ENDOTHELIAL CELL PROLIFERATION INHIBITORS DERIVED FROM HUMAN PROTHROMBIN

Abstract: The present invention provides a human prothrombin kringle-1 and 2 which have anti-endothelial cell proliferation and anti-tumor activities, cDNAs encoding the human prothrombin kringle-1 and 2 derived from human prothrombin, recombinant expression vectors comprising the cDNAs, recombinant microorganisms transformed with the recombinant expression vectors comprising the cDNA encoding the human prothrombin kringle-1 or 2, processes for preparing the human prothrombin kringle-1 and 2 from human plasma and recombinant human prothrombin kringle-2 from the recombinant microorganisms and pharmaceutical application of the said proteins. In contrast to human prothrombin, human prothrombin kringle-1 and 2 obtained as proteolytic fragments of human prothrombin display potent inhibitory effects on bovine capillary endothelial cells with a half-maximal concentration (ED50) of approximately 100nM and 120nM, respectively, and angiogenesis in the chorioallantoic membrane. The human prothrombin kringle-2 significantly suppresses tumor growth in vivo. In addition, peptides having sequences of human prothrombin kringle-2 can inhibit the endothelial cell proliferation. Since the anti-angiogenic therapy directed against tumor's endothelial cells induces little or no drug resistance, human prothrombin kringle-1 and 2 have been proposed as the most reliable candidates for cancer therapy.

Description: ENDOTHELIAL CELL PROLIFERATION INHIBITORS DERIVED FROM HUMAN PROTHROMBIN BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a human prothrombin kringle-1 and 2 which have anti-endothelial cell proliferation and anti-tumor activities, more specifically, to cDNAs encoding the human prothrombin kringle-1 and 2 derived from human prothrombin, recombinant expression vectors comprising the cDNAs, recombinant microorganisms transformed with the recombinant expression vectors comprising the cDNA encoding the human prothrombin kringle-1 or 2, processes for preparing the human prothrombin kringle-1 and 2 from human plasma and recombinant human prothrombin kringle-2 from the recombinant microorganisms and pharmaceutical application of the said proteins. Description of the Prior Art Blood coagulation is a response to vascular injury leading to the activation of platelets and coagulation factors with the ultimate formation of a fibrin plug. Several coagulation factors are zymogens of serine proteases that require vitamin K for normal biosynthesis. The active forms of these proteases and their cofactors form membrane-bound macromolecular complexes. In the final step, prothrombin is activated to thrombin by protease. Thrombin then cleaves designated peptide bonds in fibrinogen, resulting in the formation of fibrin monomers that polymerize to insoluble fibrin strands. The coagulation and anticoagulation proteases, factors VII, IX, and X, and protein C, have a common domain structure with an N-terminal gamma-carboxyglutamic acid ("Gla") -containing domain that is followed by two domains that are homologous to the epidermal growth factor ("EGF") , whereas the C-terminal half of each protein is occupied by a trypsin-like serine protease domain . Prothrombin, a 72kDa glycoprotein having 581 amino acids, is converted to thrombin on a charged membrane by the proteolytic activity of prothrombinase, an enzyme complex consisting of protease factor Xa, cofactors calcium, factor Va and membrane surface (s_e_e_: Mann, K. G. et al., Ann. Rev. Biochem. , 57:915-956(1988); Mann, K. G. et al., Blood, 76:1-16(1990)). Like other coagulation and anticoagulation proteases, prothrombin is organized into several discrete functional domains that are homologous to domains found in other plasma serine protease. Functional domains in prothrombin are composed of a Gla domain (residues 1 to 44), two contiguous kringle domains (residues 45 to 155 and residues 156 to 273) and a serine protease catalytic domain in the carboxyl-terminal region (residues 274 to 581) (see: Mann, K. G., Methods Enzymol., 45:123-156(1976)). Factor Xa in the prothrombinase complex cleaves prothrombin at two peptides bonds, one at between Arg 273 and Thr 274 and the other at between Arg 322 and lie 323, to yield an amino terminal fragment named fragment 1- 2 (residues 1 to 273) and an carboxy terminal active thrombin (residues 274-581). Thrombin formed during the activation further cleaves fragment 1-2 into fragment 1 (residues 1 to 155), which includes the Gla domain and a kringle-1 domain, and fragment 2 (residues 156 to 273), which includes a kringle-2 domain (_3_e_e: Mann, K. G., Methods Enzymol., 45:123-156(1976); Taneda, H. et al., J. Biochem. (Tokyo) , 116:589-597 (1994) ) . Although the process of prothrombin conversion into thrombin in blood