TW201533058A - Coagulation factor VII polypeptides - Google Patents

Abstract

The present invention relates to modified coagulation Factor VII polypeptides exhibiting increased resistance to antithrombin inactivation and enhanced proteolytic activity. The present invention also relates to polynucleotide constructs encoding such polypeptides, vectors and host cells comprising and expressing such polynucleotides, pharmaceutical compositions, uses and methods of treatment.

Description

Factor VII polypeptide

The present invention relates to a Factor VII (Factor VII) polypeptide having procoagulant activity. It also relates to pharmaceutical compositions, methods of treatment, and uses of such polypeptides comprising such polypeptides.

Sequence table

SEQ ID NO. 1: wild type human coagulation factor VII.

SEQ ID NO. 2: Protease domain of human coagulation factor VII.

SEQ ID NO. 3: Protease domain of ancient human (chimpanzee) factor VII.

SEQ ID NO. 4: Protease domain of canine (dog) coagulation factor VII.

SEQ ID NO. 5: Protease domain of pig (pig) coagulation factor VII.

SEQ ID NO. 6: Protease domain of bovine (bovine) coagulation factor VII.

SEQ ID NO. 7: Mouse (mouse) protease domain of Factor VII.

SEQ ID NO. 8: Mouse (rat) protease domain of Factor VII.

SEQ ID NO. 9: Rabbit (rabbit) protease domain of Factor VII.

Vascular injury activation involves a hemostatic system of complex interactions between cells and molecular components. The process that ultimately stops bleeding is called haemostasis. An important part of hemostasis is coagulation and the formation of clots at the site of injury. The coagulation process is highly dependent on the function of several protein molecules. These molecules are called coagulation factors. Inactive zymogen Or a protease present in an enzymatically active form. The zymogen form can be converted to its enzymatically active form by specific cleavage of the polypeptide chain catalyzed by proteolytically active coagulation factors.

Factor VII is a vitamin K-dependent plasma protein synthesized in the liver and secreted into the blood as a single-chain glycoprotein. Factor VII zymogen is converted to an activated form (Factor VIIa) by specific proteolytic cleavage between a single site of SEQ ID NO: 1, ie, R152 and I153, resulting in a single disulfide bond. Double-stranded molecule. The two polypeptide chains in Factor Vila are referred to as the light and heavy chains, which correspond to residues 1-152 and 153-406 of SEQ ID NO: 1 (wild type human Factor VII), respectively. Factor VII circulates primarily in the form of a zymogen, but a smaller portion is in an activated form (Factor VIIa).

The coagulation process can be divided into three phases: initiation, amplification, and spread. The initiation and propagation phases promote the formation of thrombin, a clotting factor that has many important functions in hemostasis. If the monolayer barrier of the endothelial cells lining the inner surface of the blood vessel becomes damaged, the coagulation cascade begins. This exposed blood platelets will adhere to the subendothelial cells and extravascular receptor proteins. If this occurs, the tissue factor (TF) present on the surface of the subendothelial cells becomes exposed to factor VIIa circulating in the blood. TF is a membrane-bound protein and acts as a receptor for factor VIIa. Factor VIIa is an enzyme with a very low activity, a serine protease. However, when Factor VIIa binds to TF, its activity is greatly increased. The interaction of Factor VIIa with TF also localizes Factor VIIa to the phospholipid surface of cells bearing TF and optimally positions it to activate Factor X to Xa. When this occurs, Factor Xa can be combined with Factor Va to form a so-called "prothrombinase" complex on the surface of cells bearing TF. The prothrombinase complex is then cleaved by prothrombin to produce thrombin. The pathway by exposing TF to circulating factor VIIa and causing initial thrombin activation is referred to as the TF pathway. TF: Factor VIIa complex also catalyzes the activation of Factor IX to Factor IXa. Subsequently, activation Factor IXa can diffuse to the surface of the activated platelets that adhere to the site of injury. This allows Factor IXa to be combined with FVIIIa to form an "X enzyme" complex on the surface of activated platelets. This complex plays a key role in the propagation phase due to its significant efficiency in activating factor X to Xa. Highly efficient X-enzyme-catalyzed production of factor Xa leads to efficient cleavage of prothrombin catalyzed by prothrombinase complex to thrombin.

If there is any deficiency in Factor IX or Factor VIII, it impairs important X enzyme activity and reduces the production of thrombin required for blood clotting. The thrombin initially formed by the TF pathway acts as a procoagulant signal for the recruitment, activation and aggregation of platelets that promote the site of injury. This results in the formation of loose primary platelet plugs. However, this primary platelet plug is unstable and needs to be strengthened to maintain hemostasis. Stabilization of the plug involves anchoring and entanglement of the platelets in the fibrin web.

The formation of a strong and stable clot depends on a robust burst that produces local thrombin activity. Thus, a deficiency in the process of producing thrombin after vascular injury can lead to bleeding disorders such as hemophilia A and B. People with hemophilia A and B lack functional factor VIIIa or factor IXa, respectively. The production of thrombin in the propagation phase is highly dependent on the X enzyme activity, ie both factors VIIIa and FIXa are required. Therefore, in people with hemophilia A or B, primary platelet plugs are not properly consolidated and bleeding continues.

The replacement therapy is a traditional treatment for hemophilia A and B and involves intravenous administration of Factor VIII or Factor IX. However, in many cases, the patient produces antibodies (also known as inhibitors) directed to the injected protein that reduce or counteract the therapeutic efficacy. Recombinant Factor VIIa (Novoseven®) has been approved for the treatment of hemophilia A or B patients with inhibitors, as well as for stopping bleeding events or preventing bleeding associated with trauma and/or surgery. Recombinant factor VIIa has also been approved for the treatment of patients with a congenital factor VII deficiency. It has been proposed to operate reorganization via a TF-independent mechanism FVIIa. According to this mode, recombinant FVIIa is directed to the surface of activated platelets by virtue of its Gla-domain, which in turn activates factor X proteolysis to Xa in this domain, thus circumventing the need for a functional X enzyme complex. The low enzyme activity of FVIIa in the absence of TF and the low affinity of the Gla-domain to the membrane may explain the need for a super-physiological level of circulating FVIIa required to achieve hemostasis in people with hemophilia.

Recombinant Factor VIIa has an in vivo functional half-life of 2-3 hours, which necessitates frequent administration to resolve bleeding in a patient. In addition, patients are usually treated with Factor VIIa only after they have started bleeding, rather than as a preventive measure, which usually affects the general quality of life of the patient. Recombinant Factor Vila variants with longer in vivo functional half-lives will reduce the number of administrations required, support less frequent dosing and are therefore expected to significantly improve Factor Vila treatment to benefit patients and health care holders.

WO 02/22776 discloses Factor VIIa variants having enhanced proteolytic activity compared to wild-type FVIIa. It has been shown in clinical trials that the substituted Factor VII polypeptide disclosed in WO 02/22776 shows favorable clinical results in terms of the efficacy of variants with enhanced proteolytic activity (de Paula et al. (2012) J Thromb Haemost , 10 :81-89).

WO2007/031559 discloses Factor VII variants that have less susceptibility to inhibition by antithrombin.

WO 2009/126307 discloses modified Factor VII polypeptides with altered procoagulant activity.

In general, there are many unmet medical needs in people with coagulopathy. The use of recombinant Factor VIIa to promote clot formation emphasizes the importance of its growth as a therapeutic. However, recombinant Factor VIIa treatment still retains significant unmet medical needs, with improved pharmaceuticals Retinoids, such as increased in vivo functional half-life and enhanced or higher activity, will satisfy some of these needs.

The present invention provides Factor VII polypeptides designed to have improved pharmaceutical properties. In one broad aspect, the invention relates to a Factor VII polypeptide that exhibits an increased in vivo functional half-life compared to human wild-type Factor Vila. In another broad aspect, the invention relates to a Factor VII polypeptide having enhanced activity compared to human wild-type Factor Vila. In another broad aspect, the invention relates to an agent that exhibits increased resistance to inactivation compared to human wild-type Factor VIIa by an endogenous plasma inhibitor, specifically antithrombin. Peptide.

Provided herein are Factor VII polypeptides having an increased in vivo functional half-life comprising a combination of mutations that confer minimal or no loss of resistance to antithrombin inactivation and enhanced proteolytic or proteolytic activity. In a particularly interesting aspect of the invention, the Factor VII polypeptide is coupled to one or more "half-life extending portions" to increase in vivo functional half-life.

In one aspect, the invention relates to a Factor VII polypeptide comprising two or more substitutions relative to the amino acid sequence of human Factor VII (SEQ ID NO: 1), wherein T293 is by Lys (K), Arg(R), Tyr(Y) or Phe(F) substitution and L288 is replaced by Phe(F), Tyr(Y), Asn(N) or Ala(A) and/or W201 by Arg(R), Met (M) or Lys (K) substitutions and / or K337 substitution by Ala (A) or Gly (G).

The Factor VII polypeptide of the present invention may comprise a substitution of T293 by Lys (K) and L288 by Phe (F). The Factor VII polypeptide may comprise a substitution of T293 by Lys (K) and L288 by Tyr (Y). Factor VII polypeptide may comprise replacing T293 by Arg(R) and replacing by Phe(F) L288. The Factor VII polypeptide may comprise a substitution of T293 by Arg (R) and a substitution of L288 by Tyr (Y). The Factor VII polypeptide may comprise or may further comprise a substitution of K337 by Ala (A). The Factor VII polypeptide may comprise a substitution of T293 by Lys (K) and a substitution of W201 by Arg (R).

In an interesting embodiment, the invention relates to a Factor VII polypeptide coupled to at least one half-life extending moiety.

In another aspect, the invention relates to a method of making a Factor VII polypeptide of the invention.

In another aspect, the invention relates to a pharmaceutical composition comprising a Factor VII polypeptide of the invention.

A general object of the present invention is to improve the currently available therapeutic options in humans with coagulopathy and to obtain a Factor VII polypeptide with improved therapeutic utility.

One object of the present invention is to obtain a Factor VII polypeptide having an extended in vivo functional half-life while maintaining pharmaceutically acceptable proteolytic activity. To achieve this goal, the Factor VII polypeptide of the present invention comprises a combination of mutations which confer reduced susceptibility to inactivation by plasma inhibitor antithrombin while substantially maintaining proteolytic activity; In a specific embodiment of the invention, the Factor VII polypeptide is also coupled to one or more "half-life extending portions".

The medical provision of the modified Factor VII polypeptide of the present invention provides a number of advantages over currently available treatment regimens, such as longer durations between injections, lower doses, more convenient administration, and between injections. Potentially improved hemostasis protection.

Figure 1 shows an amino acid sequence alignment of FVIIa protease domains from different species.

Figure 2 shows the correlation between antithrombin reactivity in vitro and in vivo accumulation of FVIIa antithrombin complex.

Figure 3 shows the configuration of arginine at position 201 in the FVIIa variant W201 RT293Y double mutant of the configuration of tryptophan at position 201 in FVIIa WT.

Figure 4 shows a hypothetical pattern of the interaction between tyrosine and antithrombin at position 293 of the FVIIa variant W201R T293Y double mutant. This is based on a theoretical model of the complex between the antithrombin and the FVIIa variant W201R T293Y double mutant shown in a bar graph. The anti-thrombin amino acid is depicted by the prefix "AT"; while the FVIIa amino acid is depicted by the prefix "FVIIa".

Figure 5: (A) Heparin precursor and (B) structure of a heparin precursor polymer having a maleimide functional group at the reducing end.

Figure 6: Evaluation of the purity of the conjugate by SDS-PAGE. (A) SDS-PAGE analysis of the final FVIIa conjugate. The gel was loaded with HiMark HMW standards (lane 1); FVIIa (lane 2); 13k-HEP-[C]-FVIIa (lane 3); 27k-HEP-[C]-FVIIa (lane 4); 40k-HEP -[C]-FVIIa (lane 5); 52k-HEP-[C]-FVIIa (lane 6); 60k-HEP-[C]-FVIIa (lane 7); 65k-HEP-[C]-FVIIa (lane) 8); 108k-HEP-[C]-FVIIa (lane 9) and 157k-HEP-[C]-FVIIa407C (lane 10). (B) SDS-PAGE of sugar conjugated 52k-HEP-[N]-FVIIa. The gel was loaded with HiMark HMW standards (lane 1), ST3Gal3 (lane 2), FVIIa (lane 3), asialic FVIIa (lane 4) and 52k-HEP-[N]-FVIIa (lane 5) ). [N]- represents a factor conjugate in which a heparin precursor is attached to an N-glycan. [C]- represents a factor conjugate in which a heparin precursor is attached to a cysteine residue.

Figure 7: FVIIa clot activity of heparin precursor conjugate and glycoPEGylated FVIIa reference Analysis of the level.

Figure 8: Proteolytic activity of heparin precursor conjugates and glycoPEGylated FVIIa references.

Figure 9: PK results (LOCI) in Sprague Dawley rats. Unmodified FVIIa (2 studies), 13k-HEP-[C]-FVIIa407C, 27k-HEP-[C]-FVIIa407C, 40k-HEP-[C]-FVIIa407C, 52k-HEP-[C]-FVIIa407C, 65k-HEP-[C]-FVIIa407C, 108k-HEP-[C]-FVIIa407C and 157k-HEP-[C]-FVIIa407C, sugar conjugate 52k-HEP-[N]-FVIIa and reference molecule (40kDa-PEG- Comparison of [N]-FVIIa (2 studies) and 40 kDa-PEG-[C]-FVIIa407C). Data were presented as mean ± SD (n = 3-6) in a semi-logarithmic curve. [N]- represents a factor conjugate in which a heparin precursor is attached to an N-glycan. [C]- represents a factor conjugate in which a heparin precursor is attached to a cysteine residue.

Figure 10: PK results (clot activity) in S. Pigerdog rats. Unmodified FVIIa (2 studies), 13k-HEP-[C]-FVIIa407C, 27k-HEP-[C]-FVIIa407C, 40k-HEP-[C]-FVIIa407C, 52k-HEP-[C]-FVIIa407C, 65k-HEP-[C]-FVIIa407C, 108k-HEP-[C]-FVIIa407C and 157k-HEP-[C]-FVIIa407C, sugar conjugate 52k-HEP-[N]-FVIIa and reference molecule (40kDa-PEG- Comparison of [N]-FVIIa (2 studies) and 40 kDa-PEG-[C]-FVIIa407C). Display data in a semi-logarithmic curve. [N]- represents a factor conjugate in which a heparin precursor is attached to an N-glycan. [C]- represents a factor conjugate in which a heparin precursor is attached to a cysteine residue.

Figure 11: Relationship between HEP size and mean residence time (MRT) for various HEP-[C]-FVIIa407C conjugates. The MRT values from the PK study were plotted against the heparin precursor polymer size of the conjugate. The curves indicate non-conjugated FVIIa, 13k-HEP-[C]-FVIIa407C, 27k-HEP-[C]-FVIIa407C, 40k-HEP-[C]-FVIIa407C, 52k-HEP-[C]-FVIIa407C, The values of 65k-HEP-[C]-FVIIa407C, 108k-HEP-[C]-FVIIa407C and 157k-HEP-[C]-FVIIa407C. MRT (LOCI) was calculated by a non-compartmental method using Phoenix WinNonlin 6.0 (Pharsight). [N]- represents a factor conjugate in which a heparin precursor is attached to an N-glycan. [C]- represents a factor conjugate in which a heparin precursor is attached to a cysteine residue.

Figure 12: Functionalization of glycidyl sialic acid cytidine monophosphate (GSC) via a benzaldehyde group. GSC is deuterated by 4-mercaptobenzoic acid and then reacted with heparin precursor (HEP)-amine by reductive amination.

Figure 13: Functionalization of a heparin precursor (HEP) polymer via a benzaldehyde group and subsequent reaction with glycidyl sialic acid cytidine monophosphate (GSC) in a reductive amination reaction.

Figure 14: Functionalization of glycosyl sialic acid cytidine monophosphate (GSC) via a thio group and subsequent reaction with a maleimide functionalized heparin precursor (HEP) polymer.

Figure 15: Heparin precursor (HEP)-glycosyl sialic acid cytidine monophosphate (GSC).

Figure 16: PK results (LOCI) in Spogdogi rats. 2×20K-HEP-[N]-FVIIa; 1×40K-HEP-[N]-FVIIa and 1×40k-PEG-[N]-FVIIa were compared in a semi-logarithmic curve. Data were displayed as mean ± SD (n = 3-6).

Figure 17: PK results (clot activity) in Spogdogi rats. 2×20K-HEP-[N]-FVIIa; 1×40K-HEP-[N]-FVIIa and 1×40k-PEG-[N]-FVIIa were compared in a semi-logarithmic curve.

Figure 18: Reaction scheme for the reaction of desialyl FVIIa glycoprotein with HEP-GSC in the presence of ST3GalIII sialyltransferase.

The present invention relates to the design and use of Factor VII polypeptides that exhibit improved pharmaceutical properties.

In one aspect, the invention relates to the design of a Factor VII polypeptide exhibiting increased in vivo functional half-life, reduced susceptibility to inactivation by plasma inhibitor antithrombin, and enhanced or retained proteolytic activity. use. The inventors of the present invention have found that the specific combination of mutations in human Factor VII and conjugated to half-life extending portions confers the above characteristics. The Factor VII polypeptide of the present invention has an extended functional half-life in the blood, which is therapeutically suitable for situations in which procoagulant activity is required to last for a long period of time. The Factor VII polypeptides comprise a substitution of T293 by Lys (K), Arg (R), Tyr (Y) or Phe (F). In this aspect, the invention relates to a Factor VII polypeptide comprising two or more substitutions relative to the amino acid sequence of human Factor VII (SEQ ID NO: 1), wherein T293 is by Lys (K) , Arg (R), Tyr (Y) or Phe (F) substitution and L288 is replaced by Phe (F), Tyr (Y), Asn (N), Ala (A) or Trp (W). The invention also relates to a Factor VII polypeptide comprising two or more substitutions relative to the amino acid sequence of human Factor VII (SEQ ID NO: 1), wherein T293 is by Lys (K), Arg (R), Tyr (Y) or Phe (F) substitution and W201 substitution by Arg (R), Met (M) or Lys (K). Furthermore, the present invention relates to a Factor VII polypeptide comprising two or more substitutions relative to the amino acid sequence of human Factor VII (SEQ ID NO: 1), wherein T293 is by Lys (K), Arg (R) ), Tyr (Y) or Phe (F) substitution and K337 substitution by Ala (A) or Gly (G). The Factor VII polypeptide of the present invention may further comprise, if desired, replacing Q176 by Lys (K), Arg (R) or Asn (N). The Factor VII polypeptide of the present invention may further comprise replacing Q286 by Asn(N), as needed.

In another aspect, the invention relates to the design and use of a Factor VII polypeptide exhibiting enhanced proteolytic activity. The inventors of the present invention have found that specific mutations in human Factor VII at position L288 and/or W201 confer enhanced proteolytic activity of the Factor VII polypeptide. In this aspect, the invention relates to an amino acid comprising one or more of human factor VII A substituted Factor VII polypeptide of the sequence (SEQ ID NO: 1) wherein L288 is replaced by Phe (F), Tyr (Y), Asn (N), Ala (A) or Trp (W), the restriction is polypeptide Does not have the following substitution pairs: L288N/R290S or L288N/R290T. Further, according to this aspect, the present invention relates to a Factor VII polypeptide comprising one or more substitutions with respect to the amino acid sequence of human Factor VII (SEQ ID NO: 1), characterized in that one of the substitutions is by When Arg(R), Met(M) or Lys(K) replaces W201.

