Analoques of stuart factor with modified proteasolytic site

The present invention relates to a factor X analogue, recombinant DNA, compositions, use of recombinant DNA nucleic acid and methods of making the composition.

After initiation of the blood coagulation process, the coagulation cascade proceeds through the sequential activation of various proenzymes (zymogens) in the blood to their active forms, serine proteases. These include, among others: factor XII / XIIa, factor XI / XIa, factor X / Xa, factor VII / VIIa and prothrombin / thrombin. Most of these enzymes are only active in a physiological state when they form a complex attached to the surface of the membrane. Ca ions are involved in many of these processes. Blood clotting occurs on the intrinsic pathway, in which all protein components are found in the blood, or on the extrinsic pathway, in which the tissue factor of the cell membrane plays a critical role. Wound closure eventually occurs as a result of fibrinogen to fibrin cleavage by thrombin.

The prothrombinase complex is responsible for activating prothrombin to thrombin. Thrombin is an important enzyme that can act as both a procoagulant and as an anticoagulant. The prothrombinase complex, in which factor Va (as a cofactor and factor Xa (as serine protease), among others, are involved), is part of the Ca-dependent association on the phospholipid surface. The subject of discussion is the problem of whether factor Xa is a catalytic component of the prothrombinase complex.

Factor X (Stuart / Prower factor) is a vitamin K-dependent coagulation glycoprotein that can be activated by the internal and external coagulation cascades. The original factor X translation product (prepro-FX) contains 488 amino acids and is first synthesized as a 75 kD single chain precursor protein by the liver or human hepatoma cells. In blood plasma, factor X occurs to a large extent as a double chain molecule (Fair et al., 1984, Blood 64: 194-204).

During biosynthesis, after cleavage of the pre-sequence by signal peptidase (between Ser23 / Leu24) and propeptide (between Arg40 / Ala41), a single-chain factor X molecule is cleaved by converting and removing the tripeptide Arg180-Lys181-Arg182 into a double-chain form. about 22 kD chain and about 50 kD heavy chain, which are connected to each other via a disulfide bridge (Figure 1). Therefore, factor X circulates in the plasma as a two-chain molecule.

During the blood coagulation process, factor X is converted from inactive zymogen to active factorase Xa by limited proteolysis, whereby factor X may be activated to factor Xa in 2 membrane-connected complexes: the external factor VIIa complex^/tissue factor or the internal factor VIIIa-factor IXa-phospholipid-Ca complex or "tenase complex" (Mertens et al., 1980, Biochem. J. 185: 647-658). The proteolytic cleavage between the amino acids Arg 234 / IIe235 leads to the release of the 52-amino acid activation peptide from the N-terminus of the heavy chain and, as a result, to the formation of the active enzyme, factor Xa. The factor Xa catalytic center is located on the heavy chain.

Activation by the factor VIIa-TF (external) complex leads to the development of factor Xaa (35 kD) and factor XaP (31 kD), with a 42 kD polypeptide present in the complex when the factor VIIa concentration is low. Factor Xaa arises as a result of heavy chain cleavage at Arg234 / Ile235 and represents the activation of factor X to factor Xa. The presence of factor Xap is probably the result of autocatalytic cleavage at Arg469 / Gly470 at the C-terminus of factor Xaa heavy chain and cleavage of the 4.5 kD peptide. Factor XaP, like factor Xaa, has catalytic activity. However, it was shown that as a result of the cleavage of factor Xaa to factor Xap, a plasminogen binding site is formed and factor Xap also has fibrinolytic activity or participates in fibrinolysis as a cofactor. However, the conversion of factor Xaa into factor Xaβ is slower than the formation of thrombin, which is an obstacle to the initiation of fibrinolysis before the formation of a blood clot (Pryzdial et al., 1996, J. Biol. Chem. 271: 16614-16620; Pryzdial et al., 1996, J. Biol. Chem. 271: 16621-16626).

The 42 kD polypeptide is produced by the C-terminus conversion of the heavy chain between Arg469 / Gly470 without prior conversion between Arg234 / Ile235. This product

190 734 intermediate, as well as the fragment of factor Xay, which is formed as a result of proteolysis at Lys370, 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 factor X on the intrinsic pathway is catalyzed by the factor EXa-factor VUIa complex. When activated, the same transformation products are obtained, but factor XaP as a product is obtained in greater quantity than other factor X transformation products (Jesty et al., 1974, J. Biol. Chem. 249: 5614).

In vitro, factor X can be activated, e.g., by Russell's venom (Vipera russelli) (RW) or trypsin (Bajaj et al., 1973, J. Biol. Chem. 248: 7729-7741) or purified physiological activators such as a complex FVIIa / TF or factor IXa / factor VHIa complex (Mertens et al., 1980, Biochem. J. 185: 647-658).

Commercially available factor X plasma products most often contain a mixture of factor Xaa and factor Χηβ, since after activation of factor X to factor Xa, primarily factor Xaa is formed, which in the autocatalytic process is again split into factor Xap. In order to produce factor Xa as a homogeneous product with high molecular homogeneity, EP 0 651 054 proposed to activate factor X for a long time by RW, so that the resulting end product mainly contained factor Xap. Both by-products, e.g. factor Xaa, and the protease were then removed by several chromatographic operations.

Factor X cDNA has been isolated and characterized (Leytus et al., 1984, Proc. Natl. Acad. Sci., USA, 82: 3699-3702; Fung et al., 1985, Proc. Natl. Acad. Sci., USA , 82: 3591-3595). Human factor X was expressed in vitro in various types of cells, 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 was found that when recombinantly expressed human factor X, the transformation at the Arg40 / Ala41 position, in contrast to the in vivo situation, proceeds with low yield and different N-terminus is formed in the light chain of factor X (Wolf et al., 1991, J. Biol. Chem. 266: 13726-13730). Recombinant factor X (rFX) was activated in vitro by RW to r-factor Xa (rFXa) or rFXa was directly expressed, with the activation peptide from amino acid 183 to amino acid 234 deleted and replaced by a tripeptide to allow direct conversion into the two-chain form of rFXa. The purified rFX was converted in approximately 70% into light and heavy chains, while the remaining 30% were 75 kD single-chain rFX. Although direct expression of rFXa led to the formation of active factor Xa, it also produced inactive intermediates. Wolf et al. (1991, J. Biol. Chem. 266: 13726-13730) also found less recombinant factor X activity, which was explained by the inferior activation of rFX by RW and the inactive population of proteins and polypeptides of the single chain precursor molecule. In particular, they found high instability of rFXa when expressed by recombinant cells, which was explained by the high speed of autoproteolysis.

To investigate the effect of the C-terminal peptide of factor Xaa, Eby et al. (1992, Blodd 80 (Suppl. 1): 1214A) introduced a codon retaining the Gly430 position of the factor X sequence. However, they found no difference between the degree of factor Xa activation ( FXaa) with β-peptide and the deletion mutant without β-peptide (FXaP).

Factor Xa is an important component of the prothrombinase complex, so it could be used to treat patients with blood clotting disorders, e.g., hemophilia.

However, the treatment of haemophilia patients with factor VIII or factor IX deficiency using plasma-derived factor concentrates is often difficult with longer treatment periods because antibodies are produced that inhibit these agents. Therefore, a whole range of alternative treatments for hemophilia patients using bypass factors have been developed. The use of a prothrombin complex concentrate, partially activated prothrombinase complex (APPC), factor VIIa or FEIBA has been proposed for this purpose. Commercially available factor VIIa bypass activity (FEIBA) preparations are e.g. FEIBA® or Autoplex®. FEIBA contains e.g. comparable units of factor II, factor VII, factor EX,

190 734 factor X and FEIBA, small amounts of factor VIII and factor V, and trace amounts of activated coagulation factors such as thrombin and factor Xa or factor X-like activity (Elsinger, 1982, Activated Prothrombin Complex Concentrates. Ed. Mariani, Russo, Mandelli, pp. 77-87). Elsinger draws particular attention to the importance of "factor Xa-like" activity in FEIBA. Factor VIII bypass activity has been shown in an animal model by Giles et al. (1988, British J. Haematology 9: 491-497) for a mixture of purified factor Xa and phospholipids.

There is a great demand and a number of different fields of use for factor X / X a or factor X / Xa-like proteins, alone or as part of a coagulation complex in antihemorrhagic therapy.

The half-life of factor Xa compared to zymogen is strongly reduced both in vivo and in vitro. For example, factor X in glycerin can be stored stably for 18 months, while factor Xa under the same conditions is only stable for 5 months (Bajaj et al., 1973, J. Biol. Chem 248: 7729-7241) or glycerin at 4 ° C after 8 months shows a decrease in activity by more than 60% (Teng et al., 1981, Thrombosis Res. 22: 213-220). The serum half-life of factor Xa is only 30 seconds.

Because of factor Xa instability, administration of factor X preparations has been proposed (US 4,501,731). However, in the case of life-threatening bleeding, especially in patients with haemophilia, administration of factor X is ineffective, because due to the lack of functional "tenas complex" on the intrinsic coagulation glass, sufficient activation of factor X to factor Xa cannot occur and activation on the extrinsic pathway often occurs too slow to achieve fast action.

In addition, patients with hemophilia have sufficient factor X, which, however, has a 1000-fold lower prothrombinase activity compared to factor Xa. In such cases, direct administration of activated factor Xa is required, optionally together with phospholipids, as reported by Giles et al. (1988, British J. Haematology 9: 491-497), or together with other coagulation factors, e.g. with factor VIII with activity bypass pipework.

The production of factor Xa from factor X has so far been most often carried out by activation with non-physiological activators of animal origin such as RVV or trypsin, however, it was necessary to be absolutely sure that the final product is completely free of these proteases. As mentioned above, when activating factor X into factor Xa, a large amount of partially inactive intermediates are formed (Bajaj et al., 1973, J. Bio. Chem. 248 : 7729-7741; Mertens et al., 1980, Biochem. J. 185: 647-658). The presence of such intermediates reduces the specific activity of the product and optionally such intermediates can act as antagonists of the active serine protease. Conventional methods of laborious activation and chromatographic purification are therefore needed in the production of a homogeneous, pure product with high specific activity.

The subject of the present invention is a factor X analogue characterized in that it has a modification in the region of the naturally occurring activation site for factor Xa in the factor X sequence of Gly228-R6-R5-R4-R3-IR2-Arg234-R1, which modification transformation site for protease cleaving factor X sequences unnaturally in this region, a

R1 is an amino acid selected from the group Ile, Val, Ser, Thr or Ala

R2 is an amino acid selected from the group Pro, Gly, Lys or Arg

R3 is an amino acid selected from the group Phe, Lys, Met, Gln, Glu, Ser, Val, Arg or Pro

R4 is an amino acid selected from the group Asp, Ile, Ser, Met, Pro, Thr, Arg or Lys

R5 is an amino acid selected from the group Asn, Lys, Ser, Glu, Ala, Gln, His or Arg and

R6 is an amino acid selected from the group Asp, Phe, Thr, Arg, Leu or Ser.

Preferably, the modification refers to at least one amino acid within the amino acid sequence of the activation peptide, more preferably the modification is an exchange of at least one amino acid between Gly228 and Arg 234 and optionally Ile235, with respect to the amino acid numbering of Figure 1.

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Preferably, the modification is a transformation site for a protease selected from the group of endoproteases such as e.g. kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE4, LPC / PC7, serine proteases such as e.g. factor Ha, factor XHa , factor XIa, factor Xa> or kallikrein, or a derivative of these proteases.

Preferably, the factor X analog has a modification in the C-terminal region of the factor X amino acid sequence, more preferably it has a modification in the C-terminal region of the β-peptide cleavage site, more preferably the modification is a mutation, deletion or insertion in the region of the factor X amino acid sequence between amino acid positions Arg469 and Ser476.

Preferably the modification prevents cleavage of β-peptide.

Preferably, the factor X analogue has a deletion of the factor X β-peptide.

Preferably, the factor X analog has a translation stop signal in the C-terminal region of the factor X sequence, more preferably it has a translation stop signal at amino acid position 470 of the factor X sequence.

Preferably, the factor X analog has a modification in the activation peptide region that enables the in vitro activation of the factor X analog to native factor Xa or the factor Xa analogue, more preferably the modification enables activation by a protease selected from the group of endoprotease, such as keksyna / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE4, LPC / PC7, serine protease groups such as e.g. factor Ha, factor XHa, factor XIa, factor Xa, or kallikrein, or a derivative of these proteases.

Preferably, the factor X analog has a modification that enables in vivo activation of the factor X analog to native factor Xa or factor Xa analogue, more preferably the modification enables activation by a protease selected from the group of serine proteases such as e.g. factor XHa, factor XIa, factor Ha, factor Xa, or through kallikrein.

Preferably, the factor X analogue is the factor X analogue intact (β-peptide or the factor X analog truncated at the C-terminus.

Preferably, the factor X analog is a single chain molecule.

The invention furthermore provides recombinant DNA characterized in that it encodes a factor X analog as defined above contained in a vector for the recombinant expression of the encoded protein.

The invention also relates to a composition comprising a purified factor X analogue or its precursor protein with a modification in the region of the naturally occurring factor X activation cleavage site in the factor X sequence in the sequence Gly228-R6-R5-R4-R3-R2-Arg234-R1, the modification being this is an unnatural transformation site in this region of the protease-cleaving factor X sequence, a

R1 is an amino acid selected from the group De, Val, Ser, Thr or Ala

R2 is an amino acid selected from the group Pro, Gly, Lys or Arg

R3 is an amino acid selected from the group Phe, Lys, Met, Gln, Glu, Ser, Val, Arg or Pro

R4 is an amino acid selected from the group Asp, Ile, Ser, Met, Pro, Thr, Arg or Lys

R5 is an amino acid selected from the group Asn, Lys, Ser, Glu, Ala, Gln, His or Arg and

R6 is an amino acid selected from the group Asp, Phe, Thr, Arg, Leu or Ser.

Preferably, the modification is a cleavage site for a protease selected from the group of dibasic endoproteases, such as e.g. kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE4, LPC / PC7, serine proteases such as e.g. factor IIa, factor XHa, factor XIa, factor Xa, or kallikrein.

More preferably, the factor X analog is an FXa analog.

More preferably, the factor X analog is a factor X analog shortened at the C-terminus.

Preferably, the composition contains a factor X analog as a single chain molecule in isolated form, especially a single chain factor X analog in an enzymatically inactive form with a purity of at least 80%, preferably 90%, particularly preferably 95%, and does not contain inactive, proteolytic intermediates of factor X analog / X.

Preferably the composition comprises a factor X analog as a two chain molecule in isolated form.

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Preferably, the composition comprises a factor X analog having a modification that allows in vitro activation of the factor X analog to native factor Xa or the factor Xa analog.

Preferably the composition is in the form of a pharmaceutical preparation.

Preferably, the composition is placed in a suitable device, especially in the application device, in combination with a protease selected from the group of endoprotease, e.g. keksyna / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE4, LPC / PC7, groups serine proteases, such as, for example, factor XIIa, factor XIa, factor Xa, or caloric, a derivative of these proteases, more preferably the components are spatially separated from each other.

Preferably the composition comprises a factor X analog having a modification that allows the in vivo activation of the factor X analog to native factor Xa or the factor Xa analog.

Another object of the invention is a composition characterized in that it contains a factor Xa analogue and is free of inactive factor X / Xa analogue intermediates and autoproteolytic factor X degradation products, and which can be obtained by activating the factor X analogue as defined above.

Preferably the composition comprises a physiologically acceptable carrier and is in a stable form during storage.

Preferably, the composition optionally comprises as a distal component a blood factor or an activated form of the blood factor, more preferably it comprises as a further component at least one factor VIII composition with bypass activity.

Preferably the composition is a component of the pharmaceutical kit and optionally is in the form of a multi-component formulation.

The invention also relates to the use of a composition as defined above for the preparation of a medicament.

The invention furthermore relates to the use of recombinant DNA nucleic acid encoding a factor X analogue as defined above, and contained in a vector for the recombinant expression of the encoded protein for the production of a medicament.

The invention also relates to a method of producing a composition containing a purified recombinant factor X analogue, wherein the recombination obtained is not the factor X analogue is isolated and purified by chromatography.

Preferably the method comprises the steps of:

- providing the nucleic acid as defined above

- the appropriate cell is transformed

- the factor X analogue is expressed

- optionally, the factor X analogue is incubated with the protease

- the factor X i analogue is isolated

- the factor X analogue is purified by chromatography.

Preferably, the factor X analogue is isolated as a two chain molecule.

More preferably, the two-chain factor X analogue is contacted with a protease selected from the group of endoproteases such as e.g. kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE4, LPC / PC7, serine protease groups such as e.g. factor IIa, factor Xlla, factor XIa, factor Xa, or kallikrein or a derivative of these proteases, under conditions in which the factor X analogue is activated into the native factor Xa or factor Xa analogue, and more preferably a cell that can carry out single chain cleavage is used on the light and heavy chain of factor X or the factor X analogue, it does not express and possibly has a protease deficit, and in particular a cell not expressing endoprotease such as e.g. cexin, furin, PACE or a derivative thereof.

Preferably, the factor X analogue is isolated as a single chain molecule.