coagulation has been studied intensively, the exact role of prothrombin fragment 1 and 2 remains unclear except as a structural unit and a recognition element for macromolecular assembly (,s_e_e_: Hoover, G. J. et al., Biochemistry, 32:10936-10943(1993)). Kringles are small protein domains which have a characteristic three disulfide bonded secondary structure that is found in serine proteases which are involved in blood coagulation and fibrinolysis (see: Furie, B. and Furie, B.C., Cell, 53:505-518(1988)). The kringle domains have also been found in some other proteins which have biological functions different from the protease, like apolipoprotein and hepatocyte growth factor (HGF) (≤ej≥: Ikeo, K. et al., J. Mol. Evol . , 40:331- 336(1995)). DeSerrano et al . suggested that these kringle domains appear to be independent folding unit, but the general functional role for kringles is not known (.s_e_e_: DeSerrano, V.S. et al., Arch. Biochem. Biophys., 294:282-290(1992)). In the case of the plasminogen kringle-1 and 4 domains, they were known to be involved in the binding of plasminogen to fibrin (.s_e_e.: Vali, Z. et al., J. Biol. Chem., 259:13690-13694(1984)). These kringle domains are presumed to interact with fibrin by binding to an exposed lysine side chain. However, it has been reported that prothrombin kringle domains do not adsorb to lysine-Sepharose columns in spite of a very similar amino acid sequence ( see : Arni, R.K. et al., Biochemistry, 32:4727-4737(1993)). Because of the different properties of these apparently very similar polypeptides, recent efforts have been made to apply recombinant DNA technology to express kringle domains and to attempt to understand their structure-function relationships (.s_e_e_: DeSarrano, V.S. et al., Arch. Biochem. Biophys., 294:282-290(1992)). On the other hand, Cao et al . reported that each domain of human plasminogen kringles showed anti- capillary endothelial cell proliferative activity which can be applied as an anticancer drug (s_e_e_: Cao, Y. et al., J. Biol. Chem., 271:29461-29467(1996); Boehm, T. et al . , Nature, 390:404-407(1997)). It was also demonstrated that the recombinant rabbit prothrombin kringle-2 domain has a potent endothelial cell growth inhibitory activity like that of angiostatin (see: Lee, T.H. et al . , J. Biol. Chem., 273:28805-28812(1998)). In addition, it was reported that angiostatin, an angiogenesis inhibitor which suppresses endothelial cell growth in vi tro and tumor metastases in vivo, consists of the first four kringle domains of plasminogen (1-4 kringle domains) (see: O'Reilly, M.S. et al . , Cell, 79: 315-328 (1994) ) . An endothelial cell growth is closely related to angiogenesis, and angiogenesis is quite necessary for tumor growth and maintenance (.s_e_e: Folkman, J. , Nat. Med., 1:27-31(1995)). Angiogenesis is a process of blood vessel formation in which new vessels sprout from existing blood vessels. Blood capillaries are primarily composed of endothelial cells that are normally quiescent in adult mammals under physiological conditions. Angiogenesis is required for a variety of physiological processes such as embryonic development, wound healing, tissue regeneration, and organ regeneration. Outgrowth of new blood vessels under pathological conditions can lead to the development and progression of diseases such as tumor growth, diabetic retinopathy, tissue and organ malformation, and cardiovascular disorders (see : Folkman, J. et al., J. Biol. Chem., 267:10931-10934(1992)). Therefore, extensive studies have been made on the anti-endothelial cell growth molecules related to kringle domains. In this regard, since the anti-angiogenic therapy directed against tumor' s endothelial cells induces little or no drug resistance, human prothrombin fragments 1 and 2 have been proposed as the most promising candidates for cancer therapy. SUMMARY OF THE INVENTION The present inventors isolated human prothrombin kringle-1 and 2 from human plasma and showed that the kringle domains have anti-endothelial cell proliferation activity. Then, they constructed cDNAs encoding the human prothrombin kringle-1 and 2, recombinant expression vectors comprising the cDNAs and demonstrated that the recombinant human prothrombin kringle-2 has anti-tumor activity in vivo . In addition, the inventors synthesized peptides having amino acid sequences of the human prothrombin kringle-2 and determined an active domain for endothelial cell proliferation inhibition. Accordingly, the inventors found that the said proteins or peptides may be applied for the development of an anti-tumor agent. The first object of the present invention is, therefore, to provide human prothrombin kringle-1 and 2 that have anti-endothelial cell proliferation and anti- tumor activities. The second object of the invention is