JP2002234899A - Recombinant medicine acting on thrombosis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide another method that increases the specificity of blocking the formation of active prothrombinase complex and can prolong the duration time of this action. SOLUTION: The objective medicine is the factor Xa of the blood factor that is modified so that the half-life may be transitorily prolonged in the serum and the modified blood factor gives the activated form of the blood factor in the presence of serum. The factor Xa that is the blood factor modified so that the half-life thereof may be prolonged in the serum is mixed with a pharmaceutically acceptable excipient and is used for treating hemophilia wherein the activated form of the blood factor is produced in the presence of serum.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to peptide medicine used for prevention or treatment of thrombosis. More particularly, the invention relates to factor Xa analogs that lack protease activity and inhibit the ability of endogenous factor Xa to convert prothrombin to thrombin. The present invention further provides a stable form of factor Xa for use in treating hemophilia.

[0002]

BACKGROUND OF THE INVENTION Thrombin is a multifunctional protease that regulates several important biological processes. For example, thrombin is the most potent of the known platelet enhancers. In addition, thrombin is essential for cleaving fibrinogen into fibrin and initiating clot formation. The above two components are involved in normal hemostasis, but may initiate thrombus formation in atherosclerotic arteries. This thrombus is a major factor in the etiology of vasoocclusive symptoms such as non-bleeding attacks and reocclusion of coronary arteries after treatment for myocardial infarction, unstable angina, angioplasty or thrombolysis . Thrombin is also a potent inducer of smooth cell proliferation and is therefore involved in various proliferative responses such as restenosis after angioplasty and transplant-induced atherosclerosis. In addition, thrombin plays a role in inflammation because it is chemotactic for lymphocytes (Hoover, RJ et al.
Cel1 (1978) 14: 423; Etingin,
O. R. Et al., Cell (1990) 61: 657. These observations indicate that inhibition of thrombin formation or inhibition of thrombin itself is effective in preventing or treating thrombosis, limiting restenosis and suppressing inflammation.

[0003] Thrombin is produced as a result of proteolytic cleavage of prothrombin, a pro-driving substance, at the Arg-Thr bond at positions 271-272 and the Arg-Ile bond at positions 320-321. This activation is catalyzed by the prothrombinase complex, which is composed of platelets,
It is built on the membrane surface of monocytes and endothelial cells. This complex is composed of factor Xa (serine protease), factor Va (cofactor), calcium ions and acidic phospholipid surface. Factor Xa is an activated form of its precursor factor X, which is 58 kd
And is converted to activated form of factor Xa in both the extrinsic and endogenous blood coagulation pathways.
It is known that circulating levels of Factor X and Factor V, a precursor of Factor Va, are on the order of 10-M.
The corresponding activator h and Xa levels have not yet been determined.

[0004] The complete amino acid sequences of human factor X and factor Xa are known. FIG. 1 shows Coleman, R.A. W.
Editing, Hemostasis and Thromb
osis second edition (1987) p. In 250, Davie,
E. FIG. W. Shows the complete sequence of the precursor type factor X described by E. Factor X is calcium ion binding, contains gamma-carboxyglutamyl (Gla), and is a member of the vitamin K-dependent blood coagulation glycoprotein family, which includes factor VII, factor IX, prothrombin, Protein C and Protein S (Fur
ie, B .; Cell (1988) 53: 505).

As shown in FIG. 1, the mature factor X protein is preceded by a 40 residue prepro leader sequence,
This sequence is removed during processing and secretion in the cell. The mature factor X, a precursor of factor Xa, is then
It is cleaved into a double-chain form by deletion of the three amino acids RKR shown between the C-terminus of the light chain and the N-terminus of the activation peptide / heavy chain. Finally, this double-chain factor X is referred to as the "activating peptide" (1
(Numbered ~ 52) and converted to factor Xa, the light chain shown as residues 1-139 and residues 1-254
To produce the heavy chain shown as These are linked via a single disulfide bond between position 128 of the light chain and position 108 of the heavy chain. As further shown in the figure, the light chain is Gl
a domain and a growth factor domain, while the protease active portion is in the heavy chain and histidine 8 at position 42
Aspartic acid at position 8 and serine at position 185 are circled in the figure.

There are two known routes for in vivo activation of double-chain factor X. Activation must occur before the protease is incorporated into the prothrombinase complex (Steinberg, M. et al., He.
Mostasis and Thrombosis, Co
Leman, R .; W. (1987) J. et al. B. Li
ppencott, Philadelphia, PA,
p. 112). In the intrinsic pathway, factor X
"Tenase" (tenas) composed of factor IXa, factor VIII and calcium ions and constructed on the cell surface
e) Cleaved by the complex to release the 52 amino acid activation peptide. In the extrinsic pathway, this cleavage is catalyzed by factor VIIa bound to tissue factor on the membrane. However, of interest here is the ability to convert factor X to factor Xa by cleavage in vitro using a protease such as that contained in Russell's viper venom. This protease is DiScipio,
R. G. FIG. Et al., Biomistry (1977) 6:
5253.

[0007] For some of the agents of the present invention, it is desirable to inhibit the functioning of factor Xa to prevent excessive coagulation. However, in the case of hemophiliacs, the opposite problem, which would be aided by providing a source of factor Xa independent of the activation process occurring in normal individuals. Both the usual forms of hemophilia are defective only in the intrinsic pathway of activation, but the action of the extrinsic pathway does not seem to be successful in stopping bleeding.

The most common forms of hemophilia are hemophilia A, which exhibits a deficiency in the function of factor VIII, and hemophilia B, which exhibits a deficiency in the function of factor IX (also known as Christmas factor).
It is. These forms of hemophilia are described in Levine, P .;
H. , "Plasma Coagulation Fa
ctors ”(19), edited, Public
shing Co. , P. 97-110. The deficiency of either of these factors results in an inadequate supply of factor Xa, so giving factor Xa should be effective in treating both haemophilia. In addition, some examples have been found in which factor X itself cannot provide active factor Xa. This relatively rare type of congenital disease is described, for example, in Raddy, S .; B. Bloo
d (1989) 74: 1486-1490: Watzk.
e, H .; H. Et al., J Biol Chem (1990).
285: 11982-11989: Hassan, H .;
J. Et al., Blood (1988) 71: 1353-13.
56; S. Et al., Blood, (198
9) 73: 2108-2116; and Bernard
i, F. Et al., Blood (1989) 73: 2123-.
2127.

Factor Xa has heretofore not been useful as a medicament because its half-life in serum is very short, only about 30 seconds. In the present invention described below, the half-life of the drug in serum is extended by providing a sustained release acylated form in which the acyl group attached to the active site serine is cleared. And is hydrolyzed very slowly to produce the active form of factor Xa.

The use of acylation reactions to extend the half-life of blood coagulation factors has been disclosed. For example, Ca
ssels, R.S. Et al., Biochem J (1987).
247: 359-400, various acylating agents remain bound to urokinase, tPA and the streptokinase-plasminogen activator complex, the half-life of which depends on the type and factor of the acylating group. Was found to be in the range of 40 minutes to over 1000 minutes, depending on the type of. Acylation of tPA or streptokinase is also described in US Pat. No. 4,337,244. The use of an amidinophenyl group, which functions as an arginine analog, to temporarily introduce a substituted benzoyl group into the active site for the purpose of increasing stability in serum has been described by Fears, R .; Et al, Seminars in
Thrombosis and Hemeostasis
s (1989) 15: 129-139. For a more general review of this, see Fears, R.A. Our D
rugs (1987) 33: Sup. 3 57-63
Following a brief report at Sturz
ebecher, J .; Have also converted tPA-stabilized acyl derivatives to Thrombosis Res (198).
7) Reported at 47: 699-703. Another report on the use of acylated plasminogen streptokinase activator complex (APSAC) has been published by Crabb.
e, S. J. Et al., Pharmaco-therapy (1
990) 10: 115-126.

Returning to the function of factor Xa itself, the activity of factor Xa, which converts prothrombin to thrombin, depends on its inclusion in the prothrombinase complex. The formation of a prothrompinase complex (conversion of prothrombin to thrombin is 278,000 times faster than soluble form of factor Xa) has been studied (N
esheim, H .; E. FIG. Et al., J BiolChem (1
979) 254: 10952). These studies utilize an active site-specific inhibitor, dansylglutamylglycylarginyl (DEGR) chloromethyl ketone, which covalently attaches a fluorescent reporter group to factor Xa. Factor Xa treated with this inhibitor lacks protease activity, but is stoichiometrically incorporated into the prothrombinase complex like factor Xa, with a dissociation constant of 2.7 × 10 ⁻⁶ M (Nesheim). , M .;
E. FIG. , J Boil Chem @ 1981) 256: 6.
537-6540: Skogen, W.C. F. JB
iol Chem (1984) 256: 2306-23.
10; Kishnaswamy, S .; Et al., J Bio
1 Chem (1988) 263: 3823-382.
4: Husten, E .; J. Et al., J Biol Che
m (1987) 262: 12953-12961).

