MXPA99007768A - Factor x analogues with a modified protease cleavage site - Google Patents

Abstract

The invention relates to factor X analogues which have a modification in the area of the naturally occurring factor Xa activating cleavage site, said modification representing a processing site of a protease which does not naturally cleave in this area of the factor X sequence. The invention also relates to preparations containing the innovative factor X analogues and to methods for the production thereof.

Description

ANALOGS OF THE FACTOR X THAT HAVE A PROTEASE SEGMENTATION SITE, MODIFIED Field of the Invention The invention relates to Factor X analogs having a modification in the activation peptide region, to a preparation containing the Factor X analogues according to the invention, and to a method of preparing the chain X Factor analogues. single and double chain.

Background of the Invention After the blood coagulation process has been initiated, the coagulation cascade continues through the sequential activation of several proenzymes (zymogens) in the blood to their active forms, the serine proteases. These include, inter alia, Factor XlI / XIIa, Factor Xl / XIa, Factor IX / IXa, Factor X / Xa, Factor VlI / VIIa and prothrombin / thrombin. In their physiological state, most of these enzymes are only active if they are associated with a surface of the membrane in a complex. Ref.031047 Ca ions are involved in many of these processes. Blood coagulation will follow either the intrinsic pathway, where all of the protein components are present in the blood, or the extrinsic pathway, where the tissue factor of the cell membrane plays a critical role. Finally, the wound will be closed by the fibrinogen of thrombin segmentation to fibrin. The prothrombinase complex is responsible for activating prothrombin to thrombin. Thrombin is an important enzyme which can act as a procoagulant as well as an anticoagulant. The prothrombinase complex, in which, inter alia, Factor Va (as the cofactor) and Factor Xa (as serine protease) are involved, are assembled or assembled in a Ca-dependent association on the surface of the phospholipid. It is described that Factor Xa is the catalytic component of the prothrombinase complex. Factor X (Stuart-Prower factor) is a coagulation glycoprotein dependent on vitamin K by which the coagulation cascades of intrinsic and extrinsic blood can be activated. The primary translation product of Factor X (pre-pro-FX) has 488 amino acids and is initially synthesized by the liver or human hepatoma cells as a 75 kD precursor protein of a single chain. In plasma, Factor X is widely present as a double-stranded molecule (Fair et al., 1984, Blood 64: 194-204). During biosynthesis, after segmentation of the presequence by a signal peptidase (between Ser23 / Leu24) and the propeptide (between Arg40 / Ala41), the X-Factor molecule of the single chain is cleaved by processing and removal of the tripeptide Argl80-Lysl81-Argl82 to the double-chain form consisting of the light chain of approximately 22 kD and the heavy chain of approximately 50 kD, which are connected by means of a disulfide bridge (Fig. 1). Therefore, the X Factor circulates in the plasma as a double-stranded molecule. During the process of blood coagulation, Factor X is converted from the inactive zymogen to activate Protease Factor Xa by limited proteolysis; wherein the X Factor can be activated to Factor Xa in either of the two complexes associated with the membrane: in the extrinsic Factor Vlla-factor complex of the tissue or in the complex of Factor Vlla-Factor IXa-phospholipid-intrinsic Ca, or "tenase complex" (Mertens et al., 1980, Biochem J. 185: 647-658). A proteolytic cleavage between the amino acids Arg234 / Ile235 leads to the release of an activation peptide having a length of 52 amino acids from the N-terminus of the heavy chain and consequently to the formation of the active enzyme, Factor Xa. The catalytic center of Factor Xa is located on the heavy chain. Activation by means of the Factor VIIa-TF (extrinsic) complex leads to the formation of Factor Xaa (35 kD) and Factor Xaß (31 kD), with a polypeptide of 42 (kD) that is formed, too, if the concentration of Factor Vlla in the complex is low. Xaa factor is formed by a segmentation in Arg234 / Ile235 of the heavy chain and represents the activation of Factor X to Factor Xa. The presentation of Factor Xaß presumably results from an autocatalytic cleavage of Arg469 / Gly470 at the C-terminus of the heavy chain of Factor Xaa and the removal of a 4.5 kD peptide. Similarly Factor Xaa, Factor Xaß, have catalytic activity. However, it has been shown that a plasminogen binding site is formed by the cleavage of Factor Xaa to Factor Xaß, and because Factor Xaß optionally has a fibrinolytic activity or is involved in fibrinolysis as a cofactor. The conversion of Factor Xaa to Factor Xaß, however, is slower than the formation of thrombin, thus preventing the initiation of fibrinolysis before a blood clot is formed (Pryzdial et al., 1996, J. Biol. Chem. 271: 16614-16620; Prizdial et al., 1996, J. Biol. Chem. 271: 16621-16626). The 42 kD polypeptide results from processing at the C-terminus of the heavy chain between Arg469 / Gly470 without preprocessing between Arg234 / Ile235. Similar to a fragment of Factor Xa? formed by proteolysis in Lys370, this intermediate compound has no catalytic activity (Mertens et al., 1980, Biochem J. 185: 647-658; Pryzdial et al., 1996, J. Biol. Chem. 271: 16614-16620 ). Activation of intrinsic Factor X is catalyzed by the Factor IXa-Factor Villa complex. The same processing products are obtained during activation, but the Factor Xaß product is obtained in a larger amount than the other Factor X processing products (Jesty et al., 1974, J. Biol. Chem. 249: 5614 ). In vitro, Factor X, for example, can be activated by Russell's viper venom (RW) or trypsin (Bajaj et al., 1973, J. Biol. Chem. 248: 7729-7741) or by physiological activators. purified, such as the FVIIa-TF complex or the Factor IXa-Factor Villa complex (Mertens et al., 1980, Biochem J. 185: 647-658). The X Factor products most commercially available from plasma contain a mixture of Factor Xaa and Factor Xaß, because after the activation of Factor X to Factor Xa, Factor Xaa is mainly formed, which is in turn , segmented to Factor Xaß in an autocatalytic process. To produce a uniform Factor Xa product having a high structural integrity, EP 0 651 054 suggested activating Factor X with RW for a prolonged period of time so that the resulting final product substantially contains Factor Xaß. The byproducts, for example Factor Xaa, as well as the protease were subsequently removed by several chromatographic steps. The X-Factor cDNA has been isolated and characterized (Leytus et al., 1984, Proc. Nati, Acad. Sci., USA, 82: 3699-3702; Fung et al., 1985, Proc. Nati. Acad. Sci. , USA, 82: 3591-3595). Human X Factor has been expressed in vitro in several cell types, such as human embryonic kidney cells or CHO cells (Rudolph et al., 1997, Prot. Expr. Purif. 10: 373-378; Wolf et al. , 1991, J. Biol. Chem. 266: 13726-13730). However, it has been found that in the recombinant expression of human Factor X, the processing in the Arg40 / Ala41 position is inefficient, as opposed to the in vivo situation, and that the different N terminals in the light chain of the X Factor are produced (Olf et al., 1991, J. Biol. Chem. 266: 13726-13730). Recombinant X Factor (rFX) was activated to rFactor Xa (rFXa) by RW in vitro, or rFXa was expressed directly, with the activation peptide being deleted from amino acid 183 to amino acid 234 and replaced by double-stranded rFXa for allow processing directly to a double-stranded rFXa form. Approximately 70% of the purified rFX was processed in the light and heavy chain while the remaining 30% represented the single-chain rFX of 75 kD. The direct expression of rFXa led to the formation of active Factor Xa, but also of inactive intermediates. Wolf et al. (1991, J. Biol. Chem. 266: 13726-13730) still detected a reduced activity of recombinant Factor X, which they attributed to the poorer capacity of rFX to be activated by RW and protein populations and inactive polypeptides of the single chain precursor molecule. In particular, they find a high rFXa instability when they are expressed by recombinant cells, which they attribute to the high speed of proteolysis.

To study the function of the C-terminal peptide of Factor Xaa, Eby et al. (1992, Blood 80 (Suppl 1): 1214 A) introduced a stop codon at position Gly430 of the Factor X sequence. However, they did not find a difference between the activation rate of Factor Xa (FXaa) and the β-peptide or a deletion mutant without the β-peptide (FXaβ). Factor Xa is an important component of the prothrombinase complex and can therefore be used to treat patients suffering from blood coagulation disorders, for example hemophilia. Particularly the treatment of patients with hemophilia who suffer from a deficiency of Factor VIII or Factor IX with factor concentrates produced from plasma is complicated by the formation of inhibitory antibodies against these factors in long-term therapy. Therefore, several alternatives have been developed to treat hemophiliacs with factors that have a bypass activity. The use of the prothrombin complex concentrate, the partially activated prothrombinase complex (APPC), the Vlla Factor or FEIBA, have been suggested. Commercial preparations with Factor derivation activity VIII (FEIBA) are, for example, FEIBA® or Autoplex®. FEIBA® contains comparable units of Factor II, Factor VII, Factor IX, Factor V, and traces of activated coagulation factors, such as thrombin and Factor Xa or a factor that has activity similar to Factor X ( Elsinger, 1982, Activated Prothrombin Complex Concentrates, Ed. Mariani, Russo, Mandelli, pp. 77-87). Elsinger points out in particular the importance of activity "similar to Factor Xa" in FEIBA®. Factor VIII derivatization activity was shown by Giles et al (1988, British J. Haematology 9: 491-497) for a combination of purified Factor Xa and phospholipids in an animal model. Therefore, Factor X / Xa or similar X / Xa Factor proteins, either alone or as a component of a coagulation complex, are in increasing demand and can be used in various fields of application in hemostasis therapy . In vivo as well as in vitro, the half-life of Factor Xa is considerably shorter than the half-life of the zymogen. For example, Factor X can be stored stably in glycerol for 18 months, while Factor Xa is stable for only 5 months during the same conditions (Bajaj et al., 1973, J. Biol. Chem. 248: 7729-7741 ) and shows reduced activity by more than 60% after 8 months in glycerol at 4 ° C (Teng et al., 1981, Thrombosis Res. 22: 213-220). The half-life of Factor Xa in serum is only 30 seconds. Because Factor Xa is unstable, administration of Factor X preparations have been suggested (U.S. 4,501,731). However, if the bleeding is so severe that the patient could die, particularly in hemophiliacs, the administration of Factor X is ineffective, because of the deficiency of the "tenase complex" in the intrinsic pathway of coagulation. blood, Factor X may not be sufficiently activated to Factor Xa, and activation via the extrinsic pathway is often too slow to show effects quickly. In addition, hemophiliacs have sufficient amounts of Factor X, but their prothrombinase activity is 1000 times less than that of Factor Xa. In such cases it is necessary to administer activated Factor Xa directly, optionally in combination with phospholipids, as described in Giles et al. (1988, British J. Haematology 9: 491-497) or with other coagulation factors, for example with Factor VIII derivatization activity. In the preparation of Factor Xa from Factor X, the activation has thus been carried out mostly by neophysiological activators of animal origin, such as RW or trypsin, and it was necessary to absolutely ensure that the final product is completely free of these proteases. As mentioned above, when Factor X is activated to Factor Xa, rather a number of intermediate compounds, some of them inactive, are formed (Bajaj et al., J. Bio, Chem. 248: 7729-7741).; Mertens et al., 1980, Biochem. J. 185: 647-658). The presence of such intermediates leads to a reduced specific activity of the product and can produce intermediates which can function as antagonists of active serine proteases. Therefore, the preparation of a pure, uniform product having a high specific activity according to conventional methods requires complex activation and chromatographic purification processes. Accordingly, the object of the present invention is to provide a preparation containing a polypeptide having the activity of Factor X / Xa which exhibits high stability and can be activated to Factor Xa without using any of the conventional proteases, particularly those of origin animal, such as, for example, TW or trypsin. Another object is to provide a pharmaceutical preparation having the activity of derivatizing Factor VIII.