Factor VII

Factor VII (Factor VII) is a glycoprotein mainly produced in the liver. The mature protein consists of 406 amino acid residues defined by SEQ ID NO: 1 (also disclosed, for example, in U.S. Patent No. 4,784,950) and consists of four domains. There is an N-terminal gamma-carboxy glutamic acid (Gla) enrichment domain followed by two epidermal growth factor (EGF)-like domains and a C-terminal trypsin-like serine protease domain. Factor VII circulates in plasma, mainly in the form of single-stranded molecules. Factor VII is activated as Factor VIIa by cleavage between residues Arg152 and Ile153, resulting in a double-stranded protein joined together by disulfide bonds. The light chain contains the Gla and EGF-like domains, while the heavy chain is the protease domain. The specific Glu(E) residues according to SEQ ID NO: 1 in Factor VII, ie, E6, E7, E14, E16, E19, E20, E25, E26, E29 and E35, can be translated for gamma-carboxylation. The coordination of the gamma-carboxy glutamic acid residues in the gla domain for many calcium ions is maintained by maintaining the Gla domain in a configuration that modulates interaction with the phospholipid membrane.

The terms FVII and "Factor VII" herein refer to the uncleaved single-chain enzyme factor VII and the cleavage, double-stranded and thus activated protease factor VIIa. "Factor VII" includes natural dual gene variants of Factor VII that may be present and differ from each other. The human wild-type Factor VII sequence is provided in SEQ ID NO: 1. The term "Factor VII polypeptide" as used herein refers to Factor VII (eg The uncleaved single-chain zymogen polypeptide variants described herein, as well as cleavable, double-stranded and thus activated proteases.

Factor VII and Factor VII polypeptides can be derived from plasma or produced recombinantly using well known methods of production and purification. The extent and location of glycosylation, gamma-carboxylation, and other post-translational modifications may depend on the host cell chosen and its growth conditions.

Factor VII polypeptide

The term "Factor VII" or "FVII" means a Factor VII polypeptide.

The term "Factor VII polypeptide" encompasses both wild-type Factor VII molecules as well as Factor VII variants, Factor VII conjugates, and Factor VII that have been chemically modified. Such variants, conjugates and chemically modified Factor VII may exhibit substantially the same activity as wild type human Factor Vila or relative to wild type human Factor VIIa.

The term "activity" of a Factor VII polypeptide as used herein refers to any activity exhibited by wild-type human Factor VII and includes, but is not limited to, coagulation or coagulation activity, procoagulant activity, effector X activation or factor IX activation. Proteolytic or catalytic activity; ability to bind TF, Factor X or Factor IX; and/or ability to bind to phospholipids. Acceptable assays can be used, for example, by measuring the activity in a test tube or in vivo in a coagulation tube or in vivo. The results of these analyses indicate that the polypeptide exhibits activity that is associated with the activity of the polypeptide in vivo, wherein in vivo activity can be referred to as biological activity. Analysis of the activity of Factor VII polypeptides is known to those skilled in the art. Exemplary assays for assessing FVII polypeptide activity include in vitro proteolytic assays, such as those described in the Examples below.

The term "enhanced or retained activity" as used herein refers to a Factor VIIa polypeptide that exhibits substantially the same or increased activity compared to wild-type human Factor VIIa, eg, i) Substantially the same or increased proteolytic activity compared to recombinant wild-type human Factor VIIa in the presence and/or absence of TF; ii) substantially identical or increased TF affinity compared to recombinant wild-type human Factor VIIa Factor VII polypeptide; iii) a Factor VII polypeptide having substantially the same or increased affinity for activated platelets; or iv) having substantially the same or increased binding affinity to Factor X or Factor IX as compared to recombinant wild-type human Factor VIIa / Ability Factor VII polypeptide. For example, retained activity means that the amount of activity retained is about or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80 of activity compared to wild-type human Factor VIIa. %, 90%, 100% or more. For example, enhanced activity means that the amount of activity retained is about or about 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180 of activity compared to wild-type human Factor VIIa. %, 190%, 200%, 300%, 400%, 500%, 1000%, 3000%, 5000%, 10000%, 30,000% or more.

The term "Factor VII variant" as used herein is intended to indicate a Factor VII having the sequence SEQ ID NO: 1, wherein one or more of the amino acids of the parent protein has been substituted with another naturally occurring amino acid and/or One or more amino acids of the parent protein have been deleted and/or one or more of the amino acids have been inserted into the protein and/or one or more of the amino acids have been added to the parent protein. This addition can occur at the N-terminus or C-terminus or both ends of the parent protein. In a specific example, the variant has at least 95% identity to the sequence of SEQ ID NO:1. In another embodiment, the variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the sequence SEQ ID NO:1. As used herein, any reference to a particular position refers to the corresponding position in SEQ ID NO: 1.

In some embodiments, the Factor VII variant of the invention has an enhanced or retained activity compared to wild-type human Factor VIIa.

The terms used for the amino acid substitution in this specification are as follows. The first letter indicates an amino acid naturally present at a position in SEQ ID NO: 1. Subsequent numbers represent positions in SEQ ID NO: 1. The second letter indicates a different amino acid that replaces the native amino acid. An example is K197A-Factor VII wherein the amino acid at position 197 of SEQ ID NO: 1 is replaced with alanine.

In the context of the present invention, such abbreviations are used in the conventional meaning of the three-letter or one-letter abbreviations of amino acids as indicated below. Unless otherwise indicated, the amino acid referred to herein is an L-amino acid.

Abbreviation for amino acid:

The term "Factor VII conjugate" as used herein is intended to indicate a Factor VII polypeptide wherein one or more of the amino acids and/or one or more of the attached glycan moieties have been chemically and/or enzymatically modified. , for example by alkylation, glycosylation, deuteration, ester formation, disulfide bond formation or guanamine formation.

In some embodiments, a Factor VII conjugate of the invention exhibits substantially the same biological activity as wild-type Factor VII or is enhanced relative to wild-type Factor VII.

Enhanced proteolytic activity

The inventors have surprisingly shown that certain mutant Factor VII polypeptides with residues L288 and W201 exhibit enhanced proteolytic activity.

The Factor VII variant K337A as described in WO 02/22776 has been described to have enhanced proteolytic activity. It has also been described that the Factor VII variants L305V and L305I as described in WO 03/027147 have a higher intrinsic activity.

Proteolytic activity can be determined by any suitable method known in the art as discussed further below.

For example, enhanced proteolytic activity means that the amount of activity retained is about or about 110%, 120%, 130%, 140%, 150%, 160%, 170% of activity compared to wild-type human Factor VIIa. 180%, 190%, 200%, 300%, 400%, 500%, 1000%, 3000%, 5000%, 10000%, 30,000% or more.

Resistance to half-life of inactivation by plasma inhibitors

In addition to in vivo clearance, in vivo functional half-life is important for the time when a compound is "therapeutic" in the body. The circulating half-life of recombinant human wild-type Factor VIIa is approximately 2.3 hours ("Summary Basis for Approval for NovoSeven©", FDA reference 96 0597).

The term "in vivo functional half-life" is used in its ordinary meaning, that is, the time required for the biological activity of the remaining Factor VII polypeptide remaining in the body/target organ to be reduced by 50% at the end, or the activity of the Factor VII polypeptide is 50 of its initial value. % of the time. Alternative terms for in vivo half-life include Terminal half-life, plasma half-life, circulating half-life, and elimination half-life. Suitable methods known in the art, such as those described in Example 17, and those described in Introduction to Pharmacokinetics and Pharmacodynamics: The Quantitative Basis of Drug Therapy (Thomas N. Tozer, Malcolm Rowland) The method measures half-life.

The term "increase" as used with respect to in vivo functional half-life or plasma half-life is used to indicate that the relative half-life of the polypeptide is increased relative to the half-life of a reference molecule such as wild-type human Factor VIIa as determined under comparable conditions.

In some embodiments, the Factor VII polypeptide of the invention exhibits an increased in vivo functional half-life relative to wild-type human Factor VIIa. For example, the associated half-life can be increased by at least about 25%, such as at least about 50%, such as at least about 100%, 150%, 200%, 500%, 1000%, 3000%, 5000%, 10 000%, 30 000%. Or more than 30 000%.

Despite a detailed understanding of the biochemical and pathophysiology of the coagulation cascade, the underlying mechanism of self-circulation to clear individual clotting factors remains largely unknown. A significant difference in the circulating half-life of factor VII and its activated form factor VIIa compared to the other zymogen and enzymatic forms of the vitamin K-dependent protein indicates a specific and unique clearance mechanism for factor VIIa. Two types of pathways appear to be operable in the clearance factor VIIa - one leading to the elimination of intact proteins and the other mediated by plasma inhibitors and leading to inactivation of proteolysis.

Antithrombin III (antithrombin, AT) is a rich plasma inhibitor and targets most proteases of the coagulation system including Factor VIIa. It is present in plasma at a micromolar concentration and is a serine protease inhibitor of the family of serine protease inhibitors that irreversibly binds to the target protease by suicidal stressor mechanisms and renders it inactive. Inhibition by antithrombin appears to constitute the primary clearance pathway for recombinant Factor VIIa in vivo following intravenous administration. In a recent study of the pharmacokinetics of recombinant Factor VIIa in hemophilia patients, approximately 60% of the total clearance is attributable to this pathway (Agerso et al. (2011) J Thromb Haemost , 9, 333-338).

In some embodiments, the Factor VII polypeptide of the invention exhibits resistance to an increase in inactivation by an endogenous plasma inhibitor, in particular an antithrombin, relative to wild-type human Factor VIIa.

The inventors of the present invention have found that by combining the two sets of mutations mentioned above, i.e., mutations conferring increased AT resistance and mutations conferring enhanced proteolytic activity, an increase or retention activity is achieved while maintaining The inhibitor is not activated with high resistance. That is, the Factor VII polypeptide of the present invention comprising a combination of mutations exhibits increased resistance to anti-thrombin inactivation and substantially retained proteolytic activity. When the Factor VII polypeptide of the present invention is conjugated to one or more half-life extending moieties, an unexpectedly improved effect on prolonged half-life is achieved. In view of these characteristics, the conjugated Factor VII polypeptides of the present invention exhibit an increased circulating half-life while maintaining pharmaceutically acceptable proteolytic activity. Thus, a lower dose of the conjugated Factor VII polypeptide may be desirable to achieve a functionally sufficient concentration at the site of action and thus will likely be administered to a lower dose of a subject having a bleeding event or requiring an enhanced normal hemostatic system. And / or at a lower frequency.

Other modifications

The Factor VII polypeptides of the invention may comprise additional modifications, in particular other modifications that confer additional advantageous properties to the Factor VII polypeptide. Thus, in addition to the amino acid substitutions mentioned above, the Factor VII polypeptides of the invention may, for example, comprise other amino acid modifications, such as another amino acid substitution. In one such example, the Factor VII polypeptide of the invention has another mutation or addition selected from the group consisting of R396C, Q250C and 407C as described, for example, in WO2002077218.

The Factor VII polypeptides of the invention may or may not be included in other modifications of the sequence of the Factor VII polypeptide. Other modifications include, but are not limited to, additions to the carbohydrate moiety, additions to the half-life extending moiety, such as additions to the PEG moiety, the Fc domain, and the like. For example, such other modifications can be made to increase the stability or half-life of the Factor VII polypeptide.

Half-life extension or group

The term "half-life extending moiety" is used interchangeably herein and is understood to mean one or more linked to a chain functional group such as -SH, -OH, -COOH, -CONH2, -NH2 or a plurality of amino acid sites. A chemical group that increases the in vivo functional half-life of such proteins/polypeptides when conjugated/coupled to a protein/polypeptide, and/or one or more N- and/or O-glycan structures.

The in vivo functional half-life can be determined by any suitable method known in the art as further discussed below (Example 17).

Examples of half-life extending moieties include: biocompatible fatty acids and derivatives thereof, such as hydroxyethyl starch (HES) hydroxyalkyl starch (HAS), polyethylene glycol (PEG), poly (Glyx-Sery) n ( HAP), hyaluronic acid (HA), heparin precursor polymer (HEP), phosphocholine-based polymer (PC polymer), Fleximer, polydextrose, poly-sialic acid (PSA), Fc domain, transferrin, Albumin, elastin-like peptide (ELP), XTEN polymer, PAS polymer, PA polymer, albumin binding peptide, CTP peptide, FcRn binding peptide, and any combination thereof.

In a particular embodiment of particular interest, the Factor VII polypeptide of the invention is coupled to one or more half-life extending moieties.

In one embodiment, the cysteine conjugated Factor VII polypeptide of the invention has one or more hydrophobic half-life extending moieties that are conjugated to the sulfhydryl group of the cysteine introduced into the Factor VII polypeptide. In addition, it is possible to link the half-life extending moiety to other amino acid residues.

In a specific embodiment, the Factor VII polypeptide of the invention is a disulfide linked to a tissue factor, as described, for example, in WO2007115953.

In another embodiment, the Factor VII polypeptide of the invention is a Factor VIIa variant having increased platelet affinity.

Heparin precursor conjugate

The Factor VII polypeptide heparin precursor conjugate according to the invention may have one or more heparin precursor polymers linked to any portion of the FVII polypeptide, including any amino acid residues or carbohydrate moieties of the Factor VII polypeptide ( HEP) molecule. Examples of such conjugates are provided in WO 2014/060397, which is incorporated herein by reference. Chemical and/or enzymatic methods can be used to conjugate the HEP to the glycan on the Factor VII polypeptide. Examples of enzymatic conjugation methods are described, for example, in WO03031464. The glycan may be naturally occurring or may be carried out using methods well known in the art, for example by introducing an N-glycosylation motif (NXT/S, where X is any naturally occurring amino acid) in Factor VII It is engineered in the amino acid sequence.

A "cysteine-HEP Factor VII polypeptide conjugate" according to the present invention has one or more HEP molecules conjugated to a sulfhydryl group of a cysteine residue present in or introduced into a FVII polypeptide.

In an interesting embodiment of the invention, the Factor VII polypeptide is coupled to the HEP polymer. In one embodiment, the HEP polymer coupled to the Factor VII polypeptide has a structure selected from the group consisting of 13-65 kDa, 13-55 kDa, 25-55 kDa, 25-50 kDa, 25-45 kDa, 30-45 kDa, 36-44 kDa, and 38-42 kDa. Molecular weight within the range or molecular weight of 40 kDa.

In an interesting embodiment of the invention, the Factor VII polypeptide is coupled to the HEP polymer on the N-glycan of the Factor VII polypeptide.

In another embodiment of the invention, two HEP polymers are coupled to the same Factor VII polypeptide via an N-glycan. In this particular example, each of the HEP polymers coupled to the Factor VII polypeptide has a moiety selected from the group consisting of 13-65 kDa, 13-55 kDa, 25-55 kDa, 25-50 kDa, 25-45 kDa, 30-45 kDa, 36- Molecular weight in the range of 44 kDa and 38-42 kDa or molecular weight of 40 kDa. Preferably, the polymers have the same molecular weight.

In a specific embodiment, two 20 kDa HEP polymers are coupled to the same Factor VII polypeptide via an N-glycan of a Factor VII polypeptide.

In a specific embodiment, two 30 kDa HEP polymers are coupled to the same Factor VII polypeptide via an N-glycan of a Factor VII polypeptide.

In a specific embodiment, two 40 kDa HEP polymers are coupled to the same Factor VII polypeptide via an N-glycan of a Factor VII polypeptide.

Heparin precursor polymer

The heparin precursor (HEP) is a natural sugar polymer comprising a repeating sequence of (-GlcUA-β 1,4-GlcNAc-α 1,4-) (see Figure 5A). It belongs to the family of glycosaminoglycans and is a negatively charged polymer at physiological pH. It can be found in the envelope of certain bacteria but it is also found in higher vertebrates, which act as precursors to the natural polymer heparin and acesulfate heparin in these vertebrates. Although not confirmed in detail, the salt of heparin was degraded in lysosomes. Injections of 100 kDa heparin precursor polymer labeled with Bolton-Hunter reagent have shown that heparin precursors are secreted as smaller fragments in body fluid/body waste (US 2010/0036001).

Heparin precursor polymers and methods of making such polymers are described in US 2010/0036001, the contents of which are incorporated herein by reference. According to the present invention, the heparin precursor polymer can be any heparin precursor polymer described or disclosed in US 2010/0036001.

For use in the present invention, the heparin precursor polymer can be produced by any suitable method, such as any of the methods described in US 2010/0036001 or US 2008/0109236. Heparin precursors can be produced using enzymes derived from bacteria. For example, the heparin precursor synthase PmHS1 of Pasteurella multocida polymerizes the heparin precursor sugar chain by transferring both GlcUA and GlcNAc. Escherichia coli K5 enzyme KfiA (α GlcNAc transferase) and KfiC (β GlcUA transferase) may also form a disaccharide repeat of the heparin precursor.

The heparin precursor polymer used in the present invention is typically a polymer of the formula (-GlcUA-β 1,4-GlcNAc-α 1,4-) _n .

The size of the heparin precursor polymer can be defined by the number n of repeats in this formula. The number n of such repeats can be, for example, from 2 to about 5,000. The number of repeating sequences may be, for example, 50 to 2000 units, 100 to 1000 units, or 200 to 700 units. The number of repeating sequences can be from 200 to 250 units, from 500 to 550 units, or from 350 to 400 units. Any of the lower limits of these ranges can be combined with any upper limit of these ranges to form a suitable range of unit numbers in the heparin precursor polymer.

The size of the heparin precursor polymer can be defined by its molecular weight. The molecular weight can be the average molecular weight of a population of heparin precursor polymer molecules, such as a weight average molecular mass.

The molecular weight values as described herein with respect to the size of the heparin precursor polymer may not be exactly the listed sizes in practice. Some variations are expected due to batch-to-batch variations during heparin precursor polymer production. In order to accommodate batch-to-batch variations, it should be understood that approximately +/- 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the target HEP polymer size should be expected. Or a 1% change. For example, a 40 kDa HEP polymer size represents 40 kDa +/- 10%, such as 40 kDa It may for example mean 38.8 kDa, 41.5 kDa or 43.8 kDa in practice.

The heparin precursor polymer may have a molecular weight of, for example, 500 Da to 1,000 kDa. The molecular weight of the polymer may range from 500 Da to 650 kDa, from 5 kDa to 750 kDa, from 10 kDa to 500 kDa, from 15 kDa to 550 kDa, or from 25 kDa to 250 kDa.

The molecular weight can be selected at a particular level within these ranges to achieve a suitable balance between the activity of the Factor VII polypeptide and the half-life or average residence time of the conjugate. For example, the molecular weight of the polymer can range from 15-25 kDa, 25-35 kDa, 35-45 kDa, 45-55 kDa, 55-65 kDa, or 65-75 kDa.

A more specific range of molecular weights can be selected. For example, the molecular weight can range from 20 kDa to 35 kDa, such as from 22 kDa to 32 kDa, such as from 25 kDa to 30 kDa, such as about 27 kDa. The molecular weight may range from 35 to 65 kDa, such as from 40 kDa to 60 kDa, such as from 47 kDa to 57 kDa, such as from 50 kDa to 55 kDa, such as about 52 kDa. The molecular weight can be from 50 to 75 kDa, such as from 60 to 70 kDa, such as from 63 to 67 kDa, such as about 65 kDa.

In a particularly interesting embodiment, the heparin precursor polymer of the Factor VII conjugate of the invention has a composition selected from the group consisting of 13-65 kDa, 13-55 kDa, 25-55 kDa, 25-50 kDa, 25-45 kDa, 30. Sizes in the range of -45kDa and 38-42kDa.

Any of the lower limits of such ranges of molecular weight may be combined with any upper limit of these ranges to form a suitable range of molecular weights of the heparin precursor polymer according to the present invention.

The heparin precursor polymer can have a narrow particle size distribution (i.e., monodisperse) or a broad particle size distribution (i.e., polydisperse). The polydispersity (PDI) level can be numerically represented based on the formula Mw/Mn, where Mw = weight average molecular mass and Mn = number average molecular weight. An ideal monodisperse polymer using this equation has a polydispersity value of one. Preferably, the heparin precursor polymer used in the present invention is Monodisperse. The polymer may thus have a dispersibility of about 1 and a polydispersity of less than 1.25, preferably less than 1.20, preferably less than 1.15, preferably less than 1.10, preferably less than 1.09, preferably less than 1.08, preferably less than 1.07. It is preferably less than 1.06, preferably less than 1.05.

The molecular weight particle size distribution of the heparin precursor was measured by comparison with a monodisperse particle size standard (HA Lo-Ladder, Hyalose LLC) which can be carried out on an agarose gel.