to provide cDNA sequences encoding the said human prothrombin kringle-1 and 2. The third object of the invention is to provide recombinant expression vectors comprising the said cDNAs . The fourth object of the invention is to provide recombinant microorganisms transformed with the recombinant expression vectors express human prothrombin kringle-2. The fifth object of the invention is to provide peptides which inhibit endothelial cell proliferation, which are derived from human prothrombin kringle-2 of human prothrombin. The sixth object of the invention is to provide processes for preparing human prothrombin kringle-1 and 2 from human plasma and recombinant human prothrombin kringle-2 from the recombinant microorganisms. The seventh object of the invention is to provide an anti-tumor agent comprising an active ingredient of the prothrombin kringle-1, 2 or its functionally equivalent peptides. BRTEF DESCRIPTION OF THE DRAWINGS The above and the other objects and features of the present invention will become apparent from the following descriptions given in conjunction with the accompanying drawings, in which: Figure 1 is a photograph showing purified human prothrombin derivatives analyzed on SDS-PAGE from human plasma. Figure 2 is a photograph showing Western blot analysis of human prothrombin and its derivatives . Figure 3 is a graph showing endothelial cell growth inhibitory activity of human prothrombin and its derivatives. Figure 4 shows anti-angiogenic effects of human prothrombin kringle-1 and 2 on the chick CAM. Figure 5 is a photograph showing suppression of tumors growth of human prothrombin kringle-2. Figure 6 is a graph showing suppression of tumor growth of human prothrombin kringle-2. DETAILED DESCRIPTION OF THE INVENTION The present inventors isolated human prothrombin fragments 1 and 2 comprising kringle domains from human plasma and showed that the fragments have anti- endothelial cell proliferation activity. Hereinafter, the said human prothrombin fragment 1 and 2 comprising kringle domains are referred to as "human prothrombin kringle-1" and "human prothrombin kringle-2", respectively. Human prothrombin was isolated from human plasma by barium citrate precipitation, followed by ion- exchange chromatography. Each proteolytic fragment treated with factor Xa was purified for homogeneity using Mono-Q column chromatography and identified via N-termmal ammo sequence analysis. On Westernblot and lmmunoprecipitation analysis using an anti-human prothrombin krmgle-2 antibody, human prothrombin kringle-1 showed no immunological relationship with human prothrombin fragment 2. Anti- angiostatin antibody also did not react with any fragment of prothrombin. To evaluate the ant -proliferative effect of the purified prothrombin and its derivatives, proteolytic fragments of human prothrombin purified after proteolytic cleavage of prothrombin were assayed for their inhibitory activity on BCE cell proliferation stimulated by bFGF. Not only human prothrombin krmgle-2 but also prothrombin krmgle-1 inhibited the bFGF-stimulated endothelial cell growth in a dose-dependent manner. In addition, the inhibitory effect on BCE cells was reversible after removing prothrombin krmgle-1 or 2. However, like an angiostatin (a proteolytic fragment of plasminogen including kringles 1-4 domains) , intact prothrombin did not have an anti-endothelial cell growth effect. Angiogenesis inhibitory activity of the human prothrombin krmgle-1 and 2 was also demonstrated in vivo by using the chick choπoallantoic membrane (CAM) assay. The inventors cloned cDNAs encoding the human prothrombin krmgle-1 and 2 derived from human prothrombin into E . coli expression vector pET22b T (Novagen, USA). The expression vectors comprising cDNAs encoding prothrombin krmgle-1 and prothrombin kringle-2 were designated as λ pEF-hKl' and Λ pEF-hK2', respectively. The transformed E. coli with pEF-hK2 was designated as '£. coli BL21 (DE3) ZpLysS ,phEF13 ' , and deposited with the Korean Culture Center of Microorganisms (KCCM, Department of Food Engineering, College of Eng., Yonsei University, Sodaemun-gu, Seoul 120-749, Korea), an international depository authority on August 14, 1999, as accession No. KCCM-10139. For the purification of the recombinant prothrombin kringle-2, the crude extract of cultured E. coli BL21 (DE3) /pLysS,phEF13, was chromatographed on DEAE- Sepharose and Sephacryl S-200 column. The recombinant prothrombin kringle-2 was demonstrated to suppress tumor growth in vivo . To determine an active domain for endothelial cell proliferation inhibition of human prothrombin kringle-2, the inventors synthesized 11 peptides consisting of 15 or 9 amino acids of prothrombin kringle-2. The peptides having amino acid sequences in order from N-terminal sequence of prothrombin kringle-2 