More preferably, the single-chain optionally isolated factor X analogue is contacted with a protease selected from the group of endoproteases such as kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE4, LPC / PC7, or a derivative of these proteases, under the conditions under which it cleaves a single chain factor X analogue to a double chain form factor X, more preferably a single chain factor X analogue, optionally by contacting a protease, is activated directly into factor Xa or a factor Xa analogue.

190 734

Preferably, the factor X two chain analogue is contacted with a further protease other than the previous one and activated into the factor Xa analogue or native factor Xa.

Preferably, an immobilized protease is used.

The invention also relates to a method for producing a composition comprising an active factor Xa or factor Xa analogue wherein the factor X analog obtained by the method set out above is subjected to an activation operation.

According to the invention, the factor X analogue may have a modification in the region of the naturally occurring activation factor Xa cleavage site. The modification in the region of the activation cleavage site is a new, unnaturally occurring at this position of the polypeptide, site of recognition or transformation for a protease that usually does not cleave the polypeptide at this site.

The factor X analogue of the invention may have a modification in particular in the activation peptide, which is cleaved off when factor X is activated into factor Xa. The modification refers to at least one amino acid within the amino acid sequence of the factor X activation peptide. The modification takes place in particular in the C-terminal region of the activation peptide and constitutes at least one exchange of at least one amino acid between the positions of Gly228 and Arg234 of the factor X amino acid sequence. The amino acid positions refer to the sequence numbering shown in Fig. 1 starting from Met1 and ending at Lys488.

The modification in the factor X analogue of the invention is primarily the replacement of the factor VIIa or factor IXa transformation site present in this region with an alternative protease cleavage site. The modification can be the replacement of at least one amino acid or the insertion of a peptide sequence which is the protease recognition site or cleavage site. The modification in the factor X analogue according to the invention is usually of such a nature that it constitutes a recognition or cleavage sequence for a protease from the group of endoproteases, such as e.g. kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE 4, LPC / PC7 (as described in Barr et al., 1991, Cell 66: 1-3 or US 5,460,950), serine proteases such as e.g. factor XHa, factor XIa, factor IIa, factor Xa or kallikrein, or a derivative of these proteases .

The modification is selected primarily so that transformation by one of these proteases leads to a native factor Xa polypeptide that is essentially similar to the naturally occurring factor Xa sequence and also has factor Xa activity.

In order to achieve optimal conversion, additional Ile235 amino acid replacement may be needed in individual cases. However, isoleucine, NH should usually be left after activation₂- the terminal heavy chain amino acid, as this amino acid plays an important role in forming the substrate binding pocket (Watzke et al., 1995, Molecular Basis of Thrombosis and Hemostasis ed. Katherine High & Harold Roberts). The factor X analogues of the invention show a structural difference, especially in the amino acid plane, compared to the native factor X sequence, but show comparable activity such as naturally occurring factor X or factor Xa activity after activation.

For example, a number of factor X analogues of the invention may have modifications in the activation peptide relative to the naturally occurring factor X sequence and altered protease specificity.

Modifications may occur at one or more positions in the region between the amino acids Gly228 and Arg234 and optionally Ile235, with reference to the sequence of factor X numbered from Met1 to L ^ and ^ 488 according to Fig. 1. Amino acid substitution can take place in positions Ile235 (R1), Arg234, Thr233 (R2), Leu232 (R3), Asn231 (R4), Asn230 (R5) and Asp229 (R6), but mostly Arg234 remains unchanged.

The factor X analogues of the invention usually contain the factor X sequence from Gly 228-R6-R5-R4-R3-R2-Arg234 with R1 = Ile, 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, Ile, Ser, Met, Pro, Thr, Lys or Arg; R⁵ = Asn, Lys, Ser, Glu, Ala, Gln, His or Arg and R6 = Asp, Phe, Thr, Arg, Leu or Ser.

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The preferred embodiment of the factor X analogues of the invention are factor X analogues that have the modification:

a) R1 = Val, R2 = Thr, R3 = Phe, R4 = Asp, R5 = Asn and optionally R6 = Phe (Fig. 2A) and is transformed with factor X3a;

b) R1 = Ser, R2 = Arg, R3 = Thr, R4 = Leu (Figure 2B) and it is transformed with FIIa;

c) R1 = Ile, R2 = Pro, R3 = Lys, R4 = How much and any R5 = Lys and / or r6 = Thr (Fig. 2C) or R1 - Ile, R2 = Thr, R3 = Ser, r4 = Thr and optionally R5 = Lys and / or R6 = Thr (Figure 21) and are transformed with factor XIIa;

d) R1 = Ile, R2 = Thr, R3 = Met, R4 = Cheese and optionally R5 = Cheese ί / or R6 = Leu (Fig. 2D) and is transformed with kallikrein;

e) R1 = De, R2 = Gly, R3 = Gln, R4 = Pro and possible R5 = Lys and / or R6 = Ser (Fig. 2H) or

R1 = Ile, R2 = Thr, R3 = Lys and R4 = Met (Figure 2E) or R1 = Ile, R2 = Gly, R3 = Glu and R4 = Ile (Figure 2F) and is transformed with factor Xa;

f) R1 = Ile, R2 = Lys, R3 = Arg, R4 = Arg and optionally R5 = Glu and / or R6 = Leu or

R1 = Ile, R2 = Thr, R3 = Val, R4 = Arg and optionally R5 = Ala and / or R6 = Leu or

R1 = Ile, R2 = Arg, R3 = Val, R4 = Arg and optionally R5 = Gln and / or R6 = Leu or

R1 = Ile, R2 = Arg, R3 = Arg, R4 = Arg and optionally R5 = His and / or R6 = Leu or

R1 = Ile, R2 = Lys, R3 = Pro, R4 = Arg and optionally R5 = Asn and / or R6 = Leu or

R1 = Ile, R2 = Lys, Re = Arg, R4 = Ile and optionally R5 = Arg and / or R6 = Leu or

R1 = Ile, R2 = Lys, R3 = Ser and R4 = Arg or

R1 = Ile, R2 = Thr, R3 = Val and R4 = Arg or

R1 = Ile, R2 = Lys, R3 = Leu and R4 = Arg (see all Fig. 2G), where the groups mentioned in f) are transformed with a dibasic endoprotease such as furin, PACE, kexin / Kex2, furin / PACE , PC1 / PC3, PC2, PC4, PACE4, LPC / PC7 or a derivative of one of these proteases.

A selection of possible amino acid modifications and exchanges that lead to altered protease specificities are given in Figs. 2A-I.

Modifications can be made by means of in vitro mutagenase or PGR or another gene method known in the art that is capable of specifically altering the DNA sequence to achieve the targeted amino acid exchange.

Activation of the factor X analogue of the invention to native factor Xa or the factor Xa analogue is carried out according to the present invention usually by means of a protease selected from the group of endoprotease, e.g. kexin / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE4 , LPC / PC7, serine protease groups such as, e.g., factor XIIa, factor XIa, factor Xa, factor IIa or kallikrein, or a derivative of these proteases.

One of the difficulties encountered in the production of active factor Xa is its instability, because as a result of autocatalysis it produces in addition to factor Xao and factor Xap also other inactive intermediates. In order to ensure the production of substantially intact, active factor X / Xa or factor X / Xa-like molecules, it would therefore be desirable to obtain only proteins that lead to stable end products.

It is known that the preferred cleavage site for converting factor Xaa (FXaa) into factor Xap (FXap) lies between Arg469 / Gly470. Based on the research of Eby, et al. (1992, Blood. Vol. 80, Suppl 1, 1214), in addition to the famous C-terminal peptide (amino acid radicals 476 to 487), factor X was further shorter peptide (amino acid radicals 474 to 477), which arises from the autocatalysis of the Xarn factor

In order to ensure the targeted transformation of intact factor X into essentially active factor Xa, without simultaneously obtaining inactive intermediates of this transformation, the factor X analogues of the invention may have further modifications.

According to a special embodiment of the invention, the factor X analog according to the invention has further modification in the C-terminal region of the factor X amino acid sequence.

In one embodiment of the invention, the factor X analogue of the above described type may have inactive β-peptide (FXo). The factor X analogue of the invention may be po12

190 734 sit in the modification, especially in the region of the C-terminal β-peptide cleavage site, which prevents that after the activation of factor X to factor Xa, the β-peptide is cleaved from factor X. This results in a factor Xa molecule that can be isolated in an amount up to 100% as an inactive factor Xaa molecule.

The modification may be a mutation, deletion or insertion in the region of the factor X amino acid sequence between the amino acid position Arg469 and Ser476 and optionally Lys370. However, the amino acid substitution is preferred in which no folding of the polypeptide that affects the structure and thus possibly the protein function and activity can occur as a result of the amino acid exchange.

In one embodiment of the invention, the factor X analogues of the invention may have an exchange of one of the amino acids at position Arg469 and / or Gly470, wherein Arg469 is exchanged especially for Lys, His or Ile and Gly470 especially for Ser, Ala, Val or Thr.

In addition to the mutations at position Arg469 and / or Gly470, the factor X analogues of the invention may have further mutation at positions Lys370 and / or Lys475 and / or Ser476.

By replacing the amino acid at one of these positions, the transformation of factor Xaa into factor Xae or factor Xay is avoided because the naturally occurring transformation sequence (s) is (are) so modified that it can no longer occur optional autocatalytic cleavage of the C-terminal peptide.

In another embodiment of the invention, the factor X analogue of the invention may have a deletion of the C-terminal β-peptide (ŁFi). Such a factor X analogue can be obtained when the cDNA encoding the factor X analogue is expressed in a recombinant expression system, only those sequences that encode the amino acids Met1 to Arg469 are cloned.

In a further embodiment of the invention, the factor X analog according to the invention may have a translation stop signal in the C-terminal region of the factor X sequence. The translation stop signal is usually in a position which follows the C-terminal amino acid resulting from natural transformation. The translation stop signal is therefore usually at amino acid position 470 of the factor X sequence, thereby preserving the amino acid end of factor Xa β Arg469. At the same time, the GGC codon encoding the Gly470 amino acid is replaced by TAA, TAG or TGA.

The factor X analogues of the invention may also include those factor X analogues of the invention that are activated by the appropriate protease in vitro to a native factor Xa or factor Xa analogue. Depending on the type of factor X analogue used and activated, a native factor Xa and a substantially identical polypeptide or polypeptide are obtained which, although it has factor Xa activity, have modifications relative to the sequence of native factor Xa, which, however, do not reduce its biological activity.

As a result of activation of factor X analogues that have a modification in the activation peptide region in the activation peptide sequence, only polypeptides corresponding to the native factor Xa molecule are obtained. If such a factor X analog optionally also has a signal to stop translation in the C-terminal region of the β-peptide, then factor Xaβ homologue molecules are obtained. However, if a factor X analogue is used that has modification (e) within the β-peptide sequence that causes the β-peptide not to be cleaved, then a factor Xaa analogue with amino acid exchange at the C-terminus of the molecule is obtained.

The factor X analogues of the invention only have such modifications that change the specificity of the activity but do not affect the activity. In each case, therefore, a biologically and functionally active factor Xa molecule or factor Xa analogue is obtained.

In vitro activation can be performed using a protease from the group of endoproteases, such as e.g. keksyna / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE4, LPC / PC7, serine protease groups, e.g. factor XHa, factor XIa, factor Xa, or kallikrein, or a derivative of these proteases. However, it is within the scope of the present invention to use any type of protease except RVV, trypsin, factor IXa or factor VIIa if it is capable of converting a factor X analogue into factor Xa.

190 734

The factor X analogue of the invention may contain a modification that enables the in vivo activation of the factor X analogue to factor Xa, in particular to native factor Xa. "Native" factor Xa in this context means that activated factor Xa derived from the factor X analogue of the invention has an amino acid sequence corresponding to native factor Xa and a homologous amino acid sequence and contains factor Xa activity. The modification is chosen such that the transformation of factor X into factor Xa occurs as a result of the action of a protease occurring in vivo, i.e. in the body, especially of the protease found in the blood coagulation cascade. The protease may be selected from the group of serine proteases, such as, for example, factor IIa, factor XIIa, factor XIa, factor Xa or kallikrein. Factor X analogues which, in addition to the modification in the activation peptide, have a modification in the C-terminal region of the factor X molecule, are activated, as described above, also in vivo to the corresponding factor Xa analogue.

Although e.g. Wolf et al. (1991, J. Biol. Chem. 266: 13726-13730) suppose that an endopeptidase such as Kex2, furin or PACE is involved in the transformation of the factor Xa deletion mutant described in this group, they did not provide any clues on the effect of either of these proteases on the conversion of factor X. US 5,660,950 also describes the recombinant production of PACE and the use of a protease to improve the transformation of vitamin K-dependent proteins. Factor X is also listed in a number of calculations along with other blood factors, but there is no numerical data in this relationship.

Within the framework of the present invention, it has been clearly demonstrated for the first time that the protease necessary for the maturation of factor X is a dibasic endoprotease, especially endogenously occurring furin. In vivo, the endoprotease mediates primarily the cleavage of the single-chain factor X molecule into a mature form consisting of a heavier and lighter chain. In vitro, it also mediates the cleavage of the factor X propeptide sequence (example 2).

The factor X analogues of the invention that possess it a protease cleavage site for an unnaturally occurring protease in a cell are cleaved by a selective transformation reaction only at such sites that are also cleaved in native factor X. In this way, a recombinant factor X molecule is obtained, which consists only of a 22 kD light chain and a heavy chain of about 50 kD and does not have an inactive factor X molecule resulting from non-specific transformation. These modified factor X molecules, as well as native factor X molecules, are not activated into factor Xa by an intracellular protease. They are activated into factor Xa only later by the appropriate proteases (mainly serine or subtilisin-related proteases).

The factor X analogue of the invention may thus be in the form of a two-chain factor X analogue.

The method of the invention can produce factor X analogues that exist as single chain molecules, usually in purified form. By expressing the factor X analogue in cells with a dibasic protease deficit, the X-factor is obtained in the form of a single chain molecule. The single-chain factor X molecule is characterized by high stability and molecular homogeneity. Until now, it was not possible to isolate the single-chain factor X molecule in purified form, as it is rapidly converted to the double-chain form (Fair et al., 1984, Blood 64: 194-204). The recombinant single-chain factor X analogues can be converted into the double chain form of factor X by special treatment and then activated to factor Xa or a factor Xa analogue. This can be done by contacting a single-chain recombinant factor X molecule isolated from a protease deficit cell with a dibasic protease, such as furin / PACE or Kex2, and converting it into a factor X di-chain analogue.

The factor X double chain analogue can be activated to factor Xa or Xa analogue. This can occur, for example, in such a way that the factor X analogue, which as a result of modification in the activation peptide region has a specific furin cleavage site, is extracted as a single chain molecule from a furin-deficient cell and subsequently, by contact with the endoprotease, transforms into an activated factor Xa molecule .

190 734

It is also possible to isolate the single-chain factor X analogue which has a modification in the activation peptide, enabling alternative transformation by a protease from the group of serine proteases or kallikrein, to cleave only by treatment with a dibasic endoprotease, such as furin, into a double-chain factor X molecule and the latter, then contact the serine protease so that it is activated into factor Xa or a factor Xa analogue.

The factor X analogue isolated from the cell culture in the form of a double chain molecule can be directly exposed to an activation specific protease.

The factor Xa or factor Xa analog thus obtained has, due to the selective and directed transformation reaction, high stability and structural homogeneity and in particular is free of inactive intermediates of factor X / Xa analogue and autoproteolytic degradation products.

The recombinant DNA of the invention encoding the factor X analog of the invention results in the expression of a factor X analog with the amino acid sequence corresponding to human factor X, unless it has a modification that affects the specificity of the transformation and the transformation products, which is essentially unaffected by the biological activity of the clotting factor. blood.

The recombinant DNA nucleic acid may also be contained in transformed cells.

The composition of the invention comprises a purified factor X analogue or precursor protein thereof which (a) has a modification in the region of the naturally occurring factor Xa activation site. The modification in the region of the activation cleavage site is a new unnaturally occurring at this position in the polypeptide recognition or cleavage site for the protease that normally does not convert the polypeptide at this site. The composition may be a purified composition of a single or double chain factor X analogue, wherein the polypeptides are obtained from the cell culture system after isolating them from the cell culture resource or from the cell culture extract. The pre-purified factor X analogue from the cell culture system can be further purified by a method known in the art. Chromatographic methods such as gel filtration, ion exchanger chromatography or affinity chromatography are particularly suitable for this.

In one embodiment of the invention, the composition of the invention may contain a factor X analogue especially as a single chain molecule in isolated form. A composition of this type can be prepared such that a factor X analog obtained by recombinant production as a single chain molecule from a cellular system, especially from cell cultures that do not contain endoprotease, is cleaved into a heavy and light chain.

In one embodiment of the invention, the composition may contain a single-chain factor X analogue with a modification that allows it to be activated in vitro to factor Xa by one of the proteases selected from the group of dibasic endoproteases, such as e.g. kexin / Kex2, ffyrin / PACE, PC1 / PC3 , PC2, PC4, PACE4, LPC / PC7. Activation is effected by contacting a factor X analogue with a protease, whereby the naturally occurring transformation is cleaved to the mature form of factor X and by modifying the cleavage of the activation peptide, and factor Xa or factor Xa analogue is formed.