In accordance with the present invention, the object is achieved by providing an X Factor analog having a modification in the activation factor cleavage site region of the natural Factor Xa. Modification in the region of the activation cleavage site is a novel processing and recognition site for a protease, such site is not naturally located in this position in the polypeptide, such protease could not usually segment the polypeptide at this site. The Factor X analogue according to the invention is modified particularly in the activation peptide which is removed when Factor X is activated to Factor Xa. At least one amino acid within the amino acid sequence of the activating peptide of Factor X is modified. Said modification is located particularly in the C-terminal region of the activation peptide and represents, at least, an exchange of at least one amino acid between the positions Gly228 and Arg234 of the amino acid sequence of Factor X. The position of the amino acids is based on in the numbering according to the sequence shown in Figure 1, starting with Metí and ending with Lys488. Said modification in the Factor X analogue according to the present invention is preferably an exchange of a Factor VIIa / Factor IXa processing site located in this position for an alternative cleavage site of a different protease. The modification can be a substitution of at least one amino acid, or an insertion of a peptide sequence representing a cleavage site or recognition of the protease. In the Factor X analogue according to the invention, the modification is preferably such that it represents a detection sequence or cleavage for a protease from the group of endoproteases, such as kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE 4, LPC / PC7 (as described in Barr et al., 1991, Cell 66: 1-3 or in US 5,460,950), of serine proteases, such as Factor Xlla, Factor Xla , Factor lia, Factor Xa, or kallikrein, or a derivative of these proteases. Preferably, the modification is selected such that processing by one of these proteases leads to a polypeptide corresponding to natural Factor Xa, which is substantially equal to the natural Factor Xa sequence and also exhibits Factor Xa activity. For optimal processing, it may be necessary in individual cases to also exchange the amino acid Ile235. Preferably, however, the isoleucine of the NH2 terminal amino acid of the heavy chain must still be present after activation, because this amino acid plays an essential role in the formation of the substrate binding cavity (Watzke et al. , 1995, Molecular Basis of Thrombosis and Hemostasis, ed. Katherine High &Harold Roberts). The Factor X analogues according to the invention have a structural difference, particularly over the amino acid level, when compared to a natural Factor X sequence, but can be similarly activated to natural Factor X and have the activity of Factor Xa after of the activation. The invention provides an exemplary number of Factor X analogues that have a modification in the activation peptide relative to the natural Factor X sequence and different specificity of the protease. The modifications may be located at one or more positions in the region between the amino acid Gly228 and Arg234, and optionally Ile235, based on the sequence of Factor X numbered Methyl to Lys488 according to Figure 1. The substitutions of the amino acids may be in the positions Ile235 (Rl), Arg234, Thr233 (R2), Leu232 (R3), Asn231 (R4), Asn230 (R5) and Asp229 (R6), with Arg234 which preferably remains unchanged. Preferably, Factor X analogues according to the invention contain a sequence of Factor X with Gly228-R6-R5-R4-R3-R2-Arg234, where R1 = Lie, Val, Ala, Ser or Thr; R2 = Thr, Pro, Gly, Lys or Arg; R3 = Leu, Phe, Lys, Glu, Met, Gln, Ser, Val, Arg or Pro; R4 = Asn, Asp, Lie, Ser, Met, Pro, The, Lys or Arg; R5 = Asn, Lys, Ser, Glu, Ala, Gln, His or Arg; and R6 = Asp, Phe, Thr, Arg, Leu or Ser. Preferred embodiments of Factor X analogues according to the invention are Factor X analogues that have a modification with a) R1 = Val, R2 = Thr, R30Phe , R4 = Asp, R5 = Asn and optionally R6 = Phe (Figure 2A), and processed by the Xla Factor; b) Rl = Ser, R2 = Arg, R3 = Thr, R4 = Leu (Figure 2B), and processed by Factor lia; c) Rl = Ile, R2 = Pro, R3 = Lys, R4 = Ile, and optionally R5 = Lys and / or R6 = Thr (Fig. 2C), or Rl = Ile, R2 = Thr, R3 = Ser, R4 = Thr, and optionally R5 = Lys and / or R6 = Thr (Fig. 21), and processed by the factor Xlla; d) Rl = Ile, R2 = Thr, R3 = Met, R4 = Ser, and optionally R5 = Ser and / or R6 = Leu (Figure 2D), and processed by kallikrein; e) Rl = Ile, R2 = Gly, R3 = Gln, R4 = Pro, and optionally R5 = Lys and / or R6 = Ser (Figure 2H), or Rl = Ile, R2 = Thr, R3 = Lys, and R4 = Met (Figure 2E), or Rl = Ile, R2 = Gly, R3 = Glu, and R4 = Ile (Figure 2F), and processed by Factor Xa; f) Rl = Ile, R2 = Lys, R3 = Arg, R4 = Arg, and optionally R5 = Glu and / or R6 = lg) Leu, or Rl = Ile, R2 = Thr, R3 = Val, R4 = Arg, and optionally R5 = Ala and / or R6 = Leu, or Rl = Ile, R2 = Arg, R3 = Val, R4 = Arg, and optionally R5 = Gln and / or R6 = Leu, or Rl = Ile, R2 = Arg, R3 = Arg, R4 = Arg, and optionally R5 = His and / or R6 = Leu, or Rl = Ile, R2 = Lys, R3 = Pro, R4 = Arg, and optionally R5 = Asn and / or R6 = Leu, or Rl = Ile, R2 = Lys, R3 = Arg, R4 = Ile, and optionally R5 = Arg and / or R6 = Leu, or Rl = Ile, R2 = Lys, R3 = Ser, and R4 = Arg, or Rl = Ile, R2 = Thr, R3 = Leu, and R4 = Arg (all see in Figure 2G), with those mentioned under f) _ that are processed by a dibasic endoprotease, such as furin, PACE, kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE 4, LPC / PC7, or by a derivative of one of these proteases.

Figure 2 A-I shows a possible selection of the modifications and exchanges of amino acids that lead to a different specificity of the protease. The modifications can be carried out, for example, by directed in vitro mutagenesis or PCR or other generic engineering methods known from the state of the art which are suitable for specifically changing a DNA sequence for targeted exchanges of the amino acids. According to the present invention, the Factor X analogue according to the invention is activated to natural Factor Xa or a Factor Xa analogue preferably by a protease selected from the group of endoproteases, such as kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE 4, LPC / PC7, the group of serine proteases, such as Factor Xlla, Factor Xla, Factor Xa, Factor lia, or kallikrein, or a derivative of these proteases. One of the difficulties in preparing the Active factor Xa is its instability, because apart from Factor Xaa and Factor Xaß, other inactive intermediates are formed by autocatalysis. For the preparation of active, essentially intact Factor X / Xa molecules and Factor X / Xa-like molecules, respectively, it may therefore be desirable to obtain only such proteins which lead to stable end products. It is well known that a preferred cleavage site for the processing of Factor Xaa (FXaa) to Factor Xaß (FXaß) is between Arg469 / Gly470. Based on the search by Eby et al. (1992, Blood, Vol. 80, Suppl. 1, 1214), following a peptide with prominent carboxy terminus (amino acid residues 476-487) of Factor X, other shorter peptides are found (amino acid residues 474-477) which are formed by autocatalysis of Factor Xaa. For site-directed processing of intact Factor X to essentially active Factor Xa without obtaining inactive processing intermediates, the Factor X analogues of the invention optionally have additional modifications. Therefore, according to a particular embodiment, the X-Factor analogue according to the invention has a further modification in the C-terminal region of the amino acid sequence of Factor X. According to one embodiment, an analogue of Factor X X as described above has an 'intact β-peptide (FXa). The Factor X analogue according to the invention particularly has a modification in the region of the cleavage site of the C-terminal β-peptide which prevents the cleavage of the β-peptide of Factor X after the activation of Factor X to Factor Xa . Accordingly, a Factor Xa molecule is obtained which can be isolated up to 100% as the intact Factor Xaa molecule. The modification can be a mutation, deletion or insertion in the region of the amino acid sequence of Factor X between the amino acid positions Arg469 and Ser476 and optionally Lys370. However, an amino acid substitution is preferred, which prevents the polypeptide from unfolding as a consequence of the exchange of amino acids, which could influence the structure and thus possibly the function and activity of the protein. According to one embodiment, the Factor X analogues of the invention have one of the amino acids in the Arg469 and / or Gly470 position exchanged, with Arg469 being exchanged preferably by Lys, His or lie, and Gly470 which is preferably exchanged to be , Ala, Val or Thr. In addition to a mutation in the Arg469 and / or Gly470 position, the Factor X analogues according to the invention may have another mutation in the Lys370 and / or Lys475 and / or Ser476 position. The substitution of amino acids in one of these positions prevents the processing of Factor Xaa to Factor Xaß or Factor Xa ?, respectively, because the natural processing sequence (s) is (are) modified in such a way that an occasional autocatalytic fragmentation of the carboxy-terminal peptide becomes impossible. According to an additional modality, the Factor X analogue of the invention has a deleted carboxy-terminal β-peptide (FXß). Such an X Factor analog can be prepared by expressing a cDNA encoding a Factor X analog in a recombinant expression system, cloning only those sequences encoding the amino acids Met to Arg469. According to a further embodiment, the X-Factor analogue according to the invention has a signal to stop the translation in the C-terminal region of the Factor X sequence. This signal for stopping the translation is preferably located in a position following a C-terminal amino acid formed after natural processing. Therefore, the translation stop signal is preferably at the position of amino acid 470 of the Factor X sequence, so that the terminal Arg469 of Factor Xaß is retained. For this purpose, the GGC codon encoding the amino acid Gly470 is replaced by T7? A, TAG or TGA. Another aspect of the present invention relates to Factor X analogues which are activated to natural Factor X or to Factor Xa analogs by treatment with an inappropriate protease in vitro. Depending on the activated and activated Factor X analogue, a polypeptide that corresponds to natural Factor Xa and is essentially identical, or a polypeptide having the activity of Factor Xa but having the modifications relative to the sequence of the natural Factor Xa which, without However, it does not limit its biological activity, they are obtained. When the X-Factor analogues are activated, which are modified in the activation peptide region in the activation peptide sequence, only the polypeptides corresponding to the natural Factox Xa molecules are obtained. If such an X Factor analogue additionally has a signal to stop the translation in the C-terminal region of the β-peptide, the homologous molecules of Factor Xaß are obtained. However, if the Factor X analog is employed which has modification (s) within the sequence of the β-peptide leading to the β-peptide that is not completely cleaved, a Factor Xaa analogue with an amino acid exchange in the C terminal of the molecule is obtained. The Factor X analogs of the invention only have modifications which change the specificity for the capacity that is to be activated and which have no influence on the activity. Therefore, in any case, Factor Xa or Factor Xa molecules, functionally and biologically active, are obtained. The in vitro activation can be effected by a protease selected from the group of endoproteases, such as kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE 4, LPC / PC7, the group of serine proteases, such as Factor lia, Factor Xlla, Factor Xla, Factor Xa, or kallikrein, or a derivative of these proteases. It is within the scope of the present invention to use any protease, except RW, trypsin, Factor IXa or Factor VIIA, provided that it is capable of processing the Factor X analogue of the invention to Factor Xa. According to a further embodiment of the invention, the Factor X analog contains a modification that allows the activation of the Factor X analog to Factor Xa, preferably the natural Factor Xa, in vivo. In this context, "natural" Factor Xa means that the activated Factor Xa, derived from the Factor X analogue according to the invention, has an amino acid sequence corresponding to and that is homologous with the natural Factor Xa, and has the activity of Factor Xa. Said modification is chosen in such a way that Factor X is processed to Factor Xa by a protease present in vivo, ie in the body, preferably a protease present in the blood coagulation cascade. The protease may be a protease selected from the group of serine proteases, such as Factor Xlla, Factor Xla, Factor Xa, Factor lia or kallikrein. Factor X analogs that have a modification in the C-terminal region of the Factor X molecule apart from the modification in the activation peptide are activated to the corresponding Factor Xa analogue in vivo, too, as described above. Although Wolf et al. (1991, J. Biol. Chem. 266: 13726-137309), for example, has assumed that an endopeptidase, such as Kex2, furin or PACE, is involved in the processing of the Factox Xa deletion mutant described by this group, they do not give a suggestion as to the influence of one of these proteases on the processing of Factor X. Similarly, US Pat. No. 5,660,950 describes the recombinant preparation of PACE and the use of protease to improve the processing of vitamin K-dependent proteins. In a long list of blood factors, Factor X is mentioned among others, but no data is provided to verify this foundation. The present invention unequivocally demonstrates for the first time that a protease necessary for the maturation process of Factor X is a dibasic endoprotease, particularly endogenous furin. In vivo, the endoprotease mainly measured the segmentation of the X-Factor molecule from the single chain to the mature form consisting of the light and heavy chain. In vitro, it also measured the cleavage of the propeptide sequence of Factor X (Example 2). Factor X analogs according to the present invention that have a protease cleavage site for a protease that does not naturally exist in a cell are cleaved by selective processing reactions only at those sites which are also cleaved in Factor X natural. Accordingly, the recombinant Factor X molecule is obtained which consists only of the light chain of 22 kD and the heavy chain of about 50 kD and does not have inactive X Factor molecules such as those formed by non-specific processing. Similar to the natural Factor X molecules, these modified Factor X molecules are not activated to Factor Xa by the intracellular protease. They are activated to Factor Xa only after this by the appropriate proteases (ie, preferably the serine protease or the proteases related to subtilisin). Accordingly, a double chain X-Factor analogue is provided according to one embodiment. According to a particular embodiment, Factor X analogues are provided which are preferably present in the purified form as single chain molecules. By expressing the X Factor analogues in a deficient dibasic protease cell, pro-Factor X is obtained as a single chain molecule. The single-chain Factor X molecule is characterized by high stability and molecular integrity. Until now, a single-chain X-Factor molecule has not been able to be isolated in the purified form, because it is rapidly processed into the double-stranded form (Fair et al., 1984, Blood 64: 194-204) . Recombinant single-chain X-Factor analogs can be processed by the specific processing to the double-chain X-Factor form and subsequently activated to Factor Xa or the Factor Xa analog, respectively. This can be accomplished by contacting a single chain recombinant X Factor molecule isolated from a protease deficient cell and a dibasic protease, such as furine / PACE or Kex2, and processing to a double chain X Factor analogue. . The double chain X-Factor analog can be activated to Factor Xa or to the Factor Xa analog, respectively. This can be done, for example, by isolating an X Factor analog having a specific cleavage site for the purine due to a modification in the activation peptide region., from a serine deficient cell as a single chain molecule and squently processing it to an activated Factor Xa molecule bringing it into contact with this endoprotease. Similarly, an isolated single chain X-Factor analog having a modification in the activation peptide which allows for alternative processing by a protease of the group of serine proteases or kallikrein can only be segmented to give a double-stranded X-Factor molecule by treating it with a dibasic endoprotease, such as furin, such a double-chain X-Factor molecule in the further course of events can be brought in contact with a serine protease in such a way that it occurs activation to Factor Xa or Factor Xa analog, respectively. An X Factor analog isolated from the cell culture as a double stranded molecule can be treated with the specific protease for activation. Due to the selective and targeted processing reaction, an X Factor or Factox Xa analog so obtained, has a structural integrity and high stability and, in particular, is free of inactive intermediates of the Factor X / Xa analog and the products of autoproteolytic degradation. A further aspect of the present invention relates to recombinant DNA encoding the Factor X analogues of the invention. The recombinant DNA results after expression in a Factor X analogue with an amino acid sequence corresponding to human Factor X except for a modification that influences the specificity of the processing and processing products, whereas the biological coagulating activity basically it remains without change.

According to a further aspect, also the transformed cells containing the recombinant DNA are provided. A further aspect of the invention relates to a preparation that contains a purified Factor X analog or a precursor protein thereof that has a modification in the region within the activation site of natural Factor Xa. The modification in the region of the activation cleavage site is a novel recognition and cleavage site not naturally located in this position in the polypeptide for a protease which usually does not process the polypeptide in this position. The preparation may be a purified preparation of the single or double chain X Factor analogue; the polypeptide can be obtained from a cell culture system either after isolation of the cell culture supernatant or a cell culture extract. A recombinant X Factor analogue prepurified from a cell culture system can be further purified by methods known from the prior art. Chromatographic methods are particularly suitable for this purpose, such as gel filtration, ion exchange or affinity chromatography.