were synthesized by overlapping 2 to 5 amino acids of both N-terminal and C- terminal of the peptides. As a result, the inventors found that the active domain of human prothrombin kringle-2 is located between 11th proline and 70th aspartic acid. The present invention is further illustrated in the following examples, which should not be taken to limit the scope of the invention. Example 1: Purification of human prothrombin and proteolytic fragments of the prothrombin Intact human prothrombin was purified from human plasma according to the modified method of Weinstein et a l . (see: Weinstein, R.E. et al., Thromb. Res., 59:759- 772(1990)). Sodium citrate was added to reach a concentration of 0.85% (w/v) to prevent blood coagulation and centrifuged at l,000xg for 30min to remove remaining blood cells and undissolved materials. Then, benzamidine-HCl and P-PACK (D-phenylalanyl-L-prolyl-L- arginine chloromethyl ketone) were added to reach a concentration of 5mM and 1M, respectively, and lOg QMA Acell (Pharmacia, Sweden) resin was added for ion- exchange chromatography. The mixture was incubated at ambient temperature for lhr and washed with 20mM Tris-HCl buffer (pH 7.5, containing lOOmM NaCl, 5mM benzamidine-HCl and lμM P-PACK) . Then, the isolated proteins were eluted with 20mM Tris-HCl buffer (pH 7.5, containing 0.6M NaCl, 5mM benzamidine-HCl and lμM P-PACK) . The eluted proteins were precipitated by barium chloride; dialyzed at 4°C overnight against the Tris buffer (100 mM Tris-HCl, pH 8.0, containing 150 mM NaCl and 1 mM CaCl 2 ) ; and, analyzed on SDS-PAGE(.≥≤e_: Figure 1). For the isolation of prothrombin derivatives, the lOmg of purified prothrombin was incubated with factor Xa (Sigma Chemical Co., USA) at 23°C overnight in water bath and dialyzed against lOmM Tris-HCl containing lOOmM NaCl and ImM PMSF at 4°C for 4 hr . The solution was dialyzed once more against 50mM NaPi, pH 7.0 containing ImM PMSF. Each proteolytic fragment was purified for homogeneity using Mono-Q column, previously equilibrated with starting buffer 50mM NaPi, pH 7.0; eluted with a linear gradient of 0.1M NaCl in 50mM NaPi, pH 7.0; and, identified via N-terminal amino sequence analysis. As shown in Figure 1, after overnight digestion with factor Xa, four major bands were observed on SDS gel electrophoresis : that is, prothrombin, thrombin, prothrombin kringle-1 (SEQ ID NO:l) and prothrombin kringle-2 (SEQ ID NO: 2 ) (.s_e_e_: Figure 1: 1, purified prothrombin; 2, digested with factor Xa; 3, thrombin; 4, prothrombin kringle-1; 5, prothrombin kringle-2). Compared with their molecular masses calculated from their amino acid sequences ( 17kDa and 13kDa) , prothrombin kringle-1 and 2 were migrated with a higher molecular mass of 30kDa and 19kDa, respectively, on SDS-PAGE. This phenomenon is common to several anti-endothelial cell proliferation molecules that include kringle domains, such as rabbit prothrombin kringle-2 and human angiostatin kringle-1 (.s_e_e_: Lee, T. H. et al . , J. Biol. Chem., 273:28805-27712(1998); Cao, Y. et al . , J. Biol. Chem., 271:29461-29467(1996)). Example 2 : Immunoloigical relationship between human prothrombin kringle-1 and 2 Western blot analysis was performed according to the method of Towbin et al . (see : Towbin, H. et al., Proc. Natl. Acad. Sci., USA, 76:4350-4354(1979)). Proteins were fractionated on a 15% SDS-PAGE and were transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Inc., USA) . The blots were blocked for lhr in phosphate buffered saline containing 1% bovine serum albumin (BSA) and incubated at ambient temperature for 2hr with anti- prothrombin kringle-2 antibody as a primary antibody (lOmg/ml) . After washing the unbound primary antibodies with washing buffer (PBS containing 0.5% nonidet P-40) three times for lOmin, each nitrocellulose filter was incubated with peroxidase conjugated anti-rat IgG (Sigma Chemical Co., USA) for 30min. Unbound secondary antibodies were washed with washing buffer, and then stained with 4-chloro-l-naphthol solution. Previously, it was found that anti-rabbit prothrombin kringle-2 antibody did not react with rabbit prothrombin kringle-1 (s_a≤.: Lee, T. H. et al . , J. Biol. Chem., 273:28805-28812(1998)). Like rabbit prothrombin kringle-1, human prothrombin kringle-1 showed no immunological relationship with human prothrombin kringle-2 on Westernblot analysis using an anti-human prothrombin kringle-2 antibody (.s_e_e_: Figure 2: 1, prothrombin digested with factor Xa; 2, tnrombin; 3, prothrombin kringle-1; and, 4, prothrombin kringle-2). Only intact prothrombin and human prothrombin kringle-2 reacted with anti-prothrombin kringle-2 antibody. Furthermore, anti-angiostatin antibody did not react with any fragment of prothrombin. The results of imrαunoprecipitation were the same as in Western blot