Alternatively, the particle size distribution of the heparin precursor polymer can be determined by high performance size exclusion chromatography - multi-angle laser light scattering (SEC-MALLS). This method can be used to evaluate the molecular weight and polydispersity of heparin precursor polymers.

The polymer size can be adjusted in an enzymatic production process. By controlling the molar ratio of the heparin precursor acceptor chain to the UDP sugar, it is possible to select the desired final heparin precursor polymer size.

The heparin precursor polymer can further comprise a reactive group to allow it to be linked to a Factor VII polypeptide. Suitable reactive groups can be, for example, aldehydes, alkynes, ketones, maleimide, thiols, azides, amines, hydrazines, hydroxylamines, carbonates, chelating agents, or any combination thereof. For example, Figure 5B illustrates a heparin precursor polymer comprising a maleimide group.

As stated in the examples, a maleic imide having a defined size can be prepared by using an enzymatic (PmHS1) polymerization of two molar nucleotides UDP-GlcNAc and UDP-GlcUA. Aldehyde functionalized heparin precursor polymer. Initiator trisaccharide (GlcUA-GlcNAc-GlcUA) NH 2 can be used to initiate the reaction, and the polymerization was run until the sugar nucleotide Construction block depleted. The terminal amine (derived from the primer) can then be functionalized to conjugate to a free caspase by a suitable reactive group such as a reactive group as described above, such as a maleimide functional group. The amine acid or aldehyde is reductively aminated to an amine group. The size of the heparin precursor polymer can be predetermined by the change in the stoichiometric amount of the sugar nucleotide: primer. This technique is described in detail in US 2010/0036001.

The reactive group may be present in the reducing or non-reducing end or in the entire sugar chain. When a heparin precursor polymer is conjugated to a polypeptide, it is preferred that only one such reactive group is present.

Method for preparing FVII-HEP conjugate

For example, WO 03/031464 describes a method of remodeling a glycan structure of a polypeptide such as a Factor VII or Factor Vila polypeptide and a method of adding a modifying group such as a water soluble polymer to the polypeptide. These methods can be used to attach a heparin precursor polymer to a Factor VII polypeptide according to the invention.

As stated in the examples, the Factor VII polypeptide can be conjugated to its glycan moiety using a sialyltransferase. To initiate this method, the HEP polymer first needs to be attached to sialic acid cytidine monophosphate. Glycosyl sialic acid cytidine monophosphate (GSC) is a suitable starting point for this chemical reaction, but other sialic acid cytidine monophosphates or fragments thereof can be used. The examples set forth a method of covalently linking a HEP polymer to a GSC molecule. By covalent attachment, a HEP-GSC (HEP conjugated glycosidyl sialic acid cytidine monophosphate) molecule that can be transferred to the glycan moiety of FVIIa is created.

Once the Factor VII-heparin precursor conjugate has been produced, it can be purified. For example, purification can include affinity chromatography using a fixed mAb against a Factor VII polypeptide, such as a mAb against a calcified gla-domain on FVIIa. In this affinity chromatography, the non-conjugated HEP polymer can be removed by a bottom wash of the column. FVII can be released from the column by releasing FVII from the antibody. For example, when an antibody is specific for a calcified gla-domain, release from the column can be achieved by washing with a buffer containing EDTA.

Size exclusion chromatography can be used to isolate Factor VII-heparin precursor conjugates with non-conjugated Factor VII.

The pure conjugate can be concentrated by ultrafiltration.

The final concentration of the Factor VII-heparin precursor conjugate produced by the production process can be quantified by HPLC, such as HPLC quantification, such as FVII light chain.

In connection with the present invention, it is shown that it is possible to attach a carbohydrate polymer such as HEP to a sulfur-modified GSC molecule via a maleimide group and transfer the reagent to the intact sugar on the glycoprotein by means of a sialyltransferase Base, thereby producing a bond containing a cyclic amber quinone imine group.

However, the amber imine-based bond can undergo hydrolysis opening and ringing when the conjugate is stored in an aqueous solution for a long time (Bioconjugation Techniques, GTHermanson, Academic Press, 3rd edition 2013, p. 309) and although the bond can remain intact, The ring opening reaction will add undesired heterogeneities in the form of structural isomers and stereoisomers to the final conjugate.

Following the above, preferably such that the 1) glycan protein glycan residue is retained in intact form, and 2) the half-life is extended in a manner that there is no heterogeneity in the linker portion between the intact glycosyl residue and the half-life extending portion Partially linked to glycoproteins.

There is a need in the art to conjugate two compounds, such as a method of conjugated to a protein or proteoglycan, such as a half-life extension of HEP, wherein the compounds are linked to obtain a stable and isomer-free conjugate.

In one aspect, the invention provides a stable and isomer-free linker for conjugated HEP to FVII based on glycosidic sialic acid cytidine monophosphate (GSC). The GSC starting material (Dufner, G. Eur. J. Org. Chem. 2000, 1467-1482) used in the present invention can be chemically synthesized or it can be obtained by a chemical enzyme route as described in WO07056191. The GSC structure is shown below:

In a specific example, a conjugate according to the invention comprises a linker comprising:

- Also referred to hereinafter as a sub-linker or sub-bond - which links HEP-amine and GSC in one of the following ways:

The highlighted 4-methylbenzimidyl linker thus constitutes a portion of the fully linked structure of the linked half-life extending moiety to the target protein. Sub-linkers are therefore compared to Such as a stable structure based on the substitution of an amber quinone imine linker (prepared from the reaction of maleimide with a sulfhydryl group), since the latter type of cyclic bond has been stored in the conjugate for a long time. The tendency to undergo hydrolysis and ring opening in aqueous solution (Bioconjugation Techniques, GTHermanson, Academic Press, 3rd edition 2013, p. 309). Although the bond in this condition (eg, between HEP and sialic acid on the glycoprotein) remains intact, the ring opening reaction adds heterogeneity in the form of structural isomers and stereoisomers to the final total. Yoke composition.

An advantage associated with the conjugates according to the invention is therefore a significant reduction in the tendency to obtain homogeneous compositions, i.e., isomer formation due to linker structure and stability. Another advantage is that the linkers and conjugates according to the invention can be produced in a simple manner, preferably in a one-step process.

Isomers are undesirable because such isomers can produce heterogeneous products and increase the risk of unwanted immune responses in the human body.

The 4-methylbenzhydryl-based bond used between HEP and GSC in the present invention cannot form a stereoisomer or a structural isomer. The HEP polymer can be prepared by simultaneous enzymatic polymerization as previously mentioned (US 20100036001). This method uses heparin synthase I (PmHS1) from Pasteurella multocida, which can be expressed in E. coli as a maltose binding protein fusion construct. Purified MBP-PmHS1 is capable of producing a monodisperse polymer in a synchronized, stoichiometrically controlled reaction when it is added to a molar mixture of sugar nucleotides (GlcNAc-UDP and GlcUA-UDP). A trisaccharide initiator (GlcUA-GlcNAc-GlcUA) was used to initiate the reaction, and the polymer length was determined by the primer: sugar nucleotide ratio. The polymerization will proceed until about 90% of the sugar nucleotides are consumed. The polymer and reaction mixture were separated by anion exchange chromatography and then lyophilized to a stable powder.

A method of preparing a functional HEP polymer is described in US 20100036001. Examples thereof include aldehyde, amine and maleimide functionalized HEP reagents. US 20100036001 is incorporated herein by reference in its entirety as if fully incorporated herein. A series of other functionally modified HEP derivatives are available using similar chemical methods. The HEP polymer used in certain embodiments of the present invention was originally produced by treating a primary amine at the reducing end according to the method described in US20100036001.

The amine functionalized HEP polymer prepared according to US20100036001 (i.e., HEP with amine treatment) can be converted to HEP-benzaldehyde by reaction with N-ammonium imidate of 4-methylmercaptobenzoic acid and then by reducing amine The reaction is coupled to the glycine amide group of GSC. The resulting HEP-GSC product can then be enzymatically conjugated to a glycoprotein using a sialyltransferase.

For example, the amine treatment on HEP can be converted to a benzaldehyde functional group by reaction with N-ammonium imidate of 4-methylmercaptobenzoic acid according to the following scheme:

The conversion of the HEP amine (1) to the 4-methylmercaptobenzamide compound (2) can be carried out in the above scheme by reacting with a thiol-activated form of 4-mercaptobenzoic acid.

The N-succinimide group can be selected as the sulfhydryl activating group, but a variety of other sulfhydryl activating groups are known to those skilled in the art. Non-limiting examples include 1-hydroxy-7-azabenzotriazole-, 1-hydroxy-benzotriazole-, pentafluorophenyl ester, as known from peptide chemistry.

The HEP reagent modified with a benzaldehyde functional group can remain stable for a long period of time when stored in a dry form (-80 ° C). Alternatively, the benzaldehyde moiety can be attached to the GSC compound, thereby producing a GSC-benzaldehyde compound suitable for conjugation to an amine functionalized half-life extension. This synthetic route is depicted in Figure 12.

For example, GSC can be reacted with N-succinimide 4-methylmercaptobenzoate under pH neutral conditions to provide a GSC compound containing a reactive aldehyde group (see, for example, WO2011101267). The aldehyde-derived GSC compound (GSC-benzaldehyde) can then be reacted with a HEP-amine and a reducing agent to form a HEP-GSC reagent.

The above reaction can be reversed such that the HEP-amine is first reacted with N-succinimide 4-methylmercaptobenzoate to form an aldehyde-derived HEP-polymer which is then directly reacted with GSC in the presence of a reducing agent. In practice, this eliminates the lengthy chromatographic processing of GSC-CHO. This synthetic route is depicted in Figure 13.

Thus, in one embodiment of the invention, HEP-benzaldehyde is coupled to the GSC by reductive amination.

Reductive amination is a two-step reaction which is carried out by initially forming an imine (also known as Schiff base) between the aldehyde component and the amine component (in the embodiment of the invention, the glycidyl amino group of GSC) (Schiff base)). The imine is subsequently reduced to the amine in a second step. A reducing agent is selected to selectively reduce the formed imine to an amine derivative.

A variety of suitable reducing agents are available to those skilled in the art. Non-limiting example Including sodium cyanoborohydride (NaBH3CN), sodium borohydride (NaBH4), pyridine borane complex (BH3:Py), dimethyl sulfide borane complex (Me2S: BH3) and pyrithione Compound.

Although it is possible to reductively aminated to the reducing end of the carbohydrate (e.g., to the reducing end of the HEP polymer), it is generally described as a slow and inefficient reaction (JC. Gildersleeve, Bioconjug Chem. July 2008; 19(7) :1485-1490). Side reactions such as the Amadori reaction are undesirable in the context of the present invention, in which it is also possible that the initially formed imine rearrangement to a ketoamine is possible and will result in the discussion as previously discussed. Heterogeneity.

Aromatic aldehyde (such as benzaldehyde) derivatives do not form such rearranged reactants because the imine cannot be enolized and also lack the desired adjacent hydroxyl groups typically found in carbohydrate derived imines. Aromatic aldehyde (such as benzaldehyde) derivatives are therefore particularly suitable for use in the reductive amination of HEP-GSC reagents which are free of isomers.

Excess GSC and reducing agent are used as needed to drive the reductive amination chemical reaction to complete quickly. When the reaction is complete, excess (non-reactive) GSC reagents and other small molecule components (such as excess reducing agent) can then be removed by dialysis, tangential flow filtration, or size exclusion chromatography.

The natural receptors of sialyltransferase are both charged and highly hydrophilic polyfunctional molecules. In addition, it is unstable for a long time in the solution, especially when the pH is lower than 6.0. At this low pH, the CMP activating group required for mass transfer is absent due to acid catalyzed hydrolysis of the phosphodiester. Selective modification and separation of GSC and Sia-CMP derivatives therefore requires careful pH control and a fast and efficient separation process to avoid CMP-hydrolysis.

In the present invention, the longer half-life extension is conjugated to GSC using a reductive amination chemical reaction. The half-life extension of the modification of aromatic aldehydes (such as benzaldehyde) has been found for this type The modification is optimal because it can effectively react with GSC under reductive amination conditions.

Since GSC can undergo hydrolysis in acidic media, it is important to maintain a near neutral or slightly alkaline environment during coupling to HEP-benzaldehyde. Both HEP polymers and GSC are highly water soluble and aqueous buffer systems and are therefore preferred for maintaining the pH near the neutral level. A wide variety of organic and inorganic buffers can be used, however, the buffer component should preferably not react under reductive amination conditions. This does not include, for example, organic buffer systems containing primary and minor secondary amine groups. Those skilled in the art will know what buffer is suitable and what is not suitable. Some examples of suitable buffers are shown in Table 1 below:

By applying this method, a simple method for minimizing the probability of hydrolysis of the CMP activating group can efficiently prepare and isolate a GSC reagent having a half-life extended partial modification and having a stable bond containing no isomer.

By reacting any of these compounds with each other, a HEP-GSC conjugate comprising a 4-methylbenzimidyl linker moiety can be created.

GSC can also be reacted with thiobutyrolactone to produce a thiol-modified GSC molecule (GSC-SH). As shown in the present invention, the reagents can be reacted with a maleimide functionalized HEP polymer to form a HEP-GSC reagent. This synthetic route is depicted in Figure 15. The resulting product has a linkage structure comprising amber imine.

However, the (sub) bond based on amber imine can undergo hydrolysis opening and ringing especially when the modified GSC reagent is stored in an aqueous solution for a long time and although the bond can remain intact, the ring opening reaction will be added as a structural isomer and a stereoisomer. Undesired heterogeneity of the form of the structure.

Sugar conjugation method

Conjugation of the HEP-GSC conjugate to the (poly)-peptide can be carried out via a glycan present on the residue in the (poly)-peptide main structure. The conjugate of this form is also known as sugar conjugation.

For many years, methods based on sialyltransferase have been shown to be modest and highly selective for modifying N-glycans or O-glycans on coagulation factors such as factor FVII.

The conjugation via a glycan is an attachment with less bioactivity interference than a cysteine acid alkylation, lysine deuteration, and a similar conjugation conjugate method involving an amino acid in the protein backbone. An attractive way to attach a larger structure of a polymer such as a protein/peptide fragment to a biologically active protein. This is due to the fact that the glycans are highly hydrophilic and generally tend to be remote from the surface of the protein and oriented in solution such that the binding surface important for protein activity is free of glycans.

The glycan may be naturally occurring or it may be inserted, for example, by insertion of an N-linked glycan using methods well known in the art.

GSC is a sialic acid derivative that can be transferred to a glycoprotein by using a sialyltransferase. It can be selectively modified by a substituent such as PEG on a glycidylamine group and is still enzymatically transferred to a glycoprotein by using a sialyltransferase. Can be efficiently prepared on a large scale by enzymatic methods GSC (WO07056191).

Sialyltransferase

Sialyltransferases are a class of glycosyltransferases that transfer sialic acid from a naturally activated sialic acid (Sia)-CMP (cytidine monophosphate) compound to, for example, a galactosyl moiety on a protein. Many sialyltransferases (ST3GalIII, ST3GalI, ST6GalNAcI) are capable of transferring sialic acid-CMP (Sia-CMP) derivatives that have been modified, in particular with a large population of C5 acetylamine groups, such as 40 kDa PEG (WO03031464). A broad, but non-limiting list of related sialyltransferases that can be used in the present invention is disclosed in WO2006094810, which is incorporated herein by reference in its entirety.

In one aspect of the invention, the terminal sialic acid on the glycoprotein can be removed by treatment with a sialidase to provide a asialoglyl glycoprotein. The asialoglyl glycoprotein and the GSC modified by the half-life extension will act together as a substrate for the sialyltransferase. The reaction product is a glycoprotein conjugate having a half-life extending moiety linked via an intact glycosyl linkage group (in this case, a complete sialic acid linker group). The reaction scheme is shown in Figure 18, in which the asialo-FVIIa glycoprotein is reacted with HEP-GSC in the presence of a sialyltransferase.

The term "sialic acid" refers to any member of the 9-carboxycarboxylated sugar family. The most common member of the sialic acid family is N-ethyl thioglycolic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycerol-D-galactononulopyranos-1-keto acid (usually abbreviated as Neu5Ac, NeuAc, NeuNAc or NANA.) The second member of this family is N-hydroxyethyl-neuraminic acid (Neu5Gc or NeuGc) in which the N-ethenyl group of NeuNAc is hydroxylated. The member of the sialic acid family is 2-keto-3-deoxy-nonolosonic acid (KDN) (Nadano et al. (1986) J. Biol. Chem. 261: 11550-11557; Kanamori et al., J .Biol. Chem. 265:21811-21819 (1990)). Also included A 9-substituted sialic acid such as 9-O-C1-C6 decyl-Neu5Ac, such as 9-O-chyle Neu5Ac or 9-O-ethinyl-Neu5Ac. The synthesis and use of a sialic acid compound in a sialylation procedure is disclosed in International Application No. WO 92/16640, issued Oct. 1, 1992.

The term "sialic acid derivative" refers to a sialic acid as defined above modified by one or more chemical moieties. The modifying group can be, for example, an alkyl group (such as a methyl group), an azide group, and a fluoro group, or can function as a functional group such as an amine group or a thiol group attached to a handle of another chemical moiety. Examples include 9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. The term also encompasses sialic acid which lacks one or more of the functional groups such as a carboxyl group or one or more of the hydroxyl groups. The term also encompasses derivatives in which the carboxyl group is replaced by a carbenamine or ester group. The term also refers to sialic acid in which one or more hydroxyl groups have been oxidized to a carbonyl group. Further, the term refers to sialic acid lacking a C9 carbon atom or a C9-C8 carbon chain, for example, after oxidation treatment with periodate.

Glycosyl sialic acid is a sialic acid derivative according to the above definition, wherein the N-acetyl group of NeuNAc is replaced by a glycidinyl group also known as an aminoethyl group. Glycosyl sialic acid can be represented by the following structure:

The term "CMP activation" sialic acid or sialic acid derivative refers to a sugar nucleotide containing a sialic acid moiety and cytosine monophosphate (CMP).

In the present specification, the term "glycosyl sialo cytidine monophosphate" is used to describe GSC and is synonymous with the substitution of the same CMP-activated glycosyl sialic acid. Alternative nomenclature includes CMP-5'-glycosyl sialic acid, cytidine-5'-monophosphonium-N-glycidyl tranexylamine, cytidine-5'-monophosphonium-N-gan Aminyl sialic acid.

The term "intact glycosyl linking group" refers to a linking group derived from a glycosyl moiety, wherein a sugar monomer is inserted between the polypeptides and covalently attached to the polypeptide and the HEP moiety is not formed during conjugate formation, for example by Degradation of sodium metaperiodate, such as oxidation. An "intact glycosyl-bonding group" can be derived from a naturally occurring oligosaccharide by the addition of a glycosyl unit or removal of one or more glycosyl units from a parent sugar structure.

The term "desialyl glycoprotein" is intended to include exposure of at least one from galactose or N-ethylidene groups, for example by treatment with sialidase or by chemical treatment to remove one or more terminal sialic acid residues. A glycoprotein of a galactose or N-ethylmercapto galactosamine residue ("exposed galactose residue") under the galactose.

The dotted line in the structural formula represents an open valence bond (ie, a bond between a linking structure and other chemical moieties).

Pegylated derivative

A "PEGylated Factor VII polypeptide variant/derivative" according to the invention may have one or more attachments to any portion of a FVII polypeptide, including any amino acid residues or carbohydrate moieties of a Factor VII polypeptide. Polyethylene glycol (PEG) molecule. Chemical and/or enzymatic methods can be used to conjugate PEG or other half-life extending moiety to the glycan on the Factor VII polypeptide. Examples of enzymatic conjugation methods are described, for example, in WO03031464. The glycan may be naturally occurring or it may be engineered as described above for the HEP conjugate. A "cysteine-pegylated Factor VII polypeptide variant" according to the invention has one or more PEG molecules conjugated to a sulfhydryl group of a cysteine residue present in or introduced into a FVII polypeptide .

