pro

ACA

Thr

AAG

Lys

GAA

Glu

ACT

Thr

TAT

Tyr

GGT

Gi y 110

CAA

Gln

GGA

Gl y 15

AAC

As n

TGG

T rp

GAG

Glu

GC

Al a

TTC

Phe

CAA

Gin

GCA

Al a -1

GAA

Glu

TAT

Tyr

ATG

Met

TTC

Phe

CAT

His 80

TGT

Cys

GGA

Gly

CAG

Gln

ACA

Thr

GGA

Gl y

GGC

Gly

AAG

Lys 65

TTG

Leu

GCA

Al a

ACC

Thr -15

ATC

Ile

GTC

Val

ATG

Met

TGG

T rp .50

GGA

Gl y

CAG

Gln

AGA

Arg

TCA

Ser

CAG

Gln

AAG

Lys

AAC

As n 35

ATA

Ile

CGG

Arg AT C Ile

TGG

T rp

GTC

Val 115

TTG

Leu

ATC

Ile 20

TGG.

T rp

AAC

As n

TTT

Phe-

AAG

Lys

GTA

Val1 100

ACC

Thr

GTG

Val 5

TCC

Ser

GTG

Val

ACC

Thr

GTC

Val

AAT

Asn 85

CCT

Pro

GTC

Val1

CAG

Gln

TGC

Cys

AAG

Lys

AAG

Lys

TTC

Phe 70

TTC

Phe

GGG

Gl y

TCC

Ser -10

TCT

Ser

AAG

Lys

CAG

Gln

AGT

Ser 55

TCT

Ser

AGA

Arg

ACC

Thr

TCA

Ser

GGA

Gly

GCC

Al a

GCT

Al a

GGA

Gi y

TTG

Leu

.AAT

Asn

TAT

Tyr

CCT

Pro

TCT

Ser

CCA

Pro

GAG

Gi u

GAA

Glu

GAG

Glu

GCT

Al a 105 Al a

GAG

Glu

GGG

Gl y

GGA

Gl y

CCA

Pro

ACC

Thr

GAC

ATG

Met Al a

CTG

Leu

TAT

Tyr

AAG

Lys

ACA

Thr

TCT

Ser

ACG

Thr

GAC

Asp Gln

AAG

Lys

ACC

Thr

GGT

Gly

TAT

Tyr

GCC

Al a

GOT

Ala

TAC

Tyr INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: ,4ENDED SHEET 5 03 o *r n C1 -74.15- LENGTH: 138 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: modified-site LOCATION: 2 OTHER INFORMATION: /note= (ix) FEATURE: NAME/KEY: modified-site LOCATION: 6 OTHER INFORMATION: /note= (xi) SEQUENCE DESCRIPTION: SEQ ID "Can be Ala or Asp" *000 0

S

0@S ooo o Met Leu Pro Thr 30 Lys Glu Thr Tyr Gly 110 Xaa Gin Gly 15 Asn Trp Glu Ala Phe 95 Gin Trp Val Ala Gin -1 Glu Thr Tyr Gly Met Gly Phe Lys His Leu 80 Cys Ala Gly Thr Trp Ile Val Met Trp Gly Gin Arg Ser Xaa Gin Lys Asn 35 Ile Arg Ile Trp Val 115 Leu Leu Ile 20 Trp Asn Phe Lys Val 100 Thr Leu Phe Val Gin 5 Ser Cys Val Lys Thr Lys Val Phe 70 Asn Phe 85 Pro Gly Val Ser Leu -10 Ser Lys Gin Ser 55 Ser Arg Thr Ser "Can be NO:11: Met Ala Gly Pro Ala Ser Ala Pro 40 Gly Glu Leu Glu Asn Glu Tyr Ala 105 Ala Glu Gly Gly Pro Thr Asp Met Ala Leu Tyr Lys Thr Ser Thr Asp Gln Lys Thr Gly Tyr Ala Ala Tyr Ser Lys Phe Leu Ala Ser Thr Trp Asn or Thr" INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 414 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..414 ,:iDED SHEET -74.16- (ix) FEATURE: NAME/KEY: sig peptide LOCATION: 1..57 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: ATG GMT TGG GTG TGG AMC TTG CTA TTC CTG ATG GCA GCT GCC CAA AGT 48 Met Xaa Trp Val Trp Xaa Leu Leu Phe Leu Met Ala Ala Ala Gin Ser -10 ATC CAA GCA CAG ATC CAG TTG GTG CAG TCT GGA CCT GAG CTG AAG AAG 96 Ile Gin Ala Gin Ile Gin Leu Val Gin Ser Gly Pro Glu Leu Lys Lys -1 5 CCT GGA GAG ACA GTC AAG ATC TCC TGC AAG GCT TCT GGG TAT ACC TTC 144 Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 20 ACA AAG TAT GGA ATG AAC TGG GTG AAG CAG GCT CCA GGA AAG GGT TTA 192 Thr Lys Tyr Gly Met Asn Trp Val Lys Gin Ala Pro Gly Lys Gly Leu 35 40 AAG TGG ATG GGC TGG ATA AAC ACC AAC AGT GGA GAG CCA ACA TAT GCT 240 Lys Trp Met Gly Trp Ile Asn Thr Asn Ser Gly Glu Pro Thr Tyr Ala 50 55 GAA GAG TTC AAG GGA CGG TTT GCC TTC TCT TTG GAA ACC TCT GCC AGC 288 Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser 65 70 ACT GCC TAT TTG CAG ATC AAC AAC CTC AAA AAT GAG GAC TCG GCT ACA 336 Thr Ala Tyr Leu Gin Ile Asn Asn Leu Lys Asn Glu Asp Ser Ala Thr 85 TAT TTC TGT GCA AGA TGG GTA CCT GGG ACC TAT GCT ATG GAC TAC TGG 384 Tyr Phe Cys Ala Arg Trp Val Pro Gly Thr Tyr Ala Met Asp Tyr Trp 95 100 105 GGT CAA GGA ACC .TCA GTC ACC GTC TCC TCA 414 Gly Gin Gly Thr Ser Val Thr Val Ser Ser 110 115 INFORMATION FOR SEQ ID NO:13: S(i) SEQUENCE CHARACTERISTICS: S* LENGTH: 138 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 2 OTHER INFORMATION: /note= "Can be Ala or Asp" (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 6 -74.17- OTHER INFORMATION: /note= "Can be Asn or Thr" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Met Xaa Trp Val Trp Xaa Leu Leu Phe Leu Met Ala Ala Ala Gin Ser -10 Ile Gin Ala Gin Ile Gin Leu Val Gin Ser Gly Pro Glu Leu Lys Lys -1 5 Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 20 Thr Lys Tyr Gly Met Asn Trp Val Lys Gin Ala Pro Gly Lys Gly Leu 35 40 Lys Trp Met Gly Trp Ile Asn Thr Asn Ser Gly Glu Pro Thr Tyr Ala 55 Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser 70 Thr Ala Tyr Leu Gin Ile Asn ASn Leu Lys Asn Glu Asp Ser Ala Thr 85 Tyr Phe Cys Ala Arg Trp Val Pro Gly Thr Tyr Ala Met Asp Tyr Trp 100 105 Gly Gin Gly Thr Ser Val Thr Val Ser Ser 110 115 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 414 base pairs TYPE: nucleic acid os STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..414 (ix) FEATURE: NAME/KEY: sig_peptide o, LOCATION: 1..57 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: ATG GMT TGG GTG TGG AMC TTG CTA TTC CTG ATG GCA GCT GCC CAA AGT 48 Met Xaa Trp Val Trp Xaa Leu Leu Phe Leu Met Ala Ala Ala Gin Ser -10 ATC CAA GCA CAG ATC CAG TTG GTG CAG TCT GGA CCT GAG CTG AAG AAG 96 Ile Gin Ala Gin Ile Gin Leu Val Gin Ser Gly Pro Glu Leu Lys Lys -1 5