analysis, which means the prothrombin kringle 1 and 2 are not related immunogenically, but closely related in anti- angiogenesis effects. Example 3: Endothelial cell proliferation assay To evaluate the anti-proliferative effect of the purified prothrombin and its derivatives, proteolytic fragments of human prothrombin were assayed for their inhibitory activity on BCE cell proliferation stimulated by bFGF. The endothelial cell proliferation assay was performed according to the method of O'Reilly et al. (see : O'Reilly, M. S. et al., Cell, 79:315-328(1994)). Bovine capillary endothelial ("BCE") cells were maintained in DMEM containing 10% heat-inactivated bovine calf serum, 3ng/ml recombinant human bFGF under 10% CO- atmosphere. Cells were cultured in gelatin-coated T75 flask and incubated at 37°C. Approximately 12,500 cells in 0.5ml were added to each well of gelatinized 24-well plates and incubated at 37 °C for 24hr, and then samples were added to each well. After 30min of incubation, media were added to reach a final volume of 0.5ml DMEM containing 5% bovine calf serum, and bFGF was added to a concentration of Ing/ml. After 72hr of incubation, cells were trypsinized and the cell number was determined using a hemocytometer . To ensure that any inhibition observed was not due to detachment of the cells from the plate, all wells of the assay were examined under an inverted microscope for evidence of cell detachment prior to cell counting . As shown in Figure 3, not only human prothrombin kringle-2 but also prothrombin kringle-1 inhibited the bFGF-stimulated endothelial cell growth in a dose- dependent mannerfϋ≤g: Figure 3). In Figure 3, (■) represents human prothrombin; (♦) , thrombin; (X ) , prothrombin kringle-1; and, (A), prothrombin kringle-2, respectively. The morphology of BCE cells appeared similar to that of the control cells or angiostatin-treated cells (see : Lee, T. H. et al., J. Biol. Chem., 273:28805-28812(1998); Cao, Y. et al., J. Biol. Chem., 271:29461-29467(1996)). In addition, the inhibitory effect on BCE cells was reversible after removing prothrombin kringle-1 or 2. Example 4 : Chick chorioallantoic membrane assay To investigate whether human prothrombin kringle-1 and 2 also inhibit m vivo angiogenesis, the chick chorioallantoic membrane ("CAM") assay was employed. Fertilized eggs of domestic fowl (Pulmuone, Korea) were incubated with the blunt pole uppermost at 70% relative humidity. After 3 days of incubation at 37 °C in 3% C0 2 atmosphere, a methylcellulose disc (Fischer Scientific, USA) containing either the purified prothrombin kringle-1 or prothrombin kringle-2 was applied to the chick chorioallantoic membrane of individual embryos. The discs were made by desiccation of 10ml of 0.45% methylcellulose in H 2 0 with or without prothrombin kringle-1 or 2, on Teflon rods. After 48hr of incubation, 20% of fat emulsion for intravenous injection was injected into chorioallantois of chick embryos, so that the red color of the blood vessels contrasted sharply with the white color. After the fat emulsion injection, embryos and CAMs were observed by means of a stereomicroscope (.s_e_e_: Nguyen, N.M. et al., Anticancer Res., 13:2143-2147(1993)). At doses of lOmg/disc, both the human prothrombin kringle-1 and 2 showed a potent inhibition of angiogenesis (.s_e_e_: Figure 4). However, prothrombin and thrombin showed little or no effects on BCE cell growth. As shown in Figure 4, panel A shows the PBS treated CAM with no sign of reduced angiogenesis. The results were the same with PBS, when the methylcellulose discs containing the prothrombin or thrombin were loaded to chick chorioallantoic membrane of 3-day-old chick embryos. However, lOμg/disc of prothrombin kringle-1 (panel B) and kringle-2 (panel C) reduced new blood vessel formation. Like angiostatin (a proteolytic fragment of plasminogen including kringles 1-4 domains), prothrombin fragment as a domain of intact prothrombin did not have an anti-endothelial cell growth effect. However, prothrombin kringle-1 and 2, which were cleaved by factor Xa and thrombin during the prothrombin activation, showed very strong inhibitory effects. Example 5 : Comparison of inhibitory efficacy of human prothrombin kringle-1 and 2 with other known anti-angiogenesis molecules Table 1 shows the half-maximal inhibition (ED 50 ) values of human prothrombin kringle-1, 2 and other known endothelial cell growth inhibitory molecules. The concentrations of ED 50 for human prothrombin kringle-1 and 2 are about lOOnM and 120nM, respectively. Human prothrombin kringle-1 and 2 are more potent than the other known endothelial cell growth inhibitory molecules, such as angiostatin (.s_e_e_: Cao, Y. et al., J. Biol. Chem., 271:29461-29467(1996)) and rabbit prothrombin fragment 2 (see: Lee, T. H. et al., J. Biol. Chem., 