In the composition of the invention, the X-mimic analog may exist either as a single chain molecule in the form of factor Xa (FXa) or with a β-peptide deletion. The composition may in particular contain a factor X analogue in inactive enzymatic forms with a purity of at least 80%, preferably 90%, particularly preferably 95%, and contain no inactive, proteolytic intermediates of factor XJXa analogue.

The composition according to the invention may contain a factor X analogue especially as a two-chain molecule in isolated form. For this purpose, e.g. a factor X analog obtained from a cellular system by recombinant production as a single chain molecule is cleaved into a double chain form in vitro, i.e. outside the cell, by means of a protease, especially a dibasic protease. This can be done in such a way that the prosthesis is mixed directly with the culture resource of clones expressing the analog

190 734 factor X, either by mixing a purified protease or cell culture stock from a cell culture-producing recombinant expressing protease, or by co-culturing clones expressing factor X analogue and protease.

It is also possible to contact the cell culture resource containing the factor X analogue or purified factor X analogue with the immobilized protease, which then converts into a two-chain form. In this method, the protease is predominantly bound to the matrix and the cell culture resource containing the factor X analogue is directed through the matrix. However, it is also possible to immobilize the factor X analogue while the protease is in the mobile phase. Reagents (factor X analogue and protease) can also be mixed and incubated for some time. Protease is then removed from the mixture by affinity chromatography.

The two-chain form of the factor X analogue can also be obtained by co-expressing the protease and the factor X analogue directly in the cell and optionally purifying.

The composition of the invention may contain a single or double chain factor X analogue with a modification that allows in vitro activation to factor Xa or a factor Xa analogue. Activation of the factor X analog to factor Xa or the factor Xa analogue can occur as a result of contact of the factor X analog with a protease selected from the group of dibasic endoproteases, such as e.g. keksyna / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE4 , LPC / PC7, serine protease groups, such as e.g. factor XIIa, factor XIa, factor Ra, factor Xa, or kallikrein or a derivative of these proteases. The protease may be immobilized on a support.

The composition of the invention may serve as a starting material for the production and preparation of factor Xa. To this end, a large technical device is contacted with a composition comprising a single-chain or double-chain factor X analogue, e.g. with an optionally immobilized protease under conditions that allow optimal activation of the factor X analogue to factor Xa and obtaining factor Xa or factor Xa analogue. The factor Xa / factor Xa analog thus obtained can then be used to prepare a pharmaceutical kit.

A composition of the invention comprising a purified single or double chain factor X analogue of the invention and a physiologically acceptable carrier can be formulated as a pharmaceutical preparation. The preparation can be carried out in a known manner and the pharmaceutical preparation prepared by mixing with a buffer containing salts such as NaCl, CaCF, and amino acids such as glycine and / or lysine at a pH in the range of 6 to 8. The purified composition containing the factor X analogue can be prepared as a storage product in the form of a finished solution, lyophilisate or frozen until completely consumed. The composition is mainly stored as a lyophilisate and dissolved in a suitable reconstituted solution to give a visually clear solution.

However, the composition of the present invention may also be prepared as a liquid formulation or in the form of a frozen solution. The composition according to the invention is particularly stable, i.e. it can stand for a long time in a dissolved state before it is used. It has been found that the composition according to the invention shows no loss of activity for several hours to several days.

The composition according to the invention can be placed in a suitable device, especially in an application device, in combination with a protease selected from the group of endoproteases, such as e.g. keksyna / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE4, LPC / PC7, serine protease groups, e.g., factor IIa, factor XIIa, factor XIa, factor Xa, or kallikrein or a derivative of these proteases.

The composition of the invention comprising a factor X analogue in a mixture with a protease that is able to activate the factor X analogue to factor Xa or a factor Xa analogue can be prepared as a combined preparation consisting of a reservoir containing a fixed protease on a carrier, optionally in the form of a mini column or syringe containing a protease and a reservoir containing the pharmaceutical composition with a factor X analogue. To activate the factor X analogue, a solution containing the factor X analogue is pumped, e.g. by an immobilized protease. The solution containing the factor X analogue is in pod16

190 734 storage time of the preparation spatially separated from the protease. The composition according to the invention can be placed in the same reservoir as the protease, the components however being separated from each other by a spatially impermeable partition wall which, if used, is easy to remove. The solutions can also be stored in separate tanks and only shortly before use.

It is also recommended that the protease used for activation be a serine protease that also participates in the natural state of blood coagulation, such as e.g. zinc XHa or factor XIa, which does not have to be separated from activated factor Xa or can be used with it before use.

Activation of the factor X analogue to factor Xa may occur shortly before direct use, i.e. before administration to the patient. Activation may occur by contact with the immobilized protease or by mixing the solution containing the protease with the solution containing the factor X analogue. It is therefore possible to separately hold the solutions of both components and mix them using a suitable infusion device in which the components contact each other during flow, and thus activating each molecule into factor Xa or a factor Xa analogue. The patient is therefore given a mixture of factor Xa and a further serine protease that caused the activation. Particular attention should be paid to dosage, since the endogenous factor X is also activated by the additional administration of a serine protease and thus the clotting time can be reduced.

It is particularly recommended that the composition according to the invention be placed in a suitable device, especially in the application device, either in the form of a frozen liquid or in the form of a lyophilisate. A suitable application device may be the two-chamber syringe body described in patent specification AT 366 916 or AT 382 783.

The composition of the invention may contain a factor X analog with a modification that allows in vivo activation of the factor X analog to factor Xa. The factor X analogues in the preparation according to the invention usually have a modification, which is the recognition / cleavage site for a protease selected from the group of serine proteases, such as e.g. factor XHa, factor XIa, factor Xa, or kallikrein and are cleaved in vivo to one's native factor Xa or factor Xa analogue by one of said proteases. Particularly preferred for therapeutic use are those factor X analogues that have it the recognition site / cleavage site for the protease within the coagulation cascade independent of the factor Vna / tissue complex and tenase complex. Thus, the preparation according to the invention can be used to stop bleeding in patients with a deficiency of both factor IX and factor VII as well as factor VIII. Patients who have a bleeding disorder due to factor XI or factor XII deficiency should not be administered pharmaceutical compositions containing a factor X analogue that is activated by factor XIIa or factor XIa. In the case of e.g. factor XI deficit would be advisable to use a factor X analogue with a factor XJIa cleavage site.

The composition of the invention may optionally contain as a further component a blood factor in the form of a zymogen or active serine protease. As further ingredients, ingredients with factor VIII with bypass activity are recommended. These include in particular: factor II, factor VII, factor IX, factor VIII, factor V and / or their active serine proteases. However, further components may also be phospholipids, Ca ions etc. In one embodiment of the invention, the composition of the invention may contain as at least one further factor VIII bypass activity.

The composition of the invention may be in the form of a pharmaceutical composition with factor Xa activity in the form of a one-component formulation or a mixture with other factors as a multi-component formulation.

Before preparing a pharmaceutical composition, the purified protein is subjected to the usual quality controls and is formulated for therapeutic administration. In the case of recombinant production, the purified preparation is tested for the absence of nucleic acids derived from cells and from an expression vector, mainly by the method described in EP 0 714 987.

190 734

Because virtually any biological material can be contaminated with infectious microorganisms, the composition is optionally processed to inactivate and remove viruses to produce a safe formulation.

The composition according to the invention containing a factor Xa analogue with high structural stability and homogeneity, especially free from inactive intermediates of the factor X / Xa analogue and autoproteolytic degradation products, can be obtained in such a way that the factor X analogue of the type described above is activated and a suitable composition is prepared from it .

In one embodiment of the invention, the use of a composition of the type described above is described for the manufacture of a medicament. The therapeutic agent containing the factor X analog or factor Xa analog according to the invention is particularly suitable for the treatment of patients with blood coagulation disorders, such as e.g. patients with haemophilia or patients who have developed inhibitory antibodies in their body against the factor VIII and / or IX commonly used for treatment, and in particular as a factor VIII preparation with bypass activity.

In one embodiment of the invention, the use of a DNA nucleic acid according to the invention comprising sequences encoding factor X analogues of the invention is described for the manufacture of a medicament. The nucleic acid according to the invention, if it contains the appropriate expression control sequences, can be used as the nucleic acid itself, it can be incorporated into a recombinant expression vector or connected to a carrier such as a phospholipid or viral particle. The nucleic acid according to the invention can be used in the preparation of a medicament which is particularly suitable for the treatment of patients with blood coagulation disorders, such as e.g. patients with haemophilia or haemophilic patients with inhibitory antibodies. It is also possible to use the nucleic acid of the invention in gene therapy.

The invention also describes a method of producing a factor X analog according to the invention and a composition comprising a c: zynrika X analog according to the invention. To this end, the sequence encoding the factor X analogue is placed in a suitable expression system and transfected with recombinant DNA into the appropriate cells, especially stable cell lines. Cells are cultured under optimal conditions for gene expression and the factor X analogue is isolated from the cell culture extract or from the cell culture stock. Recombinant molecules can be purified by all known chromatographic methods, such as anion or cation exchange chromatography, affinity chromatography or immunoaffinity chromatography, or a combination of these methods.

The complete cDNA encoding factor X is cloned into the expression vector for the production of factor X analogs of the invention. This is done by suitable generally known methods of cloning. The factor X iodizing nucleotide sequence is then modified so that the coding sequence is altered in the activation peptide region and possibly also in the C-terminal β-peptide region so that a factor X molecule of the type described above can be produced. This is done by gene technique methods known in the art, such as specifically regulated in vitro mutagenesis, or sequence deletion, e.g., by endonuclease restriction digestion and incorporation of another altered sequence, or by PGR. The factor X mutants thus obtained are then incorporated into an expression system suitable for recombinant expression and expressed.

The factor X analogues of the invention can also be prepared by chemical synthesis.

Factor X analogues are usually produced by recombinant expression. Production using gene technique methods can be accomplished using all possible expression systems, such as, for example, stable cell lines or viral expression systems. Stable cell lines are generated by stably incorporating foreign DNA into the host cell chromosome, e.g. from Vero, MRC5, CHO, BHK, 293, Sk-Hepl, especially liver and kidney cells, or by an episomal vector derived e.g. from the papilloma virus. Viral expression systems, e.g., such as bovine pox virus, baculovirus or retroviral systems, may also be used. Vero, MRC5, CHO, BHK, 293, Sk-Hepl, gland, liver and kidney cells are commonly used as cell lines. As eukaryotic

190 734 expression systems may also be yeast, endogenous glands (e.g., transgenic animal glands) and other types of cells. Of course, transgenic animals can also be used to express the polypeptides described herein or derivatives thereof. CHO-DHFR * cells have proved particularly useful for the expression of recombinant proteins (Uriaub et al., 1980, Proc. Natl. Acad. Sei., USA, 77: 4216-4220).

Prokarioic expression systems can also be used to recombinantly produce factor X analogues of the invention. Systems that allow expression in E. coli or B. subtilis are particularly suitable for this.

Factor X analogues are expressed in appropriate expression systems under the control of a suitable promoter. When expressed in eukaryotes, all known promoters are suitable for this, such as the SV40 promoter, CMV, RSV, HSV, EBV, the β-actin promoter, hGH or inducible promoters, such as e.g. the hsp promoter or metallothionein promoter. Factor X analogs are expressed predominantly in CHO cells under the control of the β-actin promoter

The method of producing the compositions of the invention may include operations for producing DNA encoding factor X analogue, transforming the cell with recombinant DNA, expressing the factor X analogue, optionally in the presence of a protease, isolating the act: nLka X analogue and optionally purifying by chromatography.

According to one embodiment of the method, the factor X analogue can be isolated as a two chain molecule. For this purpose, the factor X analogue is expressed in a cell that allows the pro-factor X analogue to be converted into a two-chain factor X analogue. The cell is usually a cell which, with the expression of the factor X precursor precursor capable of transformation, e.g. a dibasic protease, such like furin or its derivative. In order to increase or improve the efficiency of transformation, it is possible to modify the cells so that they express the protease more strongly. This may occur, e.g., as a result of co-expression and appropriate dibasic endoprotease, such as e.g. furin / PACE, Kex2 or a derivative thereof.

The factor X analogue of the invention can also be expressed in a cell that has normal or hence suboptimal for conversion endogenous protease concentration and consequently incomplete transformation into a double chain form. The subsequent transformation into heavy and light chains occurs in this case when the single-chain factor X analogue as described above separates from the cellular resource by co-culture with protease expressing cells or by contact with optionally immobilized protease. The cellular resource can also be pumped through a support matrix with which it is bound by a protease, resulting in a two-chain factor X analogue in the eluate. It is also possible to mix the reagents in solution, incubate for some time and then remove the protease, e.g. using an affinity matrix.

The factor X chain double analogue thus obtained can then be isolated, purified and stored stable as described above for further use.

The double-chain, optionally purified factor X analogue of the invention can be contacted in vitro with a protease selected from the group of endoproteases, such as e.g. keksyna / Kex2, furin / PACE, PC1 / PC3, PC2, PC4, PACE4, LPC / PC7 serine protease groups such such as factor XIIa, factor XIa, factor Xa, factor IIa, or kallikrein, or a derivative of these proteases, under conditions in which the factor X analogue is activated into native factor Xa or factor Xa analogue.

In one embodiment of the invention, activation can take place as part of a chromatographic operation in which the protease is immobilized on a support. For this purpose, the purified two-chain factor X analogue is directed through the matrix with which it is bound by the protease and extracts purified factor Xa from the eluate.

In another embodiment of the invention, the components can be mixed and the protease removed selectively from the mixture.

Of course, it is also possible to combine the conversion of the single-chain pro-factor X analog into the double-chain form of the factor X analog and activation into factor Xa in one process. For this purpose, the single-chain factor X analogue or its precursor is directly contacted with a dibasic protease, in particular with furin or a derivative thereof which allows conversion into a heavy and light chain and activation into factor Xa. The factor X analogue, which has no cleavage site for furin or its derivative in the activation peptide, is optionally contacted with a distal, different from the first, protease, which, however, does not allow activation. Proteases can be used in the form of a mixture, e.g. furin with factor XIa.

Activation can also be accomplished by linking both operations in devices connected in series, especially carriers, such as columns on which the protease (s) are (are) immobilized. In the first carrier, the factor X is cleaved into heavy and light chains and in the second, it is activated into factor Xa by an immobilized protease. The carriers can be coupled together by directly connecting the outlet from the first column to the inlet to the second column.

Transformation and activation reaction conditions can be easily optimized by one skilled in the art depending on the experimentally determined framework conditions. The flow rate of the reagents used is particularly important for the contact time. Ideally, it should be between 0.01 ml / min and 1 ml / min. Temperature, pH and elution conditions are important as further parameters. After flowing through the device, the activated factor Xa can optionally be further purified by selective chromatography. Performing the method with the protease placed on each of the supports is particularly recommended because the reaction device, through the use of supports, especially chromatographic columns, enables an additional purification operation.

As part of the production of the factor X analogue of the invention, the factor X analogue can be isolated as a single chain molecule. The factor X analogue is then expressed in a cell that does not help transform a single chain into a light and heavy chain. The cell usually has a dibasic endoprotease deficit, such as e.g. kexin, furin, PACE. As stated in the context of the invention, furin is an important and responsible for the cleavage of factor X into the light and heavy chain of the protease. From such a mutated endoprotease deficient cell, a factor X analog can be isolated in the form of a single chain molecule. The factor X analogue thus isolated and possibly purified is then contacted with a protease selected from the group of endoprotease, e.g. keksyna / Kex2, furyna / PACE, PC1 / PC3, PC2, PC4, PACE4, LPC / PC7, under conditions in which a single-chain factor X analogue is split into a two-chain form of factor X. Factor X analogues according to the invention having a modification in the activation peptide region which allows cleavage by one of these endoproteases, can optionally be activated to factor Xa or factor Xa analogue directly by contact with the single-chain factor X analogue endoprotease.

The factor X analogues of the invention having a modification in the activation peptide region that allows cleavage by one of the serine proteases or by kallikrein, after being converted into a factor X two-chain analogue, can be contacted with a further, different from the first, protease and activated into the factor Xa analogue.

The method of the invention can also provide a composition containing an active factor Xa or an active factor Xa analogue in such a way that the factor X analogue prepared as described above is subjected to an activation operation and the activated polypeptide is further processed into a purified composition which is optionally manufactured as a pharmaceutical kit.

By producing the factor X analogues of the invention which are activated by the above-described method for factor Xa, purified factor Xa or a factor Xa analogue is obtained having high structural stability and homogeneity and in particular free from inactive intermediates of factor X / Xa.

The invention is described in more detail by the following examples and figures of drawings, however, it is not limited to these special embodiments.

Example 1 describes the construction and expression of r-factor X; example 2 describes the conversion of r-factor X into a light and heavy chain by fiirin; example 3 describes the transformation of pro-factor X by means of an immobilized protease; Example 4 describes the in vitro converted activity of r-factor X; example 5 describes the expression of r-factor X in furin deficient cells; example 6 describes the construction and expression of r-factor X analogues; przy20

190 734 example 7 describes the determination of the N-terminus of factor X transformation products; Example 8 describes the expression and characterization of the FX analog with the furin cleavage site Arg-Arg-Lys-Arg / IUe (rFXRRKRi); example 9 describes in vitro activation of rFX protein by r-furin derivative; example 10 describes the functionality of the in vitro activated recombinant FX rFX analogue; Example 11 describes in vitro activation of an rFX analog with an FXIa Asp-Phe-Thr-Arg / Val cleavage site for FXIa.