According to one embodiment, the preparation according to the present invention contains the Factor X analog as a single chain molecule in an isolated form. Such preparation is prepared by isolating an X Factor analogue, obtained by the recombinant preparation, as a single chain molecule from the cellular system, preferably a cell culture of cells which lack the endoprotease that processes the single chain molecule into chains light and heavy. According to a particular aspect, the preparation contains a single chain X-Factor analog having a modification that allows in vitro activation of Factor Xa by one of the proteases selected from the group of dibasic endoproteases, such as kexin / Kex2. , furine / PACE, PC1 / PC3, PC2, PC4, PACE 4, LPC / PC7. The activation is carried out by bringing the X Factor analogue in contact with the protease, through which due to natural processing, a segmentation to the mature Factor X form is effected, and because of the modification, the activation peptide is segmented completely and Factor Xa or the Factor Xa analog are formed. In the preparation according to the invention, the Factor X analogue as a single chain molecule can be present either as Factor Xa (FXa) or with a deletion of the β-peptide. The preparation contains in particular the Factor X analogue in the enzymatically inactive form and has a purity of at least 80%, preferably at least 90%, particularly preferably at least 95%, and does not contain any inactive proteolytic intermediate of the analogue. of the X Factor / Xa. According to a further embodiment, the preparation according to the present invention contains the Factor X analogue preferably as a double-stranded molecule in the isolated form. For this purpose, the Factor X analog, for example, obtained by the recombinant preparation as a single-stranded molecule of a cellular system, is cleaved in vitro, ie outside the cell, by a protease, preferably a dibasic protease. , to the double chain shape. This can be done by mixing the protease directly with the culture supernatant of the clones expressing the Factor X analogues, either by mixing the purified protease or a cell culture supernatant of a cell culture expressing the protease in the recombinant form, or by the co-culture of the Factor X analog and the clones that express the protease.

Similarly, the cell culture supernatant containing the Factor X analog or the purified Factor X analog can be brought into contact with an immobilized protease., so processing occurs to the double chain shape. In this process, the protease is preferably bound to a matrix, and the cell culture supernatant or a purified preparation containing the Factor X analog is passed over this matrix. However, it is also possible to immobilize the Factor X analog while the protease is in the mobile phase. Similarly, the reagents (the Factor X analog and the protease) can be mixed and incubated for a certain period of time. Subsequently, the protease is removed from the mixture, for example by affinity chromatography. The double-stranded form of the Factor X analog can also be obtained by co-expressing the protease and the Factor X analogue directly in a given cell and optionally purifying it. According to a particular embodiment of the invention, the preparation contains a single-chain or double-chain Factor X analog having a modification that allows activation of Factor Xa or the Factor Xa analog in vitro. Activation of the Factor X analog to Factor Xa or the Factor Xa analog, respectively, can be carried out by bringing the Factor XXX analogue in contact with a protease selected from the group of dibasic endoproteases, such as kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE 4, LPC / PC7, the group of serine proteases, such as Factor Xlla, Factor Xla, Factor Xa, Factor lia or kallikrein, or a derivative of these proteases. The protease can be immobilized on a carrier. The preparation according to the invention can serve as a starting material for the preparation and production of Factor Xa. For a large-scale preparation, the preparation containing the single-chain or double-chain Factor X analogue, for example, is brought into contact with the optionally immobilized protease under conditions that allow for the optimal activation of the Factor X analog to the Factor Xa, and a Factor Xa or Factor Xa analogs are obtained. The Xa / Xa analogue thus produced can be subsequently formulated into a pharmaceutical composition in accordance with generally known methods. According to a particular embodiment, the preparation containing the purified single-chain, or double-chain, X-Factor analog contains a physiologically acceptable carrier and is optionally formulated as a pharmaceutical preparation. The formulation can be effected in accordance with a common method per se, and it can be mixed with a buffer solution containing salts, such as NaCl, CaCl 2, and amino acids, such as glycine and / or lysine, at a pH in the range from 6 to 8, and formulated as a pharmaceutical preparation. The purified preparation containing the Factor X analog can be provided as a storable product in the form of a solution made easily, lyophilized or frozen at a low temperature until its final use. Preferably, the preparation is stored in the lyophilized form and dissolved with an appropriate reconstitution solution to an optically clear solution. The preparation according to the present invention can also be provided as a liquid preparation in the form of a liquid frozen at low temperature. The preparation according to the present invention is particularly stable, ie it can be left at rest in the dissolved form for a prolonged period of time before application. The preparation according to the invention has proven to show no loss in activity for several hours to days. The preparation according to the invention can be provided in a suitable device, preferably an application device, in combination with a protease selected from the group of endoproteases, such as kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4 , PACE 4, LPC / PC7, the group of serine proteases, such as Factor Xlla, Factor Xla, Factor Xa, Factor lia or kallikrein, or a derivative of these proteases. The preparation according to the invention containing a Factor X analog in combination with a protease capable of activating the Factor X analog to Factor Xa or the Factor Xa analog can be provided as a composite preparation consisting of a container containing a protease immobilized on a carrier, optionally in the form of a small column or a syringe equipped with a protease and a container containing the pharmaceutical preparation with the Factor X analogue. For the activation of the Factor X analogue, the solution containing the X-Factor analog is, for example, passed over the immobilized protease. During storage of the preparation, the solution containing the Factor X analog is preferably kept apart from the protease. The preparation according to the invention can be present in the same container as the protease, with the components, however, which are spatially separated by an impermeable separation wall which can be easily removed in the case of use. The solutions can also be stored in individual containers and brought into contact only briefly before the application. In a particular embodiment, the protease used for activation is a serine protease naturally involved in blood coagulation, such as Factor Xlla or Factor Xla, Xa, which do not need to be separated from activated Factor Xa before the application but can be applied together with it. The Factor X analogue can be activated to Factor Xa briefly before its immediate use, ie before application to the patient. The activation can be carried out by bringing it into contact with an immobilized protease or by mixing the solutions containing a protease and the Factor X analogue. Therefore it is possible to keep the two components in solution spaced apart from each other, to be mixed by means of a appropriate infusion wherein the components are in contact with each other as they pass through, and therefore activate the respective molecule with respect to Factor Xa or the Factor Xa analogue. The patient will receive a mixture of Factor Xa and an additional serine protease which has mediated the activation. Special care must be taken with regard to dosage, because the endogenous Factor X can also be activated by the additional administration of a serine protease, which could lead to a shorter coagulation time. According to a preferred embodiment, the pharmaceutical preparation is provided in an appropriate device, preferably an application device, either in a frozen or lyophilized liquid form. A suitable application device can be a double-compartment syringe as described in AT 366 916 or AT 382 783. According to one aspect of the invention, the preparation contains an analogue of Factor X having a modification that allows the activation of the Factor X analog to a factor Xa in vivo. The Factor X analogues of the preparation according to the invention have a particular modification that represents a recognition / cleavage site for a protease selected from the group of serine proteases, such as Factor Xlla, Factor Xla, Factor Xa, Factor or kallikrein, and they are segmented in vivo by one of the proteases to the natural Factor Xa or the Factor Xa analogue. Particularly for therapeutic use, such X Factor analogues are advantageous because they have a recognition / cleavage site for a protease which is independent of the tissue / Factor Vlla complex and the tenase complex within the coagulation cascade. Therefore, the preparation according to the invention can be used to control hemorrhage in patients with Factor IX and Factor VII deficiency as well as Factor VIII. Patients suffering from a blood coagulation disorder due to Factor XI or Factor XII deficiency should not be provided with pharmaceutical preparations that contain the Factor X analogue which can be activated by Factor Xlla or Xla factor. In the case of Factor XI deficiency, for example, the Factor X analog that has an Xlla Factor cleavage site could be used. According to another aspect of the invention, the preparation according to the invention optionally contains a blood factor in the form of a zymogen or an active serine protease as an additional component. The preferred additional components are those components that have a Factor VIII derivative activation. Among them, in particular, Factor II, Factor VII, Factor IX, Factor VIII, Factor V and / or the active serine proteases thereof. The additional components can also be phospholipids, Ca ions, etc. According to a particular embodiment of the invention, the preparation according to the invention contains at least one additional component having the activity of derivatizing Factor VIII. The preparation according to the invention can be provided as a pharmaceutical preparation having Factor Xa activity as a single component preparation or in combination with other factors such as the preparation of multiple components. Prior to processing in a pharmaceutical preparation, the purified protein is subjected to the usual quality controls and brought into a therapeutically administrable form. In the recombinant production, the purified preparation is tested particularly to verify the absence of the cellular nucleic acids and derivatives of the expression vector, preferably according to a method as described in EP 0 714 987.

Since, in principle, any biological material can be contaminated with infectious agents, the preparation is optionally treated for the inactivation or depletion of the viruses to produce a safe preparation. According to a further aspect of the present invention, a preparation containing the Factor Xa analog having high structural integrity and stability is provided, which is particularly free of the intermediate compounds of the inactive Factor X / Xa analog and the self-proteolytic degradation products, and is obtainable because an X-Factor analogue of the type defined above is activated and prepared to give the appropriate preparation. A further aspect of the invention relates to the use of a preparation of the type defined above in the preparation of a pharmaceutical agent. A therapeutic agent containing the Factor X analogue or the Factor Xa analogue according to the invention is particularly useful in the treatment of patients suffering from blood coagulation disorders, such as patients suffering from hemophilia and , additionally, they may have developed inhibitory antibodies against Factor VIII and / or Factor IX, commonly used for treatment, and, in particular, as a preparation having the activity of derivatizing Factor VIII. A further aspect of the invention relates to the use of a nucleic acid containing the coding sequences of the Factor X analogues according to the invention for the preparation of a medicament. Since the nucleic acid has suitable expression control sequences, it can be applied as a pure nucleic acid, integrated into a recombinant expression vector, or linked to a carrier, either a phospholipid or a viral particle. The nucleic acid can be used for the preparation of a therapeutic agent which is particularly useful in the treatment of patients suffering from blood coagulation disorders, such as patients suffering from hemophilia or hemophilia and who have developed inhibitory antibodies. It is also possible to use the nucleic acid in gene therapy. A further aspect of the invention relates to a method for the preparation of the Factor X analogue according to the invention and to a preparation containing the Factor X analogue according to the invention. A sequence encoding the Factor X analog is inserted into an appropriate expression system, and the appropriate cells, preferably the permanent cell lines, are transfected with the recombinant DNA. The cells are cultured under optimal conditions for gene expression, and Factor X analogues are isolated either from a cell culture extract or from the cell culture supernatant. The recombinant molecule can be further purified by all known chromatographic methods, such as anionic or cation exchange, affinity or immunoaffinity chromatography or a combination thereof. For the preparation of Factor X analogues according to the invention, the complete cDNA encoding Factor X is cloned into an expression vector. This is done in accordance with generally known cloning techniques. Subsequently, the nucleotide sequence coding for Factor X is modified in such a way that the coding sequences in the region of the activation peptide and optionally also in the region of the C-terminal peptide, are modified in such a way that a Factor X molecule of the type defined above can be produced. This is effected by genetic engineering techniques known from the prior art, such as directed, specific in vitro mutagenesis, or deletion of the sequences, for example by restriction digestion by the endonucleases and insertion of other charged sequences, or by PCR. The X Factor mutants thus prepared are then inserted into an appropriate expression system for recombinant expression and are expressed. Factor X analogues according to the invention can also be prepared by chemical synthesis. Factor X analogues are preferably produced by recombinant expression. They can be prepared by genetic engineering with any usual expression systems, such as, for example, permanent cell lines or viral expression systems. Permanent cell lines are prepared by the stable integration of foreign or foreign DNA into the chromosome of the host cell, for example, Vero, MRC5, CHO, BHK, 293, Sk-Hepl, particularly liver and kidney cells, or by an episomal vector derived, for example, from the papilloma virus. Viral expression systems, such as, for example, Vaccinia virus, Baculovirus or retroviral systems, may also be employed. Like cell lines, Vero, MRC5, CHO, BHK, 293, sk-Hepl, gland, liver and kidney cells are generally used. As eukaryotic expression systems, yeasts, endogenous gland cells (for example the glands of transgenic animals) are used and other types of cells can also be used. Of course, the transgenic animals can also be used for the expression of the polypeptides according to the invention or derivatives thereof. For the expression of the recombinant proteins, the CHO-DHFR cells "have proven to be particularly useful (Urlaub et al., 1980, Proc. Nati. Acad. Sci., USA, 77: 4216-4220). Recombinant preparation of Factor X analogues according to the invention, prokaryotic expression systems can also be used Systems that allow expression in E. coli or B. subtilis are particularly useful Factor X analogues are expressed in the respective expression systems under the control of a suitable promoter For expression in eukaryotes, all known promoters are suitable, such as SV40, CMV, RSV, HSV, EBV, β-actin hGH or inducible promoters, such as, for example, hsp or the metallothionein promoter Factor X analogues are preferably expressed under the control of the β-actin promoter in the CHO cells.