Known methods for inhibiting the formation of the prothrombinase complex include treatment with heparin and heparin-like compounds. This method uses antithrombin III together with heparin to inhibit the formation of the complex. Other novel forms of factor Xa inhibition include lipoprotein binding coagulation inhibitors (LACI)
(Girard, TJ et al., Nature (198).
9) 338: 518; Grad, T .; J. Et al., Sci
ence (1990) 248: 1421), an antistatin from leech (Donwiddie, C. et al., JB).
iol Chem (1989) 264: 16694),
And TAP from ticks (Waxman, L. et al., S.
science (1990) 248: 593). Alternatively, drugs that inhibit vitamin K-dependent Gla converting enzyme, such as coumarin, have been used. These methods are unsatisfactory because of their lack of specificity, the need for large doses, toxic side effects, and prolonged delay in efficacy.

[0013]

Accordingly, the present invention provides another method for increasing the specificity of an action that inhibits the formation of an active prothrombinase complex and extending the duration of the action. .

[0014]

SUMMARY OF THE INVENTION The present invention provides effective therapeutic agents for the prevention and treatment of thrombus formation and other pathological processes in the vasculature induced by thrombin, such as restenosis and inflammation. This is very important as thrombus formation is the leading cause of death in Western societies and restenosis is a growing problem with the increasing use of angioplasty and other invasive procedures It is. Since the therapeutic agent of the present invention is an inactivated form of human factor Xa, which can nevertheless be incorporated into the prothrombinase complex, the active proton binase complex has an endogenous X
Generation from the factor a can be prevented. These medicaments are particularly useful for preventing thrombosis in acute situations. This includes prevention of thrombosis in the coronary arteries of patients with resting angina, prevention of thrombosis occurring again after thrombolysis, and prevention of thrombosis during complex angioplasty. These medicaments are also useful for preventing smooth muscle cell proliferation after angioplasty or other invasive procedures of blood vessels. The treatments of the present invention are based on current standard treatments using heparin (Hanson, RS et al., Pr.
oc Natl Acadsci (1988) 85: 3
184). The compounds of the present invention are double- or single-chain polypeptides that can participate in the prothrombinase complex but result in an inactivated complex.

In one aspect, the present invention relates to a double-chain polypeptide termed factor Xai, which polypeptide can form a prothrombinase complex but results in a complex lacking proteolytic activity. It is. The double-chain polypeptide can be produced from one of two novel precursors. One type, referred to in the present application as Factor Xi, has substantially the amino acid sequence of Factor X, but is in vitro using a normal coagulation protease or an activator of Factor X from the venom of the viper. Has been modified as described herein to produce an inactivated double-chain polypeptide, factor Xai, upon cleavage by treatment with. The other type, referred to herein as factor X'i, is a truncated form of single-chain factor X, where the proteolytic cleavage site at the C-terminus of the light chain, shown as RKR in FIG. 1 (or The part or extension) is added to the N-terminus of the activated form of the heavy chain shown in one embodiment of FIG.
(With optional addition of one or several amino residues). Upon cleavage, factor X′i also becomes a double-chain factor Xai of the invention, which becomes a prothrombinase complex that lacks proteolytic activity. Of course, the active cofactor Factor Xa can also be produced using similar precursors of the Factor X 'type, shown in FIG.

Accordingly, in another aspect, the present invention provides
Double-stranded prothrombinase complex of factor ai and Xa
The present invention relates to novel precursors of therapeutic proteins for factor i, and to DNA sequences encoding them, and generally to recombinant materials and methods by which they can be produced. Other aspects of the invention include pharmaceutical compositions of Factor Xai proteins useful for treating and thrombosis or other pathological events initiated by thrombin.
Methods of preventing or treating using these compositions are included.

The availability of recombinant Factor Xa makes it a convenient source for use in treating hemophilia. Factor Xa can be produced directly by recombinant methods, obtained by activating factor X produced by recombinant methods using, for example, Russell's viper venom, or isolated from plasma and similarly converted to factor Xa. Regardless of what they do, they can be converted to produce usable drugs by extending their half-life in serum. This conversion is
Achieved by acylating serine in the active site, resulting in a sustained release form of the active agent.

Thus, in another aspect, the present invention is directed to an agent which converts active factor Xa at an appropriate time, comprising 18
It concerns Factor Xa, wherein the serine residue at position 5 is acylated. Other aspects of the present invention include pharmaceutical compositions containing the acylated factor Xa of the present invention for the treatment of hemophilia, and methods of treating haemophilia using these compositions.

The present invention provides the following items 1 to 29. 1. Position 1-139 of the light chain and 1-2 of the heavy chain shown in FIG.
A double-chain factor Xai peptide having at least 80% homology with the amino acid sequence at position 54, wherein the factor Xai is capable of competing with factor Xa during formation of a prothrombinase complex, and A double chain factor Xai peptide, wherein the protein does not provide hydrolytic activity when factor Xai is contained in the complex. 2. Whether the serine residue corresponding to residue 185 of the light chain shown in FIG. 1 has been replaced with a different amino acid, and / or
Or the factor Xai according to item 1, wherein the aspartic acid residue corresponding to the residue at position 88 of the heavy chain shown in FIG. 1 has been substituted with another amino acid. 3. Item 3. The Xai factor according to Item 2, wherein the substitution of serine is an alanyl residue and the substitution of aspartic acid is an asparagine residue. 4. A single-chain precursor polypeptide that can be converted to factor Xai by a proteolysis reaction,
The factors are positions 1-139 of the light chain and 1-
Has at least 80% homology to the amino acid sequence at position 254, the factor Xai is capable of competing with factor Xa during formation of the prothrombinase complex, and the factor Xa is contained in the complex; A single-chain precursor polypeptide that does not confer proteolytic activity when performed. 5. Item 4. Reactive with an antibody raised against factor X.
2. The polypeptide precursor according to 1. 6. A serine residue corresponding to residue 185 of the heavy chain shown in FIG. 1 has been substituted with another amino acid, and / or an aspartic acid residue corresponding to residue 88 of the heavy chain shown in FIG. The polypeptide precursor according to item 4, wherein is substituted with another amino acid. 7. The unmodified version of light chain 1-13 shown in FIG.
Item 7. The polypeptide precursor according to Item 6, which has an amino acid sequence of the amino acid at position 9 and the amino acids at positions 1-254 of the heavy chain. 8. Item 7. The polypeptide precursor according to Item 6, wherein the light and heavy chains are linked by an amino acid sequence having at least 80% homology with the activation peptide shown in FIG. 9. Item 7. The polypeptide according to Item 6, wherein the light and heavy chains are linked by an amino acid sequence consisting essentially of a protease cleavage site. 10. Item 10. The polypeptide according to Item 9, wherein the cleavage site is RKRRRK, and the light chain and the heavy chain are linked by an amino acid sequence consisting essentially of a protease cleavage site. 11. Item 6. A DNA sequence encoding the precursor polypeptide according to Item 4. 12. A recombinant capable of expressing a DNA sequence encoding the precursor polypeptide according to item 4, when the expression system is transfected into a host cell, transformed, and the host cell is cultured under conditions favorable for the expression. Item 6. An expression vector comprising a DNA sequence encoding the precursor polypeptide according to Item 4, wherein the expression system is operably linked to a control sequence for expressing the DNA. 13. Item 13. A recombinant host cell transformed with the expression system according to Item 12. 14． A method for producing a single-chain precursor polypeptide convertible to factor Xai by a protein perhydrolysis reaction, comprising a DNA encoding the single-chain precursor polypeptide
14. culturing the cell according to item 13 under conditions preferable for expressing, and recovering the produced precursor polypeptide or a proteolytic product thereof. 15. A pharmaceutical composition for use in preventing or treating thrombosis in an animal subject, wherein the pharmaceutically acceptable amount of the factor Xai according to item 1 is effective for ameliorating or preventing thrombosis. A pharmaceutical composition comprising a mixture with various excipients. 16. A pharmaceutical composition used for preventing or treating inflammation in an animal subject, which comprises administering an effective amount of the factor Xai according to item 1 to reduce or prevent inflammation in a pharmaceutically acceptable amount. A pharmaceutical composition which is contained in a mixture with a form. 17． A pharmaceutical composition for use in preventing or treating restenosis in an animal subject, wherein the pharmaceutically acceptable amount of the factor Xai according to item 1 is effective for reducing or preventing restenosis. A pharmaceutical composition comprising a mixture with various excipients. 18. A pharmaceutical composition for preventing or treating complications at the time of transplantation surgery in an animal subject, which comprises an effective amount of the factor Xai according to item 1 for alleviating or preventing the complications at the time of transplantation surgery. , A pharmaceutical composition comprising a mixture with a pharmaceutically acceptable excipient. 19. A method for preparing factor Xai useful for treating thrombosis, comprising contacting the precursor polypeptide of item 4 with an amount of a protease effective to cleave the precursor, and forming the Xai product A method comprising recovering the factor. 20. Item 20. The method according to Item 19, wherein the protease is contained in an extract of Russell's viper venom. 21. Item 20. An Xai factor produced by the method according to item 19. 22. X modified to increase half-life in serum
Factor a, wherein the modified form of factor Xa produces active factor Xa in the presence of serum. 23. 23. The modified factor Xa according to item 22, wherein the modified factor Xa is an acylated factor Xa. 24. Claims 1. A pharmaceutical composition for treating hemophilia, which comprises:
3. A pharmaceutical composition comprising the modified factor Xa according to 2 in admixture with a pharmaceutically acceptable excipient. 25. A method for preventing or treating thrombosis in an animal subject, the subject requiring such treatment comprising:
A method comprising administering an effective amount of the factor Xai or a pharmaceutical composition thereof according to the above. 26. A method for preventing or treating inflammation in an animal subject, comprising administering to a subject in need of such treatment an effective amount of the factor Xai according to item 1 or a pharmaceutical composition thereof. 27. A method for preventing or treating restenosis in an animal subject, comprising the steps of:
A method comprising administering an effective amount of the factor Xai or a pharmaceutical composition thereof according to the above. 28. A method for preventing or treating complications at the time of transplantation surgery in an animal subject, comprising administering to a subject in need of such treatment an effective amount of factor Xai or a pharmaceutical composition thereof according to item 1. Including, methods. 29. A method of treating hemophilia in a human subject, comprising providing the subject in need of such treatment with an amount of modified serum that is effective to offset the effects of hemophilia in the subject. Administering factor Xa having an increased half-life.