The figures show:

Figure 1: Factor X-amino acid and amino acid sequence Figure 2: Schematic sketch of factor X analogues with modified protease cleavage sites in the activation peptide region Figure 3: Schematic sketch of the phAct-rFX expression vector Figure 4: Westernblot analysis of r-factor X expressed in CHO cells before and after amplification figure 5: Westernblot analysis of r-factor X after in vitro cleavage with a furin derivative figure 6: Westernblot analysis of r-factor X molecules expressed in furin-containing and furin-deficient cells figure 7: schematic sketch of the rFX / rFXa analogon construct with altered

Heavy chain C-terminus figure 8: schematic sketch of the N-terminus of r-factor X transformation products in furin-containing CHO cells and in furin deficient CHO cells before and after r-furin treatment figure 9: Westernblot analysis of r-factor fxRRKR _(; Expression and in CHO cells figure 10: Westernblot analysis of r-factor FX^RR^RR after activation in vitro a furin derivative. Figure 11: Westernblot analysis of r-factor Fx^DFI'R^vafter activation in vitro a furin derivative. Expression vectors were generated by standard cloning methods (Maniatis et al., "Molecular Cloning" - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA, 1983). DNA fragments were generated by polymerase chain reaction (PCR) by general methods (Clackson et al., 1991 m, PCR A practical approach. ED. McPherson, Quirke, Taylor, S. 187 - 214).

Example 1

Expression and conversion of single chain rFX into light / heavy chain rFX

a. Preparation of the expression vector for rFX

To obtain recombinant FX (rFX), cDNA FX was isolated from human liver from a cDNA-lambda bank as described by Messier et. al. (1991, Gene 99: 291-294). From the positive clone, PCR was amplified by oligonucleotide # 2911 (5'-ATTACTCGAGAAGCTTACCATGGGCGCCACTG-3 ') (SEQ ID No. 1) as a 5'-primer and oligonucleotide # 2912 (5'-ATTACAATrG-CTGCAGGGATATQ) ID. No. 2) as a 3'-primer, a DNA fragment that contained the 1.467 kB FX coding sequence and a 3 '39 bp untranslated region, flanked by the Xhol cleavage site at the 5'-end and the Mfel cleavage site at the 3'-end. In addition, ACC before ATG in FX was built in by primer # 2911, so that the optimal Kozak translation initiation sequence was created. This PCR product was then cloned as an XhoI / MfeI fragment into a SalI and EcoRI cleaved phAct expression vector. The resulting expression plasmid was designated as phAct-rFx (Figure 3). The phAct expression vector includes the human β-actin promoter, 78 bp 5'UTR and intron, multiple cloning site and SV40 polyadenylation site.

b. Expression of rFX in CHO cells

To establish a stable cell line expressing rFX, dhfr deficient CHO cells were co-transfected with the phAct-rFX expression plasmid and the pSV-dhfr selection marker plasmid. For all further expression and activity analyzes, cell cultures were incubated for 24 hours with plasma-free selection medium in the presence of 10 pg / ml vitamin K. Expression of rFX in the resulting cell clones was examined based on the amount of antigen (ELISA, Asserachrom, Boehringer Mannheim) and then the recombinant protein was characterized by SDS-PAGE (Figures 4A and B). In the initial clones and their subclones lies what is recognizable in Western Blot (Fig. 4A), recombinant

190 734 FX protein in the form of a 22 kD light chain (LC) and a 50 kD HC heavy chain that are identical to the plasma FX protein.

In addition, a protein streak at 75 kD that corresponds to a single chain molecule (SC) can be identified and whose presence is described in FX-transfected CHO cells (Wolf et al., J. Biol. Chem 266: 13726-13730, 1991) and in human plasma ( Fair et al., Blood 64: 194-204, 1984). To obtain highly expressed clones, initial clones were amplified with increasing amounts of methotrexate and then subcloned to stabilize. Expression could be increased from approximately 200-500 ng / 10E6 cells or Igg / ml to 78 pg / 10E6 cells or 120 pg / ml for 24 hours. Western Biot analysis of these highly expressed cellular resources (Figures 4B and 5A, trace 2) shows enrichment with single-chain rFX molecules and the presence of additional forms of light chain.

In addition to the 22 kD light chain form that corresponds to the plasma form (completely carboxylated and without propeptide), there are three further light chain variants of about 21 kD, 22.5 kD and 20 kD. The light chain heterogeneity in these clones can be explained by N-terminal sequencing of recombinant material by incomplete cleavage of the propeptide (here: about 50% rFX material) and by non-carboxylation (here: about 50% rFX). The 21 kD protein is a non-carboxylated propeptide and the 20 kD protein is a non-carboxylated propeptide-free light chain form, while the 25.5 kD streak represents the fully carboxylated but propeptide containing LC.

Example 2

Conversion of single-chain rFX to rFX with light / heavy chain by r-furin derivatives

Based on the similarity of the cleavage sites between the Χ / Ν terminus of the light chain (RYTR ^ A) and between the light / heavy chain (RXKR4rS) and the furine consensus recognition sequence (RXK / RR ^ X), it was possible to improve in vitro transformation of both single chain as well as propeptide-containing rFX molecules through r-furin derivatives. The literature mentions proteases for both transformation operations, but not furin (Rehemtulla et al., 1992, Blood 79: 2349-2355; Wallin et al., 1994, Thromb. Res. 1994: 395-403).

CHO-rFX and CHO-r-furin AATM6xHis cell culture resources (patent application EP 0 775 750 A2) and CHO-rFX and non-transfected CHO (as a negative control) were mixed in a 1: 1 ratio and incubated at 37 ° C. Samples of the reaction sets were tested before incubation (t = 0) and after different incubation times (t = 2, 4, 6 hours) after Western blot analysis on transformed rFX (Figure 5). rFX was detected in cell culture stocks with anti-human FX serum (Fig. 5 A) or a monoclonal antibody specific for the FX light chain (Figure 5B).

In contrast to the CHO-rFX / CHO mixture, the CHO-rFX / CHO-r-furin mixture after just two hours of incubation at 37 ° C (Fig. 5 A, trace 7; Fig. 5B, trace 8) has been almost completely transformed. The single-chain rFX was mostly converted into a light and heavy chain form. In the light chain region, only transformed propeptide-free forms with 22 kD (carboxylated form) and 20 kD (non-carboxylated form) in a ratio of about 50: 50 were found. By optimizing cell culture conditions, this ratio can be advantageously increased to the carboxylated form. The correctness of the split between Arg-1 and Ala + 1 and the uniformity of the light chain were found by N-terminal sequencing. In a control experiment in which CHO-rFX was mixed with CHO resources, no change in rFX band pattern was found even after a 6-hour incubation (Fig. 5 A, trace 5; Fig. 5B, trace 6). In this way, it was found that furin in the stock of CHO cells is biologically active and can carry out both the transformation of propeptide and rFX heavy / light chains.

Example 3

Transformation of factor X with r-furin immobilized on chelate tentacel gel

In order to determine whether the substrate could be cleaved by the column-bound r-furin derivative, it was examined whether it could be used as a matrix matrix in a set of

190 734 instead of Ni agarose²⁺-NTA Fractogel EMD® tantakel gel (from Merck). Because compared to agaro: oąNi²⁺-NTA metal ions here are spatially more distant from the actual column matrix, better spatial accessibility of the substrate to the bound r-furin derivative was possible. In this assumption, the pro-factor X was transformed with the r-firin derivative bound to the tantalide gel: according to the manufacturer's recipe, the Fractogel eMD® tantacel gel was loaded with Na2 + ions and equilibrated with fresh serum-free cell culture medium. The column was then loaded with a serum-free stock of r-furin derivative from CHO. Wash operations were performed with serum-free cell culture medium containing iodazole in concentrations increasing to 40 mM. Then, pro-factor X was passed through the column as a serum free CHO. The transformation of pro-factor X into a two-chain factor X was investigated in column flow by Western Biot analysis with specific factor X antiserum.

Example 4

In-vitro recombinant factor X activity

The recombinant factor X precursor was incubated with and without r-furin at 4 ° C. Samples were taken at various intervals and frozen at -20 ° C. After the incubation (after 4 days), the activity of all samples was tested using the FX-Coatest Kit (from Chromogenix). To this end, 50 μΐ of human plasma with FX deficit was added to 50 µl of each sample and according to the manufacturer's protocol, rFX was converted to rFXa by Russell's venom (RW) in the presence of CaCfe; then chromogenic substrate (S-2337) was hydrolysed with rFXa, resulting in the release of yellow colored paranitroaniline. Since the amount of rFXa and the color intensity are proportional to each other, the amount of rFX / ml of the cell culture resource activated into rFX was determined using a simple standard interpolated interpolated plasma dilution series. From these results and from known amounts of rFX antigen (ELISA data), the percentage of rFX activated to factor Xa could be calculated.

The results are shown in Table 1.

In order to exclude non-specific, proteolytic activity in CHO and CHO-r-phyrin resources, a mixture of both of these cell culture resources was also examined.

CHO-rFX incubated with CHO resources (without r-furin) as a control did not show even after 4 days no significant change in rFXa activity, which due to experimental deviations was 800 mU / ml and corresponded to 55% - 61% of functional rFX. When for comparison CHO-rFX was incubated with CHO-r-furin, there was a steady increase in rFX activity during incubation, which increased from about 61% (time T = 0) to 86% (Table 1).

Thus, it was proved that as a result of the in vitro transformation of CHO-rFX from clones with strong expression by means of the r-furin derivative, the share of rFX activated to functional rFXa was significantly increased.

Table 1

<td colspan="2">Incubation in days</td><td>Activity in mU</td><td>The amount of antigen in pg / ml</td><td>Functional rFX share in%</td>

<td>CHO-CHO + RFX</td><td> 0</td><td> 814</td><td> 14</td><td> 58</td>

<td></td><td> 1</td><td> 847</td><td> 14</td><td> 61</td>

<td></td><td> 2</td><td> 835</td><td> 14</td><td> 60</td>

<td></td><td> 3</td><td> 790</td><td> 14</td><td> 56</td>

<td></td><td> 4</td><td> 763</td><td> 14</td><td> 55</td>

<td>RFX-CHO + CHO-r-</td><td> 0</td><td> 853</td><td> 14</td><td> 61</td>

<td rowspan="2">-furyna</td><td> 1</td><td> 1018</td><td> 14</td><td> 73</td>

<td> 2</td><td> 1099</td><td> 14</td><td> 79</td>

<td></td><td> 3</td><td> 1135</td><td> 14</td><td> 81</td>

<td></td><td> 4</td><td> 1198</td><td> 14</td><td> 86</td>

<td colspan="2">CHO + CHO-r-furin</td><td> 0</td><td></td><td></td>

<td colspan="2">FX from plasma 500 mU</td><td> 585</td><td></td><td></td>

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Example 5

Expression of recombinant factor X in cells with fiirin deficiency

As shown in previous examples, for factor X precursor protein, both cleavage of the propeptide and cleavage of a single chain into a light / heavy chain in vitro is handled by furin. This suggests that these operations are also performed endogenously in the cell by everywhere furin, with varying efficiency depending on the amount of r-factor X expressed each time. However, this leads to the production of a mixture of heterogeneous forms of r-factor X.

Inhibiting the cleavage of r-factor X by endogenous proteases, especially furin, gives the possibility of producing a homogeneous and stable form of r-factor X molecules, and thus producing a functionally inactive precursor of r-factor X (which by its subsequent transformation, exactly before use, it can be transformed into its functionally active form).

This method is particularly advantageous for producing an FX analog that contains a furin cleavage site instead of the original activation site. For these constructs, the activation of such recombinant rFX mutans may occur in vivo as a result of the action of endogenous firin and lead to the secretion of activated unstable forms of rFX. Decomposition of these forms by CHO proteases, e.g. under conditions of cell culture with large cell lysis, during storage of cell culture resources or during purification operations or as a result of autoproteolysis, it can lead to inactive degradation products (Wolf et al., 1991).

This goal can be achieved, for example, by supplementing the cell culture medium with an agent that can reduce or eliminate the intracellular activity of furin.

Another possibility is the use of furin deficit cells (Mohring et al., 1983, Infect. Immun. 41: 998-1009; Ohnishi et al., 1994, J. Yirol. 68: 4075-4079; Gordon et al., 1995, Infect. Immun. 63: 82-87).

For this purpose, CHO FD11 cell clone having a fur deficit (Gordon et al., 1995, Infect. Immun. 63: 82-87) was co-transfected with 20 pg phAct-FX and 1 pg pUCSV-neo (containing the neomycin resistance gene in the pUC vector under the control of the SV40 promoter). To obtain stable clones, the medium was supplemented by adding 0.8 pg G418 / ml. By comparing the separated r-factor X molecules in the serum-free resources of CHO clones containing furin and having a furin deficit, it turned out in Western Biot that furin-deficient cells do not undergo transformation of the r-factor X precursor and only contains the single-chain factor precursor X (fig. 6); in contrast, r-factor X of "normal" cells undergoes modest expression under modest expression, but under greater expression, despite endogenous furin, it undergoes only limited transformation. That due to the low expression level of the cell clone used, the r-factor X light chain cannot be seen in Western Biot.

Example 6

Preparation of factor X analogues (most preferred embodiment)

6.1 Construction of expression plasmids for the production of factor X analogues

For the production of the recombinant r-factor X analog, the Asn-Leu-Thr-Arg / Ile cleavage site (amino acids 231 to 235), which is used to activate factor X to factor Xa, has been replaced with a cleavage site specific for another protease, such as furin, FXIa , FXa, FXIIa, FIIa or kallikrein. All expression plasmids for this factor X analogue are derived from the phAct-FX plasmid (described in Example 1).

To simplify cloning of the factor X expression plasmid, the HindIII-Nael DNA fragment from the phAct-FX expression plasmid, which includes the factor X coding region position +1 to +1116, was included in the HindII / SmaI restriction site of the pUC19 plasmid. The resulting plasmid was designated by pUC / FX.

As a result, the factor X nucleotide sequence at positions 508 to 705 (amino acids 160 to 235) could be easily removed from the pUC / FX plasmid and replaced by various mutant factor X DNA fragments. These DNA fragments are identical to the wild type factor X sequence deleted , except for positions 691 to 705 (amino acids 231 to 235), which encode new cleavage sites.

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The wild type factor X sequence was removed from the pUC / FX plasmid by restriction cleavage Bspl20I and BstXL 3'-protrusion of the BstX3 site removed in addition by Mung Bean nuclease (Biolab).

Factor X DNA fragment mutations were generated by PCR. The 5'-Primer is identical for all cloning and contains a factor X sequence from position 496 to 516. The 3'-Primers contain a factor X complementary sequence (positions 676 to 690) and a non-complementary 5'-projection that carries the sequences for the new cleavage site and restrictive cut sites. The amplified PCR product was then cleaved with the appropriate restriction enzyme (s) and cloned into the prepared pUC / FX vector (see above).

Factor X DNA fragment mutations were then cloned with HindTT-Agel from the pUC / FX plasmid into the phAct-FX vector. The end constructs are schematically shown in Figures 2.1 and 2.2. The wild type X factor is given as the reference construct. Amino acids are given in the single letter code. Positions that are mutations are additionally shaded.

To generate the Asp-Phe-Thr-Arg / Val cleavage site for FXIa, oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 ') was used as a 5'-primer (SEQ ID No. 3) and as 3 '-starter oligonucleotide # 1002 (5'-ACCA GTT AAC CCT GGT GAA GTC GTT GTC GCC CCT CTC-3') (SEQ. ID. No. 4). The Asn, Leu and Ile amino acids at positions 231, 232 and 235 of the factor X sequence were therefore replaced with Asp, Phe and Val. The PCR fragment was cut using Bstl20I and HpaI (Fig. 2A).

To generate the Arg / Ser cleavage site for FIIa, oligonucleotide # 1001 (5-CCC ACA GGG CCC TAC CCC TGT-3 ') was used as a 5'-primer (SEQ ID No. 3) and as a 3'-primer oligonucleotide # 1003 ( 5'-ACCA TCG CGA CCT GGT CAG GTT GTT GTC-3¹) (SEQ. ID. No. 5). Therefore, the amino acid Ile at position 235 was mutated to Ser. The PCR fragment was cut with Bsp 1201 and NruI (Fig. 2B).

To generate the Ile, Lys, Pro, Arg / Ile cleavage site for FXIIa, oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 ') was used as a 5'-primer (SEQ ID No. 3) and as 3 '-starter oligonucleotide # 1004 (5'-ACC AGA ATC GAT TCT GGG TTT GAT GTT GTC GCC CCT CTC-3') (SEQ. ID. No. 6). Therefore, the amino acids Asn, Leu and Thr were mutated at positions 231, 232 and 233 of the FX sequence on Ile, Lys and Pro. The PCR fragment was cut off partially with Bstl20I and XmnI (Figure 2C).

Oligos # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 ') was used as the 5'-primer for kallikrein to generate the Ser-Met-Thr-Arg / Ile cleavage site (SEQ ID No. 3) and as 3 '-starter oligonucleotide # 1005 (5'-ACC AGA ATC GAT TCT GGT CAT GCT GTT GTC GCC CCT CTC-3' (SEQ. ED. No. 7). Asn, Leu amino acid mutations at positions 231, 232 of factor X sequence were performed for Ser, Met. The PCR fragment was partially cut off with Bst120I and XmnI (Fig. 2D).