According to one embodiment of the invention, the production method of the preparation of the invention comprises the steps of: providing a DNA encoding a Factor X analog, transforming a cell with the recombinant DNA, expressing the Factor X analog, optionally in the presence of a protease, isolating the Factor X analog, and optionally purification by means of a chromatographic method. According to one embodiment of the process, the X-Factor analogue is isolated as a double-stranded molecule. The X-Factor analogue is expressed in a cell that allows the processing of the pro-Factor X analog to the double-chain Factor X analog. The cell is preferably a cell that expresses a protease capable of processing the precursor of Factor X, for example a dibasic protease, such as furin or a derivative thereof. To improve or increase the processing efficiency, the cell can be further modified in such a way that its expression of the protease is improved. For example, this can be effected by the co-expression of a corresponding dibasic endoprotease, such as furin / PACE, Kex2 or a derivative thereof. The X-Factor analogue according to the invention can also be expressed in a cell having a normal endogenous protease concentration, i.e. a suboptimal concentration for processing, which leads to incomplete processing up to the double-stranded form. In this case, since the single chain X-Factor analog is secreted into the cell supernatant as described above, the subsequent processing to a light and heavy chain is effected by co-culturing the expression cells with protease or bringing them into contact with a optionally immobilized protease. The supernatant of the cells can also be passed over a carrier matrix having the protease bound thereto, thereby giving the double chain X-Factor analog in the eluate. The reagents can also be mixed in solution, incubated for a certain period of time, and then the protease can be removed, for example by means of an affinity matrix. The double-stranded X-Factor analog thus obtained can be isolated, purified and subsequently stored stably until it is used afterwards, as described above. In a particular embodiment, the optionally double-stranded X-Factor analogue is brought into contact in vitro with a protease selected from the group of endoproteases, such as kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE 4, LPC / PC7, the group of serine proteases, such as Factor Xlla, Factor Xla, Factor Xa, Factor lia or kallikrein, or a derivative of these proteases, under conditions in which the analogue of Factor X is activated to natural Factor Xa or to an analog of Factor Xa. According to one embodiment, the activation is achieved by a chromatographic step, wherein the protease is immobilized on a carrier. The purified double-strand X-Factor analogue is passed over a matrix having the protease bound thereto, and the purified Factor Xa is isolated from the eluate. According to another embodiment, the components are mixed, and the protease is removed selectively from the mixture. Of course, also a combination of single-stranded pro-Factor X analog processing to the double-chain Factor X analog form and activation to Factor Xa in a single process is possible. The X-Factor analog of a single chain or a precursor thereof is brought directly into contact with a dibasic protease, preferably furin or a derivative thereof that allows processing to a light and heavy chain and activation to Factor Xa. The Factor X analogue having no cleavage site for furin or a derivative thereof in the activation peptide is brought into contact optionally with another protease, different from the first proteases, which allows activation. The proteases may be present in a mixture for example of furin and Factor Xla. Activation can also be effected by a combination of the two steps by means of directly interconnected and sequentially arranged or distributed devices, preferably carriers, such as columns, over which the protease (s) is (are) immobilized. In the first carrier X Factor is segmented to a heavy and light chain, and in the second carrier X Factor is activated to Factor Xa by the immobilized protease. The carriers can be coupled or attached by directly connecting the output of the first column with the input of the second column. The reaction conditions for the reaction (s) of processing and activation can be easily optimized by a person skilled in the art according to the experimental setup and the given basic conditions. For the contact time, the flow velocity of the present reagents is of particular importance. Ideally, it should be between 0.01 ml / min and 1 ml / min. Additional important parameters are temperature, pH value and elution conditions. After the passage, the activated Factor Xa can optionally be further purified by selective chromatography. It is particularly advantageous to conduct the process with the protease bound to a carrier, because when a carrier is used, preferably the chromatographic columns, the installation or adjustment of the reaction allows an additional purification step. According to a further aspect of the preparation of an X Factor analog, the Factor X analog is isolated as a single chain molecule. The Factor X analogue is expressed in a cell which does not support the processing of a single chain of Factor X in the heavy-light chain. The cell is preferably deficient in a dibasic endoprotease, such as kexin, furin, PACE. In the manufacture of the invention, it was found that one of the essential proteases responsible for the cleavage of Factor X in the light and heavy chain is furine. From such a mutant cell deficient in the endoprotease, the Factor X analog can be isolated as a single chain molecule. An X Factor analogue thus isolated and optionally purified is subsequently brought into contact with a protease selected from the group of endoproteases, such as Kexin / Kex2, Furin / PACE, PC1 / PC3, PC2, PC4, PACE 4, LPC / PC7. , under conditions in which the X Factor analogue of a single chain is segmented to the double chain X Factor form. Factor X analogs of the invention having a modification in the activation peptide region that allow segmentation by one of these endoproteases can be optionally activated directly to Factor Xa or Factor Xa analogue by this method, carrying it in contact with the endoprotease. Factor X analogues according to the invention having a modification in the activation peptide region that allow segmentation by a serine protease or kallikrein, are brought into contact with an additional protease, different from the first one, after the preparation of the double chain X Factor analog, and are activated to the Factor Xa analog. According to one aspect of the invention, in the process a preparation containing the active Factor Xa or the active Factor Xa analog is obtained by subjecting an X Factor analog prepared as described above to an activation step and the additional processing of the activated peptide to a purified preparation, which is optionally formulated as a pharmaceutical composition. With Factor X analogues according to the invention which are activated by a process as described above to Factor Xa, purified Factor Xa or the Factor Xa analog having high structural integrity and stability and which are particularly free from inactive intermediate X / Xa compounds, they are obtained. The invention is described in more detail by the following examples and figures, with the invention, however, which is not restricted to these particular examples. Example 1 illustrates the construction and expression of rFactor X; Example 2 illustrates the processing of rFactor X in a heavy and light chain by furin; Example 3 illustrates the processing of pro-Factor X by means of the immobilized protease; Example 4 illustrates the activity of rFactor X processed in vitro; Example 5 illustrates the expression of rFactor X in the furin deficient cells; Example 6 illustrates the construction of the analogs of rFactor X; Example 7 illustrates the determination of the N terminals of the Factor X processing products; Example 8 illustrates the expression and characterization of the Factor X analogue carrying the Arg-Arg-Lys-Arg / Ile cleavage site with furin (rFX1 * 71); Example 9 illustrates the in vitro activation of the rp? RRKR / I protein derived from r-furin; Example 10 illustrates the functionality of rf? RRKR I of the recombinant FX analog activated in vitro; Example 11 illustrates the in vitro activation of the rFX analog that carries the cleavage site Asp-Phe-Thr-Arg / Val for Factor Xla.

Figures: Fig. 1: Nucleotide and amino acid sequence of the Factor X Fig. 2: Schematic representation of Factor X analogs having modified protease cleavage sites in the activation peptide region Fig. 3: Schematic representation of the phAct-rFX expression vector Fig. 4: Stain analysis of Western rFactor X expressed in CHO cells before and after amplification Fig. 5: Western blot analysis of rFactor X after in vitro cleavage by furin derivatives Fig. 6: Western blot analysis of molecules of rFactor X expressed in the furin-containing and furin-deficient cells Fig. 7: Schematic representation of rFX / rFXA analog constructs having modified C-termini of the heavy chain Fig. 8: Schematic representation of the N terminals of the rFactor X processing products of CHO cells containing furine and deficient in furin prior to and after further treatment c on recombinant furin Fig. 9: Western spotting analysis of the rFactor FX RRKR / I expressed in CHO cells Fig. 10: Western blot analysis of the rFactor RRKR / I FX after in vitro activation with the furin derivative Fig. 11: Western blot analysis of rFactor FXDFTR / V after activation in vitro with the furin derivative. Expression vectors were prepared by standard cloning techniques (Maniatis et al., "Molecular Cloning" - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, E.U.A., 1983). The preparation of the DNA fragments by means of the polymerase chain reaction (PCR) followed by the general methods (Clackson et al., 1991, PCR A practical approach, Ed. McPherson, Quirke, Taylor, pp. 187- 214).

Example 1 : Expression and processing of the single-chain rFX to the light / heavy chain of rFX to. Construction of the rFX expression vector For the preparation of the recombinant FX (rFX), the FX cDNA was isolated from a lambda cDNA library of the human liver as described by Messier et al. (1991, Gene 99: 291-294). A DNA fragment was amplified from a positive clone by means of PCR with oligonucleotide # 2911 (5'-ATTACTCGAGAAGCTTACCATGGGGCGCCCACTG-3 ') (SEQ ID NO: 1) as the 5'-primer and oligonucleotide # 2912 ( 5'-ATTACAATTGCTGCAGGGATCCAC-3 ') (SEQ ID NO: 2) as the 3'-primer. This DNA fragment contains the FX coding sequence of 1,467 kB and 39 bp of the 3 'untranslated region, flanked by an Xhol cleavage site at the 5' end and an Mfel cleavage site at the 3 'end. In addition, the ACC sequence was incorporated into the front of the FX ATG by means of primer # 2911 which leads to an optimal Kozac translation initiation sequence. Subsequently, this PCR product was cloned as the XhoI / Mfel fragment in the phAct expression vector segmented with SalI and EcoRI. The resulting expression plasmid was designated as phAct-rFX (Fig. 3). The phAct expression vector comprises the 78 bp promoter of human beta-actin 5'UTR and the intron, a multiple cloning cleavage site, and the SV40 polyadenylation site. b. Expression of rFX in CHO cells To establish a stable rFX expression cell line, CHD cells deficient in dhfr were co-transfected with the phAct-rFX expression plasmid and the selection marker plasmid pSV-dhfr. For all additional performance and expression analyzes, the cells were incubated with serum free selection medium in the presence of μg / ml of vitamin K for 24 hours. The expression of rFX in the clones of the resulting cells was detected by the amount of the antigen (ELISA, Asserachrom, Boehringer Mannheim), and the recombinant protein was characterized with SDS-PAGE (Figures 4A and B). As can be seen in the Western spotting (Figure 4A), in the initial clones and subclones thereof there is the recombinant FX protein present in the form of a light chain (LC) of 22 kD and a heavy chain (HC) of about 50 kD, which are identical in size with the plasma X Factor chains. In addition, one protein band is visible at 75 kD, which corresponds to the single chain (SC) molecule and the presence of which in the CHO cells transfected with FX (Wolf et al., J. Biol. Chem. 266: 13726-13730, 1991) and in human plasma (Fair et al., Blood 64: 194-204, 1984) have been described. For the preparation of high expression clones, the initial clones were amplified with increasing amounts of methotrexate and subsequently subcloned until stabilization. Expression could be increased from approximately 200-500 ng / 10 E6 cells and 1 μg / ml, respectively, to 78 μg / 10 E6 cells and 120 μg / ml, respectively, per 24 hours. Western blot analyzes of these supernatants of the high expression cell clone (Figures 4B and 5A, band 2) show increased amounts of the single chain rFX molecule and the presence of additional light chain forms. In addition to the 22 kD form of the light chain, which corresponds to the plasma form (fully carboxylated and without propeptides), there are three additional light chain variants of approximately 21 kD, 22.5 kD, and 20 kD present. By means of the N-terminal sequencing of the recombinant material, the heterogeneity of the light chain in these clones was attributed to the incomplete segmentation of the propeptide (here: approximately 50% of the rFX material) and the hypocarboxylation (here: approximately 50% of the rFX). The 21 kD protein is a form that contains the propeptide, hypocarboxylated, and the 20 kD protein is a free form of propeptides, hypocarboxylated, of the light chain, while the band of 22.5 kD represents the fully carboxylated propeptide but containing the LC form.

Example 2: Processing of single chain rFX in the heavy / heavy chain of rFX by the furin derivatives Due to the similarity of the cleavage sites of the X-Factor propeptide / N-terminus of the light chain (RVTR ^ A) and between the light / heavy chain (RRKR> ls) to the consensus recognition sequence of furin ( RXK / RR X), it seems possible to improve the in vitro processing of the single chain as the propeptide containing the rFX molecules by the derivatives of rfurin. In the literature, proteases are suspect of the two processing steps, which, however, are not furins (Rehemtulla et al., 1992, Blood 79: 2349-2355; Wallin et al., 1994, Thromb. Res. 1994: 395-403). The cell culture supernatant of CHO-RFX and CHO-rfurin? TMdxHis (patent application EP 0 775 750 A2) as well as CHO-rFX and the non-transfected CHO (as the negative control) were mixed at a ratio of 1: 1 and incubated at 37 ° C. The aliquots of the reaction mixtures were tested for the processed rFX before incubation (t = 0) and after several incubation periods (t = 2, 4, 6 hours) by Western blot analysis (Figure 5 ). RFX was detected in the cell culture supernatants by means of an anti-human FX antiserum (Fig. 5A) and a monoclonal antibody specific for the FX light chain (Fig. 5B). As opposed to the CHO-rFX / CHO mixture, the CHO-rFX / CHO-rfurin shows almost complete processing already after 2 hours of incubation at 37 ° C (Figure 5A, strip 7, Fig. 5B, strip 8 ). The single chain rFX is widely converted into light and heavy chain form. In the area of the light chain, only the processed free propeptide forms of 22 kD (carboxylated form) and 20 kD (hypocarboxylated form) were found at a ratio of approximately 50:50. By optimizing cell culture conditions, this ratio can be improved in favor of the carboxylated form. The correct segmentation of the pro-sequence between Arg-1 and Ala + 1 and the homogeneity of the N-terminus of the light chain were determined by the sequencing of the N-terminal. In the control experiment, where the CHO-rFX was mixed with the CHO supernatants, no change in the configuration of the rFX band is visible even after 6 hours of incubation (Fig. 5A, band 5, Fig. 5B, band 6). This provides that the rfurin in the supernatant of the CHO cells is biologically active and can process the propeptide as well as the heavy / light chain of rFX.

Example 3: Processing of Factor X by means of rfurin immobilized with gel with chelate tentacles.

To investigate whether a substrate can be segmented by the rfurin derivative bound to the column, a study was carried out as to whether the Fractogel EMD® tentacle gel (Merck) can be used in an experimental facility alternatively to the Ni2 + -NTA agarose, as a matrix of the column. When the metal ions are spaced further from the matrix of the current column than in the Ni2 + -NTA agarose, the steric access of the substrate to the bound rfurin derivative could be improved. In the present installation, pro-Factor X was processed by the rfurine derivative attached to the tentacle gel: the Fractogel EMD® tentacle gel was loaded with Ni2 + ions according to the producer's instructions and equilibrated with the medium of the cell culture free of serum, fresh. Subsequently, the column was loaded with the supernatant of the serum free CHO-rfurine derivative. The washing steps were carried out by serum-free cell culture medium containing increasing concentrations of imidazole up to 40 mM. Then pro-Factor X was passed over the column as the free CHO supernatant of the serum. Processing of pro-Factor X to double-chain factor X was detected in the column effluent by Western blot analysis with the specific Factor X antiserum.