[0020]

DETAILED DESCRIPTION OF THE INVENTION In general, one aspect of the present invention includes a double-chain polypeptide useful for therapy, referred to herein as Factor Xai, and a single chain of the double-chain protein. Precursors are included. These peptides are shown in Figure 1 at positions 1-139 (light chain) and 1-254.
About 80% homologous to the amino acid sequence shown at position (heavy chain),
Preferably it is about 90% homologous. In FIG.
It should be noted that the proleader sequence is numbered from -40 to -1 before the numbering beginning at the N-terminus of the light chain. The light chains are numbered 1-139. The intervening tripeptide RKR is missing in mature factor X and is not numbered. The activating peptide beginning following this intervening tripeptide is numbered 1-52; isoleucine, hereafter referred to as "position 53" of the activating peptide, is actually an activated heavy chain Is the first amino acid. Since the numbering in the figure restarts from this amino acid, the heavy chains are numbered 1-254.

An embodiment of the double-chain peptide factor Xai comprises:
Effective in forming a prothrombinase complex,
Determined by their ability to inhibit (or compete with) the formation of the native prothrombinase complex containing factor Xa. Their ability to inhibit the formation of the prothrombinase complex is described by Krish, cited above.
naswamy, S.M. , J Biol Chem (19
88) 263: 3823-3834. However, when this factor Xai is incorporated into the prothrombinase complex, the complex cannot show proteolytic activity. This is the van. Dieijen, G .; Et al., J Biol
Chem (1981) 256: 3433, or Sk.
Ogen, W.C. F. Et al., J Biol Chem (19
84) It can be measured by the method of 256: 2306. These Factor Xai proteins are immunoreactive or not immunoreactive with native Factor Xa or antibodies raised against Factor X, including commercially available antibodies specific for human Factor X. Factor Xai protein is an antibody thrombotic substance.

The present invention also relates to precursors of the above-mentioned inactive competitors for factor Xa. One group of these precursors is a novel, modified form of factor X, termed factor X ', wherein one or more of the residues at positions 42, 88 or 185 of the heavy chain is another amino acid residue. Which results in inactivation of the proteolytic properties of the peptide. The modified factor X comprises at least a light chain sequence and a heavy chain sequence linked to the activation peptide. The intervening tripeptide (between the C-terminus of the light chain and the N-terminus of the activation peptide) and the pre-proleader sequence may or may not be present. Thus, factor X 'is a single-chain protein (if a tripeptide is included) or a double-chain precursor of factor Xa (if the tripeptide is deleted).

Preferably, the alteration of a residue in the protease active site is a deletion or conversion to a conservative, substituted amino acid in order to maintain the three-dimensional conformation of the double-stranded protein. . By the term "conservative", rather than a substitution that retains the correct activity,
It refers to a substitution that retains the proper conformation. Thus, the histidine residue at position 42 is preferably substituted with phenylalanine; the aspartic acid at position 88 is preferably substituted with asparagine or glutamine, and the serine residue at position 185 is preferably substituted with alanine or glycine. .

Another group of precursors of the antithrombotic dimeric peptide of the present invention is called factor X'i. The factor X'i precursor lacks at least a substantial portion of the activation peptide, preferably the entire activation peptide. However, the precursor of the double-chain form of factor X'i must retain a proteolytic cleavage site between the light and heavy chains. Therefore, amino acids that undergo endogenous proteolysis are conveniently included in the single-chain precursor form, which extends the carboxy terminus of the light chain to the N-terminus of the heavy chain by a cleavage site. An exemplary embodiment of a single-chain precursor (including a pre-proleader) of a double-chain factor X′i is the absence of an activating peptide sequence (and thus becomes factor Xai).
Activated automatically by the system as shown in FIG. In this embodiment, the hexapeptide sequence thigh RKRRRKR
Has the C-terminus of the light chain directly linked to the isoleucine residue at the N-terminus of the heavy chain. This single-chain factor X'i is cleaved to X'ai. In constructing such modified X'i-type precursors, a hydrophobic amino acid must be retained at the N-terminus of the heavy chain (isoleucine in the natural case). See, for example, Dayhoff, M .; O. , "Atlas of
Protein Sequence and Stru
culture "(1972) 5: 88-89 (Biome
d. Res. Foundation, Wash. D.
C. ) And Greer, J. et al. , J. et al. Molec. Bi
ol. (1981) 153: 1043-1053.

It is clear that the single-chain Factor X 'precursor is also novel, and when proteolytically cleaved, Factor Xa, a normal, enzymatically active form of the dimeric protein is obtained. Can be The corresponding configuration is shown in FIG.

Therefore, when a single-chain precursor of either factor Xa or factor Xai is produced in a suitable host cell by recombinant methods, the endogenous enzyme of the host cell will be (1) single-stranded Cleaving the precursor factor X or factor X 'to a double-stranded form and, in the case of single-chain factor X or factor X', activating the factor by further cleaving the activation peptide; In the case of a chain precursor, since there is no activating peptide, the single-chain precursor is automatically activated when it is cleaved to a dimeric peptide. For Factor X precursors, the double-chain form containing the activating peptide can also be cleaved in vitro using a suitable protease, such as the Factor X activator of Russell's viper venom. Either factor Xa or factor Xai is obtained as described below, depending on whether the active site is inactivated by changing the appropriate codons.

To summarize the terms used in this application, the following terminology may be useful: "Factor Xi"
A single or double chain factor X sequence, natural or recombinantly produced, as shown in FIG. 1, essentially a heavy chain having an activation peptide attached at least to its N-terminus, And light chains. These are shown in the figure,
It may or may not be linked by a cleavage sequence.
"RX" means this factor, especially in a recombinantly produced form.

"Factor Xi" lacks proteolytic activity by modifying the active site as described above.
It means the type X factor produced by recombinant methods. "RXi"
Is also used for this protein.

"Factor Xa" means a naturally occurring or recombinantly produced enzymatically active dimer containing only the light and heavy chains. No activating peptide is present in this complex. "RXa" refers to this complex, especially when produced by recombinant methods.

"Factor Xai" means a modified form of factor Xa, which is activated in the sense that it binds to form a prothrombinase complex, but whose serine protease activity is altered by modification of its active site. Does not have. When this protein is produced only by recombinant methods,
"RXai" is also used to refer to this complex.