^OEOSHE

r) .1 i' CCT GGA GAA ACA GTC AAG ATC TCC TGC 74.18- AAG GCT TCT GGG Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly TAT ACC TTC Tvr Thr Phe

ACA

Thr

AAG

Lys

GAA

Glu

ACT

Thr

TAT

Tyr

GGT

Gly 110

AAC

Asn

TGG

Trp

GAG

Glu

GCC

Ala

TTC

Phe

CAA

Gin

TAT

Tyr

ATG

Met

TTC

Phe

AAT

Asn

TGT

Cys

GGA

Gly

GGA

Gly

GGC

Gly

AAG

Lys

TTG

Leu

GCA

Ala

ACC

Thr

ATG

Met

TGG

Trp

GGA

Gly

CAG

Gin

AGA

Arg

TCA

Ser

AAC

Asn 35

ATA

Ile

CGG

Arg

ATC

Ile

TGG

Trp

GTC

Val 115

TGG

Trp

AAC

Asn

TTT

Phe

AAG

Lys

GTA

Val 100

ACC

Thr GTG AAG Val Lys ACC AAG Thr Lys GCC TTC Ala Phe 70 AAC CTC Asn Leu- 85 CCT GGG Pro Gly GTC TCC Val Ser

CAG

Gin

AGT

Ser 55

TCT

Ser

AAA

Lys

ACC

Thr

TCA

Ser

GCT

Ala 40

GGA

Gly

TTG

Leu

AAT

Asn

TAT

Tyr

CCA

Pro

GAG

Glu

GAA

Glu

GAG

Glu

GCC

Ala 105

GGA

Gly

CCA

Pro

ACC

Thr

GAC

Asp

ATG

Met

AAG

Lys

ACA

Thr

TCT

Ser

ACG

Thr

GAC

Asp GGT TTA Gly Leu TAT GCT Tyr Ala GCC AGC Ala Ser GCT ACA Ala Thr TAC TGG Tyr Trp 192 240 288 4oS .me.

C.

INFORMATION FOR SEQ ID SeMe

I

C

Met lie Pro Thr A SEQUENCE CHARACTERISTICS: LENGTH: 138 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 2 OTHER INFORMATION:-/note= (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 6 OTHER INFORMATION: /note= (xi) SEQUENCE DESCRIPTION: SEQ ID N Xaa Trp Val Trp Xaa Leu Leu Phe Leu M -10 31n Ala Gin Ile Gin Leu Val Gin Ser G -1 5 Gly Glu Thr Val Lys Ile Ser Cys Lys A 20 %sn Tyr Gly Met Asn Trp Val Lys Gin A 35 'Can be Ala or Asp" 'Can be O0:15: et Ala ly Pro la Ser la Pro 40 Asn or Thr" Ala Ala Gin Ser Glu Leu Lys Lys Gly Tyr Thr Phe Gly Lys Gly Leu g.ENDED

SHEET

7 a 1 7 -74.19- Lys Trp Met Gly Trp Ile Asn Thr Lys Ser Gly Glu Pro Thr Tyr Ala 55 Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser 70 Thr Ala Asn Leu Gin Ile Lys Asn Leu Lys Asn Glu Asp Thr Ala Thr 85 Tyr Phe Cys Ala Arg Trp Val Pro Gly Thr Tyr Ala Met Asp Tyr Trp 100 105 Gly Gin Gly Thr Ser Val Thr Val Ser Ser 110 115 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 411 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA o (ix) FEATURE: NAME/KEY: CDS LOCATION: 31..411 (ix) FEATURE: NAME/KEY: sig_peptide LOCATION: 31..90 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: ATTTAAATTG ATATCTCCTT AGGTCTCGAG ATG AGT GTG CTC ACT CAG GTC CTG 54 Met Ser Val Leu Thr Gin Val Leu GCG TTG CTG CTG CTG TGG CTT ACA GGT GCC AGA TGT GAC ATC CAG-ATG 102 Ala Leu Leu Leu Leu Trp Leu Thr Gly Ala Arg Cys Asp Ile Gin Met -10 -5 1 ACT CAG TCT CCA TCC TCC CTA TCT GCA TCT GTG GGA GAC AGA GTC ACC 150 S Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 10' 15 ATC ACA TGT CGA GCA AGT GGG AAT ATT CAC AAT TAT TTA GCA TGG TAT 198 Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr Leu Ala Trp Tyr 30 CAG CAG AAA CAG GGA AAA TCT CCT CAA CTC CTG GTC TAT AAT GCA AAA 246 Gin Gin Lys Gin Gly Lys Ser Pro Gin Leu Leu Val Tyr Asn Ala Lys 45 ACC TTA GCA AGT GGT GTG CCA TCA AGG TTC AGT GGC AGT GGA TCA GGA 294 Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 60 ACA GAT TTT ACT CTC ACC ATC AGC AGC CTG CAG CCT GAA GAT TTT GGG 342 .ENDED