273:28805- 28812(1998)), but less effective than plasminogen kringle-5 (.s_e_e_: Cao, Y. et al . , J. Biol. Chem., 272:22924- 22928(1997)) on bFGF-stimulated BCE cell growth inhibition. However, it has not been reported that the single kringle domains of plasminogen and angiostatin exist independently in blood, like prothrombin kringle-1 and 2. Therefore, the human prothrombin kringle-1 and 2 may be the most effective endothelial cell growth inhibitors among the naturally occurring single kringle molecules. Table 1. The half-maximal concentration (ED 50 ) of prothrombin kringle-1 and 2 and other known endothelial cell growth inhibitory molecules Protein ED 50 (nM) Human prothrombin kringle-1 100 Human prothrombin kringle-2 120 Rabbit prothrombin kringle-2 155 Angiostatin 140 Angiostatin kringle-1 320 Angiostatin kringle-3 460 Plasminogen kringle-5 50 Example 6: Expression and purification of recombinant prothrombin kringle-1 and 2 To clone cDNA encoding prothrombin kringle-1 and 2, polymerase chain reaction (PCR) was performed with a cDNA library of human prothrombin as a template. To carry out the PCR, following single stranded oligonucleotides were synthesized based on N-terminal and C-terminal amino acid sequences of human prothrombin kringle-1 and 2, respectively: Human prothrombin kringle-1: N-terminal primer: 5' -GAATTCCTTCTGGGCCAAGTACACAGC- 3' (SEQ ID N0:3) C-terminal primer: 5' -AAGCTTTTAGCGTGGAGTCATCGCTAC- 3' (SEQ ID NO:4) Human prothrombin kringle-2 : N-terminal primer: 5' -CGAATTCCGAAGGCTCCAGTGTG-3' (SEQ ID NO:5) C-terminal primer: 5' -GAAGCTTCTAACGCCCTTCGATGGC-3' (SEQ ID NO:6) PCR was performed for 30 cycles of lmin at 94 ° C, lmin at 57 ° C, and lmin at 73°C, with a 5 min pre-heat and lOmin final extension at 72 ° C. The DNA fragments thus obtained were cloned into E . coli expression vector pET22b T (Novagen, USA). The expression vector comprising cDN- encoding prothrombin kringle-1 or prothrombin kriπgle-2 was designated as ^pEF-hKl' or pEF-hK2' , respectively. The transformed E. coli with pEF-hK2 was designated as '£. coli BL21 (DE3 /pLysS,phEF13', and deposited with the Korean Culture Center of Microorganisms (KCCM, Department of Food Engineering, College of Eng., Yonsei University, Sodaemun-gu, Seoul 120-749, Korea) , an international depository authority as accession No. KCCM-10139. The transformed E . coli with pEF-hK2, E . coli BL21 (DE3) /pLysS,phEF13, were incubated at a turbidity of 0.4 to 1.4 O.D. units at 600mM in LB media, in the presence of 50μg of ampicillin per ml, before being challenged with IPTG (isopropyl β-D-thiogalactcpyranoside). IPTG was added to a final concentration of 0.1 to 5mM and the cells were further incubated for 3hr. At the end of the incubation period, bacteria were pelleted by centrifugation at 4000xg for 15min, and resuspended in a minimal volume of lOmM sodium phosphate, pH 7.0, containing 50mM NaCl and ImM phenylmethyl sulfonyl fluoride (PMSF) . Cells were then disrupted by ultrasonication (Sonics Material Inc, Danburg, USA). The cell debris were removed by centrifugation at 13,500χg for lOmin. For the purification of the recombinant prothrombin kringle-2, the crude extract was chromatcgraphed on DEAE-Sepharose column (2x12cm) preequilibrated with a buffer containing lOmM sodium phosphate, pH 7.0, containing 50mM NaCl. With lOmM sodium phosphate buffer containing 180mM NaCl, unbound and weakly bound proteins were removed. After the washing step, proteins were eluted with 0 to 500mM NaCl gradient in the above buffer at a rate of 20ml/hr. Fractions containing prothrombin kringle-2 were pooled and concentrated in the Centriprep (Amicon, USA) device. The concentrated samples were loaded on a Sephacryl S-200 column ( 1.2x100cm) equilibrated with 50mM sodium phosphate buffer (pH 7.0) and eluted at a rate of 18ml/h. In order to confirm that the cDNA sequence of human recombinant prothrombin kringle-2 was faithfully translated, the amino acid sequence analysis was performed. Example 6: Suppression of tumor growth by systemic administration of prothrombin kringle-2 Because prothrombin kringle-2 inhibited neovascularization in vivo, the inventors investigated that the antitumor activity of prothrombin kringle-2 may be applied to systemically treat C57BL6 mice bearing subcutaneously implanted primary Lewis lung carcinoma cells . Male 6 to 8 week-old C57BL6/J mice(Samyuk Animal Lab., Korea) were used. Mice were acclimated and caged in groups of four or less, and their backs were shaved. Male 6-week-old mice were housed in a barrier care facility. All mice were fed a diet of animal chow and water ad libi tum . Animals were anesthetized with an injection of 2mg of ketamine hydrochloride and 0.1 mg of xylazine hydrochloride per 20 gramed mouse. Animals were sacrificed by a lethal dose of methoxyflurane . Mice