Fusion protein

A fusion protein is a protein created by in-frame ligation of two or more DNA sequences that originally encoded an independent protein or peptide or a fragment thereof. Translation of the fusion protein DNA sequence will result in a single protein sequence that can have functional properties derived from each of the original protein or peptide. The DNA sequence encoding the fusion protein can be artificially created by standard molecular biology methods such as overlapping PCR or DNA ligation and the stop codon in the first 5'-end DNA sequence is excluded, while terminating in the 3'-end DNA sequence is retained Codons are combined. The resulting fusion protein DNA sequence can be inserted into a suitable expression vector that supports the expression of the heterologous fusion protein in a standard host organism such as a bacterial, yeast, fungal, insect cell or mammalian cell.

The fusion protein may contain a linker or spacer peptide sequence that separates the protein or peptide portion of the defined fusion protein.

In an interesting embodiment of the invention, the Factor VII polypeptide is a fusion protein comprising a Factor VII polypeptide and a fusion partner protein/peptide, such as an Fc domain or albumin.

Fc fusion protein

The term "Fc fusion protein" is intended herein to encompass a Factor VII polypeptide of the invention fused to an Fc domain that can be derived from any antibody isotype. Due to the relatively long circulating half-life of IgG antibodies, the IgG Fc domain will generally be preferred. The Fc domain can be further modified to modulate certain effector functions, such as complement binding and/or binding to certain Fc receptors. Fusion of a FVII polypeptide to an Fc domain having the ability to bind to an FcRn receptor will generally result in a circulating half-life of the fusion protein that has a longer half-life than the wt FVII polypeptide. Mutations at positions 234, 235 and 237 in the Fc Fc domain will generally result in reduced binding to the Fc gamma RI receptor and may also result in reduced binding to the Fc gamma RIIa and Fc gamma RIII receptors. These mutations do not alter binding to the FcRn receptor, which is recirculated by intracellular endocytosis The diameter promotes long cycle half-life. Preferably, the modified IgG Fc domain of the fusion protein according to the invention comprises one or more of the following mutations: mutants which result in reduced affinity to certain Fc receptors, respectively (L234A, L235E and G237A) And mutants (A330S and P331S) that result in a decrease in C1q-mediated complement fixation. Alternatively, the Fc domain can be an IgG4 Fc domain, preferably comprising the S241P/S228P mutation.

Production of Factor VII polypeptide

The Factor VII polypeptides of the invention can be produced recombinantly using well known methods of production and purification; some examples of such methods are described below; other examples of methods of production and purification are described inter alia in WO2007/031559.

In one aspect, the invention relates to a method of producing a Factor VII polypeptide. The Factor VII polypeptides described herein can be produced by means of recombinant nucleic acid techniques. In general, the human wild-type Factor VII nucleic acid sequence is modified to encode the desired protein. This modified sequence is then inserted into an expression vector which in turn is transformed or transfected into a host cell. Higher eukaryotic cells, specifically cultured mammalian cells, are preferred as host cells.

In another aspect, the invention relates to a transgenic animal comprising and expressing a polynucleotide construct.

The complete nucleotide and amino acid sequence of human wild-type Factor VII is known (see U.S. Patent 4,784,950, which describes the selection and expression of recombinant human Factor VII).

Amino acid sequence changes can be achieved by a variety of known techniques. The nucleic acid sequence can be modified by site-specific mutagenesis. Techniques for site-specific mutagenesis induction are well known in the art and are described, for example, in Zoller and Smith (DNA 3:479-488, 1984) or "Splicing by extension overlap", Horton et al, Gene 77, 1989. , pages 61-68. Therefore, the use of The nucleotides and amino acid sequences of subunit VII, we may introduce selected modifications. Likewise, procedures for preparing DNA constructs using polymerase chain reaction using specific primers are well known to those skilled in the art (see PCR Protocols, 1990, Academic Press, San Diego, California, USA).

A nucleic acid construct encoding a Factor VII polypeptide of the invention may suitably be of genomic or cDNA origin. Nucleic acid constructs encoding Factor VII polypeptides can also be prepared synthetically by established standard methods, such as the aminophosphate methods described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869. Sequences encoding human Factor VII polypeptides can also be prepared by polymerase chain reaction using specific primers, for example, as described in US 4,683,202, Saiki et al, Science 239 (1988), 487-491 or Sambrook et al., supra.

In addition, the nucleic acid construct can be a synthetic synthesis and genomic, mixed synthesis and cDNA or hybrid preparation of fragments corresponding to the entire nucleic acid construct by ligation of synthetic, genomic or cDNA source (as needed) according to standard techniques. Genomic and cDNA derived.

The nucleic acid construct is preferably a DNA construct. The DNA sequence used to produce the Factor VII polypeptide according to the invention will typically encode the amino-terminal end polypeptide of Factor VII with appropriate post-translational processing (e.g., gamma-carboxylation of glutamic acid residues) and secretion from the host cell. The propolypeptide can be Factor VII or another vitamin K-dependent plasma protein, such as a Factor IX, Factor X, Prothrombin, Protein C or Protein S polypeptide. As will be appreciated by those skilled in the art, additional modifications can be made in the amino acid sequence of the Factor VII polypeptide, wherein such modifications do not significantly impair the ability of the protein to act as a coagulant.

The DNA sequence encoding the human Factor VII polypeptide is typically inserted into a recombinant vector which can be any vector which can be conveniently subjected to recombinant DNA procedures and the choice of vector It will usually depend on the host cell into which it is to be introduced. Thus, the vector can autonomously replicate the vector, i.e., the vector in the form of an extrachromosomal entity, the vector replication being independent of chromosomal replication, such as plastids. Alternatively, the vector may be one which, when introduced into a host cell, integrates into the host cell genome and replicates along with its integrated chromosome.

The vector is preferably an expression vector in which the DNA sequence encoding the human Factor VII polypeptide is operably linked to additional fragments required for transcription of the DNA. In general, the expression vector is derived from plastid or viral DNA, or may contain elements of both. The term "operably linked" indicates that the fragment is configured to function in concert with its intended purpose, such as transcription initiated in a promoter and via a DNA sequence encoding a polypeptide.

A expression vector for expression of a Factor VIIa polypeptide variant will comprise a promoter capable of directing transcription of the cloning gene or cDNA. The promoter may be any DNA sequence which displays transcriptional activity in the host cell of choice and may be derived from a gene encoding a protein either homologous or heterologous to the host cell.

An example of a suitable promoter for directing transcription of DNA encoding a human Factor VII polypeptide in a mammalian cell is the SV40 promoter (Subramani et al, Mol. Cell Biol. 1 (1981), 854-864), MT-1. (metallothionein gene) promoter (Palmiter et al, Science 222 (1983), 809-814), CMV promoter (Boshart et al, Cell 41:521-530, 1985) or adenovirus 2 major late promoter ( Kaufman and Sharp, Mol. Cell. Biol, 2: 1304-1319, 1982).

Where necessary, the DNA sequence encoding the Factor VII polypeptide can also be operably linked to a suitable terminator, such as a human growth hormone terminator (Palmiter et al, Science 222, 1983, pages 809-814) or TPI1 (Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp. 419-434) or ADH3 (McKnight et al., The EMBO J. 4, 1985, pp. 2093-2099) Termination child. The expression vector may also contain a set of RNA splice sites located downstream of the promoter of the Factor VII sequence and upstream of the insertion site. Preferred RNA splice sites are obtainable from adenovirus and/or immunoglobulin genes. The expression vector also contains a polyadenylation signal downstream of the insertion site. Particularly good polyadenylation signals include early or late polyadenylation signals from SV40 (Kaufman and Sharp, supra), polyadenylation signals from adenovirus 5 Elb regions, human growth hormone gene terminators (DeNoto et al. Nucl. Acids Res. 9: 3719-3730, 1981) or a polyadenylation signal from the human Factor VII gene or the bovine Factor VII gene. The expression vector can also include a non-coding viral leader sequence, such as an adenovirus 2 triple leader sequence located between the promoter and the RNA splice site; and an enhancer sequence, such as the SV40 enhancer.

To direct a Factor VII polypeptide of the invention into the secretory pathway of a host cell, a secretion signal sequence (also referred to as a leader sequence, pre-sequence or pre-sequence) can be provided in the recombinant vector. The secretion signal sequence is ligated to a DNA sequence encoding a human Factor VII polypeptide in an appropriate reading frame. The secretion signal sequence is typically placed 5' to the DNA sequence encoding the peptide. The secretion signal sequence can be typically associated with a protein or can be derived from a gene encoding another secreted protein.

A procedure for ligating a DNA sequence encoding a Factor VII polypeptide, a promoter, and optionally a terminator and/or a secretion signal sequence, and inserting it into a suitable vector containing the information required for replication, is well known to those skilled in the art. Well known (see, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).

Methods for transfecting mammalian cells and expressing DNA sequences introduced into cells are described, for example, in Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J. Mol. Appl. Genet. (1982), 327-341; Loyter et al, Proc. Natl. Acad. Sci. USA 79 (1982), 422-426; Wigler et al, Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al, EMBO J. 1 (1982), 841-845.

The selected DNA sequence is transfected by, for example, calcium phosphate (Wigler et al, Cell 14: 725-732, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603-616, 1981; Graham and Van der Eb, Virology 52d: 456-467, 1973) or electroporation (Neumann et al, EMBO J. 1: 841-845, 1982) introduced into cultured mammalian cells. To identify and select cells expressing foreign DNA, a gene (optional marker) that confers an alternative phenotype is typically introduced into the cell along with the relevant gene or cDNA. Preferred selectable markers include genes that confer tolerance to drugs such as neomycin, hygromycin, and methotrexate. The selectable marker can be an amplifiable selectable marker. Preferably, the selectable marker is a dihydrofolate reductase (DHFR) sequence. The selectable marker can be introduced into the cell on the independent plastid simultaneously with the relevant gene, or it can be introduced into the same plastid. If on the same plastid, the selectable marker and related genes can be under the control of different promoters or the same promoter, and the latter configuration produces a bicistronic message. Constructs of this type are known in the art (e.g., Levinson and Simonsen, U.S. 4,713,339). It may also be advantageous to add additional DNA called "vector DNA" to the mixture introduced into the cells.

After the cells have absorbed the DNA, they are grown in a suitable growth medium, typically 1-2 days, to begin expressing the relevant genes. As used herein, the term "appropriate growth medium" means a medium containing cell growth and nutrients and other components required for expression of a related Factor VII polypeptide. The medium generally includes a carbon source, a nitrogen source, an essential amino acid, an essential sugar, a vitamin, a salt, a phospholipid, a protein, and a growth factor. For the production of γ- carboxylated proteins, the medium will contain vitamin K, preferably from about 0.1 μ g / ml to a concentration of about 5 μ g / ml of. Drug selection is then applied to select the growth of cells that display the selectable marker in a stable manner. For cells that have been transfected with a selectable marker, the drug concentration can be increased to select a colony that increases the number of copies, thereby increasing the amount of expression. The pure lines of stably transfected cells are then screened for expression of the relevant human Factor VII polypeptide.

The host cell into which the DNA sequence encoding the Factor VII polypeptide is introduced can be any cell capable of producing a translated human Factor VII polypeptide after translation and including yeast, fungi, and higher eukaryotic cells.

Examples of mammalian cell lines for use in the present invention are Chinese hamster ovary (CHO) cells (e.g., ATCC CCL 61), CHO DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77: 4216-4220, 1980), baby hamster kidney (BHK) and 293 (ATCC CRL 1573; Graham et al, J. Gen. Virol. 36: 59-72, 1977) cell line.

The transformed or transfected host cells described above are then cultured in a suitable nutrient medium under conditions permitting expression of the Factor VII polypeptide, after which all or a portion of the resulting peptide can be recovered from the culture. The medium used to culture the cells may be any conventional medium suitable for growing host cells, such as a basic or complex medium containing appropriate supplements. Suitable media are commercially available from commercial suppliers or may be prepared according to published formulations (e.g., in the catalogue of the American Type Culture Collection). The Factor VII polypeptide produced by the cell can then be recovered from the culture medium by conventional procedures, including isolation of the host cell and culture medium by centrifugation or filtration; precipitation of the supernatant or filtrate protein by means of, for example, ammonium sulfate salt Aqueous component; purification by a variety of chromatographic procedures, such as ion exchange chromatography, gel filtration chromatography, affinity chromatography or the like (depending on the type of polypeptide).

Transgenic animal technology can be used to produce the Factor VII polypeptides of the invention. Preferably, the protein is produced in the mammary gland of the host female mammal. The performance in the mammary gland and the subsequent secretion of related proteins into milk overcome many of the difficulties encountered in separating proteins from other sources. Milk is easy to receive The collection is available in large quantities and is fully characterized in biochemistry. In addition, the main milk protein is present in the milk at a high concentration (typically about 1 to 15 g/l).

From a commercial point of view, it is significantly better to use a species with a larger milk production as a host. Although smaller animals such as mice and rats can be used (and preferred at the principle verification stage), livestock mammals are preferred, including but not limited to pigs, goats, sheep, and cattle. This species is particularly preferred due to factors such as the history of sheep's transgenic genes, the amount of milk produced, the cost of equipment used to collect sheep's milk, and the ease of use (see, for example, WO 88/00239 for the selection of host species) Comparison of factors). It is generally desirable to select a host animal species that has been bred for use in dairy products, such as East Friesland sheep, or to introduce dairy animals by breeding a transgenic line at a later date. In any case, animals with known, good health conditions should be used.

To obtain performance in the mammary gland, a transcriptional promoter from the milk protein gene is used. The milk protein gene includes genes encoding casein (see U.S. 5,304,489), beta-lactoglobulin, a-lactalbumin, and whey acidic protein. The β-lactoglobulin (BLG) promoter is preferred. In the case of the sheep beta-lactoglobulin gene, at least the proximal 406 bp region of the 5' flanking sequence of the gene will generally be used, although a larger portion of the 5' flanking sequence (up to about 5 kbp) is preferred, such as An approximately 4.25 kbp DNA fragment comprising a 5' flanking promoter and a non-coding portion of the beta-lactoglobulin gene (see Whitelaw et al, Biochem. J. 286: 31-39 (1992)). Similar fragments of promoter DNA from other species are also suitable.

Other regions of the β-lactoglobulin gene can also be incorporated into the construct because the genomic region of the gene can be expressed. It is generally accepted in the art that constructs lacking introns, for example, perform poorly compared to constructs containing such DNA sequences (see Brinster et al., Proc. Natl. Acad. Sci. USA 85: 836). -840 (1988); Palmiter et al., Proc. Natl. Acad. Sci. USA 88: 478-482 (1991); Whitelaw et al, Transgenic Res. 1: 3-13 (1991); WO 89/01343; and WO 91/02318, each of which is incorporated herein by reference. . In this regard, it is generally preferred to use genomic sequences containing all or some of the natural introns encoding genes associated with the protein or polypeptide, and thus preferably further doped at least some from, for example, the beta-lactoglobulin gene. child. One region is a DNA fragment that provides intron splicing and RNA polyadenylation from the 3' non-coding region of the sheep β-lactoglobulin gene. When a native 3' non-coding sequence of a gene is substituted, the sheep beta-lactoglobulin fragment enhances and stabilizes the amount of expression of the associated protein or polypeptide. In other embodiments, the region surrounding the starting ATG of the variant Factor VII sequence is replaced by a corresponding sequence from a milk-specific protein gene. This substitution provides a putative tissue-specific starting environment to enhance performance. It is preferred to replace the entire variant Factor VII pre-sequence and the 5' non-coding sequence by, for example, the sequence of the BLG gene, although smaller regions can be replaced.

To express a Factor VII polypeptide in a transgenic animal, a DNA fragment encoding variant Factor VII is operably linked to an additional DNA fragment that is required for its performance to produce a performance unit. Such additional fragments include the promoters mentioned above, as well as sequences that provide for transcription of the mRNA and termination of polyadenylation. The expression unit will further comprise a DNA fragment encoding a secretion signal sequence operably linked to a fragment encoding a modified Factor VII. The secretion signal sequence may be a native Factor VII secretion signal sequence or may be a secretion signal sequence of another protein such as milk protein (see, eg, von Heijne, Nucl. Acids Res. 14: 4683-4690 (1986); and Meade et al. US 4,873,316, which is incorporated herein by reference.

The expression unit for use in a transgenic animal is constructed by inserting the variant Factor VII sequence into a plastid or phage vector containing the additional DNA fragment, although the expression unit can be constructed by essentially any ligation sequence. It is especially convenient to provide DNA tablets containing the encoded milk protein. The vector of the segment and the coding sequence of the milk protein are replaced by the coding sequence of the Factor VII variant; thereby generating a gene fusion comprising the expression control sequence of the milk protein gene. In any event, the selection of expression units in a plastid or other vector facilitates amplification of the variant Factor VII sequence. Amplification is carried out in a bacterial (e.g., E. coli) host cell, and thus the vector will typically include a source of replication and a selectable marker that functions in the bacterial host cell. The expression unit is then introduced into the fertilized egg (including the early embryo) of the selected host species. Heterologous DNA can be introduced by one of several pathways including microinjection (e.g., U.S. Patent No. 4,873,191), retroviral infection (Jaenisch, Science 240: 1468-1474 (1988)), or use of embryonic stems. Site-specific integration of (ES) cells (reviewed by Bradley et al, Bio/Technology 10:534-539 (1992)). The eggs are then implanted into the fallopian tubes or uterus of a pseudopregnant woman and allowed to develop. Progeny carrying the introduced DNA in the germline can transmit DNA to their offspring in a conventional, Mendelian manner, allowing the development of a population of transgenes. General procedures for producing transgenic animals are known in the art (see, for example, Hogan et al, Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986; Simons et al, Bio/Technology 6: 179- 183 (1988); Wall et al, Biol. Reprod. 32:645-651 (1985); Buhler et al, Bio/Technology 8: 140-143 (1990); Ebert et al, Bio/Technology 9: 835-838 (1991); Krimpenfort et al, Bio/Technology 9: 844-847 (1991); Wall et al, J. Cell. Biochem. 49: 113-120 (1992); US 4, 873, 191; US 4, 873, 316; WO 88/00239, WO 90/05188, WO 92/11757; and GB 87/00458). Techniques for introducing foreign DNA sequences into mammals and their germ cells were originally developed in mice (see, eg, Gordon et al, Proc. Natl. Acad. Sci. USA 77: 7380-7384 (1980); Gordon and Ruddle, Science 214: 1244-1246 (1981); Palmiter and Brinster, Cell 41: 343-345 (1985); Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438-4442 (1985); and Hogan et al. (ibid.). These techniques are then applicable to larger animals, including livestock species (see, for example, WO 88/00239, WO 90/05188, and WO 92/11757; and Simons et al, Bio/Technology 6: 179-183 (1988)). In summary, among the most effective routes currently used to produce transgenic mice or livestock, hundreds of linear molecules of the relevant DNA are injected into one of the pronuclei of the fertilized egg according to the prior art. DNA can also be injected into the cytoplasm of fertilized eggs.

purification

The Factor VII polypeptide of the present invention is recovered from the cell culture medium. Factor VII polypeptides of the invention can be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobicity, chromatofocusing, and size exclusion), Electrophoresis procedures (eg, preparative isoelectric focusing (IEF), differential solubility (eg, ammonium sulfate precipitation) or extraction (see, eg, Protein Purification, J.-C. Janson and Lars Ryden, ed., VCH Publishers, New York, 1989). Preferably, the Factor VII polypeptide can be purified by affinity chromatography on an anti-Factor VII antibody column. For example, Wakabayashi et al, J. Biol. Chem. 261: 11097-11108, (1986) and Thim et al., Biochemistry 27:7785-7793, (1988) describes the use of calcium-dependent monoclonal antibodies. It is possible to achieve additional purification by conventional chemical purification means such as high performance liquid chromatography. Other purification methods including cesium citrate precipitation are It is known in the art and can be applied to purify the novel Factor VII polypeptides described herein (see, for example, Scopes, R., Protein Purification, Springer-Verlag, NY, 1982).