"Factor X '" is a modified single-chain form of X
Meaning factor, as shown in FIG. 2, has only a light chain, a heavy chain, and an intermediate specific proteolytic cleavage site. The single-stranded precursor may also include a pre-pro sequence. If this factor is also obtained by recombinant methods alone, it is also called "rX '". This term is used again because when cleaved with a protease to activate the product is indistinguishable from factor Xa (or rXa).

Similarly, "Factor X'i" means a modified form of Factor X 'in which the catalytic site has been inactivated as described above. One version is shown in FIG. When converted to double-stranded form,
The product is indistinguishable from Xai or rXai because the activating peptide is not present in the precursor.

"Acylated factor Xa" or "AcXa"
Means, unless otherwise indicated, factor Xa, whether produced by recombinant methods or not: That is, the serine residue at position 185 is a substituent that provides factor Xa with a half-life in serum of at least 5 to 10 minutes, preferably greater than 15 minutes, and releases active factor Xa over this period. Means blocked factor Xa. Half-life in serum can be measured directly in vivo using appropriately labeled forms. However, the ability of extended life AcXa to produce an active agent in vitro within the required time frame is preferably assessed using an in vitro assay in which factor Xa is a catalyst. Under these conditions, a suitable type of AcXa for use in the present invention has a hydrolysis in an isotonic aqueous medium at pH 7.4 and 37 ° C. such that a half-life of about 5 minutes to several hours can be achieved. Having a rate constant of Its half-life can be determined directly in vitro, if desired, by measuring its ability to activate coagulation, or the rate of hydrolysis of acylated factor Xa, using the prothrombinase reaction as a measure for Xa formation. .

(Preparation of Peptide of the Present Invention) The genomic constitution and coding sequence of human factor X are known, and its cDNA has been searched and sequenced (Leyt
us, S.M. P. Et al., Proc Natl Acad S
ciUSA (1984) 81: 3699; Kaul,
R. Et al., Gene (1986) 41: 311-31.
4). The entire cDNA sequence is shown in FIG.

The full length factor X was inserted into the cDM insert using either M13mp18 or M13 to perform site-directed mutagenesis.
Subcloned into mp19 vector. (X
The correct sequence encoding the factor is confirmed by dideoxy sequencing. ) Since standard modifications are currently readily available in the art, the sequence encoding factor X can be modified to modify the sequence encoding factor X ', factor Xi, factor X'i.
NA is obtained.

The factor X ', factor Xi and the modified coding sequence of factor X'i are then ligated into a suitable expression vector in order to produce the polypeptide recombinantly. The expression vector preferably retains a preproleader sequence for expression in a compatible host cell, such as a mammalian host. If expression in bacteria or yeast is desired, it may be desirable to replace a compatible leader sequence such as a penicillinase sequence in bacteria or an α-factor sequence in yeast. Alternatively, the ATG start codon may be placed directly before amino acid 1 of the light chain encoding sequence to produce an intracellular protein.

The choice of host and expression control system will be governed by the nature of the desired result. If endogenous activation is desired by proteolytic cleavage, mammalian systems are preferred.
However, production in microorganisms that provides the convenience of cultivation can be achieved by using in vitro systems of carboxylation to produce the necessary carboxyglutamyl residues, or by microorganisms that naturally lack this post-translational processing system. Or if another host is transformed to provide it. Various expression systems for recombinant DNA sequences are known in the art.

The modified DNA encoding factor X ', factor Xi or factor X'i preferably comprises a cloning vector and a linker for binding to an expression vector. Methods for preparing such vectors are well understood in the art. Desired X 'factor, Xi
The DNA encoding the factor or factor X'i is operably linked to control sequences, including promoters, upstream enhancers, termination sequences, and the like, depending on the nature of the intended recombinant host cell. Methods for expressing heterologous genes in various hosts, including prokaryotic hosts and various eukaryotes, including yeast, mammalian or bird or insect cells and plant cells, are currently available. The types of control sequences and markers in the expression vector are appropriately selected for these hosts.

For example, for prokaryotic hosts, various promoters may be used, including inducible promoters such as the trp promoter and the phage lambda P _L promoter. A hybrid promoter such as the tac promoter can be used, but this promoter contains a trp polymerase binding region in cooperation with the lac operator. Suitable markers are generally markers for component resistance. On the other hand, promoters commonly used for mammalian cell culture are derived from viruses, such as the early and late SV40 promoter and adenovirus promoter. Mammalian regulatable promoters, such as the metallothionein-II promoter, may also be used. The metallothionein-II promoter is regulated by glucocorticoids or heavy metals. These promoter systems are compatible with typical mammalian hosts, of which the most commonly used are Chinese hamster ovary (CHO) cells.

Another commonly used system is a baculovirus expression system that is compatible with insect cells. For example, plant cells used with the nopaline synthetase promoter and yeast cells used with promoters associated with enzymes important in the glycolytic pathway can also be used. "Current Protocols in M
olecular Biology, "Ausube
1, F. M. Hen, Wiley Interscience
A number of suitable expression systems are described in the appropriate section of the latest edition of ce publication.

The AcXa of the present invention is described, for example, in Cassels, R .; Et al., Biochem J (19
87) 247: 395-400 or U.S. Pat.
Factor Xa, whether recombinantly produced or isolated from plasma, is prepared by standard acylation reactions according to methods analogous to those described or referenced in 37,244. Suitable esters include 4-toluoyl ester, 3,3-dimethylacrylyl ester, cyclohexylidine acetyl ester,
Cyclohex-1-enecarbonyl ester, 1-methylcyclohexylidine acetyl ester, 4-aminobenzoyl ester, γ-guanidinobenzoyl ester, 4-anisoyl ester, 4-N, N-dimethylaminobenzoyl ester, and PDAEB (4 -N- (2-N '(3- (2-pyridyldithio) -propenyl) amino-ethyl) aminobenzoyl esters Generally, the acylating agent is an activated, non-toxic acid which comprises Saturated wherein one carboxyl is substituted,
Provides an unsaturated or aromatic 5 or 6 carbocycle. The ring may be an amino, alkoxy, alkyl, additional ring system,
Or other substituents, such as non-interfering non-toxic substituents,
It may further include. Any compound capable of acylating the hydroxyl group of serine or otherwise blocking serine in a reversible manner is suitable for synthesizing AcXa. Generally, direct or reverse acylating agents can be used, as described in US Patent No. 4,337,244. In the case of direct acylating agents, the acylating moiety itself is attracted to the catalytic site of factor Xa, whereas in the case of reverse acylation access, the leaving group is therefore attracted. The acylated form of Xa is then purified from the reaction mixture using standard purification methods such as dialysis, chromatography, selective extraction, and the like.

The compounds of the invention which serve as medicaments of AcXa must have a suitable deacrylation rate to ensure a suitable clearance time in vivo. This deacrylation rate can be measured using a prothrombinase assay and / or a coagulation assay if it has a half-life of at least 5 minutes in vitro in buffer. The deacrylation reaction is described in Smith, R .; A. G. FIG.
Et al., "Progressin Fibronolosi
s (1985) Vol. 227-231 (Ch
urchill Livingstone) can be measured directly. Prothrombinase and coagulation assays are described in Wolf, D .; L. Et al., J Bi
ol Chem (1991) (during printing).

(Administration and Use) Since the factor Xai peptide of the present invention is a prothrombinase inhibitor, it is useful for a treatment method complicated by thrombosis and a condition whose etiology involves thrombin generation. These conditions include unstable angina (ie, resting angina) and acute vasculature during vascular interventions, including coronary and peripheral angioplasty and atherectomy, and before and after vascular bypass procedures (peripheral and coronary). Includes symptoms associated with arterial thrombosis such as closure, reocclusion after thrombolytic treatment for myocardial infarction, thrombotic attacks (gradually worsening attacks), and thrombosis due to vasculitis (Kawasaki disease). Also included are conditions associated with venous thrombosis, such as deep vein thrombosis of the lower limbs, pulmonary embolism, renal vein, hepatic vein and inferior vena cava, and cavernous sinus thrombosis. Other target symptoms include sepsis with interspersed intravascular coagulation, interspersed intravascular coagulation in other settings, thrombotic thrombocytopenic purpura, and rare symptoms of unknown etiology (anticoagulant lupus (Lupus)
anticoagulant)), which are accompanied by diffuse activation of the coagulation system.

The factor of Xai according to the invention may also be used for cardiopulmonary bypass, for organ collection, for the preparation of blood products or blood samples,
It is also useful as an anticoagulant and anti-inflammatory during organ transport and transplantation and during related treatment of the recipient. Sustained release factor Xai is available in indwelling intravascular devices (iv.
s, catheters, implants, patches).
Thrombosis is also involved in restenosis caused by the proliferation of smooth muscle cells, directly or indirectly, following vascular interventions such as angioplasty, atherectomy or endarterectomy. The factor Xai of the present invention is also useful for treating this condition.