with Lewis lung carcinoma (LLC) of 600-1200mιrr tumors were sacrificed, and the skin overlying the tumor was cleaned with betadine and ethanol. In a laminar flow food, tumor tissue was excised under aseptic conditions. A suspension of tumor cells in 0.9% normal saline was made by passage of viable tumor tissue through a sieve and series sequentially smaller hypodermic needles of a diameter of 26 gauge. The final concentration was adjusted to 1 X 10cells/ml and the suspension was placed on ice. After cleaning with ethanol, the subcutaneous dorsa of mice in the proximal midline were injected with lX10 6 cells in 0.1 ml of saline. When tumors were 1500mm 3 in size, approximately 14 days after implant, the mice were randomized into two groups. One group underwent surgical removal of the tumor. The incision was closed with simple interrupted sutures. The other group was anesthetized and had a sham surgical procedure in which tumors were manipulated but left intact. After 14 days, all mice were autopsied, and lung surface metastases were counted by means of a stereomicroscope at magnification . After resection of 1500mm 3 tumors, mice were randomized into groups that received daily subcutaneous injections with saline or various concentration of recombinant prothrombin kringle-2 (0.5mg/kg and lmg/kg) in the ventral abdomen of mice, whereas subcutaneous tumors were growing in the midline dorsum of each mouse.. One group of tumor-bearing animals had only a sham procedure and were treated with saline injections. Treatments were continued for 14days, at which point all mice were sacrificed and autopsied As shown in Figure 5, daily systemic administration of prothrombin kringle-2 resulted in a significant suppression of the growth of secondary tumors during the 14-day treatment course (.se_e: Figure 5: 1, normal lung; 2, treatment with saline; 3, 0.5mg/kg recombinant prothrombin kringle-2; 4, lmg/kg recombinant prothrombin kringle-2) . Figure 6 is a graph showing decrease the weight of lung treated with recombinant prothrombin kringle-2. At day 14 after treatment, an average of over 90% suppression of primary tumor growth was observed in the lmg/kg prothrombin kringle-2 treated mice(n=10). In contrast, tumors grew rapidly in the saline-treated animals (n=8) during the same 14-day treatment period. Example 7 : Anti-endothelial cell proliferation activity of peptides of prothrombin kringle-2 To determine the active domain for endothelial cell proliferation inhibition of human prothrombin kringle-2, 11 peptides consisting of 15 or 9 amino acids of prothrombin kringle-2 were synthesized by Peptron Corp. (Korea) . The peptides having amino acid sequences in order from N-terminal sequence of prothrombin kringle-2 were synthesized by overlapping 5 amino acids of both N-terminal and C-terminal of the peptides (see: Table 2), while the peptide of SEQ ID NO:12 was synthesized to contain only 9 amino acids because of problem in synthesis and overlap just 2 amino acids. The synthesized peptides were purified by high performance liquid chromatography (HPLC) . Table 2 Synthesi zed peptides of human prothrombin kringle-2 • PtK2: prothrombin kringle-2 The endothelial cell proliferation assay was performed in accordance with the method of Example 3. As shown in Table 2, the peptides of SEQ ID NOs : 8 to 13 inhibited the bFGF-stimulated endothelial cell growth and the inhibition activity of peptide of SEQ ID NO: 12 is the most effective. Therefore, it was clearly determined that the peptide of SEQ ID No. 12 may play a crucial role to display anti-endothelial cell growth effect . Administration and effective dose Though the effective dose of human prothrombin kringle-1, 2 or its functionally equivalent peptides is variable depending on age, body weight of patient and progression of disease, it is preferable to administer parenterally 0.8 to 2.0mg/kg/day in a single dose, and which may be individualized by experience of the skilled in the art . Acute toxicity To examine the acute toxicity of human prothrombin kringle-1, 2 or its functionally equivalent peptides, human prothrombin kringle-1, 2 or peptides was injected into male C57BL/6J mouse subcutaneously and the deads were counted to determine LD 50 for 7days. As a result, LD 50 is determined as about 5g/kg, indicating that the anti-tumor agent comprising human prothrombin kringle-1, 2 and its functionally equivalent peptides are sufficiently safe within the range of effective dose. The pharmaceutical composition of the present invention which comprises an active ingredient of human prothrombin kringle-1, 2 or its functionally equivalent peptides and pharmaceutically acceptable carrier may be administrated as an injection formula. The injection formula may further comprise isotonic aqueous solution or suspension, and the