For therapeutic purposes, the Factor VII polypeptides of the invention are preferably substantially pure. Thus, in a preferred embodiment of the invention, the Factor VII polypeptide of the invention is purified to at least about 90 to 95% homogeneity, preferably to at least about 98% homogeneity. Can be known by several of this technology The method evaluates purity, such as HPLC, gel electrophoresis, and amino terminal amino acid sequencing.

Factor VII polypeptide cleavage at its activation site is intended to convert it to its double-stranded form. It can be according to procedures known in the art, such as Osterud et al, Biochemistry 11: 2853-2857 (1972); Thomas, U.S. Patent No. 4,456,591; Hedner and Kisiel, J. Clin. Invest. 71: 1836-1841 (1983) Or; Kisil and and Fujikawa, Behring Inst. Mitt. 73:29-42 (1983) disclose their procedures for activation. Alternatively, Factor VII can be passed through an ion exchange chromatography tube such as Mono Q® (Pharmacia fine Chemicals) or the like as described by Bjoern et al. (Research Disclosure, 269 September 1986, pages 564-565). The column transmits the factor VII and is activated. The resulting activated Factor VII variant can then be formulated and administered as described below.

analysis

Suitable in vitro in vitro proteolytic and antithrombin reactivity assays for selecting preferred Factor VII polypeptides in accordance with the present invention are provided herein. These analyses are described in detail in Example 5. Briefly, the assay can be performed in a simple preliminary in-tube test format as follows: in the presence of calcium in the supported reactants and in the presence of vesicles consisting of phospholipid choline (PC) and phospholipid thioglycine (PS). The physiologically-derived plasma-derived factor X (X) was used as the proteolytic activity of the mass-measured FVIIa polypeptide at physiological pH. FVIIa and FX were analyzed by incubation at a concentration below the Km of the reactants and for a period of time sufficient to allow for the production of measurable FXa while maintaining a FX conversion of less than 20%. FXa was quantified after addition of a suitable chromogenic substrate such as S-2765 and reported relative to wild-type FVIIa after normalization according to the concentration of the tested FVIIa variant.

The antithrombin reactivity of the FVIIa polypeptide can be measured at physiological pH under pseudo-primary conditions in the presence of excess plasma-derived antithrombin, low molecular weight (LMW) heparin, and calcium. Residual FVIIa activity was measured discontinuously throughout the time course of inhibition reaction using a chromogenic substrate such as S-2288. The inhibition rate was obtained by nonlinear least squares fitting of the data with a single exponential decay function and reported relative to wild-type FVIIa after normalization of the inhibition rate according to the antithrombin concentration used. The kinetic characterization of heparin-catalyzed and uncatalyzed inhibition by coagulation enzymes of antithrombin is described in Olson et al. (1993), Methods Enzymol. 222, 525-559.

Pharmaceutical composition

In one aspect, the invention relates to compositions and formulations comprising a Factor VII polypeptide of the invention. For example, the invention provides a pharmaceutical composition comprising a Factor VII polypeptide of the invention in combination with a pharmaceutically acceptable carrier.

Accordingly, it is an object of the present invention to provide a pharmaceutical formulation comprising a Factor VII polypeptide which is present at a concentration of from 0.25 mg/ml to 100 mg/ml, and wherein the formulation has a pH of from 2.0 to 10.0. The formulation may further comprise one or more of the following: a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer, an antioxidant, or a surfactant, and various combinations thereof. Those skilled in the art are familiar with the use of preservatives, isotonic agents, chelating agents, stabilizers, antioxidants, and surfactants in pharmaceutical compositions. See Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

In one embodiment, the pharmaceutical formulation is an aqueous formulation. The formulation is typically a solution or suspension, but may also include colloids, dispersions, emulsions, and multi-phase materials. The term "aqueous formulation" is defined as a formulation comprising at least 50% w/w water. Similarly, the term "aqueous solution" is defined as a solution comprising at least 50% w/w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50% w/w water.

In another embodiment, the pharmaceutical formulation is a lyophilized formulation, physician or patient A solvent and/or a diluent is added thereto before use.

In another aspect, the pharmaceutical formulation comprises an aqueous solution of a Factor VII polypeptide and a buffer, wherein the polypeptide is present at a concentration of 1 mg/ml or more, and wherein the pH of the formulation is from about 2.0 to about 10.0.

In another aspect, the pharmaceutical formulation can be any of the formulations disclosed in WO 2014/057069, which is incorporated herein by reference; or it can be as described in Example 18. Formulation.

The Factor VII polypeptide of the invention may be administered parenterally, such as intravenously, such as intramuscularly, such as subcutaneously. Alternatively, a FVII polypeptide of the invention may be administered via a gut route, such as orally, or surface. The polypeptide of the present invention can be administered prophylactically. The polypeptides of the invention can be administered therapeutically (as needed).

Use for treatment

In a broad aspect, a Factor VII polypeptide of the invention or a pharmaceutical formulation comprising the polypeptide can be used as a pharmaceutical.

In one aspect, a Factor VII polypeptide of the invention or a pharmaceutical formulation comprising the polypeptide can be used to treat an individual having a coagulopathy.

In another aspect, a Factor VII polypeptide of the invention or a pharmaceutical formulation comprising the polypeptide can be used in the manufacture of a medicament for treating a bleeding disorder or a bleeding event or enhancing a normal hemostatic system.

In another aspect, a Factor VII polypeptide of the invention or a pharmaceutical formulation comprising the polypeptide can be used to treat hemophilia A, hemophilia B or hemophilia A or B with an acquired inhibitor.

In another aspect, a Factor VII polypeptide of the invention or a pharmaceutical formulation comprising the polypeptide can be used in a method of treating a bleeding disorder or a bleeding event in an individual or enhancing a normal hemostatic system, the method comprising administering to an individual in need thereof A therapeutically or prophylactically effective amount of a Factor VII polypeptide of the invention is administered.

The term "individual" as used herein includes any human patient, or non-human vertebrate.

The term "treatment" as used herein refers to the medical treatment of any human or other vertebrate individual in need thereof. The individual is expected to undergo a physical examination by a medical practitioner or a veterinary practitioner who gives an experimental or definitive diagnosis indicating the benefit to the health of the human or other vertebrate using the particular treatment. The timing and purpose of the treatment may vary from individual to individual depending on the health status of the individual. Thus, the treatment can be prophylactic, palliative, symptomatic, and/or curative. For the purposes of the present invention, prophylactic, palliative, symptomatic, and/or curative treatments may represent an independent aspect of the invention.

The term "coagulopathy" as used herein refers to an increased hemorrhagic tendency resulting from any qualitative or quantitative deficiency of any procoagulant component of the normal coagulation cascade or any upregulation of fibrinolysis. Such coagulopathy can be innate and/or acquired and/or iatrogenic and is identified by those skilled in the art. Non-limiting examples of congenital hypocoagulopathy are hemophilia A, hemophilia B, factor VII deficiency, factor X deficiency, factor XI deficiency, von Willebrand's disease, and thrombocytopenia, such as the Glaxo's thombasthenia And Bernard-Soulier syndrome. The clinical severity of hemophilia A or B is determined by the concentration of functional units of FIX/Factor VIII in the blood and is classified as mild, moderate or severe. Severe hemophilia is defined by a phlegm factor level of <0.01 U/ml (corresponding to 1% of <normal level), with moderate and mild hemophilia The human beings have a level of 1%-5% and >5% respectively. Hemophilia A with an "inhibitor" (ie, an alloantibodies against Factor VIII) and hemophilia B with an "inhibitor" (ie, an alloantibody against Factor IX) are partially congenital and partially acquired. A non-limiting example of a coagulopathy.

A non-limiting example of an acquired coagulopathy is a deficiency of serine protease caused by vitamin K deficiency; this vitamin K deficiency may be caused by administration of a vitamin K antagonist such as warfarin. Acquired coagulopathy can also occur after extensive trauma. In another condition called "blood vicious circle", it is characterized by hemodilution (dilute thrombocytopenia and clotting factor dilution), hypothermia, clotting factor consumption, and metabolic disorders (acidosis). Liquid therapy and increased fibrinolysis can exacerbate this condition. This bleeding can come from any part of the body.

Non-limiting examples of iatrogenic coagulopathy are overdose of anticoagulants that can be designated for the treatment of thromboembolic disorders, such as heparin, aspirin, warfarin, and other platelet aggregation inhibitors. A second, non-limiting example of a iatrogenic coagulopathy is a coagulopathy induced by excessive and/or improper fluid therapy, such as a coagulopathy induced by blood transfusion.

In one embodiment of the invention, the bleeding is associated with hemophilia A or B. In another embodiment, the bleeding is associated with hemophilia A or B with an acquired inhibitor. In another embodiment, bleeding is associated with thrombocytopenia. In another embodiment, the bleeding is associated with von Willebrand's disease. In another embodiment, bleeding is associated with severe tissue damage. In another embodiment, bleeding is associated with severe trauma. In another embodiment, the bleeding is associated with surgery. In another embodiment, the bleeding is associated with hemorrhagic gastritis and/or enteritis. In another embodiment, the bleeding is a major uterine bleeding, such as in the case of a placental pull-off. In another embodiment, the bleeding occurs in an organ with limited mechanical hemostasis, such as a skull Inside, inside or inside the eye. In another embodiment, the bleeding is associated with anticoagulant therapy.

The invention is further described by the following non-limiting list of specific examples:

Specific Example 1: A Factor VII polypeptide comprising two or more substitutions relative to the amino acid sequence of human Factor VII (SEQ ID NO: 1), wherein T293 is by Lys (K), Arg (R), Tyr (Y) or Phe (F) substitution and L288 is replaced by Phe (F), Tyr (Y), Asn (N), Ala (A) or Trp (W) and / or W201 by Arg (R), Met (M) or Lys (K) substitution and / or K337 by Ala (A) or Gly (G); where appropriate, Q176 is replaced by Lys (K), Arg (R) or Asn (N); Or Q286 is replaced by Asn(N).

Specific Example 1 (i): The Factor VII polypeptide of Specific Example 1, wherein T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F); and L288 by Phe (F), Tyr (Y), Asn (N), Ala (A) or Trp (W) substitution and / or W201 by Arg (R), Met (M) or Lys (K) and / or K337 by Ala (A ) or Gly (G) replacement.

Specific Example 1 (ii): The Factor VII polypeptide of Specific Example 1, wherein L288 is substituted by Phe (F), Tyr (Y), Asn (N) or Ala (A).

Specific Example 1 (iii): The Factor VII polypeptide of Specific Example 1, wherein W201 is substituted by Arg (R), Met (M) or Lys (K).

Specific Example 1 (iv): The Factor VII polypeptide of Specific Example 1, wherein K337 is substituted by Ala (A) or Gly (G).

Specific Example 2: The Factor VII polypeptide of Specific Example 1, wherein T293 is substituted by Lys (K), Arg (R), Tyr (Y) or Phe (F).

Specific example 2 (i): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F) and L288 is Phe(F), Tyr (Y), Asn (N), Ala (A) or Trp (W) substitution.

The factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Lys (K) and L288 is replaced by Phe (F).

The factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Lys (K) and L288 is replaced by Tyr (Y).

The factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Lys (K) and L288 is replaced by Asn (N).

Specific Example 2 (v): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Lys (K) and L288 is replaced by Ala (A).

The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Lys (K) and L288 is replaced by Trp (W).

The compound of claim VII, wherein T293 is replaced by Arg (R) and L288 is replaced by Phe (F).

Specific Example 2 (viii): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Arg (R) and L288 is replaced by Tyr (Y).

The factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Arg (R) and L288 is replaced by Asn (N).

Specific Example 2 (x): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Arg (R) and L288 is replaced by Ala (A).

The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Arg (R) and L288 is replaced by Trp (W).

Specific Example 2 (xii): As the specific factor VII of any of the specific examples 1 to 2 Peptide, wherein T293 is replaced by Tyr(Y); and L288 is replaced by Phe(F).

Specific Example 2 (xiii): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Tyr (Y); and L288 is replaced by Tyr (Y).

Specific Example 2 (xiv): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Tyr(Y) and L288 is replaced by Asn(N).

Specific Example 2 (xv): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Tyr (Y) and L288 is replaced by Ala (A).

Specific Example 2 (xvi): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Tyr(Y) and L288 is replaced by Trp(W).

Specific Example 2 (xvii): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Phe(F) and L288 is replaced by Phe(F).

Specific Example 2 (xviii): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Phe(F) and L288 is replaced by Tyr(Y).

Specific Example 2 (xix): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Phe(F) and L288 is replaced by Asn(N).

The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Phe (F) and L288 is replaced by Ala (A).

Specific Example 2 (xxi): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Phe(F) and L288 is replaced by Trp(W).

Specific Example 2 (xxii): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Lys (K) and K337 is replaced by Ala (A).

Specific Example 2 (xxiii): More than Factor VII as in any of Examples 1 to 2 Peptide, wherein T293 is replaced by Arg(R) and K337 is replaced by Ala(A).

Specific Example 2 (xxiv): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Tyr (Y) and K337 is replaced by Ala (A).

Specific Example 2 (xxv): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Phe(F) and K337 is replaced by Ala(A).

Specific Example 2 (xxvi): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Lys (K) and K337 is replaced by Gly (G).

Specific Example 2 (xxvii): The Factor VII polypeptide of any of embodiments 1 to 2, wherein T293 is replaced by Arg (R) and K337 is replaced by Gly (G).

Specific Example 2 (xxviii): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Tyr (Y) and K337 is replaced by Gly (G).

Specific Example 2 (xxix): The Factor VII polypeptide of any one of embodiments 1 to 2, wherein T293 is replaced by Phe(F) and K337 is replaced by Gly(G).

Specific Example 2 (xxx): A Factor VII polypeptide according to any one of embodiments 2 (ii) to 2 (xxii), wherein K337 is substituted by Ala (A).

Specific Example 3: The Factor VII polypeptide of Specific Example 2, wherein the polypeptide comprises one of the substitutions of the lower group: L288F/T293K, L288F/T293K/K337A, L288F/T293K/L305V, L288F/T293K/L305I, L288F/ T293R, L288F/T293R/K337A, L288F/T293R/L305V, L288F/T293R/L305I, L288F/T293Y, L288F/T293Y/K337A, L288F/T293Y/L305V, L288F/T293Y/L305I, L288F/T293F, L288F/T293F/ K337A, L288F/T293F/L305V, L288F/T293F/L305I, L288Y/T293K, L288Y/T293K/K337A, L288Y/T293K/L305V, L288Y/T293K/L305I, L288Y/T293R, L288Y/T293R/K337A, L288Y/T293R/L305V, L288Y/T293R/L305I, L288Y/T293Y, L288Y/T293Y/K337A, L288Y/T293Y/L305V, L288Y/T293Y/L305I, L288Y/T293F, L288Y/T293F/K337A, L288Y/T293F/L305V, L288Y/T293F/L305I, L288N/T293K, L288N/T293K/K337A, L288N/T293K/L305V, L288N/T293K/L305I, L288N/T293R, L288N/T293R/K337A, L288N/T293R/L305V, L288N/T293R/L305I, L288N/T293Y, L288N/T293Y/K337A, L288N/T293Y/L305V, L288N/T293Y/L305I, L288N/T293F, L288N/ T293F/K337A, L288N/T293F/L305V, L288N/T293F/L305I, L288A/T293K, L288A/T293K/K337A, L288A/T293K/L305V, L288A/T293K/L305I, L288A/T293R, L288A/T293R/K337A, L288A/ T293R/L305V, L288A/T293R/L305I, L288A/T293Y, L288A/T293Y/K337A, L288A/T293Y/L305V, L288A/T293Y/L305I, L288A/T293F, L288A/T293F/K337A, L288A/T293F/L305V or L288A/ T293F/L305I.

Specific Example 4: The Factor VII polypeptide of Specific Example 2, wherein the polypeptide has the following substitutions: L288F/T293K, L288F/T293K/K337A, L288F/T293R, L288F/T293R/K337A, L288Y/T293K, L288Y/T293K/K337A, L288Y/T293R, L288Y/T293R/K337A, L288N/T293K, L288N/T293K/K337A, L288N/T293R or L288N/T293R/K337A.

Specific Example 5: The Factor VII polypeptide of Specific Example 1, wherein Q176 is borrowed Replaced by Lys (K), Arg (R) or Asn (N).

Specific Example 6: The Factor VII polypeptide of Part 5, wherein the polypeptide comprises one of the substitutions of the lower group: L288F/Q176K/K337A, L288Y/Q176K/K337A, L288N/Q176K/K337A or L288A/Q176K/K337A.

Specific Example 7: The Factor VII polypeptide of Specific Example 1, wherein Q286 is replaced by Asn(N).

Specific Example 8: A Factor VII polypeptide comprising one or more substitutions relative to the amino acid sequence of human Factor VII (SEQ ID NO: 1), characterized in that one substitution is by Phe(F), Tyr(Y When Asn(N) or Ala(A) is substituted for L288, the restriction is that the polypeptide does not have the following substitution pair: L288N/R290S or L288N/R290T.

The Factor VII polypeptide of any one of embodiments 1 to 2 (xxx), 5, and 7 to 8, wherein the Factor VII polypeptide further comprises one or more of the following substitutions: L305I, L305V or K337A.

Specific Example 10: A Factor VII polypeptide comprising two or more substitutions relative to the amino acid sequence of human Factor VII (SEQ ID NO: 1), wherein W201 is by Arg (R), Met (M) or Lys (K) substitution and wherein T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F); wherein Q176 is replaced by Lys (K), Arg (R) or Asn (N) Or Q286 is replaced by Asn(N).

The compound of claim VII, wherein the T293 is by Lys (K), Arg (R), Tyr (Y) or Phe (F) Substitution and wherein W201 is replaced by Arg(R), Met(M) or Lys(K).

Specific Example 11: Factor VII polypeptide according to Specific Example 10, wherein T293 Replacement by Lys (K), Arg (R), Tyr (Y) or Phe (F).

The factor VII polypeptide of any one of the embodiments 1 to 2, 10 or 11, wherein the T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F) And W201 is replaced by Arg (R), Met (M) or Lys (K).

The factor VII polypeptide of any one of the embodiments 1 to 2, 10 or 11, wherein T293 is replaced by Lys (K) and W201 is replaced by Arg (R).

The factor VII polypeptide of any one of embodiments 1 to 2, 10 or 11, wherein T293 is replaced by Lys (K) and W201 is replaced by Met (M).

The factor VII polypeptide of any one of the embodiments 1 to 2, 10 or 11, wherein T293 is replaced by Lys (K) and W201 is replaced by Lys (K).

The Factor VII polypeptide of any one of embodiments 1 to 2, 10 or 11, wherein T293 is replaced by Arg (R) and W201 is replaced by Arg (R).

The factor VII polypeptide of any one of embodiments 1 to 2, 10 or 11, wherein T293 is replaced by Arg (R) and W201 is replaced by Met (M).

The factor VII polypeptide of any one of embodiments 1 to 2, 10 or 11, wherein T293 is replaced by Arg (R) and W201 is replaced by Lys (K).

The Factor VII polypeptide of any one of embodiments 1 to 2, 10 or 11, wherein T293 is replaced by Tyr (Y) and W201 is replaced by Arg (R).

A factor VII polypeptide according to any one of embodiments 1 to 2, 10 or 11, wherein T293 is replaced by Tyr (Y) and W201 is replaced by Met (M).

The Factor VII polypeptide of any one of the embodiments 1 to 2, 10 or 11, wherein T293 is replaced by Tyr (Y) and W201 is replaced by Lys (K).

A factor VII polypeptide according to any one of embodiments 1 to 2, 10 or 11, wherein T293 is replaced by Phe(F) and W201 is replaced by Arg(R).

A factor VII polypeptide according to any one of embodiments 1 to 2, 10 or 11, wherein T293 is replaced by Phe (F) and W201 is replaced by Met (M).

The factor VII polypeptide of any one of the embodiments 1 to 2, 10 or 11, wherein T293 is replaced by Phe (F) and W201 is replaced by Lys (K).