To generate the Pro-Gln-Gly-Arg / He cleavage site for FXa, oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 ') was used as a 5'-primer (SEQ ID No. 3) and as 3 '-starter oligonucleotide # 1016 (5'-ACC AGA ATC GAT TCT TCC TTG GGG GTT GTC GCC CCT CTC-3') (SEE ID No. 8). Therefore, amino acid mutations Asn, Leu and Thr were performed at positions 231, 232 and 233 of the FX protein on Pro, Gln and Gly. The PCR fragment was cut off partially with Bst120I and XmnI (Fig. 2H).

To generate the Met-Lys-Thr-Arg / Ile cleavage site for FXa, oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 ') was used as a 5'-primer (SEQ ID No. 3) and as 3 '-starter oligonucleotide # 1014 (5'-ACC AGA ATC GAT TCT CGT tTt CAT GTT GTC GCC CCT CTC-3') (SEQ. ID. No. 9). Therefore, amino acid mutations of Asn, Leu at positions 231, 232 of the FX protein on Pro, Lys were performed. The PCR fragment was partially cut off with Bst120I and Xmnf (Figure 2E).

Oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 ') was used as the 5'-primer for the FXa cleavage site Ile-Glu-Gly-Arg / Ile for FXa (SEQ. ED. No. 3) and as 3 '-starter oligonucleotide # 1015 (5'-ACC AGA ATC GAT TCT TCC CTC GAT GTT GTC GCC CCT CTC-3) (SeQ. ID. No. 10). Therefore, the amino acid mutations Asn, Leu, Thr were performed at positions 231 to 233 of the FX protein on Ile, Glu, Gly. The PCR fragment was partially cut off with Bst120I and XmnI. (Fig. 2F).

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Oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3) was used as a 5'-primer to generate the Arg-Arg-Lys-Arg / De cleavage site for Furin (SEQ ID No. 3) and as 3 ' -starter oligonucleotide # 1006 (5'-ACC AGA ATC GAT TCT TTT CCT CCT GTT GTC GCC CCT CTC-3 ') (SEQ. ID. No. 11). Therefore, the amino acids Asn, Leu and Thr were mutated at positions 231 to 233 on Arg, Arg and Lys. The PCR fragment was cut with Bspl20I and XmnI (Figure 2G).

Oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 ') was used as a 5'-primer to generate the Arg-Val-Arg-Arg / He cleavage site for furin (SEQ ID No. 3) and as 3 '-starter oligonucleotide # 1007 (5'-ACC AGA ATC GAT TCT CCT CAC CCT GTT GTC GCC CCT CTC-3') (SEQ. ID. No. 12). Therefore, the amino acids Asn, Leu and Thr were mutated at positions 231 to 233 on Arg, Val and Arg. The PCR fragment was cut off partially with Bspl20I and XmnI (Figure 2G).

Oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 ') was used as a 5'-primer to generate the Arg-Arg-Arg-Arg / Ue cleavage site for furin (SEQ ID No. 3) and as 3 '-starter oligonucleotide # 1008 (5'-ACC AGA ATC GAT TCT CCT CCT CCT GTT GTC GCC CCT CTC-3') (SEQ. ID. No. 13). Thus, the amino acids Asn, Leu and Thr were mutated at positions 231 to 233 on Arg, Arg and Arg. The PCR fragment was partially cut off with Bsp120I and XmnI (Figure 2G).

Oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 ') was used as a 5'-primer to generate the Arg-Pro-Lys-Arg / Ue cleavage site (SEQ. ID, No. 3) and as 3 '-starter oligonucleotide # 1009 (5'-ACC AGA ATC GAT TCT TTT GGG CCT GTT GTC GCC CCT CTC-3') (SEQ. ID. No. 14). Therefore, the amino acids Asn, Leu and Thr were mutated at positions 231 to 233 on Arg, Pro and Lys. The PCR fragment was cut off partially with Bspl20I and XmnI (Figure 2g).

Oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 ') was used as a 5'-primer to generate the Ile-Ag-Lys-Ag / Ue cleavage site for furin (SEQ. ID. No. 3) and as 3 '-starter oligonucleotide # 1010 (5'-ACC AGA ATC GAT TCT TTT CCT GAT GTT GTC GCC CCT CTC-3') (SEQ. ID No. 15). Therefore, the amino acids Asn, Leu and Thr were mutated at positions 231 to 233 on Ile, Arg and Lys. The PCR fragment was partially cut off with Bsp120I and XmnI (Figure 2G).

To generate the Ag-Ser-Lys-Arg / Ile cleavage site, oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 ') was used as a 5'-primer (SEQ. ID. No. 3) and as 3 '-starter oligonucleotide # 1011 (5-ACC AGA ATC GAT TCT TTT GCT CCT GTT GTC GCC CCT CTC-3') (SEQ. ID. No. 16). Therefore, the amino acids Asn, Leu and Thr were mutated at positions 231 to 233 on Arg, Ser and Lys. The PCR fragment was partially cut off with Bsp120I and XmnI (Figure 2G).

To generate the Arg-Val-Thr-Arg / Ile cleavage site, oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 ') was used as a 5'-primer (SEQ. ID. No. 3) and as 3 '-starter oligonucleotide # 1012 (5'-ACC AGA ATC GAT TCT GGT CaC CCT GTT GTC GCC CCT CTC-3') (SEQ ID No. 17). Therefore, the amino acids Asn, Leu were mutated at positions 231, 232 on Arg, Val. The PCR fragment was partially cut off with Bsp120I and XmnI (Figure 2G).

To generate the Ag-Leu-Lys-Ag / Ue cleavage site, oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 ') was used as a 5'-primer (SEQ ID No. 3) and as 3 '-starter nucleotide # 1013 (5'-ACC AGA ATC GAT TCT TTT GAG CCT GTT GTC GCC CCT CTC-3') (SEQ ID. No. 18). Therefore, the amino acids Asn and Thr were mutated at positions 231 and 233 on Arg and Lys. The PCR fragment was partially cut off with Bsp120I and XmnI (Figure 2G).

To generate the Thr-Ser-Thr-Arg / Ile cleavage site for FXIIa, oligonucleotide # 1001 (5'-CCC ACA GGG CCC TAC CCC TGT-3 '(SEQ. ID. No. 3) and 3' was used as the 5'-primer -starter oligonucleotide # 1017 (5-ACC AGA ATC GAT TCT CGT GCT CGT GTT GTC GCC CCT CTC-3 ') (SEQ. ID. No. 19). Asn, Leu amino acid mutations at positions 231, 232 of FX protein on Ile , Bal. The PCR fragment was partially cut off with Bst120I and XmnI (Figure 21).

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6.2. Contruction of expression plasmids for production of the ΕΧβ analog

These constructs were obtained from the above-described factor X analog constructs, incorporating the TGA stop codon at position 470. In addition, the amino acids from position 457 at cDNA level up to the stop codon were removed by Spel and partially digested with BstEII up to the stop codon and replaced with oligonucleotide pair # 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). A schematic diagram of the factor uβ analog constructs is shown in Figure 7. To simplify the drawing, all factor analogβ analogues are shown as one general construct in which the changed amino acids in the cleavage site regions are given as "X" shading.

6.3. Construction of expression plasmids for the production of FXa analogue

By activating factor X by cleaving the 4.5 kD activation peptide at the N-terminus of the heavy chain, factor Xaa was obtained. As a result of the autoproteolytic activity of this form and cleavage of the C-terminus of the heavy chain between Arg 469 and Gly 470 it was transformed into the ΕΧηβ form. In order to obtain the factor X expression plasmid, which, when activated, exists only in the form of FXaa with intact β-peptide, the amino acid Arg 469 has been mutated to Lys, so that no transformation can occur in the C-terminal region of the heavy chain.

In addition, the C-terminal amino acid sequence of factor X was removed from position 1363 up to the stop signal by partial digestion with BstEH-Spel and replaced with a pair of linked oligonucleotides. Oligonucleotide # 0005 (5-GTC ACC GCC TTC CTC AAG TGG ATC GAC AGG TCC ATG AAA ACC AAG GGC TTG CCC AAG-3 ') (SEQ. ED. 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 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 CT3-3 ') (SEQ ID No. 25). As a mutation of the amino acid Arg 469, a pair of nucleotides # 0005- # 0006 were introduced. Analog FX is schematically shown in Figure 7.

Example 7

Determination of the N-terminus of factor X and transformation product with and without r-furin

Recombinant factor X was expressed in CHO cells with endogenous furin (as described in Example 1) or in cells with furin deficiency (as described in Example 5). r-Factor X was isolated both from a) untreated, b) incubated for a further 12 hours at 37 ° C and c) pretreated for 12 hours at 37 ° C with r-furine from the CHO cell culture strongly expressed rFX clones in CHO, as well as d) not pretreated and e) pretreated for 12 hours at 37 ° C with r-furin in CHO cell culture stock of rFX clones with CHO-FD11. The N-terminal amino acids of factor X and the transformation products of the individual reaction sets a) to e) were determined by performing Edman analysis. The results are schematically shown in figure 8.

r-Factor X from highly expressed CHO cells was in the form of mature heavy and light chains as well as a single chain, partly containing propeptide. After incubating these cell culture resources for 12 hours at 37 ° C (b), there are additional, as already described by Wolf et al. (1991, J. Bio. Chem. 266: 13726-13730), defective light chain N-ends rFX with 3 additional amino acids Vall38-Thr39-Arg40. Such hidden ends were also found during sequencing of rFX material from untreated CHO-FD11 cells (d). This observation showed that these defective N-term can be avoided when optimal conditions or parameters of cell culture, storage and purification methods are used to transform the rFX proteolysis by CHO proteases.

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Unlike purified material from CHO (a and b) cells, rFX from non-amplified furin deficit (d) cells only occurs in the form of unconverted single chain precursors. No N-terminal sequences corresponding to propeptide contribution were found. It was thus demonstrated that the conversion of single-chain rFX precursors into light / heavy chains does not occur in furoid deficit (d) CHO cells, which leads to the conclusion that furin endoprotease plays a major role in this in vivo transformation operation.

Additionally, it has been shown that the transformation of propeptide-containing rFX molecules also occurs in furin deficient CHO cells, so furin does not play an important role in vivo in this transformation operation. After incubation in the presence of furin rFX from CHO (c) cells that only CHO-FD11 (e) cells found light and heavy chains with the correct N-terminus. Therefore, it has been proved that both single-chain FX precursors and rFX molecules containing propeptide by in vitro conversion are processed into homogeneous, mature factor X. Factor X transformed in the presence of furin has therefore extraordinary homogeneity and structural uniformity.

Example 8

Expression and characterization of the FX analog with furin cleavage site ArgArg-Lys-Arg / Ile (fETRR ^)

To obtain the recombinant protein, rFTRR was co-transfected into CHO cells with the FX expression plasmid with the Arg-Arg-Lys-Arg / Ile cleavage site (see Example 6.1, Figure 2G) and the selection plasmid pSV / dkfr as described in Example 1.

Western Biot analysis of cell culture resources (Fig. 9) showed that the recombinant protein is predominantly in the double chain form. Compared to plasma FX, the heavy chain is about 46 kD instead of 50 kD, which can probably be explained by various saccharification of the recombinant protein. In addition, small amounts of single chain precursors (SCs) as well as LC4 light chain isoforms are seen, as has already been seen with expression of wild type rFX (example 1.b.). These molecular forms of the rFX analogue indicate a reduction in both the conversion of the single chain FX precursor by endogenous proteases and light chain γ-carboxylation. Although the cleavage site inserted into the FX analog represents the consent sequence for furin, there are no visible protein bands that correspond to activated forms of the protein (35 kD, 31 kD). Both the structure in the region of the cleavage site and the surrounding amino acid sequence probably activated led to the transformation of the modified activation site by furin creating only suboptimal conditions.

Example 9

In vitro activation of rPX protein^Rx^κx^/And by r-furin derivatives

In vitro activity of the recombinant FX analogue to FXaa (35 kD) and β (: 31 kD) by r-furin was tested, as in the description for the mixed experiments in Example 2, with the difference that the purified r-furin derivative was used, and namely r-furinAACys-Spacer-10xHis, described in EP 0 75 750 A2, in 10 mM Hepes, pH 7.0, 150 mM NaCl, 2 mM CaCl₂ and 0.2% BSA instead of CHO r-furin stock. In a control experiment without r-furin, a 1: 1 ratio of rFX analogue with CHO was mixed with 10 mM Hepes buffer, pH 7.0.150 mM NaCl, 2 mM CaCl₂ and 0.2% BSA. Samples of the above kits were tested by Western Biot for activation of rFX before and after incubation times of 6, 24, 48 and 72 (t = 0, 6, 24, 48, 72) hours at 37 ° C (fg. 10). While the rFX band pattern in the absence of the r-furin derivative is unchanged even after 72 hours of incubation (Fig. 10B), in the presence of r-furin after 6 hours (Fig. 10A, trace 5) a heavy 35 kD protein band is visible which corresponds to the? α form from plasma (Fig. 10A, trace 9). During the incubation, this form a accumulates and after 72 hours of incubation (Fig. 10a, trace 8) about 50% of the starting material (HC) is processed into the activated form. An additional protein band of 31 kD, which becomes recognizable after 24 hours (Figure 10A, trace 6) and corresponds to the β-activated form of plasma FX (Figure 10a, trace 9), shows that the resulting recombinant form of analogue FX has autoproteolytic activity and is therefore functional.

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These results prove that the heterogeneous Arg-ArgLys-Arg / Ile activation cleavage site in the rFX analog is specifically recognized and correctly cleaved in vitro by the r-furin derivative, and is therefore suitable for activating the rFX analog in the FIAA molecule and the FXap molecule.

Example 10

Functionality of the in vitro activated FX rFXRRKR analogue

Samples from the mixed tests according to example 9 were tested by means of a chromogenic assay for FXa activity. For this purpose, chromogenic substrate S2337 (600 pM) in 50 mM tris, pH 7.3, 150 mM NaCl and 0.1% BSA was added to the samples. After incubation for 3 minutes at 37 ° C, the reaction was stopped with 20% acetic acid and then the optical density (OD) was measured at 405 nm. By comparison with the graph in the form of a standard straight line obtained with purified, plasma-activated RVV FXa, the values ​​of the recombinant FXa activity were determined in reaction sets. The results of this analysis, including the amounts of antigen used (ELISA values) as well as the specific activity calculated from this, are given in Table 2.

To exclude non-specific amidolytic activities in r-furin solution and in the stock from CHO cell culture, a mixture of non-transfected CHO cells was also tested for FXa activity (CHO + r-furin). In this mixture, as in the rFX analogue / buffer mixture (rFX + buffer), no FXa activity was found after 72 hours of incubation. In contrast, the reaction with r-furin had a measurable rFXa activity of 56 mU after incubation for 6 hours, which increased during further incubation and reached 133 mU after 72 hours. Although at this time, according to Western Biot, only about half of the rFX analogue was processed into activated aip forms (Fig. 10A, trace 8), the in vitro activated rFX analogue material reaching 190 mU / pg had a large, greater specific activity than fully activated using plasma RVV FX (153 mU / pg). The measured increases in activity are reflected in the occurrence of the form aip in Western Blot (Fig. 10A, traces 5-8).

In this way, it was found that heterogeneous protease cleavage sites can be embedded in FX, these sites can be recognized and cleaved by the associated protease, and recombinantly high-grade rFXa (or optionally rFXa analogues with FXa activity) can be produced in functional form.

T ab el a 2

<td colspan="2">Incubation time in hours</td><td>Activity in mU / ml</td><td>The amount of antigen in pg / ml</td><td>Specific activity in mU / pg</td>

<td>_rpx^RRKR + buffer</td><td> 0</td><td> &lt;25</td><td> 0,7</td><td> 0</td>

<td></td><td> 6</td><td> &lt;25</td><td> 0,7</td><td> 0</td>

<td></td><td> 24</td><td> &lt;25</td><td> 0,7</td><td> 0</td>

<td></td><td> 48</td><td> &lt;25</td><td> 0,7</td><td> 0</td>

<td></td><td> 72</td><td> &lt;25</td><td> 0,7</td><td> 0</td>

<td rowspan="2">RFX^RRR H-Turyna</td><td> 0</td><td> &lt;25</td><td> 0,7</td><td> 0</td>

<td> 6</td><td> 56</td><td> 0,7</td><td> 80</td>

<td></td><td> 24</td><td> 101</td><td> 0,7</td><td> 144</td>

<td></td><td> 48</td><td> 124</td><td> 0,7</td><td> 177</td>

<td></td><td> 72</td><td> 133</td><td> 0,7</td><td> 190</td>

<td rowspan="2">CHO + r-fryer</td><td> 0</td><td> &lt;25</td><td> 0</td><td></td>

<td> 6</td><td> &lt;25</td><td> 0</td><td></td>

<td></td><td> 24</td><td> &lt;25</td><td> 0</td><td></td>

<td></td><td> 48</td><td> &lt;25</td><td> 0</td><td></td>

<td></td><td> 72</td><td> &lt;25</td><td> 0</td><td></td>

<td colspan="2">Plasma FX activated by RVV</td><td> 614</td><td> 4</td><td></td>

190 734

Example 11

In vitro activation of the rFX analogue from the cleavage of FXIa Asp-Phe-Thr-Arg / Val (rFX) by FXIa from plasma

FX analogue construct was obtained by mutagenesis of the FX activation sequence to the FXIa cleavage site. Stable CHO cell clones were then generated that express these molecules. The stock of rFX CHO cell culture was mixed with purified plasma FXIa (100 pg / ml) in the presence of 10 mM tris, pH 7.3 150 mM NaCl, 8mM CaCl2, PCPS and 0.1% BSA and incubated at 37 ° C in over different times. As a negative control, the cell culture stock was only incubated with BSA containing buffer. The rFX protein and resulting activation products were characterized by Western Biot analysis (Figure 11).