[0045] Adult respiratory distress syndrome (ARDS) is a "endotoxin" in which prothrombotic endothelium appears to be present with components of inflammation and proliferation.
It is considered a disease; therefore, factor Xai also
It is also useful for treating RDS. X of the invention for use in therapy
The ai factor peptide is, for example, Remington's
Pharmaceutical Sciences,
Mack Publishing Company,
As described in the latest edition, Easton, PA,
It is typically formulated for administration using excipients commonly used to administer the protein by injection. To provide an antithrombotic effect, the Factor Xai protein is administered systemically, preferably by injection, and preferably by intravenous injection. Dosage levels will depend on many factors, including the condition of the subject and the particular Factor Xai embodiment selected. However, the appropriate dosage range is from 1 to 1 patient per continuous injection dose.
It is on the order of 50 mg. For injection, the protein is dissolved or suspended in a liquid medium such as Hanks's solution, Ringer's solution, dextrose solution, and various buffers. Additional excipients such as stabilizers may also be used.

The peptide of the present invention can be used, in addition to injection, for suppositories,
It can be administered orally, transmucosally, including intranasal spraying, and systemically via sustained release formulations. Other formulations include encapsulated formulations such as liposomes.

Factor Xai, in addition to its use as a therapeutic, generates polyclonal antisera or is fused to an immortalizing partner to create cells from which a source of monoclonal antibodies specific for this peptide may be obtained. Can be used for These antibodies are useful as passive therapeutic or diagnostic tools. The factor Xa of the present invention, which has been modified to have an increased half-life in vivo, has been shown to contribute to the hemophilia A and B, whether the cause of haemophilia is in the gene of factor X or the more widespread type. Regardless of whether
Useful for treating hemophilia. In this use, extended half-life Factor Xa is administered parenterally or systemically, using dosage levels and formulations similar to those described above for Factor Xai. Administration is typically by injection, but other systemic methods such as transmucosal, transdermal, and the like may also be employed. The formulations used for these administration routes are the same as those described above. In particular, it is advantageous to utilize various vesicles or sustained release systems as are widely known in the art. See, for example, Giles, A .; R.
Et al., Brit J Hematol (1988) 69:
491-497 describes blending factor Xa with phosphatidylcholine-phosphatidylserine vesicles. Similarly, extended life factor Xa can be administered in this manner.

The following examples are intended to illustrate, but not limit, the invention.

[0049]

EXAMPLES (Example 1) (As a catalyst, inactive recombinant human factor X (rXi)
Of DNA encoding human factor X)
The clone was designated R. Dr. Church, Univer
Obtained from the city of Vermont (FIG. 4). This cDNA encodes the amino acid sequence of FIG. 1 or a variant of the allele. This human factor X cDNA
With the E of the vector pBSII (Stratagene)
Cloning into the coRI site yielded pBSX. All X
Hind of pBSX containing the coding region of the factor
The III-XbaI fragment was transformed with the vector M13mp
19 (Mp19X) at the HindIII-XbaI site. Next, Kunkel, T .; A.
Et al., Methods in Enzymol (198
7) Oligonucleotide site-directed mutagenesis was performed as described in 154: 367.

The following forms were produced:

Oligomer TGC CGA GGG GA
Using the C GCC GGG GGC CCG CAC, the serine (S
₁₈₅ ) was converted to alanine (A ₁₈₅ ) to give rXiA ₁₈₅ .

Oligomer ACC TAT GAC TT
Using C AAC ATC GCC GTG CTC
Asparagine at position 88aa of the heavy chain of factor X
Acid (D ₈₈) Is converted to asparagine (N%) to give rXiN
₈₈Was obtained.

Using both oligomers, rXiN ₈₈ A
_The gene encoding ₁₈₅ was obtained (see FIG. 1 for the location of these sites). Confirmation of oligonucleotide-directed mutagenesis was performed by dideoxy sequencing.

Example 2 (Construction of DNA Encoding Precursor (rX ') Truncating Human Factor Xa) The activating peptide was deleted,
Oligonucleotide site-directed mutagenesis (Kunk
e1, T. A. Et al, Methods in Enzym
ol (1987) 154: 367), the cDNA of human factor X (Mp19X) was converted to encode various truncated forms of human factor Xa (collectively referred to as rX's). Subsequent oligonucleotides employed in the corresponding amino acid changes are as follows:

RX '△ 0: ACC CTG GAAC
GC AGG AAG AGG ATC GTG GG
A GGC CAG GAA TGC, which is arginine (R ₁₄₂₎ following the C-terminus of the light chain of Factor X, 53aa isoleucine (I ₅₃₎ of the activation peptide of factor X (1aa chain) and arranged on a straight line the did.

RX ′ △ 1: ACC CTG GAAC
GC AGG AAG AGG AGA ATC ATC GT
G GGA GGC CAG GAA TGC, which is the above R _142, arranged in a straight line and arginine (R52) of the activation peptide of Factor X.

RX ′ △ 2: ACC CTG GAAC
GC AGG AAG AGG CGG CGG AA
A AGA ATC GTG GGA GGC CAG
GAA TGC, which is two amino acid R ₁₄₂ following the light chain of Factor X, arginine (R143) and lysine (K
Extended by _144), an array of the R ₅₂ of the activation peptide of the terminal and Factor X in a straight line (Figure 2).

RX ′ △ 3: ACC CTG GAAC
GC AGG AAG AGG CCT AGG CC
A TCT CGG AAA CGC AGG ATC
GTG GGA GGC CAG GAA TGC,
This is described in Ehrich, H .; J. Et al., J Biol
Chem (1989) 264: 14298 followed light chain of Factor X R ₁₄₂ a 7tsu amino acids as described in, proline (P _143), arginine (R _144),
Proline (P ₁₄₅ ), Serine (S ₁₄₆ ), Arginine (R
₁₄₇ ), lysine (K ₁₄₈ ), arginine (R ₁₄₉ ) and then extend this terminus and the factor X activation polypeptide R
₅₂ and were arranged in a straight line.

Confirmation of the oligonucleotide-directed mutagenesis described above was made by dideoxy sequencing.

As described further below, rX ′ △ 2
Precursor derived from the recombinant form of rXa is directly cleaved endogenously when produced recombinantly in CHO cells.
was gotten. The precursor from rX '△ 0 was not cleaved endogenously in CHO cells when produced by recombinant methods. The precursor from rX '△ 1 or rX' △ 3 was incompletely cleaved. rX '△ 0, rX'
Dimeric peptides from Δ1 and rX′Δ3 were not active as enzymes.

Example 3 (Construction of DNA Encoding Truncated Precursor (rX'i) Inactive as Catalyst) cDNA described in Example 2
The factor X 'construct was converted to encode a catalytically inactive form of X'(rX'i) by oligonucleotide site-directed mutagenesis as described in Example 1. These structures include rX′i (△ 2) as shown in FIG.
N ₈₈ and rX'i (△ 2) include N ₈₈ A _185.

(Example 4) (Expression of genes encoding precursors (rX and rX ')) Expression vector pRC / CMV (Invitrog)
en) and its CMV promoter by the SRα promoter (Takabe, Y. et al., Molec Cell Bi).
ol (1988) 8: 466). S
The ClaI-XbaI fragment filled into the ClaI site containing the Rα promoter with Klenow polymerase was expressed in the expression vector pBJ1 (Lin, A. et al.
science (1990) 249: 677 and M.C. D
avis, available from Stanford University) and subcloned into the NruI-Xbal site of pRC / CMV to generate the expression vector pBN. P containing the SRα promoter, bovine growth hormone polyadenylation site and M13 origin of replication
The StuI fragment of BN was subcloned into the StuI site of pSV2DHFR generating the expression vector pBD. Mp1 of the precursor DNA described in the Examples
The 9SmaI-EcoRV fragment was constructed from XBN of pBN and pBD filled with Klenow polymerase.
It was subcloned into the aI site.

The resulting expression vector was ligated with lipofection (BRL)
Transfected in HO. Selection of transfected clones was performed with 1 mg / ml G418 neomycin (Gibco) or 25 ng / ml methotrexate (Sigma). Single clones were isolated and expanded by cloning cylinders and expression levels were determined by Harlow, E. et al. And Lane, D.M. ,
Antibodies (1988), ColdSpri
ng Harbor Laboratory, New
Measured for 24 hours on serum-free media by standard solid phase antibody capture assay (ELISA) as described in York. In this ELISA method, rabbit polyclonal anti-human factor X (STAGO, America)
n Diagnostics Inc. ) And a rabbit-specific secondary antibody of peroxidase-conjugated goat IgG. Structures pBNX, pBNX ′ △ 0,
pBNX '△ 1, pBNX'2 and PBNX' △ 3
The derived clones were expanded to confluence in T-75 tissue culture flasks containing RPMI medium supplemented with 10% calf serum, penicillin, streptomycin, glutamine and 10 μg / ml vitamin K, and serum-free medium And incubated overnight with serum-free medium.