pharmaceutically acceptable carrier covers preservatives, stabilizers, wetting agents, emulsifiers, salts for changing osmotic pressure or buffers. While the present invention has been shown and described with reference to the particular embodiments employing Lewis lung carcinoma as a tested tumor, the anti-tumor agent of the invention may be effective for various tumors such as skin cancer, laryngeal cancer, uterine cancer, colon cancer, lung cancer and bone marrow cancer. As clearly illustrated and demonstrated as above, the present invention provides human prothrombin kringle- 1, 2 and its functionally equivalent peptides having amino acid sequences of human prothrombin kringle-2 which have anti-endothelial cell proliferation, cDNAs encoding the human prothrombin kringle-1 and 2 derived from human prothrombin, recombinant expression vectors comprising the cDNAs, recombinant microorganisms transformed with the recombinant expression vectors expressing human prothrombin kringle-2, processes for preparing human prothrombin kringle-1 and 2 from human plasma and recombinant human prothrombin kringle-2 from the recombinant microorganisms and pharmaceutical application of the said proteins. Since an anti-angiogenic therapy directed against tumor's endothelial cells should induce little or no drug resistance, the anti-endothelial cell growth molecules, human prothrombin kringle-1, 2 and its functionally equivalent peptides, have been as the most reliable candidates for cancer therapy. BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL FORM To. Souπg Soo Kim Department of Biochemistry RECEIPT IN THE CASE OF AN ORIGINAL College of Science and Bioproduct issued pursuant to Rule 7. 1 by the Research Center Yonsei University INTERNATIONAL DEPOSITARY AUTHORITY Seoul 120-749, Korea identified at the bottom of this page L. -J 1 Where Rule 5. 4 (d) applies, such date is the date on which the status of international depositary authority was acquired : where a deposit made outside the Budapest Treaty after the acquisition of the status cf i nternational depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by the international depositary autnouity. Form BP/4 (KCCM Form 17) Sole page

Claims: WHAT IS CLAIMED IS: 1. An amino acid sequence of human prothrombin kringle-1 represented as SEQ ID NO:l. 2. A cDNA(SEQ ID NO: 18) encoding the human prothrombin kringle-1 of claim 1. 3. An amino acid sequence of human prothrombin kringle-2 represented as SEQ ID NO: 2. 4. A cDNA(SEQ ID NO: 19) encoding the human prothrombin kringle-2 of claim 3. 5. A recombinant expression vector pEF-hKl comprising a cDNA(SEQ ID NO: 18) which expresses human prothrombin kringle-1. 6. A recombinant expression vector pEF-hK2 comprising a cDNA(SEQ ID NO: 19) which expresses human prothrombin kringle-2. 7. E. coli BL21 (DE3) ZpLysS, phEF13 (KCCM-10139) which is transformed with the recombinant expression vector pEF-hK2 of claim 6. 8. A peptide which inhibits endothelial cell proliferation, which is derived from human prothrombin kringle-2 of human prothrombin. 9. The peptide of claim 8, which has a sequence of 5 to 20 amino acids in order from 11th proline to 70th aspartic acid of human prothrombin kringle-2 . 10. A process for preparing prothrombin kringle-1 and 2 from human plasma which comprises the steps of: (i) isolating and purifying human prothrombin from human plasma; (ϋ) treating the human prothrombin with factor Xa to give prothrombin kringle-1 and 2; and, (iii) isolating the prothrombin kringle-1 and 2 by employing ion-exchange chromatography. 11. A process for preparing a recombinant human prothrombin kringle-2 which comprises the steps of: (i) culturing recombinant microorganism transformed with an expression vector comprising the cDNA of claim 4; and, (ii) applying the culture to ion-exchange and gel filtration chromatography to obtain the recombinant human prothrombin kringle-2. 12. The process for preparing a recombinant human prothrombin kringle-2 of claim 11, wherein the recombinant microorganism is E . coli BL21(DE3)/ pLysS,phEF13 (KCCM-10139) . 13. An anti-tumor agent comprising an active ingredient of the human prothrombin kringle-1 or 2, or its functionally equivalent peptide and pharmaceutically acceptable carrier. 14. The anti-tumor agent of claim 13 which is effective for skin cancer, laryngeal cancer, uterine cancer, colon cancer, lung cancer and bone marrow cancer.

Inventors: KIM EUN KYUNG, PARK CHAN SOO, RHIM TAI YOUN, KIM, EUN, KYUNG, PARK, CHAN, SOO, RHIM, TAI, YOUN
Applicants: GENOTECH CORP, KIM EUN KYUNG, KIM SOUNG SOO, PARK CHAN SOO, RHIM TAI YOUN, GENOTECH CORP., KIM, EUN, KYUNG, KIM, SOUNG, SOO, PARK, CHAN, SOO, RHIM, TAI, YOUN
Application year: 1999
Main classification: C12N9
IPC codes: A61K38, A61P35, C07K14, C12N1, C12N5, C12N9, C12N15, C12P21, C12R1