Example 4 Activity of recombinant factor X processed in vitro The recombinant Factor X precursor was incubated with and without rfurin at 4 ° C. At different time intervals, the samples were taken and frozen at -20 ° C. After the incubation was complemented (after 4 days), all samples were tested to verify FX activity using the Coatest FX Set or Set (Chromogenix). 50 μl of each supernatant were mixed with 50 μl of human plasma deficient in FX, and rFX was activated with viper venom (RW) to rFXa in the presence of CaCl2 according to the instructions of the producer; the rFXa is then hydrolysed to the chromogenic substrate (S-2337) and leads to the release of the yellow-colored paranitroaniline. When the amount of rFXa and the intensity of the color are proportional to each other, the amount of the cell culture supernatant of rFX / ml which is activated to rFXa, can be determined by means of a calibration line interpolated from the values of a series. of plasma dilutions. Using these results and the known amount of rFX antigen (ELISA data), the ratio of rFactor X activated to Factor Xa can be calculated in%. The results are presented in Table 1. To exclude the non-specific proteolytic activity in the supernatants of CHO and CHO-rfurin, the mixture of these two supernatants of the cell culture was also tested. Even after 4 days, the CHO-RFX incubated with CHO supernatants (without rfurin) as the control, did not exhibit substantial change in rFXa activity, which was approximately 800 mU / ml and corresponded to 55% - 61% of functional rFX due to experimental variations. When, in comparison, the CHO-rFX was incubated with CHO-rfurin, the activity of rFX increased gradually during the incubation, rising from 61% (T = 0) to 86% (Table 1). This proves that in the in vitro processing of CHO-rFX from the high expression clones using the rfurin derivative, the proportion of rFX that can be activated to the functional rFXa is substantially improved.

Table 1 Example 5: Expression of Recombinant Factor X in Furin Deficient Cells As shown in the previous examples, in the case of the Factor X precursor protein, furin measured the cleavage of the propeptide as well as the cleavage of the single chain to the light / heavy chain in vitro. This suggests that these segmentations are effected endogenously in the cells by the appropriate furin with a variable efficiency depending on the amount of rFactor X expressed. This in turn leads to the production of a mixture of heterogeneous rFactor forms. One way to prepare a form of rFactor X molecules which is as homogeneous as possible and also stable, is to prevent cleavage of rFactor X by endogenous proteases, particularly furin, and consequently produce the inactive precursor of rFactor X functionally (which can be transformed into its functionally active forms later by means of downstream processing, ideally directly before its use). This method will be particularly useful in the preparation of FX analogs containing a furin cleavage site in place of the original activation site. In these constructions, such recombinant rFX mutant could be activated in vivo by the endogenous furin and lead to the secretion of more unstable, activated forms of rFX. The degradation of these forms, for example under cell culture conditions of elevated cell lysis by CHO proteases during the storage of cell culture supernatants or the purification process, or by autoproteolysis, could lead to inactive degradation products ( Wolf et al., 1991). This object can be achieved, for example, by supplementing the cell culture medium with agents which can reduce or prevent the activity of intracellular furin. Another way is to use cells which are deficient in a priori furine (Mdhring et al., 1983, Infect. Immun. 41: 998-1009; Ohnishi et al., 1994, J. Virol. 68: 4075-4079; Gordon et al., 1995, Infect. Immun. 63: 82-87). For this purpose, a furin deficient CHO cell clone FDll (Gordon et al., 1995, Infect.Immun 63: 82-87) was co-transfected with 20 μg phAct-FX and 1 μg pUCSV-neo (containing the resistance gene of neomycin in the pUC vector under the control of the SV40 promoter). To obtain stable clones, the medium is supplemented with 0.8 μg of G418 / ml. By comparing rFactor X molecules secreted in the free supernatants of the serum of a furin containing, and a CHO clone deficient in furin, the Western blot shows that the precursor of rFactor X is not processed in the furin deficient cells and only the precursor of the X Factor of a single chain is present (Figure 6); on the contrary, rFactor X is completely processed by "normal" cells to a modest expression, but is processed only to a very limited degree with the highest expression despite endogenous furin. Due to the level of expression of low rFX of the cell clone used for this analysis, the light chain of rFactor X is not visible in this staining.

Example 6: Preparation of the Factor X analogues (at the time of filing the application, the applicant considers this as the best way to carry out the invention). 6. 1. Construction of expression plasmids for the preparation of the Factor X analogue For the preparation of the recombinant rFactor X analogue, the cleavage site of Asn-Leu-Thr-Arg / Ile (amino acids 231 to 235) which serves for activation from Factor X to Factor Xa was replaced by a specific cleavage site for a different protease, such as furin, FXIa, FXa, FXIIa, Fila or kallikrein. The expression plasmids for these Factor X analogues are all derived from the phAct-FX plasmid (described in Example 1). To simplify the cloning of the Factor X expression plasmids, the HindlII-Nael DNA fragment of the phAct-FX expression plasmid, which comprises the coding region of Factor X from the position +1 to +1116, was inserted in the cleavage site of the HindIII / Smal restriction of plasmid pUC19. The resulting plasmid was designated püC / FX. Accordingly, the X-Factor sequence of the nucleotide at positions 508 to 705 (amino acids 160 235) could be easily removed from the pUC / FX plasmid and replaced by several DNA fragments of the mutated Factor X. These DNA fragments are identical with the wild-type X Factor sequence for positions 691 to 705 (amino acids 231 to 235) which code for new cleavage sites.

The wild-type X Factor sequence was removed from the pUC / FX plasmid by means of the digestions of the Bspl20I and BstXI constraints. The 3 'pendant portion of the BstXI site was further removed with the nuclease of the ung seed. (variety of chickpea from tropical Asia) (Biolab). The mutated Factor X DNA fragments were prepared by PCR. The 5 'primer is identical for all cloning and contains the sequence of Factor X from positions 496 to 516. The 3' primers contain a sequence complementary to Factor X (positions 676 to 690) and a non-complementary 5 'end which carries the sequences for a new segmentation site and a restriction targeting site. The amplified PCR product was subsequently digested by the appropriate restriction enzyme (s) and cloned into the prepared pUC / FX vector (see above). Subsequently, the mutated Factor X DNA fragments were recloned by means of HindIII-Agel from the pUC / FX plasmids in the phAct-FX vector. The final constructions are represented schematically in Figures 2.1 and 2.2. The wild type of Factor X is given as a reference construction. The amino acids are given in the form of a one letter code, the imitated positions are further shaded. To prepare the FXIa cleavage site of Asp-Phe-Thr-Arg / Val, oligonucleotide # 1001 (5 '-CCC ACA GGG CCC TAC CCC TGT-3') (SEQ ID No. 3) was used as a 5 'primer, and oligonucleotide # 1002 (5'-ACCA GTT AAC CCT GGT GAA GTC GTT GTC GCC CCT CTC-3') (SEQ ID No. 4) was used as a 3 'primer. Accordingly, the amino acids of Asn, Leu e lie at positions 231, 232 and 235 of the Factor X sequence were replaced by Asp, Thr and Val. The PCR fragment was cut by means of Bstl20I and Hpal (Fig. 2A). To prepare a Fila / Arg / Ser cleavage site, oligonucleotide # 1001 (5 '-CCC ACA GGG CCC TAC CCC TGT-3') (SEQ ID No. 3) was used as a 5 'primer, and Oligonucleotide # 1003 (5'-ACCA TCG CGA CCT GGT CAG GTT GTT GTC3 ') (SEQ ID No. 5) was used as a 3' primer. Accordingly, the amino acid at position 235 was mutated into Ser. The PCR fragment was cut by means of Bspl20I and NruI (Fig. 2B). To prepare an FXIIa cleavage site of Ile-Lys-Pro-Arg / Ile, oligonucleotide # 1001 (5'-CCC AC GGG CCC TAC CCC TGT-3 ') (SEQ ID No. 3) was used as a 5 'primer, and oligonucleotide # 1004 (5' -ACC AGA ATC GAT TCT GGG TTT GAT GTT GTC GCC CCT CTC-3 ') (SEQ.

ID. No. 6) was used as the 3 'primer. Accordingly, amino acids Asn, Leu and Thr at positions 231, 232 and 233 of the FX sequence were mutated into Lie, Lys and Pro. The PCR fragment was cut with Bstl20I and partially with Xmnl (Fig. 2C). To prepare the segmentation site of Ser-Met-Thr-Arg / Ile kallikrein, oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 ') (SEQ ID No. 3) was used as the Oligonucleotide # 1005 (5 '-ACC AGA ATC GAT TCT GGT CAT GCT GTT GTC GCC CCT CTC-3') (SEQ ID No. 7) was used as a 3 'primer. Accordingly, amino acids Asn, Leu at positions 231, 232 of the Factor X sequence were mutated in Ser, Met. The PCR fragment was digested with Bstl20I and partially with Xmnl (Fig. 2D). To prepare the FXa fragmentation site of Pro-Gln-Gly-Arg / Ile, oligonucleotide # 1001 (5'-CCC AC GGG CCC TAC CCC TGT-3 ') (SEQ ID No. 3) was used as a 5 'primer, and oligonucleotide # 1016 (5' -ACC AGA ATC GAT TCT TCC TTG GGG GTT GTC GCC CCT CTC-3 ') (SEQ ID No. 8) was used as the 3' primer. Accordingly, amino acids Asn, Leu and Thr at positions 231, 232 and 233 of the FX protein were mutated to Pro, Gln and Gly. The PCR fragment was trimmed with Nstl20I and partially with Xmnl (Fig. 2H).

To prepare a FXa cleavage site of Met-Lys-Thr-Arg / Ile, oligonucleotide # 1001 (5 '-CCC ACA GGG CCC TAC CCC TGT-3') (SEQ ID No. 3) was used as a 5 'primer, and oligonucleotide # 1014 (5' -ACC AGA ATC GAT TCT CGT TTT CAT GTT GTC GCC CCT CTC-3 ') (SEQ ID No. 9) was used as a 3' primer. Accordingly, amino acids Asn, Leu at positions 231, 232 of the FX protein were mutated to Met, Lys. The PCR fragment was cut with Bstl20I and partially with Xmnl (Fig. 2E). To prepare an FXa cleavage site of Ile-Glu-Gly-Arg / Ile, oligonucleotide # 1001 (5 '-CCC ACA GGG CCC TAC CCC TGT-3') (SEQ ID No. 3) was used as a 5 'primer, and oligonucleotide # 1015 (5' -ACC AGA ATC GAT TCT TCC CTC GAT GTT GTC GCC CCT CTC-3 ') (SEQ ID No. 10) was used as a 3' primer. Accordingly, amino acids Asn, Leu, Thr at positions 231 to 233 of the FX protein were mutated to lie, Glu, Gly. The PCR fragment was trimmed with Bstl20I and particularly with Xmnl (Figure 2F). To prepare a furin cleavage site of Arg-Arg-Lys-Arg / Ile, oligonucleotide # 1001 (5 '-CCC ACA GGG CCC TAC CCC TGT-3') (SEQ ID NO: 3) was used as a 5 'primer, and oligonucleotide # 1006 (5' -ACC AGA ATC GAT TCT TTT CCT CCT GTT GTC GCC CCT CTC-3 ') (SEQ ID No. 11) was used as a 3' primer. Accordingly, the amino acids Asn, Leu and Thr at positions 231 to 233 were mutated to Arg, Arg and Lys. The PCR fragment was cut with Bspl20I and partially with Xmnl (Fig. 2G). To prepare a furin cleavage site of Arg-Val-Arg-Arg / Ile, oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 ') (SEQ ID No. 3) was used as a 5 'primer, and oligonucleotide # 1007 (5' -ACC AGA ATC GAT TCT CCT CAC CCT GTT GTC GCC CCT CTC-3 ') (SEQ ID No. 12) was used as a 3' primer. Accordingly, amino acids Asn, Leu and Thr at positions 231 to 233 were mutated to Arg, Val and Arg. The PCR fragment was trimmed with Bspl20I and partially with Xmnl (Figure 2G). To prepare a furin cleavage site of Arg-Arg-Arg-Arg / Ile, oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 ') (SEQ ID No. 3) was used as a 5 'primer, and the oligonucleotide # 1008 (5' -ACC AGA ATC GAT TCT CCT CCT CCT GTT GTC GCC CCT CTC-3 ') (SEQ ID No. 13) was used as a 3' primer. Accordingly, amino acids Asn, Leu and Thr at positions 231 to 233 were mutated to Arg, Arg "and Arg. The PCR fragment was trimmed with Bspl20I and partially with Xmnl (Fig. 2G).