Specific Example 12: The Factor VII polypeptide of Embodiment 11, wherein the polypeptide comprises one of the substitutions of the lower group: W201R/T293K, W201R/T293K/K337A, W201R/T293K/L305V, W201R/T293K/L305I, W201R/ T293R, W201R/T293R/K337A, W201R/T293R/L305V, W201R/T293R/L305I, W201R/T293Y, W201R/T293Y/K337A, W201R/T293Y/L305V, W201R/T293Y/L305I, W201R/T293F, W201R/T293F/ K337A, W201R/T293F/L305V, W201R/T293F/L305I, W201K/T293K, W201K/T293K/K337A, W201K/T293K/L305V, W201K/T293K/L305I, W201K/T293R, W201K/T293R/K337A, W201K/T293R/ L305V, W201K/T293R/L305I, W201K/T293Y, W201K/T293Y/K337A, W201K/T293Y/L305V, W201K/T293Y/L305I, W201K/T293F, W201K/T293F/K337A, W201K/T293F/L305V, W201K/T293F/ L305I, W201M/T293K, W201M/T293K/K337A, W201M/T293K/L305V, W201M/T293K/L305I, W201M/T293R, W201M/T293R/K337A, W201M/T293R/L305V, W201M/T293R/L305I, W201M/T293Y, W201M/T293Y/K337A, W201M/T293Y/L305V, W201M/T293Y/L305I, W201M/T293F, W201M/T293F/K337A, W201M/T293F/L305V or W201M/T293F/L305I.

Specific Example 13: The Factor VII polypeptide of Specific Example 11, wherein the polypeptide has the following substitutions: W201R/T293K, W201R/T293K/K337A, W201R/T293R, W201R/T293R/K337A, W201R/T293Y, W201R/T293F, W201K/ T293K or W201M/T293K.

Specific Example 14: The Factor VII polypeptide of Embodiment 10, wherein Q176 is substituted by Lys (K), Arg (R) or Asn (N).

Specific Example 15: The Factor VII polypeptide of Embodiment 14, wherein the polypeptide comprises one of the subgroup substitutions: W201R/Q176K, W201R/Q176R, W201K/Q176K, W201K/Q176R, W201M/Q176K or W201M/Q176R.

Specific Example 16: The Factor VII polypeptide of Embodiment 10, wherein Q286 is replaced by Asn(N).

The Factor VII polypeptide of any one of embodiments 10 to 11, 14 and 16, wherein the Factor VII polypeptide further comprises one or more of the following substitutions: L305I, L305V or K337A.

Specific Example 18: A Factor VII polypeptide comprising one or more substitutions relative to the amino acid sequence of human Factor VII (SEQ ID NO: 1), characterized in that one substitution is by Arg(R), Met (M ) or Lys (K) when W201 is replaced.

Specific Example 19: A Factor VII polypeptide comprising two or more substitutions relative to the amino acid sequence of human Factor VII (SEQ ID NO: 1), wherein L288 is via Phe (F), Tyr (Y), Asn(N) or Ala(A) substitution; wherein W201 is by Arg(R), Met(M) or Lys (K) substitution, and if necessary, where T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F); Q176 by Lys (K), Arg (R) or Asn ( N) permutation; or Q286 is replaced by Asn(N).

Specific Example 20: The Factor VII polypeptide of Embodiment 19, wherein the polypeptide comprises one of the subgroup substitutions: L288F/W201K, L288F/W201R, L288F/W201M, L288N/W201K, L288N/W201R, L288N/W201M, L288Y/W201K, L288Y/W201R, L288Y/W201M, L288A/W201K, L288A/W201R, L288A/W201M, L288F/W201K/T293K, L288F/W201K/T293Y, L288F/W201R/T293K, L288F/W201R/T293Y, L288F/ W201M/T293K, L288F/W201M/T293Y, L288N/W201K/T293K, L288N/W201K/T293Y, L288N/W201R/T293K, L288N/W201R/T293Y, L288N/W201M/T293K, L288N/W201M/T293Y, L288A/W201K/ T293K, L288A/W201K/T293Y, L288A/W201R/T293K, L288A/W201R/T293Y, L288A/W201M/T293K, L288A/W201M/T293Y, L288Y/W201K/T293K, L288Y/W201K/T293Y, L288Y/W201R/T293K, L288Y/W201R/T293Y, L288Y/W201M/T293K or L288Y/W201M/T293Y.

The factor VII polypeptide of any one of the preceding embodiments, wherein the Factor VII polypeptide further comprises one or more of the following substitutions: R396C, Q250C or 407C.

The factor VII polypeptide of any one of the preceding embodiments, wherein the Factor VII polypeptide is a cleavage, double-stranded Factor Vila polypeptide.

Specific Example 22 (i): The Factor VII polypeptide according to any one of the preceding embodiments, comprising two amino acid residues relative to the amino acid sequence of human Factor VII (SEQ ID NO: 1) generation.

Specific Example 22 (ii): The Factor VII polypeptide of any of the preceding embodiments, comprising three amino acid substitutions relative to the amino acid sequence of human Factor VII (SEQ ID NO: 1).

Specific Example 22 (iii): The Factor VII polypeptide of any of the preceding embodiments, comprising four amino acid substitutions relative to the amino acid sequence of human Factor VII (SEQ ID NO: 1).

The compound VII polypeptide according to any one of the preceding embodiments, comprising five amino acid substitutions relative to the amino acid sequence of human Factor VII (SEQ ID NO: 1).

Specific Example 22 (v): The Factor VII polypeptide of any one of embodiments 22 (i) to 22 (iv) comprising up to ten amino acid sequences relative to human Factor VII (SEQ ID NO: 1) Substituted by an amino acid.

Specific Example 22 (vi): The Factor VII polypeptide according to any one of the preceding specific examples, wherein the proteolytic activity is wild-type human Factor VIIa as measured by in-tube proteolytic analysis in the absence of soluble tissue factor At least 110% of the proteolytic activity, such as at least 120%, such as at least 130%, such as at least 140%, such as at least 150%, such as at least 160%, such as at least 170%, such as at least 180%, such as at least 190%, such as At least 200%, such as at least 300%, such as at least 400%, such as at least 500%, such as at least 1000%, such as at least 3000%, such as at least 5000%, such as at least 10 000%, such as at least 30 000%.

Specific Example 22 (vii): The Factor VII polypeptide according to any one of the preceding specific examples, such as anti-thrombin in the presence of low molecular weight heparin and in the absence of soluble tissue factor Analytical assay, which has less than 20%, such as less than 19%, such as less than 18%, such as less than 17%, such as antithrombin reactivity of wild-type human Factor VIIa (SEQ ID NO: 1), such as Less than 16%, such as less than 15%, such as less than 14%, such as less than 13%, such as less than 12%, such as less than 11%, such as less than 10%, such as less than 9%, such as less than 8%, such as less than 7%, such as Less than 6%, such as less than 5% antithrombin reactivity.

The method of any one of the preceding embodiments, wherein the Factor VII polypeptide is coupled to at least one half-life extending moiety.

Specific Example 24: The Factor VII polypeptide of Embodiment 23, wherein the half-life extending moiety is selected from the group consisting of biocompatible fatty acids and derivatives thereof, such as hydroxyethyl starch (HES) hydroxyalkyl starch (HAS), polyethyl b. Glycol (PEG), poly(Glyx-Sery)n (HAP), hyaluronic acid (HA), heparin precursor polymer (HEP), phosphorylcholine-based polymer (PC polymer), Fleximer, polydextrose, poly Sialic acid (PSA), Fc domain, transferrin, albumin, elastin-like peptide (ELP), XTEN polymer, PAS polymer, PA polymer, albumin binding peptide, CTP peptide, FcRn binding peptide and any combination.

Specific Example 25: The Factor VII polypeptide of Embodiment 24, wherein the half-life extending moiety is a heparin precursor polymer.

Specific Example 26: The Factor VII polypeptide of Embodiment 25, wherein the heparin precursor polymer has a moiety selected from the group consisting of 13-65 kDa, 13-55 kDa, 25-55 kDa, 25-50 kDa, 25-45 kDa, 30-45 kDa, and 38- Molecular weight in the range of 42 kDa or molecular weight of 40 kDa.

The FVII polypeptide of any one of the specific examples 25 to 26, which comprises the structural fragment shown in Formula I,

Wherein n is an integer from 95 to 115.

The compound of claim VII, which has a half-life of at least 100% greater than wild-type human Factor VIIa (SEQ ID NO: 1).

The method of any one of the preceding embodiments, wherein the Factor VII polypeptide is a disulfide linked to a tissue factor.

The method of any one of the preceding embodiments, wherein the polypeptide has other amino acid modifications that increase the platelet affinity of the polypeptide.

The compound of claim VII, wherein the polypeptide is a fusion conjugate protein comprising a Factor VII polypeptide according to any one of the specific examples 1 to 22 and, for example, an Fc domain or albumin. / peptide fusion protein.

Specific Example 30: A polynucleotide encoding a Factor VII polypeptide as defined in any one of Specific Examples 1 to 22 and 28 to 29.

Specific Example 31: A recombinant host cell comprising the polynucleotide of Specific Example 30.

Specific Example 32: A method of producing a Factor VII polypeptide as defined in any one of Embodiments 1 to 22 and 28 to 29, which method comprises allowing expression of the structure of the polynucleotide The cells are cultured in an appropriate medium under conditions and the resulting polypeptide is recovered from the medium.

Specific Example 33: A pharmaceutical composition comprising a Factor VII polypeptide as defined in any one of Specific Examples 1 to 29 and a pharmaceutically acceptable carrier.

Specific Example 34: A method for treating a bleeding disorder or a bleeding event in an individual or enhancing a normal hemostatic system, the method comprising administering to a subject in need thereof a therapeutic or prophylactically effective amount of any one of the specific examples 1 to 29 A defined Factor VII polypeptide.

Specific Example 35: The Factor VII polypeptide according to any one of Embodiments 1 to 26, which is for use as a pharmaceutical.

Specific Example 35 (i): The Factor VII polypeptide of any of embodiments 1 to 26 for use in the treatment of a coagulopathy.

Specific Example 36: The Factor VII polypeptide of Specific Example 35 (i) for use as a medicament for the treatment of hemophilia A or B.

The invention is further illustrated by the following examples, which are not to be construed as limiting the scope of the invention. The features disclosed in the foregoing description and the following examples can be used to implement the materials of the present invention in various forms, alone and in any combination thereof.

Example

protein

Human plasma source factor X (FX) and factor Xa (FXa) were obtained from Enzyme Research Laboratories, Inc. (South Bend, IN). Soluble tissue factor 1-219 (sTF) or 1-209 was prepared according to the published procedure (Freskgard et al., 1996). The performance and purification of recombinant wild-type FVIIa was performed as previously described (Thim et al., 1988; Persson and Nielsen, 1996). Heparin agarose chromatography (GE Healthcare) according to published procedures (Olson et al., 1993) Human plasma-derived antithrombin (Baxter) was purified. Bovine serum albumin (BSA) was obtained from Sigma Aldrich (St. Louis, MO).

Example 1 - FVIIa variant design

To design a FVIIa variant with high proteolytic activity on FX as a substrate, a two-way strategy was employed. The FVIIa loop and the single amino acid surrounding the active site region were selected for exchange with the corresponding FVII amino acids (Fig. 1) from different species and point mutation induction. The FVIIa proteolytic activity was measured as outlined in Example 5. The proteolytic activity of the tricyclic exchange FVIIa variant is shown in Table 1, wherein when the amino acid at position 288 was changed, the residues at positions 287 and 289 were mutated to sulphonic acid and glutamic acid, respectively. It was observed that a change occurred at position 288 while maintaining the same amino acid at positions 287 and 289 significantly affected proteolytic activity. It was also observed that the amino acid at the substitution position 201 affects the proteolytic activity regardless of the leucine acid carried by the rat and the rabbit FVII or the arginine carried by the bovine FVII. Furthermore, it was observed that the amino acid at position 337 of substitution, whether by horse-loaded glutamic acid or a smaller amino acid such as alanine, affects proteolytic activity (Table 1). These observations indicate that amino acids at positions 288 and 201 may be involved in FX recognition and activation. Therefore, positions 288 and 201 were further investigated by saturation mutation and the representative results are summarized in Table 2.

Table 1. Proteolytic activity of selected FVIIa variants. Results are shown as percentage (%) relative to wild-type FVIIa.

Example 2 - Selection of FVIIa variants

Using KOD Xtreme ^TM Hot Start DNA Polymerase from Stratagene or from Novagen the QuickChange® of site-directed mutagenesis kit, using PCR-based site-directed mutagenesis method of introducing mutations into the mammalian expression vector encoding the FVII cDNA. The pQMCF expression vector from Icosagen Cell Factory (Estonia) and CHOEB NALT85 were used as expression systems. The introduction of the desired mutation was verified by DNA sequencing (MWG Biotech. Germany).

Example 3 - FVIIa performance

FVII variants were expressed in CHOEBNALT85 cells from Icosagen Cell Factory (Estonia). Briefly, CHOEBNALT85 suspension cells were transiently transfected by electroporation (Gene Pulse Xcell, Biorad, Copenhagen, DK). Transfected cells were selected by 700 μg/l Geneticin® (Gibco, Life Technologies) and extended to obtain a total of 300 ml to 10 liters of supernatant. The cells were cultured in a medium (Sigma-Aldrich) supplemented with 5 mg/l of vitamin K1 according to the manufacturer's instructions. Depending on the size, the cells were cultured in shake flasks (37 ° C. 5% - 8% CO ₂ and 85-125 rpm) or shake culture bags (37 ° C. 5% CO ₂ and 30 rpm). The small-scale supernatant was collected by centrifugation followed by filtration through a 0.22 μm PES filter (Corning; Fischer Scientific Biotech, Slangerup, DK) and the larger volume was further filtered by depth filtration followed by 0.22 μm absolute filtration (3 μm Clarigard, Opticap XL10; 0.22 μm Durapore, Opticap XL10, Merck Millipore, Hellerup, DK).

Example 4 - Purification and Concentration Determination of FVIIa

FVII variants were purified by antibody affinity chromatography of the directed Gla-domain essentially as described elsewhere (Thim et al. 1988). In short, the program consists of 3 steps. In step 1, 5 mM CaCl _{2 was} added to the modified medium and the sample was loaded onto the affinity column. After washing with 10 mM His, 2 M NaCl, 5 mM CaCl ₂ , 0.005% Tween 80, pH 6.0, the bound protein was eluted to (step 2) anion exchange tube with 50 mM His, 15 mM EDTA, 0.005% Tween 80, pH 6.0. Column (Source 15Q, GE Healthcare). After washing with 20 mM HEPES, 20 mM NaCl, 0.005% Tween 80, pH 8.0, the bound protein was eluted with 20 mM HEPES, 135 mM NaCl, 10 mM CaCl ₂ , 0.005% Tween 80, pH 8.0 (Step 3) CNBr-Sepharose Fast On the Flow column (GE Healthcare), human plasma source FXa was coupled to the column at a density of 1 mg/ml according to the manufacturer's instructions. The flow rate is optimized to ensure that the purified zymogen variant is substantially fully activated to an activated form. For FVIIa variants with enhanced activity, capable of autoactivation in an improved medium or anion exchange column, step 2 and/or step 3 are omitted to prevent proteolytic degradation. The purified protein was stored at -80 °C. Protein quality was assessed by SDS-PAGE analysis and the concentration of functional molecules was quantitatively quantified by active site titration or by light chain content of rpHPLC as described below.

Determination of FVIIa variant concentration by active site titration

Basically, as described elsewhere (Bock PE, 1992. J. Biol. Chem. 267. 14963-14973), the sub-chemically calculated content of d-Phe-Phe-Arg-chloromethylketone (FFR-) was determined by active site titration. Cmk; Bachem) The irreversible loss of the indoleamine hydrolysis activity after titration was determined by the concentration of functional molecules in the purified preparation. Briefly, all proteins were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM CaCl ₂ , 1 mg/mL BSA, and 0.1% w/v PEG 8000). FVIIa variants with a final concentration of 150 nM were pre-incubated with 500 nM soluble tissue factor (sTF) for 10 minutes, followed by FFR-cmk at a final concentration of 0-300 nM (n=2) in a total reaction volume of 100 μL in a 96-well plate. . The reaction was incubated overnight at room temperature. In a 96-well plate, 20 μL of each reaction was diluted 10 times in assay buffer containing 1 mM S-2288 (Chromogenix, Milano, Italy). The absorbance was continuously measured at 405 nM for 10 minutes in a Spectramax 190 microplate spectrophotometer equipped with SOFTmax PRO software. The indole hydrolysis activity is reported as the slope of the linear progression curve after removal of the blank. The active site concentration was determined by extrapolation as the minimum concentration of FFR-cmk required to completely eliminate the guanamine hydrolysis activity.

The FVIIa variant concentration was measured from the light chain content using reverse phase HPLC - in an alternative method, the concentration of the functional FVIIa molecule in the purified formulation was determined by quantifying the FVIIa light chain (LC) content by reverse phase HPLC (rpHPLC). A calibration curve for wild-type FVIIa was prepared using FVIIa concentrations in the range of 0 to 3 [mu]M while samples of unknown concentration (n=2) were prepared at an estimated concentration of 1.5 [mu]M. A 1:1 mixture of 0.5 M gin (2-carboxyethyl) phosphine (TCEP; Calbiochem/Merck KGaA, Darmstadt, Germany) and formic acid added to the sample to achieve a concentration of 20% (v/v), followed by All samples were reduced by heating the sample at 70 ° C for 10 minutes. The reduced FVIIa variant was loaded onto a C4 column (Vydac. 300Å, particle size 5 μM, 4.6 mm, 250 mm) maintained at 30 °C. The mobile phase consisted of 0.09% TFA (solvent A) in water and 0.085% TFA (solvent B) in acetonitrile. After 80 μL of sample was injected, the system was run at 25% solvent B for 4 minutes followed by a linear gradient of 25%-46% B for 10 minutes. The peaks were detected by fluorescence using excitation and emission wavelengths of 280 and 348 nm, respectively. Light chain quantification was performed by peak integration and the relative amount of FVIIa variants was calculated based on the wild-type FVIIa standard curve.

Example 5 - Screening for mutations that confer increased activity

As outlined in Example 1, summarized in Table 1, and in order to evaluate locations 201 and 288 The action of the FVIIa amino acid; subjecting these positions to a strict site-directed saturation mutation induction. To further identify FVIIa variants with enhanced proteolytic activity, other amino acid positions 305 and 337 were also selected for saturation mutation induction. Briefly, activity measurements were the ability of each variant to proteolytically activate macromolecular receptor factor X in the presence of phospholipid vesicles (in vitro in vitro proteolytic analysis). Each reaction was carried out in the presence or absence of cofactor tissue factor (sTF) to mimic the possible TF-dependent and independent mechanism of action of recombinant FVIIa. In addition, in order to understand the effect of these substitutions on FVIIa inhibition by antithrombin; anti-thrombin inhibition was quantified in the presence of low molecular weight heparin under pseudo-primary conditions to mimic endogenous heparin-like glycosaminoglycans ( GAG) The ability to accelerate the response in vivo. These results are summarized in Table 2. As shown in Figure 2, the in vitro in vivo antithrombin reactivity of the assay was found to correlate with the in vivo accumulation of the FVIIa-antithrombin complex, thus verifying the predictability of the in vitro assay procedure.

The amino acid including branic acid, tyrosine, methionine, lysine, and arginine at position 201 is required to obtain proteolytic activity against FX as a substrate in the presence of phospholipids. of. W201R provides the greatest increase in proteolytic activity in the presence of phospholipids and in the absence or presence of sTF. On the other hand, amino acids including phenylalanine, leucine and aspartame reduce proteolytic activity compared to FVIIa WT. In the case of position 288, alanine, aspartame, serine, tryptophan, phenylalanine and tyrosine are provided in the presence of phospholipids Increased proteolytic activity against FX as a substrate. L288F and L288Y provide the greatest increase in proteolytic activity in the presence of phospholipids. The data presented in Table 2 indicates a priori prediction of the proteolytic activity and the challenge of antithrombin reactivity. The method induced by the use of saturation mutations has therefore been rationalized to explore the full effect of the different amino acids on the activity produced in the FVIIa variant.