As can be recognized in the mixture without FXIa before incubation (t = 0), the recombinant protein (Figure 11, trace 5) is secreted in a two-chain form almost identical to plasma FX (trace 2) except that the heavy chain (HC) , as already observed in rFXRRR * of Example 8, has a molecular weight slightly less than 50 kD. When incubating this mixture at 37 ° C (Figure 11, trace 6), no significant change in the band pattern is visible. The CHO-rFX / FXIa mixture can be identified, shortly before the addition of purified FXIa but before the actual incubation of the cell culture stock (Fig. 11, trace 3), 35 kD and 31 kD protein bands, which correspond in size to plasma forms ai β heavy chain (Figure 11, track 9). Both forms increase strongly after a 4-hour incubation with FXIa (Fig. 11, trace 4).

Thus, it has been shown that the FX analog, which includes a heterogeneous protease cleavage site for a proteolytic enzyme active in the coagulation cascade, can be successfully transformed by the latter.

By the occurrence of rFXaβ bands, the functional activity of the resulting rFXaa was successfully demonstrated, in addition to the result of the auto-proteolytic activity of the rFXaa analogue.

190 734

Sequence list (1) General data (s) Applicant (A) Name: Baxter AG (B) Street: Industriestrasse 67 (C) Town: Vienna (D) Federal country: Austria (E) Country: AusAia (F) Postal code : 1220 (A) Name: Michele Himmelspach (B) Street; Breitstetten 19 (C) Town: Leopoldsdorf (D) Federal country: Austria (E) Country: Austria (F) Postal code: 2285 (A) Name: Uwe Schlokat (B) Street: Hauptstrasse 51 (C) Town: Orth / Donau (D) Federal country: Austria (E) Country: Austria (F) Postal code: 2304 (A) Name: Andreas Fisch (B) Street: Tellstrasse 19 (c) Town: St. Gallen (D) Federal country: Austria (E) Country: Switzerland (F) Postal area code: 9000 (A) Name: Friedrich Domer (B) Street: Peterlinigasse 17 (C) Town: Vienna (D) Federal country: Austria (E) Country: Austria (F) Postal area code: 1230 (A) Name: Johann Eibl (B) Street: Gustav Tschermakgasse 2 (C) Town: Vienna (D) Federal country: Austria (E) Country: Austria (F) Postal code: 1180 (ii) Title of the invention: Factor X analogue, recombinant DNA, compositions, use of recombinant DNA nuicleic acid and methods of making the composition (iii) Number of sequences: 27 (iv) Computer readable text:

(A) Data carrier: floppy disk (B) Computer: IBM PC compatible (C) Operation system: PC-DOS / MS-DOS (D) Software: Patentln edition # 1.0, version # 1.30 (EPA)

190 734 (2) Data for SEQ ID NO: 1:

(i) Sequence features:

(A) Length: 34 base pairs (B) Type: nucleotide (C) Thread form: single strand (D) Topology: linear (ii) Type of molecule: genomic DNA (xi) sequence description: SEQ ID NO: 1:

ATTACTCGAG AAGCTTACCA TGGGGCGCCC ACTG 34 (2) Data for SEQ ID. No: 2:

(i) Sequence features (A) Length: -24 base pairs (B) Type: nucleotide (C) Strand form: single strand (D) Topology: linear (ii) Type of molecule: genomic DNA (xi) Sequence description: SEQ ID NO: 2:

ATTACAATTG CTGCAGGGAT CCAC 24 (2) Data for SEQ ID NO: 3:

(i) Sequence features:

(A) Length: 21 base pairs (B) Genus: nucleotide (C) Thread form: single strand (D) Topology: linear (ii) Molecule type: genomic DNA (xi) Sequence description: SEQ ID NO: 3:

CCCACAGGGC CCTACCCCTG T 21 (2) Data for SEQ ID NO: 4:

(i) Sequence features:

(A) Length: 37 base pairs (B) Type: nucleotide (C) Thread form: single strand (D) Topology: linear (ii) Type of molecule: genomic DNA (xi) Sequence description: SEQ ID NO: 4:

ACCAGTTAAC CCTGGTGAAG TCGTTGTCGC CCCTCTC 37 (2) Data for SEQ ID NO: 5:

(i) Sequence features:

(A) Length: 28 base pairs (B) Genus: nucleotide (C) Strand form: single strand (D) Topology: linear (ii) Molecule type: genomic DNA (xi) Sequence description: SEQ ID NO: 5:

ACCATCGCGA CCTGGTCAGG TTGTTGTC 28 (2) Data for SEQ ID NO: 6:

(i) Sequence features:

190 734 (A) Length: 39 base pairs (B) Type: nucleotide (C) Thread form: single strand (D) Topology: linear (ii) Molecule type: genomic DNA (xi) Sequence description: SEQ ID NO: 6:

ACCAGAATCG ATTCTGGGTT TGATGTTGTC GCCCCTCTC 39 (2) Data for SEQ ID NO: 7:

(i) Sequence features:

(A) Length: 39 base pairs (B) Type: nucleotide (C) Thread form: single strand (D) Topology: linear (ii) Type of molecule: genomic DNA (xi) Sequence description: SEQ ID NO: 7:

ACCAGAATCG ATTCTGGTCA TGCTGTTGTC GCCCCTCTC 39 (2) Data for SEQ ED NO: 8:

(i) Sequence features:

(A) Length: 39 base pairs (B) Genus: nucleotide (C) Thread form: single strand (D) Topology: linear (ii) Molecule type: genomic DNA (xi) Sequence description: SEQ ID NO: 8:

ACCAGAATCG ATTCTTCCTT GGGGGTTGTC GCCCCTCTC 39 (2) Data for SEQ ID NO: 9:

(i) Sequence features:

(A) Length: 39 base pairs (B) Genus: nucleotide (C) Thread form: single strand (D) Topology: linear (ii) Molecule type: genomic DNA (xi) Sequence description: SEQ ED NR: 9:

ACCAGAATCG ATTCTCGTTT TCATGTTGTC GCCCCTCTC 39 (2) Data for SEQ ED NO: 10:

(i) Sequence features:

(A) Length: 39 base pairs (B) Type: nucleotide (C) Thread form: single strand (D) Topology: linear (ii) Type of molecule: genomic DNA (xi) Sequence description: SEQ ID NO: 10:

ACCAGAATCG ATTCTTCCCT CGATGTTGTG GCCCCTCTC 39 (2) Data for SEQ ID NO: 11:

(i) Sequence features:

(A) Length: 39 base pairs (B) Type: nucleotide (C) Thread form: single strand (D) Topology: linear

190 734 (ii) Type of molecule: genomic DNA (xi) Sequence description: SEQ ID NO: 11:

ACCAGAATCG ATTCTTTTCC TCCTGTTGTC GCCCCTCTC 39 (2) Data for SEQ ID NO: 12:

(i) Sequence features:

(A) Length: 39 base pairs (B) Genus: nucleotide (C) Strand form: single strand (D) Topology: linear (ii) Molecule type: genomic DNA (xi) Sequence description: SEQ ID, NO: 12:

ACCAGAATCG ATTCTCCTCA CCCTGTTGTC GCCCCTCTC 39 (2) Data for SEQ ID NO: 13:

(i) Sequence features:

(A) Length: 39 base pairs (B) Type: nucleotide (C) Thread form: single strand (D) Topology: linear (ii) Molecule type: genomic DNA (xi) Sequence description: SEQ ID NO: 13:

ACCAGAATCG ATTCTCCTCC TCCTGTTGTC GCCCCTCTC 39 (2) Data for SEQ ID NO: 14:

(i) Sequence features:

(A) Length: 39 base pairs (B) Genus: nucleotide (C) Strand form: single strand (D) Topology: linear (ii) Molecule type: genomic DNA (xi) Sequence description: SEQ ID NO: 14:

ACCAGAATCG ATTCTTTTGG GCCTGTTGTC GCCCCTCTC 39 (2) Data for SEQ ID NO: 15:

(i) Sequence features:

(A) Length: 39 base pairs (B) Type: nucleotide (c) Thread form: single strand (D) Topology: linear (ii) Type of molecule: genomic DNA (xi) Sequence description: SEQ ID NO: 15:

ACCAGAATCG ATTCTTTTCC TGATGTTGTC GCCCCTCTC 39 (2) Data for SEQ ID NO: 16:

(i) Features se ^ e ^ k ^^^^ c ^:

(A) Length: 39 base pairs (B) Type: nucleotide (c) Thread form: single strand (D) Topology: linear

190 734 (ii) Type of molecule: genomic DNA (xi) Sequence description: SEQ ID NO: 16:

ACCAGAATCG ATTCTTTTGC TCCTGTTGTC GCCCCTCTC 39 (2) Data for SEQ ID NO: 17:

(i) Sequence features:

(A) Length: 39 base pairs (B) Genus: nucleotide (C) Strand form: single strand (D) Topology: linear (ii) Molecule type: genomic DNA (xi) Sequence description: SEQ ID NO: 17:

ACCAGAATCG ATTCTGGTCA CCCTGTTGTC GCCCCTCTC 39 (2) Data for SEQ ID NO: 18:

(i) Sequence features:

(A) Length: 39 base pairs (B) Type: nucleotide (C) Strand form: single strand (D) Topology: linear (ii) Type of molecule: genomic DNA (xi) Sequence description: SEQ ID NO: 18:

ACCAGAATCG ATTCTTTTGA GCCTGTTGTC GCCCCTCTC 39 (2) Data for SEQ ID NO: 19:

(i) Sequence features:

(A) Length: 39 base pairs (B) Type: nucleotide (C) Thread form: single strand (D) Topology: linear (ii) Type of molecule: genomic DNA (xi) Sequence description: SEQ ID NO: 19:

ACCAGAATCG ATTCTCGTGC TCGTGTTGTC GCCCCTCTC 39 (2) Data for SEQ ID NO: 20:

(i) Sequence features:

(A) Length: 49 base pairs (B) Type: nucleotide (C) Thread form: single strand (D) Topology: linear (ii) Type of molecule: genomic DNA (xi) Sequence description: SEQ DD NR: 20:

GTCACCGCCT TCCTCAAGTG GATCGACAGG TCCATGAAAA CCAGGTGAA 49 (2) Data for SEQ ID NO: 21:

(i) Features of selection:

(A) Length: 48 base pairs (B) Type: nucleotide (C) Thread form: single strand (D) Topology: linear

190 734 (ii) Type of molecule: genomic DNA (xi) Sequence description: SEQ DD NR: 21:

CTAGTTCACC TGGTTTTCAT GGACCTGTCG ATCCACTTGA GGAAGGCG 48 (2) Data for SEQ ID NO: 22:

(i) Sequence features (A) Length: 57 base pairs (B) Type: nucleotide (C) Strand form: single strand (D) Topology: linear (ii) Molecule type: genomic DNA (xi) Sequence description: SEQ ED NR : 22:

GTCACCGCCT TCCTCAAGTG GATCGACAGG TCCATGAAAA CCAAGGGCTT GCCCAAG 57 (2) Data for SEQ ID NO: 23:

(i) Sequence features:

(A) Length: 57 base pairs (B) Genus: nucleotide (C) Strand form: single strand (D) Topology: linear (ii) Molecule type: genomic DNA (xi) Sequence description: SEQ ID NO: 23:

TTGGCCTTGG GCAAGCCCTT GGTTTTCATG GACCTGTCGA TCCACTTGAG GAAGGCG 55 (2) Data for SEQ ID NO: 24:

(i) Sequence features:

(A) Length: 55 base pairs (B) Genus: nucleotide (C) Strand form: single strand (D) Topology: linear (ii) Molecule type: genomic DNA (xi) Sequence description: SEQ ID NO: 24:

GCCAAGAGCC ATGCCCCGGA GGTCATAACG TCCTCTCCAT TAAAGTGAGA TCCCA 55 (2) Data for SEQ ID NO: 25:

(i) Sequence features:

(A) Length: 54 base pairs (B) Genus: nucleotide (C) Strand form: single strand (D) Topology: linear (ii) Molecule type: genomic DNA (xi) Sequence description: SEQ ID NO: 25:

190 734

CTAGTGGGAT CTCACTTTAA TGGAGAGGAC GTTATGACCT CCGGGGCATG GCTC 54 (2) Data for SEQ ID NO: 26:

(i) Sequence features:

(A) Length: 1467 base pairs (B) Type: nucleotide (C) Thread form: single strand (D) Topology: linear (ii) Type of molecule: genomic DNA (ix) Characteristics:

(A) Name / key: CDS (B) Location: 1..1467 (xi) Sequence description: SEQ ID NO: 26:

<td>ATG</td><td>GGG</td><td>CGC</td><td>CCA</td><td>CTG</td><td>CAC</td><td>CTC</td><td>GTC</td><td>CTG</td><td>CTC</td><td>AGT</td><td>GCC</td><td>TCC</td><td>CTG</td><td>GCT</td><td>GGC</td><td> 48</td>

<td>Underworld</td><td>Gly</td><td>Arg</td><td>Pro</td><td>Leu</td><td>His</td><td>Leu</td><td>val</td><td>Leu</td><td>Leu</td><td>Cheese</td><td>ala</td><td>Cheese</td><td>Leu</td><td>ala</td><td>Gly</td><td></td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td><td></td>

<td>CTC</td><td>CTG</td><td>CTG</td><td>CTC</td><td>GGG</td><td>GAA</td><td>AGT</td><td>CTG</td><td>TTC</td><td>ATC</td><td>CGC</td><td>AGG</td><td>GAG</td><td>CAG</td><td>GCC</td><td>AAC</td><td> 96</td>

<td>Leu</td><td>Leu</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Glu</td><td>Cheese</td><td>Leu</td><td>phe</td><td>How much</td><td>Arg</td><td>Arg</td><td>Glu</td><td>Gln</td><td>ala</td><td>own</td><td></td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td><td></td>

<td>AAC</td><td>ATC</td><td>CTG</td><td>GCG</td><td>AGG</td><td>GTC</td><td>ACG</td><td>AGG</td><td>GCC</td><td>AAT</td><td>TCC</td><td>TTT</td><td>CTT</td><td>GAA</td><td>GAG</td><td>ATG</td><td> 144</td>

<td>own</td><td>How much</td><td>Leu</td><td>ala</td><td>Arg</td><td>val</td><td>Thr</td><td>Arg</td><td>ala</td><td>own</td><td>Cheese</td><td>phe</td><td>Leu</td><td>Glu</td><td>Glu</td><td>Underworld</td><td></td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td><td></td>

<td>AAG</td><td>AAA</td><td>GGA</td><td>CAC</td><td>CTC</td><td>GAA</td><td>AGA</td><td>GAG</td><td>TGC</td><td>ATG</td><td>GAA</td><td>GAG</td><td>ACC</td><td>TGC</td><td>TCA</td><td>TAC</td><td> 192</td>

<td>lys</td><td>lys</td><td>Gly</td><td>His</td><td>Leu</td><td>Glu</td><td>Arg</td><td>Glu</td><td>Cys</td><td>Underworld</td><td>Glu</td><td>Glu</td><td>Thr</td><td>Cys</td><td>Cheese</td><td>Tyr</td><td></td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td><td></td>

<td>GAA</td><td>GAG</td><td>GCC</td><td>CGC</td><td>GAG</td><td>GTC</td><td>TTT</td><td>GAG</td><td>GAC</td><td>AGC</td><td>GAC</td><td>AAG</td><td>ACG</td><td>AAT</td><td>GAA</td><td>TTC</td><td> 240</td>

<td>Glu</td><td>Glu</td><td>ala</td><td>Arg</td><td>Glu</td><td>val</td><td>phe</td><td>Glu</td><td>Asp</td><td>Cheese</td><td>Asp</td><td>lys</td><td>Thr</td><td>own</td><td>Glu</td><td>phe</td><td></td>

190 734

<td>TGG</td><td>AAT</td><td>AAA</td><td>TAC</td><td>AAA</td><td>GAT</td><td>GGC</td><td>GAC</td><td>CAG</td><td>TGT</td><td>GAG</td><td>ACC</td><td>AGT</td><td>CCT</td><td>TGC</td><td>CAG</td><td> 288</td>

<td>Trp</td><td>own</td><td>lys</td><td>Tyr</td><td>lys</td><td>Asp</td><td>Gly</td><td>Asp</td><td>Gln</td><td>Cys</td><td>Glu</td><td>Thr</td><td>Cheese</td><td>Pro</td><td>Cys</td><td>Gln</td><td></td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td><td></td>