To prepare a furin cleavage site of Arg-Pro-Lys-Arg / Ile, oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 ') (SEQ ID No. 3) was used as a 5 'primer, and oligonucleotide # 1009 (5 '-ACC AGA ATC GAT TCT TTT GGG CCT GTT CTC GCC CCT CTC-3') (SEQ ID No. 14) was used as a 3 'primer. Accordingly, amino acids Asn, Leu and Thr at positions 231 to 233 were mutated to Arg, Pro and Lys. The PCR fragment was cut with Bspl20I and partially with Xmnl (Fig. 2G). To prepare a furin cleavage site of Ile-Arg-Lys-Arg / Ile, oligonucleotide # 1001 (5 '-CCC ACA GGG CCC TAC CCC TGT-3') (SEQ ID No. 3) was used as a 5 'primer, and oligonucleotide # 1010 (5' -ACC AGA ATC GAT TCT TTT CCT GAT GTT GTC GCC CCT CTC-3 ') (SEQ ID No. 15) was used as a 3' primer. Accordingly, the amino acids Asn, Leu and Thr at positions 231 to 233 were mutated to lie, Arg and Lys. The PCR fragment was cut with Bspl20I and partially with Xmnl (Fig. 2G). To prepare a furin cleavage site of Arg-Ser-Lys-Arg / Ile, oligonucleotide # 1001 (5'-CCC AC GGG CCC TAC CCC TGT-3 ') (SEQ ID No. 3) was used as a 5 'primer, and oligonucleotide # 1011 (5' -ACC AGA ATC GAT TCT TTT GCT CCT GTT GTC GCC CCT CTC-3 ') (SEQ ID No. 16) was used as a 3' primer. Accordingly, the amino acids Asn, Leu and Thr at positions 231 to 233 were mutated to Arg, Ser and Lys. The PCR fragment was cut with Bspl20I and partially with Xmnl (Figure 2G). To prepare a furin cleavage site of Arg-Val-Thr-Arg / Ile, oligonucleotide # 1001 (5 '-CCC ACA GGG CCC TAC CCC TGT-3') (SEQ ID No. 3) was used as a 5 'primer, and oligonucleotide # 1012 (5' -ACC AGA ATC GAT TCT GGT CAC CCT GTT GTC GCC CCT CTC-3 ') (SEQ ID No. 17) was used as a 3' primer. Accordingly, amino acids Asn, Leu at positions 231, 232 were mutated to Arg, Val. The PCR fragment was cut with Bspl20I and partially with Xmnl (Fig. 2G). To prepare a furin cleavage site of Arg-Leu-Lys-Arg / Ile, oligonucleotide # 1001 (5'-CCC AC GGG CCC TAC CCC TGT-3 ') (SEQ ID No. 3) was used as a 5 'primer, and oligonucleotide # 1013 (5' -ACC AGA ATC GAT TCT TTT GAG CCT GTT GTC GCC CCT CTC-3 ') (SEQ ID No. 18) was used as a 3' primer. Therefore, the amino acids Asn and Thr in the positions 231 and 233 were mutated to Arg and Lys. The PCR fragment was cut with Bspl20I and partially with Xmnl (Fig. 2G). To prepare a Thr-Ser-Thr-Arg / Ile cleavage site, oligonucleotide # 1001 (5 '-CCC ACA GGG CCC TAC CCC TGT-3') (SEQ ID No. 3) was used as a primer 5 ', and oligonucleotide # 1017 (5' -ACC AGA ATC GAT TCT CGT GCT CGT GTT GTC GCC CCT CTC-3 ') (SEQ ID No. 19) was used as a 3' primer. Accordingly, amino acids Asn, Leu at positions 231, 232 of the FX protein were mutated to lie, Lys. The PCR fragment was cut with Bstl20I and partially with Xmnl (Fig. 21). 6. 2. Construction of the expression plasmids for the preparation of the FXß analog These constructs were derived from the Factor X analogue constructs described above by introducing a TGA stop codon at position 470. The amino acids from position 457 to the stop codon at the cDNA level were removed by Spel digestion and partial digestion. of BstEII and replaced by the pair of oligonucleotides # 0003 (5 '-GTC ACC GCC TTC CTC AAG TGG ATC GAC AGG TCC ATG AAA ACC AGG TGA A-3') (SEQ ID No. 20) and # 0004 (5 '-CTA GTT CAC CTG GTT TTC ATG GAC CTG TCG ATC CAC TTG AGG AAG GCG-3') (SEQ ID No. 21). Figure 7 is a schematic representation of the constructs of the Factor Xß analog. To simplify the Figure, all Xβ analogs are represented as a general construct in which the variable amino acids in the regions of the cleavage site are shown as a shaded "X". 6. 3. Construction of the expression plasmids for the preparation of the FXa analog The activation of Factor X by the removal of the activation peptide of 4.5 kDa at the N-terminal end of the heavy chain, leads to the generation of the Factor Xaa form. This form is subsequently converted into the FXaß form by autoproteolytic activity and cleavage at the C-terminus of the heavy chain between Arg469 and Gly470. For the preparation of the X-Factor expression plasmids that lead to the production of Factor X, which will be present after activation exclusively in the FXaa form with the intact β-peptide, the amino acid Arg469 was used to Lys, so that this region of the heavy chain can not be further segmented. For this purpose, the C-terminal amino acid sequence of Factor X from position 1363 to the stop signal was removed by digestion with partial BstEII-Spel and replaced by two pairs of ligated oligonucleotides. Oliglonucleotide # 0005 (5 '-GTC ACC GCC TTC CTC AAG TGG ATC _GAC AGG TCC ATG AAA ACC AAG GGC TTG CCC AAG-3') (SEQ ID No. 22) and oligonucleotide # 0006 (5 '-TTG GCC TTG GGC AAG CCC TTG GTT TTC ATG GAC CTG TCG ATC CAC TTG AGG AAG GCG-3 ') (SEQ ID No. 23) were ligated with the oligonucleotide # 0007 (5'-GCC AAG AGC CAT GCC CCG GAG GTC ATA ACG TCC TCT CCA TTA AAG TGA GAT CCC A-3 ') (SEQ ID No. 24) and oligonucleotide # 0008 (5' -CTA GTG GGA TCT CAC TTT AAT GGA GAG GAC GTT ATG ACC TCC GGG GCA TGG CTC-3 ') (SEQ ID No. 25). The mutation of the amino acid Arg469 is introduced by the pair of oligonucleotides # 0005- # 0006. Figure 7 shows a representation of the FX analogs.

Example 7: Determination of the N terminals of the X Factor and processing products with and without r-furin Recombinant Factor X was expressed in the CHO cells with the endogenous furin, as described in Example 1, and in the furin deficient cells, as described in Example 5. rFactor X was isolated from the cell culture supernatant of the high-expressing CHO-rFX clones, which a) were not treated, b) were incubated at 37 ° C for an additional 12 hours, c) were incubated with the CHO-rfurin supernatant at 37 ° C for a 12-hour period, as well as from the cell culture supernatant of the CH0-FD11 clones which d) were not treated, and e) were incubated with the supernatant of CHO-rfurine at 37 ° C for a period of 12 hours . The N-terminal amino acids of Factor X and the processing products of the individual reaction mixtures a) to e) were determined by the Edman analysis. Figure 8 shows a schematic representation of the results. The rFactor X of CHO cells that are highly expressed is present in the form of light and heavy chains, mature, as well as in the form of a single chain, which still partially contains, the propeptide. After incubation of these cell culture supernatants for 12 hours at 37 ° C (b), the additional defective N terminals of the rFX light chain having 3 additional Val38-Thr39-Arg40 amino acids are formed, as already described by Wolf et al. (1991, J. Bio, Chem. 266: 13726-13730). These cryptic ends are also found when the rFX material of untreated CH0-FD11 cells (d) is subjected to sequencing. This observation shows that the formation of these defective N terminals can be prevented by optimized conditions, i.e. cell culture conditions, storage and purification processes to minimize the proteolysis of rFX by CHO proteases. Contrary to the purified material of the CHO (a and b) cells, the rFX of the non-amplified furin deficient (d) cells are only present in the form of the unprocessed single-chain precursors. which correspond to the portion of the propeptide are not found either.This shows that a single-chain rFX precursor is no longer processed to the light / heavy chain in the CHO cells deficient in furin (d), which suggests a central role of the endoprotease furin in this in vivo processing step, furthermore, it is shown that the rFX molecules containing the propeptide are also processed in the CHO cells deficient in furin, ie that furin not only plays a role essential role in this in vivo processing step After the incubation of rFX from CHO cells and from CHO-FD11 (e) cells in the presence of furin, only c light and heavy chains that have the correct N terminals. This provides that the single-stranded FX precursors as well as the rFX molecules containing the propeptides are converted to mature, homogeneous Factor X by in vitro processing. Accordingly, the X Factor processed in the presence of furin exhibits exceptional homogeneity and structural integrity.

Example 8: Expression and characterization of the FX analogue of the furin cleavage site Arg-Arg-Lys-Arg / Ile (rFXRRKR I) The FX expression plasmid having the cleavage site Arg-Arg-Lys-Arg / Ile (see Example 6.1, Fig. 2G) and the psV / dhfr of the selection plasmid, were co-transfected into the CHO cells, as was described in Example 1, to prepare the protein of r? RRKR /? recombinant. Western blot analysis of cell culture supernatants (Fig. 9) shows that the recombinant protein is present mainly in the double-stranded form. When compared to plasma FX, the heavy chain runs at 46 kD instead of 50 kD, which can be attributed to changes in the glycosylation of the recombinant protein. In addition, the small amounts of the single chain precursor (SC) and the LC4 isoform of the light chain become apparent, as was already observed when the wild-type rFX is expressed (Example l.b.). These molecular forms of the rFX analog suggest that the processing of the single chain FX precursor by the endogenous proteases as well as the? -carboxylation of the light chain are limited. Although the cleavage site t introduced in the FX analog represents a furin consensus sequence, no protein band is visible which could correspond to the activated forms of the protein (35 kD, 31 kD). The structure of the region of the cleavage site or the next amino acid sequence appears to represent a suboptimal configuration for the processing of the activation site modified by furin in vivo.

Example 9: In vitro activation of rFXRSKR '1 protein by r-furin derivatives The ability of the recombinant FX analog to be activated to the (35 kD) and β (31 kD) forms by r-furin in vitro was tested as described in Example 2 by blending experiments. The tests were different, however, in that the purified r-furin derivatives of rfurincys-spacer-lOxHis, described in patent application EP 0 775 750-A2, in hepes1 10 mM pH 7.0, 150 mM NaCl, 2 mM CaCl2 and 0.2% BSA were used in place of the CHO-rfurin supernatants. In the control experiment without r-furin, the supernatant of the CHO-rFX analog was mixed with the buffer solution 10 mM hepes pH 7.0, 150 mM NaCl, 2 mM CaCl2 and 0.2% BSA at a ratio of 1: 1. The aliquots of the reaction mixtures before and after an incubation period of 6, 24, 48 and 72 hours (t = 0, 6, 24, 48, 72) at 37 ° C were tested to verify activation of rFX by Western blotting (Figure 10). Although the band configuration of rFXRRKR / I remained unchanged in the absence of the r-furin derivative even after 72 hours of incubation (Figure 10B), in the presence of r-furin, a heavy protein band of 35% was detected. kD that corresponds to the form a of the plasmatic FX (Fig. 10A, strip 9) already appears after 6 hours (Figure 10A, strip 5). In the course of incubation, this form a accumulates and after 72 hours of incubation (Figure 10A, strip 8), approximately 50% of the starting material (HC) has been converted to the activated form. The additional 31 kD heavy protein band, which appears after 24 hours (Figure 10A, strip 6) and corresponds to the β form of the activated plasma FX (Figure 10A, strip 9), shows that the form a generated from of the recombinant FX analogue has autoproteolytic activity and is therefore functional. These results prove that the Arg-Arg-Lis-Arg / Ile fragmentation site of heterologous activation in the rFX analog is specifically recognized and correctly segmented by the r-furin derivatives in vitro and is therefore adapted to activate the analog of rFX to α- and β-FXa molecules.

Example 10: Functionality of rFXHBKR I in vitro activated recombinant FX analog The aliquots of the mixing experiment of Example 9 were tested to verify FXa activity by means of a chromogen test. The aliquots were mixed with the chromogen substance S2337 (600 μM) in 50 mM Tris pH 7.3, 150 mM NaCl, 0.1% BSA. After an incubation period of 3 minutes at 37 ° C, the reaction was stopped by means of 20% acetic acid, and then the OD was measured at 405 nm. The amount of recombinant FXa activity in the reaction mixtures was determined by comparison with a calibration curve, prepared by means of the FXa of the RW-activated plasma. The results of this analysis, the amounts of the antigen used (ELISA data), and the specific activity calculated from it are presented in Table 2. To exclude undesirable amidolytic activities in the r-furin solution and the supernatant of the CHO cell culture, the mixture of the supernatants of the CHO cells not transfected with the purified r-furin derivative was tested to verify the activity of FXa, also (CHO + rfurin). In this mixture, exactly as in the mixture of rFX buffer / analog solution (rF? RRKR /? + Buffer), no FXa activity was detected even after 72 hours of incubation. In contrast, in the case of the reaction of rFXRRKR I / rfurin, a rFXa activity of 56 mU was already detectable after 6 hours of incubation, which increased steadily during the incubation and was quantified up to 133, mU after 72 hours. Although at this point, in accordance with Western blotting, only about half of the rFX analog has been reacted with the α- and β-activated forms (Figure 10A, strip 8), the rFX analogue material activated in vitro showed a much higher specific activity of 190 U / μg than the plasma FX completely activated with RW (153 mü / μg). The increments measured in the activity correspond to the output of the forms a and ß in the Western stain (Figure 10A, strips 5-8). This proves that the cleavage sites of the heterologous protease can be incorporated into the FX, which are recognized and segmented by the respective protease, and that the high quality rFXa (or, optionally, the rFXa analogs having FXa activity. ) in the functional form, can be prepared by recombinant technology.