Factor X was used as the mass-measured proteolytic activity (in-tube proteolytic analysis) - plasma-derived factor X (FX) was used as the proteolytic activity of the FVIIa variant. All proteins were diluted in 50 mM HEPES pH 7.4, 100 mM NaCl, 10 mM CaCl ₂ , 1 mg/mL BSA and 0.1% w/v PEG 8000. By using a total reaction volume of 100 μL in a 96-well plate at room temperature in the presence of 25 μM 75:25 phospholipid choline: phospholipidinoic acid (PC:PS) phospholipid (Haematologic technologies, Vermont, USA) The relative proteolytic activity (n=2) was determined by incubating 1 to 10 nM of each FVIIa conjugate with 40 nM FX for 30 minutes. FX activation (n=2) in the presence of sTF was determined by incubating 5 pM of each FVIIa conjugate with 30 nM FX for 20 minutes in the presence of 25 μM PC:PS phospholipid at a total reaction volume of 100 μL. After the incubation, the reaction was stopped by adding 100 μL of 1 mM S-2765 (Chromogenix, Milano, Italy) in stop buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 80 mM EDTA). Immediately after the discontinuation, the increase in absorbance was continuously measured at 405 nM in an Envision microplate reader (PerkinElmer, Waltham, MA). All additions, incubations, and disk movements were performed by a Hamilton Microlab Star robot robot (Hamilton, Bonaduz, Switzerland) coupled to the Envision microplate reader on-line. Since the FX acceptor concentration ([S]) is lower than the K _{m of the} activation reaction, the data is fitted to the Michaelis Menten equation (v=k _cat *[S]*[E]/K _m ) by linear regression. The apparent catalyzed rate value (k _cat /K _m ) was evaluated. The amount of FXa produced was evaluated from a standard curve prepared by human plasma source FXa under the same conditions. After generating the normalized rate of FXa measurement of the concentration of FVIIa variant with respect to the use of a wild-type evaluation reports FVIIa k _cat / K _m values. The results are provided in Table 1, Table 2, Table 3 and Table 7.

The FVIIa inhibition -discontinuous method was measured by antithrombin for use in human plasma-derived antithrombin under pseudo-primary conditions in the presence of low molecular weight (LMW) heparin (Calbiochem/Merck KGaA, Darmstadt, Germany) AT) The amount of test tube inhibition rate. The assay was performed in a 96-well plate using a buffer containing 50 mM HEPES pH 7.4, 100 mM NaCl, 10 mM CaCl ₂ , 1 mg/mL BSA, and 0.1% w/v PEG 8000 in a total reaction volume of 200 μL. 5 μM antithrombin was added to a mixture of 200 nM FVIIa and 12 μM LMW heparin, and the final reaction volume was 100 μL. At different times, by transferring 20 μL of the reaction mixture to another trace containing 180 μL of sTF (200 nM), condensed amine (0.5 mg/mL; Hexadimethrine bromide, Sigma-Aldrich) and S-2288 (1 mM) The reaction was stopped in the titration tray. Immediately after the transfer at different times, the substrate was monitored for cleavage at 405 nm for 10 minutes in an Envision microplate reader. The pseudo first-order rate constant (k _obs ) is obtained by nonlinear least squares fitting of the data and the exponential decay function, and the second-order rate constant (k) is obtained from the following relation: k=k _obs /[AT]. All additions, incubations, and disk shifts were performed by a Hamilton Microlab Star robot (Hamilton, Bonaduz, Switzerland) coupled to an Envision microplate reader (PerkinElmer, Waltham, MA). The rate of inhibition was reported relative to wild-type FVIIa. The results are provided in Tables 2, 3 and 7.

Example 6 - Combining FVIIa mutations conferring increased activity and antithrombin resistance.

In order to design a FVIIa variant having high proteolytic activity and antithrombin resistance, selected variant variants that promote FVIIa proteolytic activity are combined with FVIIa variants that confer antithrombin resistance. In particular, FVIIa combinatorial variants are made by substitution at positions 293 and 201, 288, 305, 337, 176 and/or 286. Using the test tube described in Example 5 Proteolysis and antithrombin inhibition assays The characterization of the combined FVIIa purified protein preparations is summarized in Table 3.

Table 3 shows that some combinations produce FVIIa variants that exhibit the desired high activity while having the desired low antithrombin reactivity. For example, the FVIIa variant L288F T293K shows 600% proteolytic activity in the presence of phospholipids and only 6% antithrombin reactivity in the presence of low molecular weight heparin compared to wild-type FVIIa. Similarly, the FVIIa variant L288Y T293K showed 447, 8% proteolytic activity in the presence of phospholipids and only 5,8% antithrombin reactivity in the presence of low molecular weight heparin compared to wild-type FVIIa. Furthermore, W201R T293K showed 609% proteolytic activity in the presence of phospholipids and only 9% antithrombin reactivity in the presence of low molecular weight heparin compared to wild-type FVIIa.

Interestingly, the combination of the two FVIIa mutations L288F and K337A provided greatly enhanced activity, with a proteolytic activity increased by 2646% compared to wild-type FVIIa. After further introduction of the mutant T293K, the enhanced activity is retained while achieving low antithrombin reactivity. This variant showed 1310% proteolytic activity in the presence of phospholipids and only 17% antithrombin reactivity in the presence of low molecular weight heparin compared to wild-type FVIIa.

In conclusion, it can be inferred that when the T293K, T293R and T293Y mutations are combined with W201R or L288F, the anti-thrombin reactivity is effectively reduced compared to wild-type FVIIa, while providing higher proteolytic activity compared to wild-type FVIIa.

Example 7 - Evaluation of FVIIa potency and plasma level

Commercial FVIIa specific coagulation assays were used; STACLOT ^® VIIa-rTF from Diagnostica Stago was used to assess potency. The analysis is based on the method published by JHMorrissey et al. Blood. 81:734-744 (1993). It measures the sTF-initiating FVIIa activity-dependent time for the formation of fibrin clots in FVII-deficient plasma in the presence of phospholipids. Clot time was measured on an ACL9000 (ILS) coagulation instrument and the results were calculated using linear regression on a log-log scale based on the FVIIa calibration curve. The same analysis was used to measure FVIIa clot activity in plasma samples from animal PK studies. The lower limit of quantitation (LLOQ) in plasma was estimated to be 0.25 U/ml. The plasma activity level is converted to nM using specific activity.

Example 8 - Crystallographic Analysis of FVIIa Variants

To investigate the mechanism by which the identified substitutions affect proteolytic activity and antithrombin recognition, the crystal structures of representative FVIIa variants L288Y T293K, L288F T293K, W201R T293K, W201R T293Y and L288F T293K K337A were determined.

The 1DAN structure of wild-type (WT) FVIIa that is mismatched with soluble tissue factor when comparing structures on a 3-dimensional scale [Banner, DW et al, Nature, (1996) Vol. 380, 41-46] has been based on SEQ The ID NO:1 numbering scheme renumbers the heavy chain residues of FVIIa.

Purification with soluble tissue factor (fragment 1-219) by hanging drop method according to [Kirchhofer, D et al., Proteins Structure Function and Genetics, (1995), Vol. 22, pp. 419-425] H- _D- Phe-Phe-Arg chloromethyl ketone (FFR-cmk; Bachem, Switzerland) Active site-inhibited FVIIa variant crystals. The protein buffer solution was a mixture of 10 mM Tris pH 7.5, 100 mM NaCl, 15 mM CaCl ₂ at 25 °C. The protein concentration of the FVIIa variant together with the precipitant solution and mixing conditions are shown in Table 4. A hanging drop method using a 24-well VDX disk and a 1.0 ml well solution was used. A drop was established by a mixture of 1.5 μl of protein solution and 0.5 μl of a well solution. Streak seeding was used to initiate nucleation.

The low temperature conditions are shown in Table 4. Soak the crystals in a low temperature solution for about 30 seconds, After this, the crystals were transferred to liquid nitrogen and rapidly frozen therein. The software is simplified by using the XDS data of the analytical cutoff described by Karplus et al. [Karplus, PA et al., Science (New York, NY), (2012), Vol. 336, pp. 1030-1033] [Kabsch, W. Acta Crystallographica Section D Biological Crystallography, (2010), Vol. 66, pp. 125-132] Processing crystallographic data.

It will be based on the 1DAN entry from the Protein Library (PDB) [Berman, HM et al, Nucleic Acids Res., (2000), Vol. 28, pp. 235-242] [Banner, DW et al, Nature, (1996) , vol. 380, pp. 41-46] The internally generated coordinates (unpublished) of the crystallographic coordinates are used for the PHENIX kit software [Adams, PD et al., Acta Cryst. D, (2010), Vol. 66, p. Molecular displacement calculations in phenix.phaser [Mccoy, AJ et al., J. Appl. Crystallogr., (2007), vol. 40, pp. 658-674] or by phenix.refine software [pp. 213-221] Afonine, PV et al., Acta Crystallogr. Sect. D-Biol. Crystallogr., (2012), Vol. 68, pp. 352-367] The initial model of direct refinement. Refinement followed by computer graphics software COOT [Emsley, P. et al., Acta Crystallogr. Sect. D-Biol. Crystallogr., (2010), 66 Interaction model correction in Volumes, pages 486-501. The crystallographic data, refinement and model statistics of the 5 FVIIa variants are shown in Table 5.

3-dimensional structural analysis

In general, there is no significant difference between the structure of the wild type (WT) FVIIa molecule 1 DAN [Banner, DW et al, Nature, (1996), Vol. 380, pp. 41-46] and the structure of the FVIIa variant. 1DAN FVIIa heavy chain calculated by gesamt [Krissinel, E., Journal of Molecular Biochemistry, (2012), Vol. 1, pp. 76-85] with L288Y T293K, L288F T293K, W201R T293K, W201R T293Y and L288F T293K K337A The total root mean square deviation (RMSD) between the FVIIa variants was 0.424, 0.365, 0.451, 0.342, and 0.289 Å, respectively. The number of C _α -atoms used in the calculations were 254, 254, 251, 254 and 254, respectively.

W201R T293Y FVIIa variant

Mutant FVIIa W201R:

In detail, the heavy chain FVIIa Arg 201 residue of the double mutant is located in the "60-loop" (chymotrypsin number). In the likelihood-weighted 2mFo-DFc electron density map, at the 1.0 σ cutoff, there is evidence of stretching of the backbone loop, while the side chain of the Arg 201 residue (Trp residue in wild-type FVIIa) and before and after This sign is not visible in the side chains of the residues (Asn and Arg residues, respectively). This indicates the high flexibility of their side chains. To aid in structural interpretation, the observed structural factors from internal wild-type protein crystals were compared with those from FVIIa using software from the CCP4 software package [Collaborative Computational Project, N. Acta crystallographica, Part D, Biological crystallography, 1994, 50, 760-763]. The difference electron density map [F _obs (WT FVIIa/sTF)-F _obs (FVIIa W201R T293Y/sTF)] was calculated between the observed structural factors of the mutant. Similar differences were generated using the data from wild-type data or double mutant data. On the positive side of the difference map, the side chain of the Trp residue from wild-type FVIIa was clearly visible (using the phase from the wild-type data, the maximum peak was at the 5.6 σ level), but not on the negative side of the difference map. There are clear signs of Arg side chains. This also demonstrates that the Arg residue is more flexible than the Trp residue of the wild type protein. However, it should be noted that using the likelihood-weighted 2mFo-DFc electron density map, the side chains before or after the Trp residue are not apparently observed in the 1DAN structure, which is similar to the result from the FVIIa W201R T293Y/sTF crystal structure. The location of Trp 201 is clearly visible in the FVIIa WT structure.

Regarding the orientation of the main chain of the study, the likelihood weighted 2mFo-DFc electron density map and phenix.refine refinement placed 200, 201 and 202 residues closer to the position of the replacement Trp side chain residue than the published 1DAN structure. [Banner. DW et al, Nature, 1996, 380, 41-46]. Certain words, residues Asn 200 and which has been moved to the _C α position and its wild type structure of FIG. 3 in the position apart 3.1Å. Further, in the description of the [F _obs (WT FVIIa/sTF)-F _obs (FVIIa W201R T293Y/sTF)] difference map, there is a peak indicating the movement of the residue Asn 200. A 5.7 σ positive peak is close to the position of the wild type loop configuration and the other 4.3 σ negative peak is slightly located inside the refinement double mutant configuration. This supports the view that the backbone is moved closer to the WT Trp side chain and relatively closer to the center of the FVIIa heavy chain.

The structural differences between the wild-type structure and the double mutant protein visible for residues 200, 201 and 202 of heavy chain FVIIa may depend on by filling the major hydrophobic volume in the FVIIa protein and thereby anchoring the wild-type structure Stabilization of the inward Trp 201 residue side chain in the WT structure of the loop. The side chain of the corresponding residue Arg in FVIIa W201R T293Y does not form the same rigid structure as the tightly bound side chain, but is more flexible, and thus does not anchor the ring in the same manner as the WT FVIIa structure.

Figure 3 shows two crystal structures: 1) FVIIa wild-type protein in a complex with a tissue factor by light-colored carbon atoms, using an internal data set from the same type of crystal as the PDB structure 1DAN [Banner.DW Et al, Nature, 1996, 380, 41-46], and 2) a bar graph of the comparison of the FVIIa double mutant W201R T293Y in a complex with a tissue factor by a dark carbon atom. Some of the residues are marked by a single amino acid code and end with "-wt" or "-m" for 1) and 2) respectively. A plurality of side chains have been truncated (removed outside the _C β atoms), since the likelihood 2mFo-DFc weighted electron density map of electron density does not show any of their side chains. For example, residues N200, R201 and R202 of the FVIIa double mutant W201R T293Y are truncated for this reason. The pattern was prepared by the molecular pattern software PyMOL [The PyMOL Molecular Graphics System. Version 1.6.0.0 Schrödinger, LLC].

Mutant FVIIa T293Y:

The heavy chain FVIIa Tyr 293 residue is located in the activation loop 1. Likelihood weighted 2mFo-DFc electron density map 1.0 The σ cutoff clearly shows the main chain and side chain of the Tyr residue of the refinement structure. The Tyr side chain atom C _β -C _γ is in the same direction for the C _β -C _{γ 2} atom in the wild type Thr residue. The CC _α -C _β -C _γ and CC _α -C _β -C _γ2 dihedral angles were 165° and 173° for the double mutant and the FVIIa residue 293 of the WT form, respectively. Thereby, the Tyr 293 residue of the double mutant directs its side chain along the catalytic domain direction and toward the binding site of the FFR-cmk binding inhibitor. The calculated [F _obs (WT FVIIa/sTF)-F _obs (FVIIa W201R T293Y/sTF)] difference map confirms the orientation of the Tyr side chain, has a negative peak (4.7 σ height) at the Tyr loop system and is in the absence of Thr O γ ₁ atom has a positive peak (4.2 σ height).

To investigate the possible interaction between antithrombin and the FVIIa mutant T293Y molecule, Factor Xa molecular complex and antithrombin PDB-encoding 2GD4 were performed on the FVIIa double mutant [Johnson. DJD et al., Embo J. , 2006, 25, 2029-2037] superposition. The molecular graphic software PyMOL [The PyMOL Molecular Graphics System, version 1.6.0.0 Schrödinger, LLC] was used to superimpose FXa and FVIIa molecules and produced an RMSD of 0.769 Å for 1194 atoms. Self-riding antithrombin molecular pattern, then it is apparent that the Tyr 293 residue of the FVIIa W201R T293Y mutant in the theoretical molecular complex (FVIIa W201R T293Y/anti-thrombin III) is produced, especially with Figure 4 The residues of the antithrombin molecule, Leu 395 and Arg 399, form a spatial overlap. This was confirmed by calculation of the distance between the Tyr 293 of the FVIIa double mutant and the riding antithrombin molecule by the software of the CCP4 program. A cutoff distance of 3.5 Å was used between the Tyr 293 residue in the mutant FVIIa molecule and the antithrombin molecule and the results are shown in Table 6 . FVIIa (W201R T293Y) and FXa were used as usual, after the FXa complex was superimposed on the FVIIa mutant (W201R T293Y)/sTF structure, the residue Tyr 293 of the FVIIa W201R T293Y double mutant and the anti-thrombin-S195A All distances between 3.5 Å or 3.5 Å between FXa-pentaose complex antithrombin PDB: 2GD4 [Johnson. DJD et al, Embo J., 2006, 25, 2029-2037] are summarized in Table 6 . . Spatial overlap will most likely adversely affect the likelihood that antithrombin will place its central loop of reaction (RCL) into the active site of FVIIa. Thereby, the T293Y mutant FVIIa molecule will be less susceptible to inhibition by antithrombin. This is consistent with and explained by experimental observations showing the increased resistance to inactivation by antithrombin and the extended half-life.

Figure 4 is a bar graph of the theoretical mode of the complex between antithrombin (indicated by light carbon atoms) and FVIIa W201R T293Y double mutant (indicated by dark carbon atoms). The relative positions of residues Tyr 293, Gln 255, Lys 341, Gln 286 and antithrombin molecular residues Leu 395, Arg 399, Glu 295, Tyr 253 and V317 of FVIIa mutant W201R T293Y are shown and labeled. A structural construct model based on antithrombin/FXa complex [Johnson. DJD et al, Embo J., 2006, 25, 2029-2037], PDB encodes 2GD4, in which the FXa molecule that rides antithrombin has been Superimposed on the heavy chain of the FVIIa W201R T293Y variant molecule. The residues and antithrombins of FVIIa W201R T293Y have the prefixes of "FVIIa" and "AT", followed by the one-letter amino acid code and the number of residues. The pattern was prepared by the molecular pattern software PyMOL [The PyMOL Molecular Graphics System, version 1.6.0.0 Schrödinger, LLC].

W201R T293K FVIIa variant

Pressure domain around residue 201 of FVIIa: In detail, the heavy chain FVIIa Arg 201 residue of the double mutant is located in the "60-loop" (chymotrypsin number). The main chain loop stretching was clearly observed at the 1.0 σ cutoff in the likelihood weighted 2mFo-DFc electron density map. The side chain of the Arg 201 residue (Trp residue in wild type FVIIa) was also apparently observed. However, the outer portion of the Arg 202 residue has a missing electron density in the likelihood-weighted 2mFo-DFc electron density map and at the selected cutoff, indicating higher mobility or disorder. Regarding the orientation of the backbone of the study loop ("60-ring"), it shows the transformation between the W201R T293K and the 1DAN structure [Banner, DW et al., Nature, (1996), Vol. 380, pp. 41-46] . After superimposing the two structures, it can be seen that when moving along the polypeptide from residue 197 toward 203, there is a difference of 0.64, 2.48, 3.63, 6.41, 4.15, and 0.81 Å at the equivalent C _? positions, respectively. The main chain of the loop moves closer to the position of the 1DAN WT Trp side chain position [Banner, DW et al., Nature, (1996), Vol. 380, pp. 41-46] and also towards the center of the FVIIa heavy chain, the catalytic domain shifts. The Arg 201 residue of W201R T293K FVIIa is placed in a superimposed structure at the position of the displaced WT Trp side chain residue of the published 1DAN structure.

The structural differences seen between the wild-type structure of the heavy chain "60-loop" and the W201R T293K FVIIa variant may depend on the primary hydrophobic body in the FVIIa protein. Stabilization of the inward Trp 201 residue side chain in the WT structure of the loop in the wild type structure is thereby accumulated. The side chain of the corresponding smaller residue Arg in FVIIa W201R T293K does not anchor the ring in the same manner as Trp in the WT FVIIa structure.