<td>AAC</td><td>CAG</td><td>GGC</td><td>AAA</td><td>TGT</td><td>AAA</td><td>GAC</td><td>GGC</td><td>CTC</td><td>GGG</td><td>GAA</td><td>TAC</td><td>ACC</td><td>TGC</td><td>ACC</td><td>TGT</td><td> 336</td>

<td>own</td><td>Gln</td><td>Gly</td><td>lys</td><td>Cys</td><td>lys</td><td>Asp</td><td>Gly</td><td>Leu</td><td>Gly</td><td>Glu</td><td>Tyr</td><td>Thr</td><td>Cys</td><td>Thr</td><td>Cys</td><td></td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td>

<td>TTA</td><td>GAA</td><td>GGA</td><td>TTC</td><td>GAA</td><td>GGC</td><td>AAA</td><td>AAC</td><td>TGT</td><td>GAA</td><td>TTA</td><td>TTC</td><td>ACA</td><td>CGG</td><td>AAG</td><td>CTC</td><td> 384</td>

<td>Leu</td><td>Glu</td><td>Gly</td><td>phe</td><td>Glu</td><td>Gly</td><td>lys</td><td>own</td><td>Cys</td><td>Glu</td><td>Leu</td><td>phe</td><td>Thr</td><td>Arg</td><td>lys</td><td>Leu</td><td></td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td><td></td>

<td>TGC</td><td>AGC</td><td>CTG</td><td>GAC</td><td>AAC</td><td>GGG</td><td>GAC</td><td>TGT</td><td>GAC</td><td>CAG</td><td>TTC</td><td>TGC</td><td>CAC</td><td>GAG</td><td>GAA</td><td>CAG</td><td> 432</td>

<td>Cys</td><td>Cheese</td><td>Leu</td><td>Asp</td><td>own</td><td>Gly</td><td>Asp</td><td>Cys</td><td>Asp</td><td>Gln</td><td>phe</td><td>Cys</td><td>His</td><td>Glu</td><td>Glu</td><td>Gln</td><td></td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td><td></td>

<td>AAC</td><td>TCT</td><td>GTG</td><td>GTG</td><td>TGC</td><td>TCC</td><td>TGC</td><td>GCC</td><td>CGC</td><td>GGG</td><td>TAC</td><td>ACC</td><td>CTG</td><td>GCT</td><td>GAC</td><td>AAC</td><td> 480</td>

<td>own</td><td>Cheese</td><td>val</td><td>val</td><td>Cys</td><td>Cheese</td><td>Cys</td><td>ala</td><td>Arg</td><td>Gly</td><td>Tyr</td><td>Thr</td><td>Leu</td><td>ala</td><td>Asp</td><td>own</td><td></td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td><td></td>

<td>GGC</td><td>AAG</td><td>GCC</td><td>TGC</td><td>ATT</td><td>CCC</td><td>ACA</td><td>GGG</td><td>CCC</td><td>TAC</td><td>CCC</td><td>TGT</td><td>GGG</td><td>AAA</td><td>CAG</td><td>ACC</td><td> 528</td>

<td>Gly</td><td>lys</td><td>ala</td><td>Cys</td><td>How much</td><td>Pro</td><td>Thr</td><td>Gly</td><td>Pro</td><td>Tyr</td><td>Pro</td><td>Cys</td><td>Gly</td><td>lys</td><td>Gln</td><td>Thr</td><td></td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td><td></td>

<td>CTG</td><td>GAA</td><td>CGC</td><td>AGG</td><td>AAG</td><td>AGG</td><td>TCA</td><td>GTG</td><td>GCC</td><td>CAG</td><td>GCC</td><td>ACC</td><td>AGC</td><td>AGC</td><td>AGC</td><td>GGG</td><td> 576</td>

<td>Leu</td><td>Glu</td><td>Arg</td><td>Arg</td><td>lys</td><td>Arg</td><td>Cheese</td><td>val</td><td>ala</td><td>Gln</td><td>ala</td><td>Thr</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Gly</td><td></td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td colspan="2"> 185</td><td></td><td></td><td></td><td> 190</td><td></td><td></td><td></td>

<td>GAG</td><td>GCC</td><td>CCT</td><td>GAC</td><td>AGC</td><td>ATC</td><td>ACA</td><td>TGG</td><td>AAG</td><td>CCA</td><td>TAT</td><td>GAT</td><td>GCA</td><td>GCC</td><td>GAC</td><td>CTG</td><td> 624</td>

<td>Glu</td><td>ala</td><td>Pro</td><td>Asp</td><td>Cheese</td><td>How much</td><td>Thr</td><td>Trp</td><td>lys</td><td>Pro</td><td>Tyr</td><td>Asp</td><td>ala</td><td>ala</td><td>Asp</td><td>Leu</td><td></td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td><td></td>

<td>GAC</td><td>CCC</td><td>ACC</td><td>GAG</td><td>AAC</td><td>CCC</td><td>TTC</td><td>GAC</td><td>CTG</td><td>CTT</td><td>GAC</td><td>TTC</td><td>AAC</td><td>CAG</td><td>ACG</td><td>CAG</td><td> 672</td>

<td>Asp</td><td>Pro</td><td>Thr</td><td>Glu</td><td>own</td><td>Pro</td><td>phe</td><td>Asp</td><td>Leu</td><td>Leu</td><td>Asp</td><td>phe</td><td>own</td><td>Gln</td><td>Thr</td><td>Gln</td><td></td>

210

215

220

190 734

<td>CCT GAG</td><td>AGG GGC</td><td>GAC</td><td>AAC AAC CTC</td><td>ACC AGG</td><td>ATC</td><td>GTG GGA</td><td>GGC CAG GAA</td><td> 720</td>

<td>Pro Glu</td><td>Arg Gly</td><td>Asp</td><td>Asn Asn Leu</td><td>Thr Arg</td><td>How much</td><td>Val Gly</td><td>Gly Gln Glu</td><td></td>

<td> 225</td><td></td><td></td><td> 230</td><td></td><td> 235</td><td></td><td> 240</td><td></td>

<td>TGC AAG</td><td>GAC GGG</td><td>GAG</td><td>TGT CCC TGG</td><td>GCC CAG</td><td>CTG</td><td>CTC ATC</td><td>AAT GAG GAA</td><td> 768</td>

<td>Lys</td><td>Asp Gly</td><td>Glu</td><td>Cys Pro Trp</td><td>Gln Ala</td><td>Leu</td><td>Leu Ile</td><td>Asn Glu Glu</td><td></td>

<td></td><td></td><td> 245</td><td></td><td> 250</td><td></td><td></td><td> 255</td><td></td>

<td>AAC GAG</td><td>GGT TTC</td><td>TGT</td><td>GGT GGA ACT</td><td>ATT CTG</td><td>AGC</td><td>TTC GAG</td><td>TAC ATC CTA</td><td> 816</td>

<td>Asn Glu</td><td>Gly Phe</td><td>Cys</td><td>Gly Gly Thr</td><td>How much leu</td><td>Cheese</td><td>Glu Phe</td><td>Tyr Ile Leu</td><td></td>

<td></td><td> 260</td><td></td><td></td><td> 265</td><td></td><td></td><td> 270</td><td></td>

<td>ACG GCA</td><td>GCC CAC</td><td>TGT</td><td>CTC TAC CAA</td><td>GCC AAG</td><td>AGA</td><td>TTC AAG</td><td>GTG AGG GTA</td><td> 864</td>

<td>Thr Ala</td><td>Ala His</td><td>Cys</td><td>Leu Tyr Gln</td><td>Ala Lys</td><td>Arg</td><td>Phe Lys</td><td>Val Arg Val</td><td></td>

<td></td><td> 275</td><td></td><td> 280</td><td></td><td></td><td> 285</td><td></td><td></td>

<td>GGG GAC</td><td>CGG AAC</td><td>ACG</td><td>GAG CAG GAG</td><td>GAG GGC</td><td>GGT</td><td>GCG GAG</td><td>GTG CAC GAG</td><td> 912</td>

<td>Gly Asp</td><td>Arg Asn</td><td>Thr</td><td>Glu Gln Glu</td><td>Glu Gly</td><td>Gly</td><td>Glu Ala</td><td>Val His Glu</td><td></td>

<td> 290</td><td></td><td></td><td> 295</td><td></td><td></td><td> 300</td><td></td><td></td>

<td>GTG GAG</td><td>GTG GTC</td><td>ATC</td><td>AAG CAC AAC</td><td>CGG TTC</td><td>ACA</td><td>AAG GAG</td><td>ACC TAT GAC</td><td> 960</td>

<td>Val Glu</td><td>Val Val</td><td>How much</td><td>Lys His Asn</td><td>Arg Phe</td><td>Thr</td><td>Lys Glu</td><td>Thr Tyr Asp</td><td></td>

<td> 305</td><td></td><td></td><td> 310</td><td></td><td> 315</td><td></td><td> 320</td><td></td>

<td>TTC GAC</td><td>ATC GCC</td><td>GTG</td><td>CTC CGG CTC</td><td>AAG ACC</td><td>CCC</td><td>ATC ACC</td><td colspan="2">TTC CISC ATG 1008</td>

<td>Phe Asp</td><td>How much ala</td><td>val</td><td>Leu Arg Leu</td><td>Lys Thr</td><td>Pro</td><td>How much thr</td><td>Phe Arg Met</td><td></td>

<td></td><td></td><td> 325</td><td></td><td> 330</td><td></td><td></td><td> 335</td><td></td>

<td>AAC GTG</td><td>GCG CCT</td><td>GCC</td><td>TGC CTC CCC</td><td>GG CGT</td><td>GAC</td><td>TGG GCC</td><td colspan="2">GAG TCC ACG 1056</td>

<td>Asn Val</td><td>Ala Pro</td><td>ala</td><td>Cys Leu Pro</td><td>Glu Arg</td><td>Asp</td><td>Trp Ala</td><td>Glu Ser Thr</td><td></td>

<td></td><td> 340</td><td></td><td></td><td> 345</td><td></td><td></td><td> 350</td><td></td>

<td>CTG ATG</td><td>ACG CAG</td><td>AAG</td><td>ACG GGG ATT</td><td>GTG AGC</td><td>GGC</td><td>TTC GGG</td><td colspan="2">CGC ACC CAC 1104</td>

<td>Leu Met</td><td>Thr Gln</td><td>lys</td><td>Thr Gly Ile</td><td>Val Ser</td><td>Gly</td><td>Phe Gly</td><td>Arg Thr His</td><td></td>

355

360

365

190 734

<td>GAG AAG GGC CGG</td><td>CAG TCC ACC AGG CTC AAG ATG</td><td>CTG GAG</td><td>GTG CCC TAC 1152</td>

<td>Glu Lys Gly Arg</td><td>Gln Ser Thr Arg Leu Lys Met</td><td>Leu Glu</td><td>Val Prc Tyr</td>

<td> 370</td><td> 375</td><td> 380</td><td></td>

<td>GTG GAC CGC AAC</td><td>AGC TGC AAG CTG TCC AGC AGC</td><td>TTC ATC</td><td>ATC ACC CAG 1200</td>

<td>Val Asp Arg Asn</td><td>Cheese Cys Lys Leu Cheese Cheese Cheese</td><td>Phe Ile</td><td>Ile Thi Gln</td>

<td> 385</td><td> 390 395</td><td></td><td> 400</td>

<td>AAC ATG TTC TGT</td><td>GCC GGC TAC GAC ACC AAG CAG</td><td>GAG GAT</td><td>GCC TGC CAG 1248</td>

<td>Asn Met Phe Cys</td><td>Ala Gly Tyr Asp Thr Lys Gln</td><td>Glu Asp</td><td>Ala Cys Gln</td>

<td></td><td> 405 410</td><td></td><td> 415</td>

<td>GGG GAC AGC GGG</td><td>GGC CCG CAC GTC ACC CGC TTC</td><td>AAG GAC</td><td>ACC TAC TTC 1296</td>

<td>Gly Asp Cheese Gly</td><td>Gly Pro His Val Thr Arg Phe</td><td>Lys Asp</td><td>Thr Tyr Phe</td>

<td> 420</td><td> 425</td><td></td><td> 430</td>

<td>GTG ACA GGC ATC</td><td>GTC AGC TGG GGA GAG AGC TGT</td><td>GCC CGT</td><td>AAG GGG AAG 1344</td>

<td>Val Thr Gly Ile</td><td>Val Ser Trp Gly Glu Cheese Cys</td><td>Ala Arg</td><td>Lys Gly Lys</td>

<td> 435</td><td> 440</td><td> 445</td><td></td>

<td>TAC GGG ATC TAC</td><td>ACC AAG GTC ACC GCC TTC CTC</td><td colspan="2">AAG TGG ATC GAC AGG 1392</td>

<td>Tyr Gly Ile Tyr</td><td>Thr Lys Val Thr Ala Phe Leu</td><td>Lys Trp</td><td>Ile Asp Arg</td>

<td> 450</td><td> 455</td><td> 460</td><td></td>

<td>TCC ATG AAA ACC</td><td>AGG GGC TTG CCC AAG GCC AAG</td><td colspan="2">AGC CAT GCC CCG GAG 1440</td>

<td>Cheese Met Lys Thr</td><td>Arg Gly Leu Pro Lys Ala Lys</td><td>His cheese</td><td>Ala Pro Glu</td>

<td> 465</td><td> 470 475</td><td></td><td> 480</td>

<td>GTC ATA ACG TCC</td><td>TCT CCA TTA AAG TGA</td><td></td><td> 1467</td>

<td>Val Ile Thr Ser</td><td>Pro Leu Lys cheese *</td><td></td><td></td>

485 (2) Data for SEQ ID NO: 27:

(i) Sequence features:

(A) Length :: 489 amino acids (B) Type: amino acid

190 734 (D) Topology: linear (ii) Type of molecule: protein

<td></td><td>(xi) Description</td><td>sequence and:</td><td>SEQ</td><td>ID NO:</td><td> 27:</td>

<td>Underworld</td><td>Gly Arg Pro</td><td>Leu His Leu</td><td>val</td><td>Leu Leu</td><td>Cheese Ala Cheese Leu Ala Gly</td>

<td> 1</td><td></td><td> 5</td><td></td><td> 10</td><td> 15</td>

<td>Leu</td><td>Leu Leu Leu</td><td>Gly Glu Cheese</td><td>Leu</td><td>Phe Ile</td><td>Arg Arg Glu Gln Ala Asn</td>

<td></td><td> 20</td><td></td><td></td><td> 25</td><td> 30</td>

<td>own</td><td>Ile Leu Ala</td><td>Arg Val Thr</td><td>Arg</td><td>Ala Asn</td><td>Phe Leu Glu Glu Cheese Met</td>

<td></td><td> 35</td><td></td><td> 40</td><td></td><td> 45</td>

<td>lys</td><td>Lys Gly His</td><td>Leu Glu Arg</td><td>Glu</td><td>Cys Met</td><td>Glu Glu Thr Cys Ser Tyr</td>

<td></td><td> 50</td><td> 55</td><td></td><td></td><td> 60</td>

<td>Glu</td><td>Glu Ala Arg</td><td>Glu Val Phe</td><td>Glu</td><td>Asp Ser</td><td>Asp Lys Thr Asn Glu Phe</td>

<td> 65</td><td></td><td> 70</td><td></td><td></td><td> 75 80</td>

<td>Trp</td><td>Asn Lys Tyr</td><td>Lys Asp Gly</td><td>Asp</td><td>Gln</td><td>Glu Thr Ser Pro Cys Gln</td>

<td></td><td></td><td> 85</td><td></td><td> 90</td><td> 95</td>

<td>own</td><td>Gln Gly Lys</td><td>Cys Lys Asp</td><td>Gly</td><td>Leu Gly</td><td>Glu Tyr Thr Cys Thr Cys</td>

<td></td><td> 100</td><td></td><td></td><td> 105</td><td> 110</td>

<td>Leu</td><td>Glu Gly Phe</td><td>Glu Gly Lys</td><td>own</td><td>Cys Glu</td><td>Leu Phe Thr Arg Lys Leu</td>

<td></td><td> 115</td><td></td><td> 120</td><td></td><td> 125</td>

<td>Cys</td><td>Leu Asp cheese</td><td>Asn Gly Asp</td><td>Cys</td><td>Asp Gln</td><td>Phe Cys His Glu Glu Gln</td>

<td></td><td> 130</td><td> 135</td><td></td><td></td><td> 140</td>

<td>own</td><td>Val Val cheese</td><td>Cys Cheese Cys</td><td>ala</td><td>Arg Gly</td><td>Tyr Thr Leu Ala Asp Asn</td>

<td> 145</td><td></td><td> 150</td><td></td><td></td><td> 155 160</td>

<td>Gly</td><td>Lys Ala Cys</td><td>Ile Pro Thr</td><td>Gly</td><td>Pro Tyr</td><td>Pro Cys Gly Lys Gln Thr</td>

<td></td><td></td><td> 165</td><td></td><td> 170</td><td> 175</td>

<td>Leu</td><td>Glu Arg Arg</td><td>Lys Arg Cheese</td><td>val</td><td>Ala Gln</td><td>Ala Thr Ser Ser Ser Gly</td>