SHEET

-74.20 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 75 AGT CAT TAC TGT CAA CAT TTT TGG ACC ACT Ser His Tyr Cys Gin His Phe Trp Thr Thr 90 GGC ACC AAG CTG GAA ATC AAA Gly Thr Lys Leu Giu Ile Lys 105 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 127 amino acids TYPE: amino acid TOPOLOGY: linear Gin Pro Giu Asp Phe Gly CCG TGG ACG TTC GGT GGAL Pro Trp Thr Phe Giy Giy 95 100 (ii) MOLECULE TYPE: protein 0000 0 *000 0 000 00 00 00 0 0 0* 0 000 0 000

S

000000 0 000* 0000 0000 0 0000

S

000000 0 00 00 0 0 0

S

000 Met -20 Gi y Al a Ile Gin Arg Thr Ser Al a 5cr His Leu Phe Leu Thr xi) Val Arc Val2 As n Leu Ser Gin Pro 95

SEQUENCE

*Leu Thr Cys Asp 1 *Giy Asp Tyr Leu *Vai Tyr *Giy Ser 65 Pro Giu 80 Trp Thr DESCRIPTION: SEQ ID Gin Val Leu Ala Leu -15 Ile Gin Met Thr Gin Arg Val Thr Ile Thr 20 Ala Trp Tyr Gin Gin 35 Asn Ala Lys Thr Leu 50 Giy Ser Giy Thr Asp 70 Asp *Phe GiV Ser His Phe Gly Gly Gly Thr 100 NO: 17: Leu Leu Ser Pro Cys Arg Lys Gin Ala Ser 55 Phe Thr Tyr Cys Lys Leu Leu Ser Al a Gi y Gly Leu Gin Giu 105 Leu Leu Gi y Ser Pro Ile Phe Lys Th 3cr As n Pro Ser Ser Tr= INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 417 base pairs TYPE: nucieic acid STRANDEDNESS: doubie TOPOLOGY: iinear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: l. .417 1 7. ;i SPEET -74.2 1- (ix) FEATURE: NAME/KEY: sig peptide LOCATION: 1. (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ATG AGT GTG CTC ACT GAG GTC CTG GCG TTG CTG CTG CTG TGG CTT ACA Met Ser Val Leu Thr Gin Val Leu Ala Leu Leu Leu 0S* 0 0

GG-T

Gi y

AAG

Lys

TTC

Phe

TTA

Leu

GCT

Al a

ACC

Thr

GTG

Val

TGG

T rp,

GCC

Ala

CCT

Pro

ACA

Thr

GAG

Giu

GAA

Giu

ACT

Thr

TAT

Tyr

GGT

Gi y 110 ',GA G Arg Cys GGA CC Gly Ala AAC TAT Asn Tyr TGG ATG Trp Met GAG TTC Giu Phe CCC TAT Ala Tyr TTC TGT Phe Cys 95 CAA GGA Gin Cly

CAG

Gin 1 T CA Ser

GGA

Gi y

GGC

Ci y

AAC

Lys

TTG

Leu

CCA

Al a

ACC

Thr -is AT C Ile

CTC

Val

ATG

Met

TCC

T rp 50

CGA

Cl y

GAG

Gin

AGA

Arg

ACG

Thr CAG TTG Gin Leu AAG ATC Lys Ile 20 AAC TG Asn Trp 35 ATA AAC Ile Asn CCC TTT Arg Phe ATC AGC Ile Ser TCG GTA Trp Vai i00 GTC ACC Vai Thr 115 -r' Val 5

TCC

Ser

GTG

Val

ACC

Thr

CTC

Vai

AC

Ser 85

CCT

Pro

GTC

Val -10

TCT

Ser

AAG

Lys

GAG

Gin

ACT

Ser 55

TCT

Ser Lys

ACC

Thr

TCA

Ser rG? Gly

GCT

Al a

CCT

Al.a

GCA

Ciy

TTG

Leu

GCT

Al a

TAT

Tyr Leu

TCT

Ser

TCT

Ser

CCA

Pro

GAG

Giu

GAG

Asp

GAG

Clu

CC

Al a 105 T rp

GAG

Ci u

CCC

Giy

GCA

Ci y

CCA

Pro

ACC

Thr

GAG

Asp

ATG

Met Leu Leu

TAT

Tyr

CAA

Gin

ACA

Thr

TCT

Ser

ACG

Thr

GAG

Asp Thr Lys

ACC

Thr

GGT

Civ

TAT

Tvr 00

GTC

Val.