After incubation, the medium is harvested and
Centrifugation was performed at 0 rpm, and 2 ml was precipitated with 10% trichloroacetic acid (TCA). 100 times the TCA pellet
Wash with 3% acetone and 0.05 ml SDS-PA
Resuspended in GE sample buffer or 0.05m SDS-PAGE sample buffer containing 1M β-mercaptoethanol. Two of each 10 μl of the resulting suspension was added to 12%
After electrophoresis on SDS polyacrylamide gel, Imm
Obilon filter (Millipore). Primary human factor X polyclonal rabbit serum (ST
AGO, American Diagnostics,
Inc. ), 1% nonfat dry milk, 0.1% NP40, 1
0MM Tris-HCl pH 7.5, and 150 mm
Western blot analysis was performed on a 1/4000 dilution with a solution consisting of NaCl. The secondary antibody was a ¹²⁵ I-labeled Fab donkey anti-rabbit IgG (Amersha
m). Autoradiography was performed overnight at -70 ° C using an intensifying screen.

The pattern of antibody reactivity indicated that the expected product had been formed. All five products, r
X, rX '△ 0, rX' △ 1, rX '△ 2, and rX'
The product from Δ3 was positive in the above ELISA based on rabbit polyvalent human Factor X antiserum. The ELISA method uses a mouse monoclonal antibody, Mab323,
Mab743 and Mab325 were also performed.
Mab 323 is particularly reactive towards the activating peptide. Mab743 is reactive to either active or inactive human factor X. Mab325 is calcium-dependent and specific for the light chain, and this antibody reacts with the γ-carboxylated region.

[0066] Any supernatant from the culture containing the above five structures was subjected to ELISA with Mab743 and Mab743.
325 was positive. Therefore, the translated GLA
Processing was shown in all cases. All of the rX 'mutants failed to react with Mab323, confirming the absence of the activating peptide in this mutant.

FIG. 5 shows that rX, rX ′ △ 0, rX ′ △ 1,
Figure 3 shows the results of Western blot analysis performed with polyclonal rabbit antisera on products from rX '△ 2, rX' △ 3 and CHO control medium. Rabbit polyclonal antiserum to X was not effective in localizing the fully processed heavy chain of human factor Xa. Thus, in all cases, the position expected to be occupied by the activated heavy chain is not shown. FIG. 5a shows the reduced forms of these recombinant proteins, and FIG. 5b shows the non-reduced forms of these proteins. Lane 1 shows 0.7 μg of native human factor X (Dr. C. Esom, OMRF, Uni).
lane 2 (version of Oklahoma);
Is rX; lane 3 is rX '△ 0; lane 4 is rX' △
1; lane 5 shows rX '△ 2; lane 6 shows rX' △ 3; and lane 7 shows a CHO control medium.

FIG. 5 a shows that the recombinant product of rX and rX ′ △ 2 is a dimeric protein that can be separated under reducing conditions. The expression products of rX '△ 0, rrX' △ 1 and rX '△ 3 are clearly largely single-stranded products. The untreated factor X 'single strand appears to irregularly co-migrate with the heavy chain, as shown in lanes 3-7, clearly indicating a new cleavage site. This is due to the degree of proteolytic processing. The lack of proper processing of these X 'precursor proteins was consistent with the results of the coagulation assay described in Example 8. Example 8 shows that Factor Xa, RVV-activated or recombinant Factor X, and X '△ 2 are equally active, while the products secreted by the remaining X' are dramatically more efficient. Low, at least about five orders of magnitude lower. Table 1 shows data on the enzyme activities.

[0069]

[Table 1] The column of Table 1 described as "catalyst efficiency" indicates that R
Various factors activated by VV, amidolytic subatrateactivities
v.). The proposed catalyst efficiency is kcat / Km
And normalized to the results for plasma Factor X. As shown in the table, both recombinant factor X and X '△ 2 were active in the factor X-dependent 2PT coagulation assay, while the enzymatic activity of the remaining recombinant proteins was four orders of magnitude lower.

From FIG. 5b, it is clear that the expression product of the gene encoding X 'has a lower molecular weight than rX or native factor X.

Example 5 (Purification of rX and X ′ △ 2) Both recombinant factor X and X ′ △ 2 were purified to homogeneity as follows. That is, after growing to a confluent state, CHO cells transfected with pBNX or pBNX '# 2 were washed 4 to 5 times with a serum-free medium. Next, the cells were cultured continuously at 37 ° C. for 24 hours in a medium from which serum supplemented with 4 μg / ml of vitamin K3 had been removed.

The harvested medium was centrifuged at 15,000 × g for 20 minutes, and the supernatant was filtered through a 0.2 μm filter. Add Tris HCl pH 7.5 to the medium for 20 minutes.
mM and NaEDTA up to 10 mM, and the resulting product is Q-Sepharose Fast Fl.
ow (Pharmacia). All steps of the chromatography were performed at 4 ° C.
The column was loaded with 20 mM Tris, pH 7.5, 10 mM E
Wash thoroughly with DTA, 0.15 M NaCl and remove proteins from 20 mM Tris pH 7.5, 0.5 M N
aCl, and eluted with 5mM CaCl _2. The peak fractions are pooled and stored frozen at -20 ° C or Ch
urch, W, R.A. Et al., Throm Res (198
5) Directly applied to an anti-Factor X monoclonal antibody affinity column as described in 38: 417-424. The antibody used for isolation (aHFX-1d, M
abB12-A3) is specific for human factor X, not affected by Ca ² +, cut Factor X and X
Binds to both factor a (unpublished data). The rX 'factor was further analyzed by Krishnaswamy et al., J Bio.
1 Chem (1987) 262: 3291-3299.
Purified on a benzamidine-sepharose column (Pierce) as described in. Protein concentrations were determined by quantitative ELISA, protein colorimetric assay (Harlow, E. et al., Antibodies, A.
Laboratory Manual "(198
8), ColdSpring Harbor Labo
ratories, Cold Spring Har
bor, New York) and an extinction coefficient of 11.6,
280 using molecular weight of 58,900 for factor X and 46,000 for X '△ 2.
Measured by absorbance at nm.

The purified factor was subjected to SDS-reducing under reducing conditions.
When PAGE was performed and stained with silver, the recombinantly produced factor X contained the full-length precursor (75 kD), the heavy chain (45 kD) containing the activation peptide and the light chain (22 kD).
kD). Electrotransfer of the nylon filter followed by sequence analysis of the amino terminus revealed that the light chain was heterogeneous,
27% start with Val ₃₇ and 73% start with Ala ₄₁ , while the 75 KD species is also heterogeneous, 41% Val
It begins with a _37, and 59% has been found that starting with Ala _41.

Example 6 Inactivated Recombinant Human Factor X (rXi and rX'i)
(Expression of the gene encoding) The X ′ type selected for conversion to the inactive type was the rX ′ △ 2 type shown in FIG.
rX, rX ′ (△ 2), rXiN ₈₈ A ₁₈₅ , rXi
A ₁₈₅ , rX′i (△ 2) N ₈₈ A ₁₈₅ and rX′i (△ 2)
2) The cell lines induced with pBN for N _88, were grown to confluence in 800 cm ² Rorabin as described in Example 4, was washed 4 times with serum was removed culture broth, 50M1 Was incubated overnight with the culture medium from which the serum was removed. The culture was replenished and harvested daily.

The continuous crop was pooled and 3000 rpm
In centrifugation, then, C · E _SMON Dr. (0MRF, Un
The cells were directly passed through a factor X-specific monoclonal antibody (Mab) affinity column (Mab717) provided by the University of Oklahoma.
The bound "Factor X" is treated with 80% ethylene glycol in M
Elution from ab717 column, 10 mM Tris HC
l Dialysis against pH 7.5, 150 mM NaCl, Centricon 10 permeator (Amicon)
And concentrated. The concentration of the "Factor X" protein was determined in Example 4
A standard formulation of human factor X (HaematologicTechno) using serial dilutions as described in
logs, Inc. , C.I. Esmon, OMR
F, University of Oklahoma)
Was measured by an ELISA method in comparison with

The purified protein was characterized by Western blot analysis as outlined in Example 4. FIG.
Is reduced by these β-lecaptoethanols and Ma
Figure 5 shows a Western blot of recombinant human factor X analog purified in b717. Lane 1 contains 0.1 μg of human X
(Haematologic Technology
s, Inc. ); Lane 2 shows 0.1 μg of human Xa (H
aematologic Technologies, I
nc. ); Lane 3 is 0.1 μg rX Lane 4 is 0.
16 μg of rX ′ △ 2; Lane 5 is 0.13 μg of rX
iN ₈₈ rA ₁₈₅ ; lane 6 contains 0.15 μg of rXi
A ₁₈₅ ; Lane 7 is 0.187 μg of rX′i (△ 2)
N ₈₈ ; lane 8 contains 0.05 μg of rX′i (△ 2) N ₈₈
A ₁₈₅ .