<td></td><td> 180</td><td></td><td></td><td> 185</td><td> 190</td>

<td>Glu</td><td>Ala Pro Asp</td><td>Cheese Ile Thr</td><td>Trp</td><td>Lys Pro</td><td>Tyr Asp Ala Ala Asp Leu</td>

195

200

205

190 734

<td>Asp</td><td>Pro 210</td><td>Thr</td><td colspan="3">Glu Asn Pro</td><td colspan="2">Phe Asp 215</td><td colspan="3">Leu Leu Asp</td><td>phe 220</td><td>own</td><td>Gln</td><td>Thr</td><td>Gln</td>

<td>Pro</td><td>Glu</td><td>Arg</td><td>Gly</td><td>Asp</td><td>own</td><td>own</td><td>Leu</td><td>Thr</td><td>Arg</td><td>How much</td><td>val</td><td>Gly</td><td>Gly</td><td>Gln</td><td>Glu</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Cys</td><td>lys</td><td>Asp</td><td>Gly</td><td>Glu</td><td>Cys</td><td>Pro</td><td>Trp</td><td>Gln</td><td>ala</td><td>Leu</td><td>Leu</td><td>How much</td><td>own</td><td>Glu</td><td>Glu</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>own</td><td>Glu</td><td>Gly</td><td>phe</td><td>Cys</td><td>Gly</td><td>Gly</td><td>Thr</td><td>How much</td><td>Leu</td><td>Cheese</td><td>Glu</td><td>phe</td><td>Tyr</td><td>How much</td><td>Leu</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Thr</td><td>ala</td><td>ala</td><td>His</td><td>Cys</td><td>Leu</td><td>Tyr</td><td>Gln</td><td>ala</td><td>lys</td><td>Arg</td><td>phe</td><td>lys</td><td>val</td><td>Arg</td><td>val</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Gly</td><td>Asp</td><td>Arg</td><td>own</td><td>Thr</td><td>Glu</td><td>Gln</td><td>Glu</td><td>Glu</td><td>Gly</td><td>Gly</td><td>Glu</td><td>ala</td><td>val</td><td>His</td><td>Glu</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>val</td><td>Glu</td><td>val</td><td>val</td><td>How much</td><td>lys</td><td>His</td><td>own</td><td>Arg</td><td>phe</td><td>Thr</td><td>lys</td><td>Glu</td><td>Thr</td><td>Tyr</td><td>Asp</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>phe</td><td>Asp</td><td>How much</td><td>ala</td><td>val</td><td>Leu</td><td>Arg</td><td>Leu</td><td>lys</td><td>Thr</td><td>Pro</td><td>How much</td><td>Thr</td><td>phe</td><td>Arg</td><td>Underworld</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>own</td><td>val</td><td>ala</td><td>Pro</td><td>ala</td><td>Cys</td><td>Leu</td><td>Pro</td><td>Glu</td><td>Arg</td><td>Asp</td><td>Trp</td><td>ala</td><td>Glu</td><td>Cheese</td><td>Thr</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>Leu</td><td>Underworld</td><td>Thr</td><td>Gln</td><td>lys</td><td>Thr</td><td>Gly</td><td>How much</td><td>val</td><td>Cheese</td><td>Gly</td><td>phe</td><td>Gly</td><td>Arg</td><td>Thr</td><td>His</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>Glu</td><td>lys</td><td>Gly</td><td>Arg</td><td>Gln</td><td>Cheese</td><td>Thr</td><td>Arg</td><td>Leu</td><td>lys</td><td>Underworld</td><td>Leu</td><td>Glu</td><td>val</td><td>Pro</td><td>Tyr</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>val</td><td>Asp</td><td>Arg</td><td>own</td><td>Cheese</td><td>Cys</td><td>lys</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>phe</td><td>How much</td><td>How much</td><td>Thr</td><td>Gln</td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>own</td><td>Underworld</td><td>phe</td><td>Cys</td><td>ala</td><td>Gly</td><td>Tyr</td><td>Asp</td><td>Thr</td><td>lys</td><td>Gln</td><td>Glu</td><td>Asp</td><td>ala</td><td>Cys</td><td>Gln</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>Gly</td><td>Asp</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>Pro</td><td>His</td><td>val</td><td>Thr</td><td>Arg</td><td>phe</td><td>lys</td><td>Asp</td><td>Thr</td><td>Tyr</td><td>phe</td>

420 425 430

190 734

<td>val</td><td>Thr</td><td>Gly</td><td>How much</td><td>val</td><td>Cheese</td><td>Trp</td><td>Gly</td><td>Glu</td><td>Cheese</td><td>Cys</td><td>ala</td><td>Arg</td><td>lys</td><td>Gly</td><td>lys</td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td>

<td>Tyr</td><td>Gly</td><td>How much</td><td>Tyr</td><td>Thr</td><td>lys</td><td>val</td><td>Thr</td><td>ala</td><td>phe</td><td>Leu</td><td>lys</td><td>Trp</td><td>How much</td><td>Asp</td><td>Arg</td>

<td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td>

<td>Cheese</td><td>Underworld</td><td>lys</td><td>Thr</td><td>Arg</td><td>Gly</td><td>Leu</td><td>Pro</td><td>lys</td><td>ala</td><td>lys</td><td>Cheese</td><td>His</td><td>ala</td><td>Pro</td><td>Glu</td>

<td> 465</td><td></td><td></td><td></td><td colspan="2"> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td> 480</td>

<td>val</td><td>How much</td><td>Thr</td><td>Cheese</td><td>Cheese</td><td>Pro</td><td>Leu</td><td>lys</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

485

190 734 (-40)

Met Gly Arg Pro Leu Hi3 Leu Val Leu Leu Cheese Ala Ser Leu Ala Gly Leu Leu Leu ATG GGG CGC CCA CTG CAC CTC GTC CTG CTC AGT GCC TCC CTG GCT GGC CTC CTG CTG

<td></td><td> 9</td><td> 18</td><td> 27</td><td> 36</td><td> 45</td><td> 54</td>

<td></td><td></td><td></td><td></td><td></td><td> (-4)</td><td> (-1</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td> 40</td>

<td colspan="7">Leu Gly Glu Cheese Leu Phe Ile Arg Arg Glu Gln Ala Asn Asn Ile Leu Ala Arg Val Thr Arg</td>

<td colspan="7">CTC GGG GAA AGT CTG TTC ATC CGC AGG GAG CAG GCC AAC AAC ATC CTG GCG AGG GTC ACG AGG</td>

<td></td><td> 66 75</td><td> • 84</td><td>ί 93</td><td>and 102</td><td> 111</td><td> 120</td>

<td colspan="2">(+ D</td><td></td><td></td><td></td><td></td><td></td>

<td> 41</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>ala</td><td>. Asn Ser Phe Leu Glu</td><td>Glu Met Lys</td><td>Lys Gly His</td><td>Leu Glu Arg Glu</td><td>Cy3 Met Glu</td><td>Glu Thr</td>

<td>GCC</td><td>AAT TCC TTT CTT GAA</td><td>GAG ATG AAG</td><td>AAA GGA CAC</td><td>CTC GAA AGA GAG</td><td>: TGC ATG GAA</td><td>GAG ACC</td>

<td></td><td> 129 138</td><td> 147</td><td> 156</td><td> 165</td><td> 174</td><td> 183</td>

<td>Cys</td><td>Tyr Glu Glu Ala cheese</td><td>Arg Glu Val</td><td>Phe Glu Asp</td><td>Cheese Asp Lys Thr</td><td>Asn Glu Phe</td><td>Trp. Asn</td>

<td>TGC</td><td>TCA TAC GAA GAG GCC</td><td>CGC GAG GTC</td><td>TTT GAG GAC</td><td>AGC GAC AAG ACG</td><td>AAT GAA TTC</td><td>TGG AAT</td>

<td></td><td> 192 201</td><td> 210</td><td> 219</td><td> 228</td><td> 237</td><td> 246</td>

<td>lys</td><td>Tyr Lys Asp Gly Asp</td><td>Gln Cys Glu</td><td>Thr Ser Pro</td><td>Cys Gln Asn Gln</td><td>Gly Lys Cys</td><td>Lys Asp</td>

<td>AAA</td><td>TAC AAA GAT GGC GAC</td><td>CAG TCT GAG</td><td>ACC AGT CCT</td><td>TGC CAG AAC CAG</td><td>GGC AAA TCT</td><td>AAA GAC</td>

<td></td><td> 255 264</td><td> 273</td><td> 282</td><td> 291</td><td> 300</td><td> 309</td>

<td>Gly</td><td>Leu Gly Glu Tyr Thr</td><td>Cys Thr Cys</td><td>Leu Glu Gly</td><td>Phe Glu Gly Lys</td><td>Asn Cy3 Glu</td><td>Leu Phe</td>

<td>GGC</td><td>CTC GGG GAA TAC ACC</td><td>TGC ACC TCT</td><td>TTA GAA GGA</td><td>TTC GAA GGC AAA</td><td>AAC TGT GAA</td><td>TTA TTC</td>

<td></td><td> 318 327</td><td> 336</td><td> 345</td><td>3S4</td><td> 363</td><td> 372</td>

<td>Thr</td><td>Arg Lys Leu Cys Ser</td><td>Leu Asp Asn</td><td>Gly Asp Cys</td><td>Asp Gln Phe Cys</td><td>His Glu Glu</td><td>Gln Asn</td>

<td>ACA</td><td>CGG AAG CTC TGC AGC</td><td>CTG GAC AAC</td><td>GGG GAC TCT</td><td>GAC CAG TTC TGC</td><td>CAC GAG GAA</td><td>CAG AAC</td>

<td></td><td> 381 390</td><td> 399</td><td> 408</td><td> 417</td><td> 426</td><td> 435</td>

<td>Cheese</td><td>Val val Cys Cheese Cys</td><td>Ala Arg Gly</td><td>Tyr Thr Leu</td><td>Ala Asp Asn Gly</td><td>Lys Ala Cys</td><td>How much Pro</td>

<td>TCT</td><td>GTG GTG TGC TCC TGC</td><td>GCC CGC GGG</td><td>TAC ACC CTG</td><td>GCT GAC AAC GGC</td><td>AAG GCC TGC</td><td>ATT CCC</td>

<td></td><td> 444 453</td><td> 462</td><td> 471</td><td> 480</td><td> 489</td><td> 498</td>

<td></td><td></td><td></td><td> 178</td><td> 179 180 181 182</td><td> 183</td><td></td>

<td>Thr</td><td>Gly Pro Tyr Pro Cys</td><td>Gly Lys Gln</td><td>Thr Leu Glu</td><td>Arg Arg Lys Arg</td><td>Val Ala cheese</td><td>Gln Ala</td>

<td>ACA</td><td>GGG COC TAC CCC TGT</td><td>GGG AAA CAG</td><td>ACC CTG GAA</td><td>OGC AGG AAG AGG</td><td>TCA GTG GCC</td><td>GCC CAG</td>

<td></td><td> 507 516</td><td> 525</td><td> 534</td><td> 543</td><td> 552</td><td> 561</td>

<td>Thr</td><td>Cheese Cheese Cheese Gly Glu</td><td>Ala Pro Asp</td><td>Cheese Ile Thr</td><td>Trp Lys Pro Tyr</td><td>Asp Ala Ala</td><td>Asp Leu</td>

<td>ACC</td><td>AGC AGC AGC GGG GAG</td><td>GCC CCT GAC</td><td>AGC ATC ACA</td><td>TGG AAG CCA TAT</td><td>GAT GCA GCC</td><td>GAC CTG</td>

<td></td><td> 570 579</td><td> 588</td><td> 597</td><td> 606</td><td> 615</td><td> 624</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td>R6</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td> 229</td>

<td>Asp</td><td>Pro Thr Glu Asn Pro</td><td>Phe Asp Leu</td><td>Leu Asp Phe</td><td>Asn Gln Thr Gln</td><td>Pro Glu Arg</td><td>Gly Asp</td>

<td>GAC</td><td>CCC ACC GAG AAC CCC</td><td>TTC GAC CTG</td><td>CTT GAC TTC</td><td>AAC CAG ACG CAG</td><td>CCT GAG AGG</td><td>GGC GAC</td>

<td></td><td> 633 642</td><td> 651</td><td> 660</td><td> 669</td><td> 678</td><td> 687</td>

<td>R5</td><td>R4 R3 R2 Rl</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 234 235</td><td></td><td></td><td></td><td></td><td></td>

<td>own</td><td>A3n Leu Thr Arg Ile</td><td>Val Gly Gly</td><td>Gln Glu Cys</td><td>Lys Asp Gly Glu</td><td>Cys Pro Trp</td><td>Gln Ala</td>

<td>AAC</td><td>AAC CTC ACC AGG ATC</td><td>GTG GGA GGC</td><td>CAG GAA TGC</td><td>AAG GAC GGG GAG</td><td>TGT CCC TGG</td><td>GCC CAG</td>

<td></td><td> 696 705</td><td> 714</td><td> 723</td><td> 732</td><td>74 L.</td><td> 750</td>

Fig 1-1

190 734

Leu Leu Ile CTG CTC ATC

759

Asn Glu Glu AAT GAG GAA

768

Asn Glu Gly AAC GAG GGT

777

Phe Cys Gly TTC TGT GGT

786

Gly Thr Ile GGA ACT ATT

795

Leu Ser Glu CTG AGC GAG

804

Phe Tyr Ile TTC TAC ATC

813

Leu Thr Ala CTA ACG GCA

822

Ala His Cy3 GCC CAC TGT

831

Leu Tyr Gln CTC TAC CAA

840

Ala Lys Arg GCC AAG AGA

849

Phe Lys Val TTC AAG GTG

858

Arg Val Gly AGG GTA GGG

867

Asp Arg Asn GAC CGG AAC

876

Thr Glu Gln ACG GAG CAG

885

Glu Glu Gly GAG GAG GGC

894

Gly Glu Ala GGT GAG GCG

903

Val His Glu GTG CAC GAG

912

Val Glu Val GIG GAG GTG

921

Val Ile Lys GTC ATC AAG

930

His Asn Arg CAC AAC CGG

939

Phe Thr Lys TTC ACA AAG

948

Glu Thr Tyr GAG ACC TAT

957

Asp Phe Asp GAC TTC GAC

966

Ile Ala Val ATC GCC GTG

975

Leu Arg Leu CTC CGG CTC

984

Lys Thr Pro AAG ACC CCC

993

Ile Thr Phe ATC ACC TTC

1002

Arg Met Asn CGC ATG AAC

1011

Val Ala Pro GTG GCG OCT

1020

Ala Cys Leu GOC TGC CTC

1029

Pro Glu Arg CCC GAG CGT

1038

Asp Trp Ala GAC ​​TGG GCC

1047

Glu Ser Thr GAG TCC ACG

1056

Leu Met Thr CTG ATG ACG

1065

Gln Lys Thr CAG AAG ACG

1074

Gly Ile Val GGG ATT GTG

1083

Cheese Gly Phe AGC GGC TTC

1092

Gly Arg Thr GGG CGC ACC

1101

His Glu Lys CAC GAG AAG

1110

Gly Arg Gln GGC CGG CAG

1119

Cheese Thr Arg TCC ACC AGG

1128

Leu Lys Met CTC AAG ATG

1137

Leu Glu Val CTG GAG GTG

1146

Pro Tyr Val COC TAC GTG

1155

Asp Arg Asn GAC CGC AAC

1164

Cys Lys AGC TGC AAG

1173

Leu Ser Cheese CTG TCC AGC

1182

Phe Ile AGC TTC ATC

1191

Ile Thr Gln ATC ACC CAG

1200

Asn Met Phe AAC ATG TTC

1209

Cys Ala Gly TGT GCC GGC

1218

Tyr Asp Thr TAC GAC ACC

1227

Lys Gln Glu AAG CAG GAG

1236

Asp Ala Cys GAT GCC TGC

1245

Gln Gly Asp CAG GGG GAC

1254

Gly Gly AGC GGG GGC

1263

Pro His Val CCG CAC GTC

1272

Thr Arg Phe ACC CGC TTC

1281

Lys Asp Thr AAG GAC ACC

1290

Tyr Phe Val TAC TTC GTG

1299

Thr GLy Ile ACA GGC ATC

1308

Val Ser Trp GTC AGC TGG

1317

Gly Glu Cheese GGA GAG AGC

1326

Cys Ala Arg TGT GCC CGT

1335

Lys Gly Lys AAG GGG AAG

1344

Tyr Gly Ile TAC GGG ATC

1353

Tyr Thr Lys TAC ACC AAG

1362

Val Thr Ala GTC ACC GCC

1371

Phe Leu Lys TTC CTC AAG

1380

Trp Ile Asp TGG ATC GAC

1389

Arg Ser Met AGG TCC ATG

1398

Ile Thr Ser ATA ACG TCC

1452

Pro Leu TCT CCA TTA cheese

1461

469 Lys Thr Arg AAA ACC AGG 1407

488

Lys TER AAG TGA

1467

470

Gly Leu Pro GGC TTG CCC

1416

475 Lys Ala Lys AAG GCC AAG 1425

476

His His Ala AGC CAT GCC

1434

480 Pro Glu Val CCG GAG GTC

1443

Pre- / propeptide Peptide combining activation peptide

Fig 1-2

190 734 ftf

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UP Department of Publications. Circulation of 50 copies

Price PLN 6.00.