GCT

Al a

TAC

ITyr 144 192 240 INFORMATION FOR SEQ ID NO:i9: SEQUENCE CHARACTERISTICS: LENGTH: 139 amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Met Ser Vai Leu Thr Gin Vai Leu Ala Leu Leu Leu Leu Trp Leu Thr -i5 -10 Z Gly Ala Arg Cys Gin Ile Gln Leu Val Gin Ser Gly Ser Giu Leu Lys 1 5 Lys Pro Gly Ala Ser Vai Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr -74.22- 20 Phe Thr Asn Tyr Gly Met Asn Trp Val Arg Gin Ala Pro Gly Gin Gly 35 Leu Glu Trp Met Gly Trp Ile Asn Thr Lys Ser Gly Glu Pro Thr Tyr 50 55 Ala Glu Glu Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val 70 Thr Thr Ala Tyr Leu Gin Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala 85 Val Tyr Phe Cys Ala Arg Trp Val Pro Gly Thr Tyr Ala Met Asp Tyr 100 105 Trp Gly Gin Gly Thr Thr Val Thr Val- Ser Ser 110 115 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 447 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear e (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 31..447 (ix) FEATURE: NAME/KEY: sig_peptide LOCATION: 31..90 (xi) SEQUENCE DESCRIPTION: SEQ ID CGATTGGAAT TCTTGCGGCC GCTTGCTAGC ATG AGT GTG CTC ACT CAG GTC CTG 54 e .Met Ser Val Leu Thr Gin Val Leu GCG TTG CTG CTG CTG TGG CTT ACA GGT GCC AGA TGT CAG ATC CAG TTG 102 A la Leu Leu Leu Leu Trp Leu Thr Gly Ala Arg Cys Gin Ile Gin Leu S -10 -5 1 GTG CAG TCT GGA GCT GAG GTG AAG AAG CCT GGA GCC TCA GTC AAG ATC 150 Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Ile 10 15 TCC TGC AAG GCT TCT GGG TAT ACC TTC ACA AAC TAT GGA ATG AAC TGG 198 Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp 30 GTG CGA CAG GCT CCA GGA CAA GGT TTA GAG TGG ATG GGC TGG ATA AAC 246 Val Arg Gin Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Asn 45 AMENDED

SHEET

ACC AAG Thr Lys ACC TTC Thr Phe AGC CTC Ser Leu CCT GGG Pro Gly GTC TCC Val Ser

AGT

Ser

ACC

Thr

AGA

Arg

ACC

Thr

TCA

Ser GGA GAG Gly Giu TTG GAC Leu Asp TCT GAC Ser Asp TAT GCC Tyr Ala 105

CCA

Pro

ACC

Thr

GAC

Asp 90

ATG

Met

TAT

Tyr 60

ACG

Thr

GCT

Al a

TAC

Tyr

GCT

Al a

AGC

Ser

GTG

Val

TGG

T rp -74.23-

GAA

Giu G ACT G *Thr A TAT T Tyr P GGT C.

Giy G 110

'AG

I u cc l a

TC

he 95

AA

in

TTC

Phe

TAT

Tyr

TGT

Cys

GGA

Gly

AAG

Lys

TTG

Leu

GCA

Al a

ACC

Thr

GGA

Gly

GAG

Giu

AGA

Arg

ACG

Thr

CGG

Arg

ATC

Ile

TGG

T rp

GTC

Val 115

TTT

Phe

AGG

Ara

GTA

Val 100

ACC

Thr 294 342 390 438 *.eO 0 e.g.

S.

C

*5* 0* CS

S

S.

0@ *5B 0S

C

C..

0

C

S.C.

CS..

0 0*CS 0 06 S

C

0@C

C

INFORMATION FOR SEQ ID NO:21: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 139 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Met Ser Val Gly Ala Arg Lys Pro Gly 15 Phe Thr Asn 30 Leu Glu Trp 45 Ala Glu Glu Ser Thr Ala Val Tyr Phe rrp Gly Gln 110 Leu Thr Gin -15 Cys Gin Ile 1 Ala Ser Vai Tyr Gly Met Met Gly Trp 50 Phe Lys Gly 65 Tyr Leu Glu Cys Ala Arg Gly Thr Thr Val Gin Lys As n 35 Ile Arg Ile T rp Val1 115 Le u Leu Ile 20 T rp As n Phe Arg Val 100 Thr Ala Leu Val Gln 5 Ser Cys Val Arg Thr Lys Thr Phe 70 Ser Leu 85 Pro Gly Val Ser Leu -10 Ser Lys Gln Ser 55 Thr Arg Thr Ser Leu Gl y Al a Al a Gly Leu Ser Tyr Leu Ala Ser Pro Gi u As p Asp Al a 105 T rp Gi u Gi v Gi y Pro Th r Asp Met Leu Thr Val Lvs Tyr Thr Gln Gly Thr Tyr Ser Thr Thr Ala Asp Tyr INFORMATION FOR SEQ ID 110:22: AMENDED SHEET I r 1 11 O4, .1 ,J S

S.

-74.24- SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 0O*0 *60: 0000 00 0 00 SS0 0

S

0O

OS

000

S.

0 SEQUENCE DESCRIPTION: SEQ ID NO:22: NNNNNNGAAT TCACTGGATG GTGGGAAGAT GGA INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: NNNNNNGAAT TCAYCTCCAC ACACAGGRRC CAGTGGATAG AC INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 0S5@ 0 *e 0 0 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: ACTAGTCGAC ATGAGTGTGC TCACTCAGGT CCTGGSGTTG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 88 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA m C "EDED SHEET 4 -74.25- (xi) SEQUENCE DESCRIPTION: SEQ ID TAGGGAGACC CAAGCTTGGT ACCAATTTAA ATTGATATCT CCTTAGGTCT CGAGTCTCTA GATAACCGGT CAATCGATTG GGATTCTT 88 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 88 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: GACACTATAG AATAGGGCCC TTCCGCGGTT GGATCCAACA CGTGAAGCTA GCAAGCGGCC GCAAGAATTC CAATCGATTG ACCGGTTA 88 S INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA o (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GATCTGCTAG CCCGGGTGAC CTGAGGCGCG CCTTTGGCGC C 41 0..0 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: *o*oo LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: GATCGGCGCC AAAGGCGCGC CGCAGGTCAC CCGGGCTAGC A 41 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: AMENDED

SHEET

-74.26- LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: CCGGGCCTCT CAAAAAAGGG AAAAAAAGCA TG 32 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA *0O (xi) SEQUENCE DESCRIPTION: SEQ ID CTTTTTTTCC CTTTTTTGAG AGGC 24 S INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 74 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 0e (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: CGCGCCGGCT TCGAATAGCC AGAGTAACCT TTTTTTTTAA TTTTATTTTA TTTTATTTTT GAGATGGAGT TTGG 74 S(2) INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 72 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA AMENDED SHEET -74.27- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: CGCCAAACTC CATCTCAAAA ATAAAATAAA ATAAAATTAA AAAAAAAGGT TACTCTGGCT ATTCGAAGCC GG INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA -:86 :74 :0.

a 06.

S*

*so* a *04 0 a

S.