Table 2 Example 11: In vitro activation of the rFXa analog carrying the FXIa cleavage site of Asp-Phe-Thr-Arg / Val (rF? DFTR / vj by F? Ia d? L plasma A construction of the FX analog was prepared by mutagenesis of the FX activation sequence to a FXIa cleavage site. Subsequently, stable CHO cell clones were established, which express these molecules. The supernatant (from the CHO cell culture containing the rF? DFTR v was mixed with the purified plasma FXIa (100 μg / ml) in the presence of 10 mM Tris pH 7.3, 150 mM NaCl, 8 mM CaCl2, PCPS and 0.1% BSA, and incubated at 37 ° C for several periods of time.As the negative control, the cell culture supernatant was incubated only with the buffer solution containing BSA.The rp? DFTRv protein and the resulting activation products were analyzed by Western blot analysis (Fig. 11) As we can see err the mixture without FXIa before incubation (t = 0), the recombinant protein (Fig. 11, strip 5) is almost identical to plasma FX ( strip 2) in the double-chain form, the only difference is that the heavy chain (HC) has a molecular weight slightly lower than 50 kD, as already observed in the case of rp? RRKR in e? Example 8. During the incubation of this mixture at 37 ° C (Figure 11, strip 6), no significant change in the c appears onfiguration of the band. In the mixture of the CHO-rFX / FXIa analog, the 35 kD and 31 kD protein bands appear rapidly after the addition of the purified FXIa, but prior to the actual incubation of the cell culture supernatant (Fig. 11, strip 3). ). These bands correspond by their size to the forms a and ß of the heavy chain (Fig. 11, strip 9). These two forms increase considerably after 4 hours of incubation with FXIa (Fig. 11, fa to 4). This shows that an FX analog carrying the heterologous protease cleavage site for an active proteolytic enzyme in the coagulation cascade can also be successfully processed by the latter. In addition, the functional activity of the resulting rFXaa analog is successfully demonstrated by the presentation of the rFXaß band, the result of the autoproteolytic activity of the rFXaa analog.

LIST OF SEQUENCES (1. GENERAL INFORMATION: i) APPLICANT: (TO NAME: IMMUNO AG (B STREET: Industriestrasse 67 (C CITY: Vienna (D STATE: Austria (E COUNTRY: Austria (F ZIP CODE: 1220 (A NAME: Michele Himmelspach (B STREET: Breitstetten 19 (C CITY: Leopoldsdorf (D STATE: Austria (E COUNTRY: Austria (F ZIP CODE): 2285 (A NAME: Uwe Schlokat (B STREET: Hauptstrasse 51 (C CITY: Orth / Donau (D STATE: Austria (E COUNTRY: Austria (F ZIP CODE: 2304 (TO NAME: Andreas Fisch (B STREET: Wiener Strasse 14 (C CITY: Orth / Donau (D STATE: Austria (E COUNTRY: Austria (F ZIP CODE): 2304 (A NAME: Friedrich Dorner (B STREET: Peterlinigasse 17 (C CITY: Vienna (D STATE: Austria (E COUNTRY: Austria (F ZIP CODE): 1238 (A NAME: Johann Eibl (B STREET: Gustav Tschermakgasse 2 (C CITY: Vienna (D STATE: Austria (E COUNTRY: Austria (F ZIP CODE): 1180 (ii) TITLE OF THE INVENTION: X Factor Analogs having a modified protease cleavage site (iii) SEQUENCE NUMBER: 27 (iv) READABLE FORM BY THE COMPUTER: (A) TYPE OF MEDIA: Flexible magnetic disk (B) COMPUTER: compatible with IBM PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patentln Relay # 1.0, Version # 1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) [xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 ATTACTCGAG AAGCTTACCA TGGGGCGCCC ACTG 34 [2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear(ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: ATTACAATTG CTGCAGGGAT CCAC 24 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: CCCACAGGGC CCTACCCCTG T 21 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4 ACCAGTTAAC CCTGGTGAAG TCGTTGTCGC CCCTCTC 37 (2) INFORMATION FOR SEQ ID NO: 5: i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic; (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: ACCATCGCGA CCTGGTCAGG TTGTTGTC 28 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) [xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: ACCAGAATCG ATTCTGGGTT TGATGTTGTC GCCCCTCTC 39 INFORMATION FOR SEQ ID NO: 7: [i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7 ACCAGAATCG ATTCTGGTCA TGCTGTTGTC GCCCCTCTC 39 (2) INFORMATION FOR SEQ ID NO: i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) [xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: ACCAGAATCG ATTCTTCCTT GGGGGTTGTC GCCCCTCTC 39 (2) INFORMATION FOR SEQ ID NO: 9: i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: ACCAGAATCG ATTCTCGTTT TCATGTTGTC GCCCCTCTC 39 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) ) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: ACCAGAATCG ATTCTTCCCT CGATGTTGTC GCCCCTCTC 39 (2) INFORMATION FOR SEQ ID NO: 11 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: ACCAGAATCG ATTCTTTTCC TCCTGTTGTC GCCCCTCTC 39 (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12 ACCAGAATCG ATTCTCCTCA CCCTGTTGTC GCCCCTCTC 39 (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) ) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13 ACCAGAATCG ATTCTCCTCC TCCTGTTGTC GCCCCTCTC 39 (2) INFORMATION FOR SEQ ID NO: 14 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic! (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14 ACCAGAATCG ATTCTTTTGG GCCTGTTGTC GCCCCTCTC 39 (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic; (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15 ACCAGAATCG ATTCTTTTCC TGATGTTGTC GCCCCTCTC 39 (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) [xi] ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: ACCAGAATCG ATTCTTTTGC TCCTGTTGTC GCCCCTCTC 39 [2) INFORMATION FOR SEQ ID NO: 17 [i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) [xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17 ACCAGAATCG ATTCTGGTCA CCCTGTTGTC GCCCCTCTC 39 (2) INFORMATION FOR SEQ ID NO: l (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l ACCAGAATCG ATTCTTTTGA GCCTGTTGTC GCCCCTCTC 39 (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) ) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) [xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19: ACCAGAATCG ATTCTCGTGC TCGTGTTGTC GCCCCTCTC 39 [2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 49 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 20: GTCACCGCCT TCCTCAAGTG GATCGACAGG TCCATGAAAA CCAGGTGAA 49 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21: CTAGTTCACC TGGTTTTCAT GGACCTGTCG ATCCACTTGA GGAAGGCG 48 [2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single ( D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22: GTCACCGCCT TCCTCAAGTG GATCGACAGG TCCATGAAAA CCAAGGGCTT GCCCAAG 57 (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23: TTGGCCTTGG GCAAGCCCTT GGTTTTCATG GACCTGTCGA TCCACTTGAG GAAGGCG 57 (2) INFORMATION FOR SEQ ID NO: 24 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 24: GCCAAGAGCC ATGCCCCGGA GGTCATAACG TCCTCTCCAT TAAAGTGAGA TCCCA 55 [2) INFORMATION FOR SEQ ID NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: genomic DNA) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25: CTAGTGGGAT CTCACTTTAA TGGAGAGGAC GTTATGACCT CCGGGGCATG GCTC 54 (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1467 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 1..1467 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 26: ATG GGG CGC CCA CTG CAC CTC GTC CTG CTC AGT GCC TCC CTG GCT GGC 48 Met Gly Arg Pro Leu His Leu Val Leu Leu Ser Ala 'Ser Leu Ala Gly 1 5 10 15 CTC CTG CTG CTC GGG GAA AGT CTG TTC ATC CGC AGG GAG CAG GCC AAC 9 Leu Leu Leu Glu Glu Ser Leu Phe lie Arg Arg Glu Gln Wing Asn 20 25 30 AAC ATC CTG GCG AGG GTC ACG AGG GCC AAT TCC TTT CTT GAA GAG ATG 14 Asn lie Leu Ala Arg Val Thr Arg Ala Asn Ser Phe Leu Glu Glu Met 35 40 45 AAG AAA GGA CAC GAA GAA AGA GAG TGC GAA AGG GAG TGC TCA TAC 19 Lys Lys Gly His Leu Glu Arg Glu Cys Met Glu Glu Thr Cys Ser Tyr 50 55 60 GAA GAG GCC CGC GAG GTC TTT GAG GAC AGC GAC AAG ACG AAT GAA TTC 24 Glu Glu Wing Arg Glu Val Phe Glu Asp Ser Asp Lys Thr Asn Glu Phe 65 70 75 80 TGG A? T AAA TAC AAA GAT GGC GAC CAG TGT GAG ACC AGT CCT TGC CAG 288 Trp Asn Lys Tyr Lys Asp Gly Asp Gln Cys Glu Thr Ser Pro Cys G n 85 90 95 AAC CAG GGC AAA TGT AAA GAC GGC CTC GGG GAA TAC ACC TGC ACC TGT 33 Asn Gln Gly Lys Cys Lys Asp Gly Leu Gly Glu Tyr Thr Cys Thr Cys 100 105 110 TTA GAA GGA TGA GAA GGC AAA AAC TGT GAA TTA TTC ACA CGG AAG CTC 384 Leu Glu Gly Phe Glu Gly Lys Asn Cys Glu Leu Phe Thr Arg Lys Leu 115 120 125 TGC AGC CTG GAC AAC GGG GAC TGT GAC CAG TTC TGC CAC GAG GAA CAG 432 Cys Ser Leu Asp Asn Gly Asp Cys Asp Gln Phe Cys His Glu Glu Gln 130 135 140 AAC TCT GTG GTG TGC TCC TGC GCC CGC GGG TAC ACC CTG GCT AAC 480 Asn Ser Val Val Cys Ser Cys Ala Arg Gly Tyr Thr Leu Ala Asp Asn 145 150 155 160 GGC AAG GCC TGC ATT CCC ACA GGG CCC TAC CCC TGT GGG AAA CAG ACC 528 Gly Lys Wing Cys lie Pro Thr Gly Pro Tyr Pro Cys Gly Lys Gln Thr 165 170 175 CTG GAA CGC AGG AAG AGG TCA GTG GCC CAG GCC ACC AGC AGC GGG 576 Leu Glu Arg Arg Lys Arg Ser Val Wing Gln Wing Thr Ser Ser Ser Gly 180 185 190 GAG GCC CCT GAC AGC ATC ACE TGG AAG CCA TAT GAT GCC GCC GAC CTG 624 Glu Ala Pro Asp Ser lie Thr Trp Lys Pro Tyr Asp Wing Wing Asp Leu 195 200 205 GAC CCC ACC GAG AAC CCC TTC GAC CTG CTT GAC TTC AAC CAG ACG CAG 6 ?; Asp Pro Thr Glu Asn Pro Phe Asp Leu Leu Asp Phe Asn Gln Thr Gln 210 215 220 CCT GAG AGG GGC GAC AAC AAC CTC ACC AGG ATC GTG GGA GGC CAG GAA 720 Pro Glu Arg Gly Asp Asn Asn Leu Thr Arg lie Val Gly Gly Gln Glu 225 230 • 235 240 TGC AAG GAC GGG GAG TGT CCC TGG CAG GCC CTG CTC ATC AAT GAG GAA 768 Cys Lys Asp Gly Glu Cys Pro Trp Gln Ala Leu Leu lie Asn Glu Glu 245 250 255 AAC GAG GGT TTC TGT GGT GGA ACT ATT CTG AGC GAG TTC TAC ATC CTA 816 Asn Glu Gly Ghe Phe Cys Gly Gly Thr lie Leu Ser Glu Phe Tyr lie Leu 260 265 270 ACG GCC GCC CAC TGT CTC TAC CAA GCC AAG AGA TTC AAG GTG AGG GTA 864 Thr Ala Ala His Cys Leu Tyr Gln Ala Lys Arg Phe Lys Val Arg Val 275 280 285 GGG GAC CGG AAC ACG GAG CAG GAG GAG GGC GGT GAG GCG GTG CAC GAG 912 Gly Asp Arg Asn Thr Glu Gln Glu Glu Gly Gly Glu Ala Val His Glu 290 295 300 GTG GAG GTG GTC ATC AAG CAC AAC CGG TTC ACÁ AAG GAG ACC TAT GAC 960 Val Glu Val Val lie Lys His Asn Arg Phe Thr Lys Glu Thr Tyr Asp 305 310 315 320 TTC GAC ATC GCC GTG CTC CGG CTC AAG ACC CCC ATC ACC TTC CGC ATG 1008 Phe Asp lie Wing Val Leu Arg Leu Lys Thr Pro lie Thr Phe Arg Met 325 330 335 AAC GTG GCG CCT GCC TGC CTC CCC GAG CGT GAC TGG GCC GAG TCC ACG 1056 Asn Val Wing Pro Wing Cys Leu Pro Glu Arg Asp Trp Wing Glu Ser Thr 340 345 350 CTG ATG ACG CAG AAG ACG GGG ATT GTG AGC GGC TTC GGG CGC ACC CAC 1104 Leu Met Thr Gln Lys Thr Gly lie Val Ser Gly Phe Gly Arg Thr His 355 360 365 GAG AAG GGC CGG CAG TCC ACC AGG CTC AAG ATG CTG GAG GTG CCC TAC 1152 Glu Lys Gly Arg Gln Ser Thr Arg Leu Lys Met Leu Glu Val Pro Tyr 370 375 380 GTG GAC CGC AAC AGC TGC AAG CTG TCC AGC AGC TTC ATC '' ATC ACC CAG 120C Val Asp Arg Asn Ser Cys Lys Leu Ser Ser Be Phe He He Thr Gln 385 390 395 400 AAC ATG TTC TGT GCC GGC TAC GAC ACC AAG CAG GAG GAT GCC TGC CAG 1248 Asn Met Phe Cys Wing Gly Tyr Asp Thr Lys Gln Glu Asp Wing Cys Gln 405 410 415 GGG GAC AGC GGG GGC CCG CAC GTC ACC CGC TTC AAG GAC ACC TAC TTC 1296 Gly Asp Ser Gly Gly Pro His Val Thr Arg Phe Lys Asp Thr Tyr Phe, 420 425 430 '. GTG ACA GGC ATC GTC AGC TGG GGA GAG AGC TGT GCC CGT AAG GGG AAG 1344 Val Thr Gly laugh Val Ser Trp Gly Glu Ser Cys Wing Arg Lys Gly Lys 435 440 5 TAC GGG ATC TAC ACC AAG GTC ACC GCC TTC CTC AAG TGG ATC GAC AGG 139 Tyr Gly lie Tyr Thr Lys Val Thr Wing Phe Leu Lys Trp lie Asp Arg 450 455 460 TCC ATG AAA ACC AGG GGC TTG CCC AAG GCC AAG AGC CAT GCC CCG GAG 144 Ser Met Lys Thr Arg Gly Leu Pro Lys Ala Lys Ser His Wing Pro Glu 465 470 475 480 GTC ATA ACG TCC TCT CCA TTA AAG TGA 1467 Val lie Thr Ser Ser Pro Leu Lys * 485 (2) INFORMATION FOR SEQ ID NO: 27 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 489 amino acids (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein [xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 27: Met Gly Arg Pro Leu His Leu Val Leu Leu Ser Wing Be Leu Wing Gly 1 5 10 15 Leu Leu Leu Gly Glu Be Leu Phe lie Arg Arg Glu Gln Wing Asn 20 25 30 Asn He Leu Wing Arg Val Thr Arg Wing Asn Being Phe Leu Glu Glu Met, 35 40 45 Lys Lys Gly His Leu Glu Arg Glu Cys Met Glu Glu Thr Cys Ser Tyr 50 55 60 Glu Glu Wing Arg Glu Val Phe Glu Asp Ser Asp Lys Thr Asn Glu Phe 65 70 75 80 Trp Asn Lys Tyr Lys Asp Gly Asp Gln Cys Glu Thr Ser Pro Cys Gln 85 90. 95 Asn Gln Gly Lys Cys Lys Asp Gly Leu Gly Glu Tyr Thr Cys Thr Cys 100. 105 110 Leu Glu Gly Phe Glu Gly Lys Asn Cys Glu Leu Phe Thr Arg Lys Leu 115 120 125 Cys Ser Leu Asp Asn Gly Asp Cys Asp Gln Phe Cys His Glu Glu Gln 130 135 140 Asn Ser Val Val Cys Ser Cys Ala Arg Gly Tyr Thr Leu Wing Asp Asn 145 150 155 160 Gly Lys Wing Cys He Pro Thr Gly Pro Tyr Pro Cys Gly Lys Gln Thr 165 170 175 Leu Glu Arg Arg Lys Arg Ser Val Wing Gln Wing Thr Ser Ser Ser Gly 180 185 190 Glu Ala Pro Asp Ser He Thr Trp Lys Pro Tyr Asp Wing Wing Asp Leu 195 200 205 15 Asp Pro Thr Glu Asn Pro Phe Asp Leu Leu Asp Phe Asn Gln Thr Gln 210 215 220 Pro Glu Arg Gly Asp Asn Asn Leu Thr Arg He Val Gly Gly Gln Glu 225 230 235 240 Cye Lys Asp Gly Glu Cys Pro Trp Gln Wing Leu Leu He Asn Glu Glμ 245 250 255 Asn Glu Gly Phe Cys Gly Gly Thr lie Leu Ser Glu Phe Tyr He Leu 260 265 270 20 Thr Wing Wing His Cys Leu Tyr Gln Wing Lys Arg Phe Lys Val Arg Val 275 280 285 Gly Asp Arg Asn Thr Glu Gln Glu Glu Gly Gly Glu Wing Val His Glu 290 295 300 Val Glu Val Val He Lys His Asn Arg Phe Thr Lys Glu Thr Tyr Asp 305 310 315 320 Phe Asp He Wing Val Leu Arg Leu Lys Thr Pro He Thr Phe Arg Met 325 330 335 Asn Val Wing Pro Wing Cys Leu Pro Glu Arg Asp Trp Wing Glu Ser Thr 340 345 350 Leu Met Thr Gln Lys Thr Gly He Val Ser Gly Phe Gly Arg Thr His 355 360 365 Glu Lys Gly Arg Gln Ser Thr Arg Leu Lys Met Leu Glu Val Pro Tyr 370 375 380 Val Asp Arg Asn Ser Cys Lys Leu Ser Ser Be Phe He He Thr Gln 385 390 395 400 Asn Met Phe Cys Wing Gly Tyr Asp Thr Lys Gln Glu Asp Wing Cys Gln 405 410 415 Gly Asp Ser Gly Gly Pro His Val Thr Arg Phe Lys Asp Thr Tyr Phe 420 425 430 Val Thr Gly He Val Ser Trp Gly Glu Ser Cys Ala Arg Lys Gly Lys 435 440 445 Tyr Gly He Tyr Thr Lys Val Thr Wing Phe Leu Lys Trp He Asp Arg 450 455 460 Ser Met Lys Thr Arg Gly Leu Pro Lys Wing Lys Ser His Wing Pro Glu 465 470 475 480 Val He Thr Ser Ser Pro Leu Lys * 485 It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in * the following