Under reducing conditions, human X and human Xa are in dimeric form; human Xa has no activating peptide,
It is clear that it shows a low molecular weight form of the heavy chain. Lane 3
Of human X is similar to natural human X, but is clearly a single-chain precursor. Lane 4 shows that recombinant rX ′ △ 2 also cleaves into heavy and light chains. In lanes 5 and 6, the modified recombinant Xi protein behaves in a manner similar to recombinant human X. As expected, lanes 7 and 8 show the presence of monomers, heavy and light chains induced by proteolytic cleavage of X'i.

Example 7 Oxygen Analysis of Recombinant Human Factor X Natural Human Factor X;
Factor Xa, recombinant X (rX), rX ′ {0, rX ′}
1, rX '△ 2, rX' △ 3, rXiN ₈₈ A ₁₈₅ , rX
iN ₈₈ , rX′i (△ 2) N ₈₈ A ₁₈₅ , and inactive bovine Xa, C.I. Dr. Emon (OMRF, Univer
X provided by the site of Oklahoma)
ai-APMSF (Skogen, WF, et al., JB
iol Chem (1984) 259: 2306), Chromozym X (N-methoxycarbonyl-D-norleuc-glycyl-arginine-4-nitranilidoacetate, Boehring)
The kinetic measurement of the hydrolysis of E. Mannheim was determined at room temperature by Molecular Devices V
The max spectrophotometer was tested in lung 96 microtiter plates. The absorbance at 405 nM was continuously monitored and the reaction rate was measured directly by the machine,
Fitter program (Elsevier Pres
s). Protein concentration is ELI
It was measured by the SA method (Example 6). All enzymes are
O. It was diluted to an appropriate concentration with 1% bovine serum albumin (BSA), 50 mM Tris-HCl, pH 8.0, 150 mM NaCl. 50 mM Tris HC1, pH 8.0,
The reaction was performed twice in 150 mM NaCl and 2.5 mM CaCl2. All recombinant human factor X '
RX ′ △ 0, rX ′ △ 1 and rX′-purified and concentrated using QAE-Sepharose (Pharmacia)
Except for X ′ △ 3, all were purified by Mab717 (Example 6) (Skogen, WF et al. J Biol.
Chem (1984) 259: 2306).

Recombinantly produced peptides derived from vectors containing rX, rXiN ₈₈ A ₁₈₅ , rXiN ₈₈ and rXiN ₈₈ A ₁₈₅ are treated with a Russell's viper venom for 5 minutes prior to treatment to form Xa or Xa Converted to Xai type. rX '△ 0, r
Peptides from vectors containing X '△ 1, rX' △ 2 and rX '△ 3 were not treated in this manner.

FIG. 7 is a Lineweaver-Burk plot comparing native human factor X and factor Xa with activated forms derived from recombinant human rX and rX '. FIG.
FIG. 7b is human Xa; FIG. 7c is human rX (treated with Russell's viper venom protease); FIG.
d is human rX '(not treated with protease).

Table 2 shows the values of Kcat and Km for human factor Xs produced by the recombinant method and Haematol.
organic Technologies, Inc. 3 shows a comparison with native human factor X and factor Xa supplied by S.A.

[0082]

[Table 2] Of course, none of the inactivated forms showed a value; only rX '△ 2 among the rX' forms showed activity.

Example 8 (Human X, Xa and recombinant human rX and rX ')
One activity of factor X-dependent prothrombinase complex) factor X
Activity of the child-dependent prothrombinase complex
Chromodim TH (tosyl-glycyl-prolyl)
-Arginine-4-nitroanilide acetate, Boe
(Hringer Mannheim)
To the Molecular Devices Vmax
In a 96-well microtiter plate of a photometer
And was determined by measuring at room temperature. 405 nM
Of the reaction for the first minute.
Is measured directly on the machine and the Enzfitter
Plotted using gram (Elsevier). Anti
The reaction mixtures were made in triplicate with the following composition. That is, ELI
0.05 × 10 measured by SA method (Example 6)^-FourM
1.5X10^-9"Factor X" of M, 0.5X10 ^-6M hee
Toprothrombin (STAGO, American D
iagnostics, Inc. ), 7.5X10^-9M
Human Va factor (HaematologicTechno)
logs, Inc. ), 20X10^-6M phosphoricoli
/ Phosphoserine 75% / 25% (PCPS) (W.
R. Dr. Church, University of
(Provided by Vermont) or an equivalent amount of rabbit brain sef
(Sigma) (Example 9), 0.1% BSA
(Sigma), 0.1X10 ^-3M Chromodymium TH (B
oehringer Mannheim), 25M
Squirrel HCl pH 7.5, 150 mM NaCl
And 5 mM CaCl_TwoThe composition of

The activity of the prothrombinase complex dependent on human rX and rX 'factors utilized PCPS, and the activity of the prothrombinase complex dependent on human factor X and factor Xa utilized cephalin. Human factor X and rX are Russell's viper venom (Haematol).
organic Technologies, Inc. ) For 5 minutes. The hydrolysis of chromozym TH by thrombin, as measured by the increase in the fluorescence signal, was linear throughout the experimental protocol. RXiN ₈₈ A at a concentration of 59.2 × 10 ⁻⁴ M
₁₈₅ , 10.2 × ^{10 19} M concentration of bXai-APMSF
Did not show an observable rate. 8a to 8d
Are human X (FIG. 8a), human Xa (FIG. 8b) (Haem
atologic Technologies, In
c. ), Recombinant human rX (after treatment with protease)
(FIG. 8c) and Factor X-dependent prothrombinase complex activity of recombinant human rX ′ お よ び 2 (after no treatment with protease) (FIG. 8d). All have equal activity.

(Example 9) (Coagulation of plasma) rXa and rXa purified by Mab717
Xa was assayed for plasma clotting activity in an automated two-step prothrombin assay using MLA Electra 800 fibrome overnight. Enzyme protein concentration is ELIS
Measured by Method A (Example 6), 0.1
% BSA, diluted in 150 mM NaCl. Bovine factor X and factor VII deficient plasma (Sigma)
As well as rabbit brain cephalin (Sigma) was prepared according to the manufacturer's instructions. Russell's viper venom 0.1 μg / ml was added to the human X and rX assays. The reaction mixture contains 0.1 ml of factor X, 0.1 ml
150 mM NaCl, 0.1 ml cephalin and 0.1 ml 25 mM CaCl2. Each concentration was tested twice and the average of the results of the two experiments was calculated. FIG. 9 compares the plasma clotting activity of human X, human Xa, human rX and human rXa. As a result of the calculation, the activity of human rX is 45% of that of human X,
a had an activity of 32% of human Xa.

[0086] (Example 10) (rXiN ₈₈ A _185, human _{rX'i (△ 2) N 88 A} 185 and inhibition of prothrombinase complex activity by bovine bXai-APMSF) by human rXiN ₈₈ A _185, native human inhibition of factor X dependent proto lobby kinase complex activity, as well as human _{rX'i (△ 2) N 88 a} 185 and bovine bXai-APMSF (C.Esmon, OMRF,
The inhibition of native factor Xa (5 × 10 ⁻⁹ M) -dependent prothrombinase complex activity by University of Oklahoma was tested as described in detail in Example 8. Because of the kinetic factors and the strength of the complex formed once, X and Xi and Xa and Xai
Need to be compared directly. Human rXiN ₈₈ A ₁₈₅
Preincubation for 5 minutes with 0.1 μg / ml Russell's viper venom. The concentration of human factor X and factor Xa was 1 × 10 ⁻⁹ M.

FIG. 10 shows that bXai-APMSF and r
X'i (△ 2) N₈₈A, human factor Xa-dependent pro
Concentration-dependent inhibition of thrombinase complex, and rX
iN ₈₈A₁₈₅Factor X-dependent prothrombiner by
4 shows inhibition of the zeta complex. 5 by bXai-APMSF
0% inhibition is 0.9 × 10^-9RX'i
(△ 2) N₈₈A₁₈₅50% inhibition by 6X10^-9M's
Concentration, and rXiN₈₈A₁₈₅50% blocking by
Harm is 10.6X10^{One nine}M concentration.