0O *00 004

S

B. *S ar

S

a,.

S

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: ATCGATGCTA GCACCAAGGG CCCA INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: CTCGAGGGGT CACCACGCTG CTGA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID AACAGCTATG ACCATGATTA C INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid A4.;,DED

SHEET

S

3I n1~7) ~a o S 2 0J 3 6a S S S S S. *S -74.28- STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: CACCCAGCCT GTGCCTGCCT G INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single- TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA SoS

S

bOS I. Sr *n

S.

*0

S

.15.0

OS.,,

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: CGATTGGAAT TCTTGCGGCC GCTTGCTAGC INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 5500

S

9 005660

S

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: CTTGCGGCCG CTTGCTAGCA TGGATTGGGT GTGGAACTTG CTATTCCTGA TGGCAGCTGC CCAAAGTATC CAAGCACAGA INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: ?Q <'E~S3HUET -74.29- CTTGACTGTT TCTCCAGGCT TCTTCAGCTC AGGTCCAGAC TGCACCAACT GGATCTGTGC TTGGATACTT TGGGCAGCTG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (il) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID CTGAAGAAGC CTGGAGAAAC AGTCAAGATC TCCTGCAAGG CTTCTGGGTA TACCTTCACA AACTATGGAA TGAACTGGGT INFORMATION FOR SEQ ID NO:41: e SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear S. (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: TCTTGGTGTT TATCCAGCCC ATCCACTTTA AACCCTTTCC TGGAGCCTGC TTCACCCAGT TCATTCCATA GTTTGTGAAG Pee INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: Oeo*** LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: AGTGGATGGG CTGGATAAAC ACCAAGAGTG GAGAGCCAAC ATATGCTGAA GAGTTCAAGG GACGGTTTGC CTTCTCTTTG INFORMATION FOR SEQ ID 110:43: AMENDED

SHEET

-74.30- SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEOUENCE DESCRIPTION: SEO ID NO:43: TCCTCATTTT TGAGGTTCTT GATCTGCAAA TTGGCAGTGC TGGCAGAGGT TTCCAAAGAG AAGGCAAACC GTCCCTTGAA INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 0 *o (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: GCAGATCAAG AACCTCAAAA ATGAGGACAC GGCTACATAT TTCTGTGCAA GATGGGTACC TGGGACCTAT GCCATGGACT INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid -STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID TGGGCCCTTG GTGCTAGCTG AGGAGACGGT GACTGAGGTT CCTTGACCCC AGTAGTCCAT GGCATAGGTC CCAGGTACCC INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear AMENDED SHEET (1 I -74.31- (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: GGGAAGACGG ATGGGCCCTT GGTGCTAGC 29 INFORMATION FOR SEQ ID NO:47: (i qSEnOTFIrC. CrRTTRRTSTTr.C~: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: o. ATTTAAATTG ATATCTCCTT AGGTCTCGAG *OoO INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid So(C) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: ATTTAAATTG ATATCTCCTT'AGGTCTCGAG ATGAGTGTGC TCACTCAGGT CCTGGCGTTG CTGCTGCTGT GGCTTACAG 79 INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 78 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: AGATGCAGAT AGGGAGGCTG GAGACTGAGT CATCTGGATG TCACATCTGG CACCTGTAAG "7:3ED SHEET 2 b> -74.32- CCACAGCAGC AGCAACGC 78 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 78 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GTCTCCAGCC TCCCTATCTG CATCTGTGGG AGAAACTGTC ACCATCACAT GTCGAGCAAG TGGGAATATT CACAATTA 78 INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 78 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: STATAGACCAG GAGCTGAGGA GATTTTCCCT GTTTCTGCTG ATACCATGCT AAATAATTGT GAATATTCCC ACTTGCTC 78 INFORMATION FOR SEQ ID NO:52: 0 4* SEQUENCE CHARACTERISTICS: LENGTH: 78 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: AAATCTCCTC AGCTCCTGGT CTATAATGCA AAAACCTTAG CAGATGGTGT GCCATCAAGG TTCAGTGGCA GTGGATCA 78 INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: r) v T C n -74.33- LENGTH: 78 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: CTCCCAAAAT CTTCAGGCTG CAGGCTGTTG ATCCTGAGAG AAAATTGTGT TCCTGATCCA CTGCCACTGA ACCTTGAT 78 INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 78 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear :7 (ii) MOLECULE TYPE: cDNA 0 000 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: GCCTGCAGCC TGAAGATTTT GGGAGTCATT ACTGTCAACA TTTTTGGACC ACTCCGTGGA CGTTCGGTGG AGGCACCA 78 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 81 base pairs *o TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 6066 (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID TTCCAATCGA TTGACCGGTT ATCTAGAGAC TCGAGACTTA CGTTTGATTT CCAGCTTGGT GCCTCCACCG AACGTCCACG G 81 INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear AMENDED SHEET .4 *4 -74.34- (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: TCGATTGACC GGTTATCTAG AGACTCGAGA INFORMATION FOR SEQ ID NO:57: SEQUENCE CHARACTERISTICS: LENGTH: aU base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: S CTTGCGGCCG CTTGCTAGCA TGAGTGTGCT CACTCAGGTC CTGGCGTTGC TGCTGCTGTG GCTTACAGGT GCCAGATGTC S INFORMATION FOR SEQ ID NO:58: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: o GACTGAGGCT CCAGGCTTCT TCAGCTCAGA TCCAGACTGC ACCAACTGGA TCTGACATCT GGCACCTGTA AGCCACAGCA INFORMATION FOR SEQ ID NO:59: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRAINDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: AMENDED SHEET -74.35- GAGCTGAAGA AGCCTGGAGC CTCAGTCAAG ATCTCCTGCA AGGCTTCTGG GTATACCTTC ACAAACTATG GAATGAACTG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID TGGTGTTTAT CCAGCCCATC CACTCTAAAC CTTGTCCTGG AGCCTGTCGC ACCCAGTTCA TTCCATAGTT TGTGAAGGTA INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: 00006: STAGAGTGGAT GGGCTGGATA AACACCAAGA GTGGAGAGCC AACATATGCT GAAGAGTTCA AGGGACGGTT TGTCTTCTCT INFORMATION FOR SEQ ID NO:62: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid S* 0 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: TCAGCTTTGA GGCTGCTGAT CTGCAAATAG GCAGTGCTGA CAGAGGTGTC CAAAGAGAAG ACAAACCGTC CCTTGAACTC INFORMATION FOR SEQ ID 110:63:

AM

4 1ENED SHE'T -74.36- SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEOUENCE DESCRIPTTON: SEO TO NO:63: TTTGCAGATC AGCAGCCTCA AAGCTGAGGA CACGGCTGTG TATTTCTGTG CAAGATGGGT ACCTGGGACC TATGCCATGG INFORMATION FOR SEQ ID NO:64: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: GCCCTTGGTG CTAGCTGAGG AGACGGTGAC CGTGGTTCCT TGACCCCAGT AGTCCATGGC ATAGGTCCCA GGTACCCATC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single 0000 TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 0:0 (xi) SEQUENCE DESCRIPTION: SEQ ID TGCTGTGGCT TACAGGTGCC AGATGTCAGA TCCAGTTGGT GCAGTCTGGA GCTGAGGTGA AGAAGCCTGG AGCCTCAGTC INFORMATION FOR SEQ ID NO:66: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear AMENDED SHEET -74.37- (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: TAGAGTGGAT GGGCTGGATA AACACCAAGA GTGGAGAGCC AACATATGCT GAAGAGTTCA AGGGACGGTT TACCTTCACC INFORMATION FOR SEO ID NO:67: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 0*@ (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: TCAGATCTGA GGCTCCTGAT CTCCAAATAG GCAGTGCTCG TAGAGGTGTC CAAGGTGAAG GTAAACCGTC CCTTGAACTC INFORMATION FOR SEQ ID NO:68: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear *0SS*S (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: 0@*OeO TTTGGAGATC AGGAGCCTCA GATCTGACGA CACGGCTGTG TATTTCTGTG CAAGATGGGT ACCTGGGACC TATGCCATGG INFORMATION FOR SEQ ID NO:69: SEQUENCE CHARACTERISTICS: LENGTH: 78 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA AMENDED SHEET -74.38- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: AGATGCAGAT AGGGAGGATG GAGACTGAGT CATCTGGATG TCACATCTGG CACCTGTAAG CCACAGCAGC AGCAACGC 78 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 78 base pairs TYPE: nucleic acid 'TRAMnnFl.T.S S .qin-le TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GTCTCCATCC TCCCTATCTG CATCTGTGGG AGACAGAGTC ACCATCACAT GTCGAGCAAG TGGGAATATT CACAATTA 78 INFORMATION FOR SEQ ID NO:71: SEQUENCE CHARACTERISTICS: LENGTH: 78 base pairs TYPE: nucleic acid STRANDEDNESS: single Ss TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: AAATCTCCTC AACTCCTGGT CTATAATGCA AAAACCTTAG CAAGTGGTGT GCCATCAAGG TTCAGTGGCA GTGGATCA 78 INFORMATION FOR SEQ ID NO:72: SEQUENCE CHARACTERISTICS: LENGTH: 78 base pairs TYPE: nucleic acid o(C) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: CTCCCAAAAT CTTCAGGCTG CAGGCTGCTG ATGGTGAGAG TAAAATCTGT TCCTGATCCA CTGCCACTGA ACCTTGAT 78 AENi\DED

SHEET

-74.39- INFORMATION FOR SEQ ID NO:73: SEQUENCE CHARACTERISTICS: LENGTH: 18 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: Tyr Pro Arg Ser Ile Tyr Ile Arg Arg Arg His Pro Ser Pro Ser Leu 1 5 10 Thr Thr INFORMATION FOR SEQ ID NO:74: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: not relevant *se (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: Gly Gly Ser Gly Ser Gly Gly Ser Gly Ser Gly Gly Ser Gly Ser 1 5 10 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: o* LENGTH: 107 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Asp Ile Gin Met Thr Gin Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 Glu Thr Val Thr Xaa Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr 25 Leu Ala Trp Tyr Gin Gln Lys Gin Gly Lys Ser Pro Gin Leu Leu Val 40 AMENDED

SHEET

-74.40- Tyr Asn Ala Xaa Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 55 Ser Gly Ser Gly Thr Gin Xaa Ser Leu Xaa Ile Asn Ser Leu Gin Pro 70 75 Glu Asp Phe Gly Ser Xaa Tyr Cys Gin His Phe Trp Xaa Xaa Pro Trp 90 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 INFORMATION FOR SEQ ID NO:76: SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: S" Asp Ile Gin Met Thr Gin Ser Pro Ala Ser Leu Ser Ala Ser Val Giv 1 5 10 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr 25 Leu Ala Trp Tyr Gin Gin Lys Gin Gly Lys Ser Pro Gin Leu Leu Val 40 Tyr Asn Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 Ser Gly Ser Gly Thr Gin Phe Ser Leu Xaa Ile Asn Ser Leu Gln Pro 70 75 Glu Asp Phe Gly Ser His Tyr Cys Gin His Phe Trp Thr Thr Pro Trp 90 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 0* *e INFORMATION FOR SEQ ID NO:77: SEQUENCE

CHARACTERISTICS:

LENGTH: 107 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: AMENDED SHEET -74.4 1- Asp Ile Gin Met Thr Gin Ser Pro Xaa Ser Leu Ser Ala Ser Vai Giy 1 5 10 Xaa Xaa Vai Thr Xaa Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr 25 Leu Ala Trp Tyr Gin Gin Lys Gin Gly Lys Ser Pro Gin Leu Leu Val 40 Tyr Asn Ala Xaa Thr Leu Ala Xaa Giy Val Pro Ser Arg Phe Ser Giy 55 Ser Gly Ser Giy Thr Xaa Xaa Xaa Leu Xaa Ile Xaa Ser Leu Gin Pro 70 75 Giu Asp Phe Gly Ser Xaa Tyr Cys Gin His Phe Trp Xaa Xaa Pro Trp 90 Thr Phe Gly Gly Gly Thr Lys Leu Giu Ile Lys 100 105 INFORMATION FOR SEQ ID NO:73: SEQUENCE CHARACTERISTICS: LENGTH: 119 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: Gin Ile Gin Leu Val Gin Ser Gly Xaa Giu Xaa Lys Lys Pro Gly Ala 1 5 10 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 25 S. Gly Met Asn Trp Vai Arg Gin Ala Pro Gly Gin Gly Leu Giu Trp Met .5.35 40 5 Gly Trp Ile Asn Thr Lys Ser Gly Giu Pro Thr Tyr Ala Giu Giu Phe 55 Lys Gly Arg Phe Xaa Phe Xaa Leu Asp Thr Set Xaa Set Thr Ala Tyr 70 75 Leu Xaa Ile Xaa Ser Leu Xaa Xaa Xaa Asp Thr Ala Val Tyr Phe Cys 90 Ala Arg Trp Val Pro Gly Thr Tyr Ala Met Asp Tyr Trp Gly Gin Gly 100 105 110 Thr Thr Val Thr Vai Ser Set 115 INFORMATION FOR SEQ ID 1,:79: AMENDED SHCC tT -74.42- SEQUENCE CHARACTERISTICS: LENGTH: 119 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide

@OSS

S**

(2)

S

S

a.

SEQUENCE DESCRIPTION: 3EQ ID NO:79: Gln Ile Gln Leu Val Gln Ser Gly Pro Glu 1 5 10 Thr Val Lys Ile Ser Cys Xaa Ala Ser Gly 25 Gly Met Asn Trp Val Lys Gln Ala Pro Gly 40 Gly Trp Ile Asn Thr Xaa Ser Gly Glu Pro 50 55 Lys Gly Arg Phe Xaa Phe Ser Leu Glu Thr 65 70 Leu Gln Ile Xaa Asn Xaa Xaa AsnGlu Asp 85 90 Ala Arg Trp Val Pro Gly Thr Tyr Ala Met 100 105 Thr Ser Val Thr Val Ser Ser 115 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 119 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide Leu Tyr Lys Thr Ser 75 Xaa Asp Lys Thr Gly Tyr Ala Ala Tyr Lys Phe Leu Ala Ser Thr Trp Pro Thr Lys Glu Thr Tyr Gly 110 Gly Xaa Trp Glu Ala Phe Gln Glu Tyr Met Phe Xaa Cys Gly (xi) Gln 1 Thr Gly Gly SEQUENCE DESCRIPTION: SEQ ID Ile Gln Leu Val Gin Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 5 10 Val Lys Ile Ser Cys Xaa Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 25 Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 40 Trp Ile Asn Thr Lys Ser Gly Glu Pro Thr Tyr Ala Glu Glu Phe AMENDED SHEET -74.43- 55 Lys Gly Arg Phe Xaa Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Xaa 70 75 Leu Gin Ile Lys Asn Xaa Xaa Asn Glu Asp Thr Ala Thr Tyr Phe Cys 90 Ala Arg Trp Val Pro Gly Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 INFORMATION FOR SEQ ID NO:81: SEQUENCE CHARACTERISTICS: LENGTH: 119 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: Gln Ile Gln Leu Val Gln Ser Gly Xaa Glu Xaa Lys Lys Pro Gly Xaa 1 5 10 *00 Xaa Val Lys Ile Ser Cys Xaa Ala Ser Gly Tyr Thr Phe Thr Xaa Tyr 25 65 70 75 Leu eaa Ile Xaa Xaa Xaa Xa Xaa Xaa Asp Xaa Ala Xaa Tyr Phe Cys 90 T Ala I Arg Trp Val Pro Gly Thr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Xaa Val Thr Val Ser Ser 115 e~1g.

AMENDED SHEET

Claims (28)

1. An immunologic molecule wherein said immunologic molecule is capable of binding to both human and nonhuman circulating a2-antiplasmins and human and nonhuman fibrin crosslinked c2-antiplasmins.

2. The immunologic molecule is a chimeric antibody.

3. The immunologic molecule is a humanized antibody. of claim 1, wherein said immunologic molecule of claim 1, wherein said immunologic molecule of claim 1, wherein said immunologic molecule of claim 1, wherein said immunologic molecule 000'* a" *0 c 0 0 a 00 g a Si... 0 The immunologic molecule is an antibody fragment. The immunologic molecule is a monoclonal antibody.

6. The immunologic molecule of claim 1, wherein said immunologic molecule comprises amino acids 1 to 107 of SEQ ID NO:9 and amino acids 1 to 119 of SEQ ID

7. The immunologic molecule of claim 1, wherein said immunologic molecule comprises amino acids 1 to 107 of SEQ ID NO:5 and amino acids 1 to 119 of SEQ ID NO:11.

8. The immunologic molecule of claim 1, wherein said immunologic molecule comprises amino acids 1 to 107 of SEQ ID NO:7 and amino acids 1 to 119 of SEQ ID NO:13. ck12330oct3.claims WO 98/12329 PCT/US97/16122 -76-

9. The immunologic molecule of claim 1, selected from the group consisting of an immunologic molecule, wherein the CDRI region of the light chain of said immunologic molecule comprises amino acids 26 to 32 of SEQ ID an immunologic molecule, wherein the CDR2 region of the light chain of said immunologic molecule comprises amino acids 50 to 52 of SEQ ID an immunologic molecule, wherein the CDR3 region of the light chain of said immunologic molecule comprises amino acids 91 to 96 of SEQ ID an immunologic molecule, wherein the CDRI region of the heavy chain of said immunologic molecule comprises amino acids 26 to 32 of.SEQ ID NO:79; an immunologic molecule, wherein the CDR2 region of the heavy chain of said immunologic molecule comprises amino acids 53 to 56 of SEQ ID NO:79; and an immunologic molecule, wherein the CDR3 region of the heavy chain of said immunologic molecule comprises amino acids 100 to 107 of SEQ ID NO:79. The immunologic molecule of claim 1, selected from the group consisting of: 2&000 an immunologic molecule, wherein the CDR1 region of the light chain of said immunologic molecule comprises amino acids 26 to 32 of SEQ ID NO: 76; S S an immunologic molecule, wherein the CDR2 region of the light chain of said immunologic molecule comprises amino acids 50 to 52 of SEQ ID NO:76; WO 98/12329 PCT/US97/16122 -77- an immunologic molecule, wherein the CDR3 region of the light chain of said immunologic molecule comprises amino acids 91 to 96 of SEQ ID NO:76; an immunologic molecule, wherein the CDRI region of the heavy chain of said immunologic molecule comprises amino acids 26 to 32 of SEQ ID an immunologic molecule, wherein the CDR2 region of the heavy chain of said immunologic molecule comprises amino acids 53 to 56 of SEQ ID NO:80; and an immunologic molecule, wherein the CDR3 region of the heavy chain of said immunologic molecule comprises amino acids 100 to 107 of SEQ ID