Claims (51)

1. An analogue of Factor X, characterized in that it has a modification in the site of activation cleavage site of natural Factor Xa, the modification represents a processing site for a protease that is not naturally segmented in this region of the Factor sequence. X.

2. An analogue of Factor X according to claim 1, characterized in that said! "modification" refers to at least one amino acid within the amino acid sequence of the activation peptide.

3. An X Factor analogue according to claim 1 or 2, characterized in that the i modification represents an exchange of at least one amino acid between Gly228 and Arg234, and optionally Ile235, relative to the numbered amino acid as shown in Figure 1.

4. The Factor X analogue according to any of claims 1 to 3, characterized in that it contains a sequence of Factor X having Gly228-R6-R5-R4-R3-R2-Arg234-R1, where a) R1 is an amino acid selected from the group of lie, Val, Ser, Thr or Ala, b) R2 is an amino acid selected from the group of Pro, Gly, Lys or Arg, c) R3 is an amino acid selected from the group of Phe, Lys, Met, Gln, Glu, Ser, Val, Arg or Pro, d) R4 is an amino acid selected from the group of Asp, Lie, Ser, Met, Pro, Thr, Arg or Lys, e) R5 is an amino acid selected from the group of Asn, Lys, Ser, Glu, Ala, Gln, His or Arg, and f) R6 is an amino acid selected from the group of Asp, Phe, Thr, Arg, Leu or Ser.

5. The Factor X analogue according to any of claims 1 to 4, characterized in that the modification represents a processing site for a protease selected from the group of endoproteases, such as kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE 4, LPC / PC7, serine erin proteases, such as Factor lia, Factor Xlla, Factor Xla, Factor Xa or kallikrein, or a derivative of these proteases.

6. The Factor X analogue according to any of claims 1 to 5, characterized in that it has a further modification in the amino acid sequence region of the C-terminal X Factor.

7. The Factor X analogue according to claim 6, characterized in that it has a modification in the C-terminal region of the β-peptide cleavage site.

8. The Factor X analogue according to claim 7, characterized in that the modification is a mutation, deletion or insertion in the amino acid sequence region of Factor X between the amino acid positions of Arg469 and Ser476.

9. The Factor X analogue according to any of claims 6 to 8, characterized in that the modification prevents the β-peptide from being removed by cleavage.

10. The Factor X analogue according to claim 6, characterized in that it has a deletion of the β peptide of Factor X.

11. The Factor X analogue according to claim 6, characterized in that it has a stop signal of the translation in the C-terminal region of the Factor X sequence.

12. The Factor X analogue according to claim 11, characterized in that it has a stop signal of translation at the position of amino acid 470 of the Factor X sequence.

13. The Factor X analogue according to any of claims 1 to 12, characterized in that the modification in the activation peptide region allows the in vitro activation of the analogue of the Factor X to natural Factor Xa or an analog of Factor Xa, respectively.

14. An X Factor analog according to claim 13, characterized in that the modification allows activation by a protease selected from the group of endoproteases, such as kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE 4, LPC / PC7, the group of serine proteases, such as Factor lia, Factor Xlla, Factor Xla, Factor Xa, or kallikrein, or a derivative of these proteases. i

15. The X-Factor analogue according to any of claims 1 to 12, characterized in that the modification allows the in vivo activation of the Factor X analogue to the natural Factor Xa or a Factor Xa analogue, respectively.

16. The Factor X analogue according to claim 15, characterized in that the modification allows activation by a protease selected from the group of serine proteases, such as Factor Xlla, Factor Xla, Factor a Factor Xa, or kallikrein.

17. The Factor X analogue according to any of claims 1 to 16, characterized in that it is provided as an analogue of Factor X i having an intact β-peptide or as an analogue of the X factor that has a shortened C-end.

18. The Factor X analogue according to any of claims 1 to 17, characterized in that it is provided as a single chain molecule.

19. A recombinant DNA encoding an X Factor analogue according to any one of claims 1 to 18, characterized in that it is contained in a vector for the recombinant expression of the encoded protein.

20. A preparation containing a purified Factor X analogue or a precursor protein thereof having a modification in the site of the activation site of natural Factor Xa, with the modification representing a processing site of a protease that is not naturally segmented in this region of the X Factor sequence.

21. A preparation according to claim 20, characterized in that the modification is a cleavage site for a protease selected from the group of dibasic endoproteases, such as kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE 4, LPC / PC7, serine proteases, such as Factor lia, Factor Xlla, Factor Xla, Factor Xa or kallikrein.

22. A preparation according to any of claims 20 or 21, characterized in that the Factor X analog is provided as the Fxa analog.

23. A preparation according to any of claims 20 or 21, characterized in that the Factor X analog is provided as an analogue of Factor X having a shortened C-tip.

24. A preparation according to any of claims 20 to 23, characterized in that it contains the Factor X analog as a single chain molecule in the isolated form.

25. The preparation according to any of claims 20 to 24, characterized in that it contains a single chain X-Factor analog, in the enzymatically inactive form having a plurality of at least 80%, preferably 90%, particularly in a manner 95% preferable, and because it does not contain inactive proteolytic intermediates of the Factor X / Xa analog.

26. The preparation according to any of claims 20 to 25, characterized in that it contains the Factor X analog as a double chain molecule in the isolated form.

27. The preparation according to any of claims 20 to 26, characterized in that it contains the Factor X analog having a: modification which allows the in vitro activation of the Factor X analogue to the natural Factor Xa or a Factor Xa analogue , respectively.

28. A preparation according to any of claims 20 to 27, characterized in that it is formulated as a pharmaceutical preparation.

29. A preparation according to any of claims 20 to 28, characterized in that it is provided in a suitable device, preferably an application device, 'in combination with a protease selected from the group of endoproteases, such as kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE 4, LPC / PC7, from the group of serine proteases, such as Factor Xlla, Factor Xla, Factor Xa, Factor lia or kallikrein, or a derivative of these proteases.

30. The preparation according to claim 29, characterized in that the components are provided separately.

31. The preparation according to any of claims 20 to 26, characterized in that it contains an analogue of Factor X having a modification that allows in vivo activation of the Factor X analogue to the natural Factor Xa or the Factor Xa analogue, respectively .

32. A preparation containing the Factor Xa analog, which has a high structural integrity and stability, characterized in that it is particularly free of the intermediate compounds of the inactive Factor X / Xa analog and the products of the autoproteolytic Factor X degradation, obtainable by the activation of an X Factor analogue according to any of claims 1 to 18.

33. The preparation according to any of claims 20 to 32, characterized in that it contains a physiologically acceptable carrier and is provided in a stable storable form.

34. The preparation according to any of claims 20 to 33, characterized in that it optionally contains a blood factor or an activated form of a blood factor as an additional component.

35. The preparation according to claim 34, characterized in that it contains at least one component having the activity of derivatization of Factor VIII as an additional component.

36. The preparation according to any of claims 20 to 35, characterized in that it is formulated as a pharmaceutical composition and is optionally provided as a preparation of multiple compounds.

37. The use of a preparation according to any of claims 20 to 36, in the preparation of a pharmaceutical agent.

38. The use of a nucleic acid according to claim 19 in the preparation of a pharmaceutical agent.

39. A process for preparing a preparation containing the recombinant Factor X analog, characterized in that an X Factor analogue obtained by the recombinant preparation is isolated and purified by means of a chromatographic process.

40. A process according to claim 39, characterized in that it comprises the following steps: - providing a nucleic acid according to claim 19 transformation of an appropriate cell expression of the Factor X analogue optional incubation of the Factor analogue with a protease isolation of the Factor X analog, and purification of the Factor X analog by means of a chromatographic process.

41. The process according to claim 40, characterized in that the Factor X analog is isolated as a double chain molecule.

42. A process according to any of claims 39 to 41, characterized in that the double chain X-Factor analogue is brought into contact with a protease selected from the group of endoproteases, such as kexin / Kex2, furin / PACE, PC1 / PC3 , PC2, PC4, PACE 4, LPC / PC7, the group of serine proteases, such as Factor lia, Factor Xlla, Factor Xla, Factor Xa, or kallikrein, or a derivative of these proteases, under conditions in which the X-Factor analogue is segmented to natural Factor Xa or to an analogue of Factor Xa.

43. The process according to claim 42, characterized in that the cell is a cell that does not express a protease which can segment the light chain into the light and heavy chain of Factor X or the Factor X analog, and such cell is optionally deficient in protease.

44. The process according to claim 43, characterized in that the cell 'does not express an endoprotease, such as kexin, furin, PACE, or a derivative thereof.

45. A process according to any one of claims 43 or 44, characterized in that the Factor X analog is isolated as a single chain molecule.

46. The process according to claim 45, characterized in that the optionally isolated, single chain X-Factor analogue is brought into contact with a protease selected from the group of endoproteases, such as kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE 4, LPC / PC7, or a derivative of these proteases, under conditions in which the single chain X Factor analogue is cleaved to the double chain X Factor form.

47. The process according to: claim 46, characterized in that the analog of the X Factor of a single chain is activated directly to the Factor Xa or the Factor Xa analog, respectively, optionally carrying it in contact with a protease.

48. The process according to claim 46, characterized in that the double chain X-Factor analog is brought into contact with another protease different from the first one and is activated to the Factor Xa analogue or the natural Factor Xa.

49. The process according to any of claims 39 to 48, characterized in that the protease is immobilized.

50. A preparation process that contains the Factor Xa or the active Factor Xa analogue, respectively, characterized in that an analogue of Factor X prepared according to a process according to any of claims 39 to 46 is subjected to an activation step.

51. The process according to claim 50, characterized in that a purified Factor Xa or natural Factor Xa analog having high structural integrity and stability is obtained, which is particularly free of the inactive intermediate X / Xa intermediate compounds.