Example 11 Preparation of Acylated Factor Xa Human Recombinant Xa (rXa)
Was prepared as described in Example 5 and cleaved with Russell's viper venom. rXa was added to physiological saline solution (2μ
mol / 25 ml) and treated with 5 μmol of p-nitrophenyl ester of p-toluoyl acid while stirring at 4 ° C. for 20 minutes. The resulting reaction mixture was dialyzed against glycerin / saline and the resulting dialysate was
Store at ° C.

Analogs of factor Xa (factor Xai), which are inactive as proteases in the prothrombinase reaction, are useful for treating diseases characterized by thrombosis. These antithrombotic agents can be conveniently prepared using recombinant techniques. In addition, modified forms of factor Xa that produce factor Xa in serum but have an extended half-life are useful for treating the symptoms of hemophilia. These modified forms of X
Factor a is typically an acylated form of the active site serine residue and is slowly deacylated in vivo.

[0090]

[Sequence List] SEQUENCE LISTING <110> COR Therapeutics, Inc.WOLF, David <120> Recombinant Agents Affecting Thrombosis <130> 44481-5002-JP <140> JP 3-517527 <141> 1991-09-04 <150> PCT / US91 / 06337 <151> 1991-09-04 <160> 2 <170> PatentIn version 3.1 <210> 1 <211> 139 <212> PRT <213> Artificial sequence <220> <223> Mutant Factor Xa: Light Chain <220> <221> misc_feature <222> (63) .. (63) <223> X can be beta-hydroxy aspartic acid <220> <221> misc_feature <222> (6) .. (39) < 223> X can be gamma-carboxy glutamic acid <400> 1 Ala Asn Ser Phe Leu Xaa Xaa Met Lys Lys Gly His Leu Xaa Arg Xaa 1 5 10 15 Cys Met Xaa Xaa Thr Cys Ser Tyr Xaa Xaa Ala Arg Xaa Val Phe Xaa 20 25 30 Asp Ser Asp Lys Thr Asn Xaa Phe Trp Asn Lys Tyr Lys Asp Gly Asp 35 40 45 Gln Cys Glu Thr Ser Pro Cys Gln Asn Gln Gly Lys Cys Lys Xaa Gly 50 55 60 Leu Gly Glu Tyr Thr Cys Thr Cys Leu Glu Gly Phe Glu Gly Lys Asn 65 70 75 80 Cys Glu Leu Phe Thr Arg Lys Leu Cys Ser Leu Asp Asn Gly Asp Cys 85 90 95 Asp Gln Phe Cys His Glu Glu Glu Gln Asn Se r Val Val Cys Ser Cys Ala 100 105 110 Arg Gly Tyr Thr Leu Ala Asp Asn Gly Lys Ala Cys Ile Pro Thr Gly 115 120 125 Pro Tyr Pro Cys Gly Lys Gln Thr Leu Glu Arg 130 135 <210> 2 <211> 254 <212> PRT <213> Artificial sequence <220> <223> Mutant Factor Xa: Heavy Chain <400> 2 Ile Val Gly Gly Gln Glu Cys Lys Asp Gly Glu Cys Pro Trp Gln Ala 1 5 10 15 Leu Leu Ile Asn Glu Glu Asn Glu Gly Phe Cys Gly Gly Thr Ile Leu 20 25 30 Ser Glu Phe Tyr Ile Leu Thr Ala Ala His Cys Leu Tyr Gln Ala Lys 35 40 45 Arg Phe Lys Val Arg Val Gly Asp Arg Asn Thr Glu Gln Glu Glu Glu Gly 50 55 60 Gly Glu Ala Val His Glu Val Glu Val Val Ile Lys His Asn Arg Phe 65 70 75 80 Thr Lys Glu Thr Tyr Asp Phe Asn Ile Ala Val Leu Arg Leu Lys Thr 85 90 95 Pro Ile Thr Phe Arg Met Asn Val Ala Pro Ala Cys Leu Pro Glu Arg 100 105 110 Asp Trp Ala Glu Ser Thr Leu Met Thr Gln Lys Thr Gly Ile Val Ser 115 120 125 Gly Phe Gly Arg Thr His Glu Lys Gly Arg Gln Ser Thr Arg Leu Lys 130 135 140 Met Leu Glu Val Pro Tyr Val Asp Arg Asn Ser Cys Lys Leu Ser Ser 145 150 155 160 Ser Phe Ile Ile Thr Gln Asn Met Phe Cys Ala Gly Tyr Asp Thr Lys 165 170 175 Gln Glu Asp Ala Cys Gln Gly Asp Ala Gly Gly Pro His Val Thr Arg 180 185 190 Phe Lys Asp Thr Tyr Phe Val Thr Gly Ile Val Ser Trp Gly Glu Gly 195 200 205 Cys Ala Arg Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Thr Ala Phe 210 215 220 Leu Lys Trp Ile Asp Arg Ser Met Lys Thr Arg Gly Leu Pro Lys Ala 225 230 235 240 Lys Ser His Ala Pro Glu Val Ile Thr Ser Ser Pro Leu Lys 245 250

[Brief description of the drawings]

FIG. 1 shows the structure of human factor X and its associated cleavage sites as described in the prior art.

FIG. 2 shows the structure of one embodiment of a single-chain factor X ′, which is a precursor to obtain a double-strand cleavage product involved in the formation of a prothrombinase complex. The form shown in this figure produces a double-stranded peptide that retains proteolytic activity in the complex; the modified form shown below is catalytically inactive.

FIG. 3 shows one embodiment of factor X′i.

FIG. 4 shows the cDNA sequence encoding factor X.

FIG. 5 is a Western plot of potentially active factor X and factor Xa produced by recombinant methods.

FIG. 6 is a Western plot of inactive Factor X and Factor Xa produced by recombinant methods.

FIGS. 7a-7d are a series of Lineweaver-Burk plots showing the enzymatic activity of native Factor X converted to the activated form and Factor X produced by recombinant methods.

Figures 7a-7d are a series of Lineweaver-Burk plots showing the enzymatic activity of native Factor X converted to the activated form and recombinantly produced Factor X.

Figures 7a-7d are a series of Lineweaver-Burk plots showing the enzymatic activity of native Factor X converted to the activated form and Factor X produced by recombinant methods.

Figures 7a-7d are a series of Lineweaver-Burk plots showing the enzymatic activity of native Factor X converted to the activated form and Factor X produced by recombinant methods.

8a-8d compare the activity of various factor X-type prothrombinase complexes.

8a-8d compare the activity of various factor X-type prothrombinase complexes.

8a to 8d compare the activity of various factor X-type prothrombinase complexes.

8a to 8d compare the activity of various factor X-type prothrombinase complexes.

FIG. 9 shows the results of performing a two-step prothrombin coagulation assay on various types of factor X.

FIG. 10 shows the inhibition of prothrombin complex formation by an inactive form of factor X.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI theme coat ゛ (reference) // C12N 15/09 C12N 15/00 A F term (reference) 4B024 AA01 AA11 BA07 CA04 DA02 EA04 GA13 HA06 4C084 AA02 BA01 BA08 BA21 BA22 BA23 BA44 CA53 CA59 CA62 DC10 NA14 ZA362 ZA392 ZA512 ZA542 4H045 AA10 BA10 BA32 BA35 CA42 DA65 EA24 FA74 GA26 HA06

Claims (7)

[Claims] Claims 1. A factor Xa which is a blood factor modified to transiently prolong its half-life in serum, wherein the modified blood factor has an activity of the blood factor in the presence of serum. Factor Xa that produces the morphology. 2. The modified blood factor according to claim 1, which is an acylated blood factor. 3. The blood factor according to claim 2, wherein said modified blood factor is factor Xa. 4. A pharmaceutical composition for treating hemophilia, comprising factor Xa, which is a blood factor modified to increase the half-life in serum, in admixture with a pharmaceutically acceptable excipient. A composition, wherein the modified blood factor produces an active form of the blood factor in the presence of serum. 5. A modified factor Xa having an extended plasma half-life, said modification comprising a serine residue reversibly blocked in the proteolytic active site of said modified factor Xa, And a modified factor Xa, wherein the modified factor Xa produces an active factor Xa in the presence of plasma. 6. The modified factor Xa according to claim 5, which is an acylated factor Xa. 7. The blood factor according to claim 5, wherein said modified blood factor is factor Xa.