11. The monoclonal antibody of claim 5, wherein said monoclonal antibody is 77A3.

12. The monoclonal antibody of claim 5, wherein said monoclonal antibody is 49C9.

13. The monoclonal antibody of claim 5, wherein said monoclonal antibody is 70B 11. 0006 0 S S 0 0055

14. comprising: thereof; hybridoma cell A method of making the monoclonal antibody of claim immunizing an animal with a2-antiplasmin or fragment fusing cells from the animal with tumor cells to make a S S S line; (c) cloning the hybridoma cell line; WO 98/12329 PCT/US97/16122 -78- selecting for the monoclonal antibody capable of binding to both human and nonhuman circulating a2-antiplasmins and human and nonhuman fibrin crosslinked a2-antiplasmins; and obtaining the monoclonal antibody. A hybridoma cell line which produces the monoclonal antibody of claim

16. The hybridoma cell line of claim 15, wherein said hybridoma cell line is ATCC Accession No. HB-12192.

17. A method of making the hybridoma cell line of claim comprising: immunizing an animal with a2-antiplasmin or fragment thereof; fusing the cells from the animal with tumor cells to make the hybridoma cell line; and obtaining the hybridoma cell line which produces the monoclonal antibody capable of binding to both human and nonhuman circulating a2-antiplasmins and human and nonhuman fibrin crosslinked a2- antiplasmins.

18. A nucleic acid molecule, selected from the group consisting of: a nucleic acid molecule comprising a nucleotide sequence encoding for amino acids 1 to 107 ofSEQ ID a nucleic acid molecule comprising a nucleotide sequence S* encoding for amino acids 1 to 107 of SEQ ID NO:7; a nucleic acid molecule comprising a nucleotide sequence encoding for amino acids 1 to 107 of SEQ ID NO:9; a nucleic acid molecule comprising a nucleotide sequence encoding for amino acids 1 to 107 of SEQ ID WO 98/12329 PCT/US97/16122 -79- a nucleic acid molecule comprising a nucleotide sequence encoding for amino acids 1 to 119 of SEQ ID NO: 11; a nucleic acid molecule comprising a nucleotide sequence encoding for amino acids 1 to 119 of SEQ ID NO:13; a nucleic acid molecule comprising a nucleotide sequence encoding for amino acids 1 to 119 of SEQ ID NO: 15; and a nucleic acid molecule comprising a nucleotide sequence encoding for amino acids 1 to 119 of SEQ ID NO:79.

19. A method for treating pulmonary embolism, myocardial infarction, or thrombosis in a patient comprising administering a therapeutically effective amount of an immunologic molecule of claim 1 to said patient.

20. The method of claim 19, wherein said immunologic molecule is a 1 monoclonal antibody.

21. The method of claim 20, wherein said monoclonal antibody is 77A3.

22. The method of claim 19, wherein said immunologic molecule is administered by continuous intravenous infusion or by bolus.

23. A method of treatment for pulmonary embolism, myocardial infarction, or thrombosis in a patient which comprises co-administering to a patient in need of such treatment: a therapeutically effective amount of an immunologic molecule of claim 1; and a therapeutically effective amount of a thrombolytic agent, wherein said immunologic molecule is different from said thrombolytic agent thereby treating said patient. WO 98/12329 PCT/US97/16122

24. The method of claim 23, wherein said immunologic molecule is a monoclonal antibody. The method of claim 24, wherein said monoclonal antibody is 77A3.

26. The method of claim 23, wherein said thrombolytic agent is plasmin.

27. The method of claim 23, wherein said thrombolytic agent is an anti- coagulant which inhibits fibrin.

28. The method of claim 27, wherein said anti-coagulant is selected from the group consisting of heparin, hirudin and activated protein C. i*

29. The method of claim 23, wherein said thrombolytic agent is an anti- coagulant which inhibits platelets. S The method of claim 23, wherein said thrombolytic agent is a plasminogen activator.

31. The method of claim 30, wherein said plasminogen activator is selected from the group consisting of streptokinase, prourokinase, urokinase, tissue-type plasminogen activator, staphylokinase, and vampire bat plasminogen activator. S0 0 0 0

32. The method of claim 23, wherein both said immunologic molecule and said thrombolytic agent are provided to said patient by an intravenous infusion or by an intravenously injected bolus. wo 9812329PCT1US97/16122 -81-

33. The method of claim 23, wherein said patient is provided with a first bolus containing said immunologic molecule and a subsequently administered second bolus containing said thrombolytic agent

34. The method of claim 23, wherein: said immunologic molecule is provided to said patient at a dose of between 3 to 600 nimole per kg of patient weight; and said thrombolytic agent is provided to said patient at a dose of between 0.01 to 3.0 mg per kg of patient weight. A kit useful for carrying out the method of claim 23, being compartmentalized in close confinement to receive two or more container means therein, which comprises: *0eS(1) a first conitainer containing a therapeutically effective amount of said immunologic molecule and a second container containing a therapeutically effective amount of said thrombolytic agent wherein said immunologic molecule is different from said thrombolytic agent DATED this 3 rd day of October, 2001. a a~ THE GENERAL HOSPITAL CORPORATION and #-*aTHE PRESIDENT ANT) FELLOWS OF HARVARD COLLEGE -000 By their Patent Attorneys: 0 0 CALLiINAN LAWRIE to de .0