The region around residue 293 of FVIIa : the heavy chain FVIIa Lys 293 residue is located in the activation loop 1. The 1.0 σ cutoff at the likelihood weighted 2mFo-DFc electron density map clearly shows the main chain and side chains of the Lys residue in the refinement structure. The Lys side chain atom C _β -C _γ is in the same direction for the C _β -C _{γ 2} atom in the wild type Thr residue. The CC _α -C _β -C _γ and CC _α -C _β -C _γ2 dihedral angles were 169° and 173° for the double mutant and the FVIIa residue 293 of the WT form, respectively. Lys 293 shows "mttt" rotamer orientation, as by computer graphics software COOT [Emsley, P. et al., Acta Crystallogr. Sect. D-Biol. Crystallogr., (2010), Vol. 66, pp. 486- Page 501] The most common rotamer orientation of Lys is visible [Lovell, SC et al., Proteins, (2000), Vol. 40, pp. 389-408]. In addition, the Lys 293 residue N _ζ atom of the W201R T293K FVIIa variant forms a strong hydrogen bond with the residue Gln 176 O _ε1 atom, which has a distance of 2.68 Å, thereby stabilizing the two side chains. Compared to the WT FVIIa 1DAN structure, the Gln 176 residue thus altered its side chain configuration to optimize its hydrogen bond formation with the Lys 293 residue in the W201R T293K FVIIa variant. The rotamer is changed from the "tt0°" configuration of the WT structure to the rotamer configuration that is not in the standard configuration. [Lovell, SC et al., Proteins, (2000), Vol. 40, pp. 389-408 Page]. Thereby, the Lys 293 residue of the double mutant directs its side chain along the catalytic domain direction and toward the binding site of the FFR-cmk binding inhibitor and fills the initiation site of the FVIIa active site gap.

L288Y T293K FVIIa variant

The area around residue 288 of FVIIa:

This region is clearly visible at the 1.0 sigma cutoff in the crystal structure likelihood weighted 2mFo-DFc electron density map. Residues in the loop following the Tyr 288 residue, residues 289 to 292 in the heavy chain of the FVIIa L288Y T293K FVIIa variant show a change in the backbone configuration with the largest difference at the residue Arg 290 where the C _alpha atom is The superposition molecule of the FVIIa L288Y T293K FVIIa variant differs from the WT structure 1DAN of FVIIa by 2.87 Å [Banner, DW et al, Nature, (1996), Vol. 380, pp. 41-46]. Equivalent atoms _C α atoms of residues Tyr288 superimposed WT FVIIa in the residue Leu 288 of the display 0.80Å difference. The side chain rotamer of Tyr 288 in the FVIIa L288Y T293K FVIIa variant is "p90°", while the side chain rotamer of the Ley side chain rotamer of the WT FVIIa 1DAN structure shows "mt" orthotropy Body [Lovell, SC et al., Proteins, (2000), Vol. 40, pp. 389-408]. It produces two equivalent side chain points in different directions, showing the difference in CC _α -C _β -C _γ dihedral angles for L288Y T293K FVIIa variant and WT FVIIa, respectively: -69° and 157°. The hydroxyl group of the Tyr 288 side chain in the L288Y T293K FVIIa variant advantageously interacts with surrounding water molecules which are ordered in the crystal structure and the side chain is folded by the ring after Tyr 288 of the FVIIa L288Y T293K variant. . The structural backbone change of the loop following residue 288 and the mutation of residue 288 itself can at least partially explain the activity improvement seen by this FVIIa variant.

The area around residue 293 of FVIIa:

The 3D structure of this residue and other residues in contact therewith is highly similar to the structure described for the W201R T293K FVIIa variant. Therefore, all the conclusions about the T293K mutation of this variant also apply to the T293K mutation of the L288Y T293K FVIIa variant.

L288F T293K FVIIa variant

The area around residue 288 of FVIIa:

This region is clearly visible at the 1.0 σ cutoff in the crystal structure likelihood weighted 2mFo-DFc electron density map. The 3D structure of this region is highly similar to the L288F T293K FVIIa variant. For example, the two variants share the same backbone orientation. One difference between the two FVIIa variants is that the Phe 288 side chain has another preferred rotamer ("m-85°") about its side chain, actually with the Leu 288 side chain of WT FVIIa. Point to the same direction. The unusual property of the Phe 288 side chain of the L288F T293K FVIIa variant is that it is unusually exposed for Phe residues (according to AREAIMOL by the CCP4 crystallography program set to 145 Å ² [Bailey, S., Acta Crystallogr .Sect. D-Biol. Crystallogr., (1994), Vol. 50, pp. 760-763]).

The area around residue 293 of FVIIa:

The 3D structure of this residue and other residues in contact therewith is highly similar to the structure described for the W201R T293K FVIIa variant. Therefore, all the conclusions about the T293K mutation of this variant also apply to the T293K mutation of the L288F T293K FVIIa variant.

L288F T293K FVIIa variant

The area around residue 288 of FVIIa:

This region is clearly visible at the 1.0 sigma cutoff in the crystal structure likelihood weighted 2mFo-DFc electron density map. The 3D structure of this region is highly similar to the L288F T293K FVIIa variant. For example, the two variants share the same backbone orientation. One difference between the two FVIIa variants is that the Phe 288 side chain has another preferred rotamer ("m-85°") about its side chain, actually with WT FVIIa and L288F T293K FVIIa variants. The Leu 288 side chain of the body points in the same direction. Therefore, all the conclusions obtained from the L288F mutation of this variant also apply to the L288F mutation of the L288F T293K K337A FVIIa variant.

The region around residue 293 of FVIIa : the 3D structure of this residue and other residues in contact therewith is highly similar to the structure described for the W201R T293K FVIIa variant. Therefore, all the conclusions about the T293K mutation of this variant also apply to the T293K mutation of the L288F T293K K337A FVIIa variant.

The area around residue 337 of FVIIa:

This region was clearly observed at the 1.0 sigma cutoff in the crystal structure likelihood weighted 2mFo-DFc electron density map. The 3D structure of this region is similar to the WT structure 1DAN of FVIIa [Banner, DW et al, Nature, (1996), Vol. 380, pp. 41-46] and is similar to the other FVIIa variants of this example. The overall backbone and side chains are oriented close to the WT FVIIa 1DAN structure and other FVIIa variant structures, however, there is a small difference based on the calculated value of the maximum likelihood of phenix.refine which is slightly larger than the 0.40Å coordinate error of the crystal structure. Equivalent residues _C β atoms of between 337 1DAN FVIIa structure of WT and L288F T293K K337A FVIIa variant spaced 0.8Å. The C _α atoms of the same residue are separated by 0.4 Å. The equivalent C _α atom of residue 336 is 0.6 Å apart between the FVIIa superimposed WT structure 1DAN and the L288F T293K K337A FVIIa variant. For the Phe 332 residue, the side chain moves approximately 0.5 Å toward the Ala 337 residue of the L288F T293K K337A FVIIa variant compared to the WT structure 1DAN of FVIIa [Banner, DW et al, Nature, (1996), 380 Volume, pp. 41-46]. It can also be inferred that the other FVIIa variants of this example show substantially the same deviation from the L288F T293K K337A FVIIa variant as the WT structure 1DAN of FVIIa. In addition, other FVIIa variants clustered much closer to the WT FVIIa 1DAN structure than the L288F T293K K337A FVIIa variant. This can explain, at least in part, the changing properties of this variant.

Chemical modification of Example 9-14-FVIIa variant

Abbreviations used in Examples 9-14:

AUS: Arthrobacter ureafaciens sialidase

CMP-NAN: Cytosine-5'-monophosphate-N-acetyl-neuraminic acid

CV: column volume

GlcUA: glucuronic acid

GlcNAc: N-acetylglucosamine

GSC: 5'-glycosyl sialyl cytidine monophosphate

GSC-SH: 5'-[(4-mercaptobutyl) glycine thiol] sialic acid cytidine monophosphate

HEP: Heparin precursor polymer

HEP-GSC: GSC functionalized heparin precursor polymer

HEP-[N]-FVIIa: Heparin precursor conjugated to FVIIa via N-glycans.

HEPES: 2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethanesulfonic acid

His: histidine

PABA: p-aminobenzamide

ST3GalIII N-glycan specific a2,3-sialyltransferase

TCEP: ginseng (2-carboxyethyl) phosphine

UDP: uridine diphosphate

Quantitative methods used in Examples 9-14:

The purity of the conjugate of the invention was analyzed by HPLC. HPLC was also used to quantify the amount of isolated conjugate based on the FVIIa reference molecule. Samples were analyzed in a non-reduced or reduced form. A Zorbax 300SB-C3 column (4.6 x 50 mm; 3.5 [mu]m Agilent, catalog number: 865973-909) was used. The column was operated at 30 °C. A 5 μg sample was injected and the column was eluted with a water (A)-acetonitrile (B) solvent system containing 0.1% trifluoroacetic acid. The gradient program was as follows: 0 minutes (25% B); 4 minutes (25% B); 14 minutes (46% B); 35 minutes (52% B); 40 minutes (90% B); 40.1 minutes (25%) B). A reduced sample was prepared by adding 10 μl of TCEP/formic acid solution (70 mM ginseng (2-carboxyethyl)phosphine and 10% formic acid in water) to 25 μl/30 μg of FVIIa (or conjugate). The reaction was allowed to stand at 70 ° C for 10 minutes and then analyzed on HPLC (5 μl injection).

Starting materials used in Examples 9-14:

HEP-m-butylene imine and HEP-benzaldehyde polymer

A defined size of maleimide and aldehyde functionalized HEP polymers are prepared by enzymatic polymerization as described in US 2010/0036001. Two sugar nucleotides (UDP-GlcNAc and UDP-GlcUA) and a trisaccharide (GlcUA-GlcNAc-GlcUA) NH _{2 were} used for the initiation of the reaction, and polymerization was carried out until the sugar nucleotide construct block was depleted. The process produces a HEP polymer having a single terminal amine group. The size of the HEP polymer was determined by the ratio of sugar nucleotide to primer. The terminal amine (derived from the primer) is then functionalized with a maleimide functional group to be conjugated to GSC-SH, or functionalized with a benzaldehyde functional group to reductive amination chemical reaction to the glycine thiol end of the GSC .

HEP-benzaldehyde can be prepared by reacting an amine functionalized HEP polymer with excess N-succinimide-4-methylmercaptobenzoic acid in a neutral aqueous solution ( Nano Letters (2007), 7(8), 2207-2210). The benzaldehyde functionalized polymer can be separated by ion exchange chromatography, size exclusion chromatography or HPLC.

It can be prepared by reacting an amine functionalized HEP polymer with excess N-HEP-maleimidobutylbutylidene-oxysuccinimide (GMBS; Fujiwara, K. et al. (1988), J. Immunol. Meth. 112, 77-83).

The benzaldehyde or maleimide functionalized polymer can be separated by ion exchange chromatography, size exclusion chromatography or HPLC. Any terminal functional group by an amine (HEP-NH ₂₎ HEP of functionalized polymers can be used in embodiments of the present invention. The two choices are shown below:

The terminal sugar residue in the non-reducing end of the polysaccharide may be N-acetylglucosamine or glucuronic acid (glucuronic acid is drawn above). Typically, if a molar amount of UDP-GlcNAc and UDP-GlcUA has been used in the polymerization, it is expected that the mixture of the two sugar residues will be in the non-reducing end.

5 ' -Glycidyl sialo cytidine monophosphate (GSC):

The GSC starting material (Dufner, G. Eur. J. Org. Chem. 2000, 1467-1482) used in the present invention can be chemically synthesized or it can be obtained by a chemical enzyme route as described in WO07056191. The GSC structure is shown below:

Example 9 - Preparation of 38.8k-HEP-[N]-FVIIa L288F T293K

Step 1: Synthesis of [(4-mercaptobutyl) glycine thiol] sialic acid cytidine monophosphate (GSC-SH)

Glycosyl sialic acid cytidine monophosphate (200 mg; 0.318 mmol) was dissolved in water (2 mL), and thiobutyrolactone (325 mg; 3.18 mmol) was added. The two phase solution was gently mixed at room temperature for 21 h. The reaction mixture was then diluted with water (10 mL) and applied to a reverse phase HPLC column (C18, 50 mm x 200 mm). The column was eluted with a gradient system of water (A), acetonitrile (B) and 250 mM ammonium bicarbonate (C) at a flow rate of 50 ml/min as follows: 0 min (A: 90%, B: 0%, C: 10%) 12 min (A: 90%, B: 0%, C: 10%); 48 min (A: 70%, B: 20%, C: 10%). The eluted fraction (20 ml size) was collected and analyzed by LC-MS. The pure eluted fractions were collected and transferred slowly via a short pad of Dowex 50W x 2 (100-200 mesh) resin in sodium form, followed by lyophilization to a dry powder. The content of the title material in the lyophilized powder was then determined by HPLC using the absorbance at 260 nm and using glycidyl sialic acid cytidine monophosphate as a reference material. For HPLC analysis, a Waters X-Bridge phenyl column (5 μm 4.6 mm x 250 mm) and a water acetonitrile system (linear gradient from 0-85% acetonitrile over 30 min, containing 0.1% phosphoric acid) was used. Yield: 61.6 mg (26%). ^{LCMS: 732.18 (MH +);} 427.14 (MH + -CMP). The compound was stable for a long time (>12 months) when stored at -80 °C.

Step 2: Synthesis of 38.8 kDa HEP-GSC reagent with amber quinone imine bond

Coupling from GSC-SH ([(4-mercaptobutyl) glycine) sialyl cytosine monophosphate) from step 1 with HEP-m-butyleneimine by 1:1 molar ratio is as follows Preparation of HEP-GSC reagent: Add 35 mg of GSC-SH (0.68 mg) dissolved in 50 mM Hepes, 100 mM NaCl, pH 7.0 (50 μl) to 38.8 dissolved in 50 mM Hepes, 100 mM NaCl, pH 7.0 (1, 35 ml). k-HEP-m-butyleneimine. The clear solution was allowed to stand at 25 ° C for 2 hours. Unreacted GSC-SH was removed by dialysis using a Slide-A-Lyzer card (Thermo Scientific) at a cutoff of 10 kD. The dialysis buffer was 50 mM Hepes, 100 mM NaCl, 10 mM CaCl ₂ , pH 7.0. The reaction mixture was dialyzed twice for 2.5 hours. As in step 4 below, it is assumed that the quantitative reaction between GSC-SH and HEP-m-butyleneimine uses recycled materials. The HEP-GSC reagent prepared by this procedure will contain an HEP polymer attached to sialic acid cytidine monophosphate via an amber quinone imine bond.

Step 3: Desialylation of FVIIa L288F T293K

The sialidase ( AUS, 100 ul, 20 U) in 10 mM His, 100 mM NaCl, 60 mM CaCl ₂ , 10 mM PABA pH 5.9 (10 mL) was added to FVIIa L288F T293K (30 mg), and allowed to stand at room temperature for 1 hour. The reaction mixture was then diluted with 50 mM HEPES, 100 mM NaCl, 1 mM EDTA, pH 7.0 (30 mL) and cooled on ice. A 250 mM EDTA solution (2.6 mL) was added in small portions, and the pH was maintained neutral by the addition of sodium hydroxide. The EDTA treated sample was then applied to a 2 x 5 ml HiTrap Q FF ion exchange distillation column (Amersham Biosciences, GE Healthcare) equilibrated with 50 mM HEPES, 100 mM NaCl, 1 mM EDTA, pH 7.0. Non-binding protein was eluted with 50 mM HEPES, 100 mM NaCl, 1 mM EDTA, pH 7.0 (4 CV) followed by 50 mM HEPES, 150 mM NaCl, pH 7.0 (8 CV), followed by 50 mM HEPES, 100 mM NaCl, 10 mM CaCl ₂ , pH 7.0 ( 20 CV) elution of sialyl-based FVIIa L288F T293K. Disialyl FVIIa L288F T293K was isolated in 50 mM Hepes, 150 mM NaCl, 10 mM CaCl ₂ , pH 7.0. Yield (19.15 mg) was determined by quantifying the FVIIa L288F T293K light chain content against the FVIIa standard after reduction of ginseng (2-carboxyethyl)phosphine using reverse phase HPLC.

Step 4: Synthesis of 38.8 kDa HEP-[N]-FVIIa L288F T293K with amber quinone imine bond

Addition to 50 mM Hepes, 100 mM NaCl, 10 mM CaCl ₂ , pH 7.0 to asialo FVIIa L288F T293K (19.2 mg) in 50 mM Hepes, 100 mM NaCl, 10 mM CaCl ₂ , 10 mM PABA, pH 7.0 (18.0 mL) 38.8 kDa-HEP-GSC (35 mg from step 2) in 2.3 mL), and rat ST3GalIII enzyme (5 mg; 1.1 units) in 20 mM Hepes, 120 mM NaCl, 50% glycerol, pH 7, 0 (7.2 mL) /mg). The reaction mixture was incubated overnight at 32 ° C with slow rotation. The reaction mixture was then applied to a FVIIa-specific affinity column (CV = 24 mL) modified with a Gla-domain specific antibody and first with 2 column volumes of buffer A (50 mM Hepes, 100 mM NaCl, 10 mM CaCl ₂ , pH 7.4), followed by stage elution with 2 column volumes of Buffer B (50 mM Hepes, 100 mM NaCl, 10 mM EDTA, pH 7.4). This method essentially follows the principles described by Thim, L et al. Biochemistry (1988) 27, 7785-779. The product with the unfolded Gla-domain was collected and applied directly to a 2 x 5 ml HiTrap Q FF ion exchange distillation column (Amersham Biosciences, GE Healthcare). The column was washed with 10 mM His, 100 mM NaCl, 0.01% Tween 80, pH 7.5 (3 column volumes) and 10 mM His, 100 mM NaCl, 10 mM CaCl ₂ , 0.01% Tween 80, pH 7.5 (3.5 column volumes). The pH was then lowered to 6.0 by 10 mM His, 100 mM NaCl, 10 mM CaCl ₂ , 0.01% Tween 80, pH 6.0 (3 column volumes), and with 5 column volumes of 60% buffer A (10 mM His, 100 mM). Pretreatment of heparin by buffer mixture consisting of NaCl, 10 mM CaCl ₂ , 0.01% Tween 80, pH 6.0) and 40% buffer B (10 mM His, 1 M NaCl, 10 mM CaCl ₂ , 0.01% Tween 80, pH 6.0) material. The recovered asialo FVIIa L288F T293K (unmodified) was recycled, i.e., heparin was again protonated as described in step 4 and purified in the same manner as just described. Pooled extracts from two heparin pre-treatments were collected and concentrated by ultrafiltration (Millipore Amicon Ultra, cut off 10 kD).

Step 5: Blocking of the monosaccharide conjugated heparin precursor 38.8k-HEP-[N]-FVIIa L288F T293K

Finally, the non-sialylated N-glycan of 38.8k-HEP-[N]-FVIIa L288F T293K was capped (ie, sialylated) by ST3GalIII enzyme and CMP-NAN as follows: by ST3GalIII (0.18mg) at 32 °C /ml); CMP-NAN (4.98 mM) vs. 38.8k-HEP-[N]-FVIIa L288F T293K (5.85 mg) in 8.4 ml of 10 mM His, 100 mM NaCl, 10 mM CaCl ₂ , 0.01% Tween 80, pH 6.0 Insulation for 1 h. The reaction mixture was then applied to a FVIIa-specific affinity column modified with a Gla-domain specific antibody and first with 2 column volumes of buffer A (50 mM Hepes, 100 mM NaCl, 10 mM CaCl ₂ , pH 7.4), followed by Stage elution was carried out with 2 column volumes of Buffer B (50 mM Hepes, 100 mM NaCl, 10 mM EDTA, pH 7.4). The collected fractions containing 38.8k-HEP-[N]-FVIIa L288F T293K were combined and dialyzed against a cut-off of 10 kD using a Slide-A-Lyzer card (Thermo Scientific). The dialysis buffer was 10 mM His, 100 mM NaCl, 10 mM CaCl ₂ , 0.01% Tween 80, pH 6.0. Protein concentration was determined by light chain HPLC analysis after TCEP reduction. The total yield of 38.8k-HEP-[N]-FVIIa L288F T293K was 2.46 mg (13%).

Example 10 - Preparation of 41.5 kDa-HEP-[N]-FVIIa L288F T293K K337A

Step 1: Synthesis of 41.5 kDa HEP-GSC reagent with 4-methylbenzhydryl linkage

41.5 kDa HEP-benzaldehyde (99.7) was added to 5.0 ml of 50 mM Hepes, 100 mM NaCl, 10 mM CaCl ₂ buffer, glycine sialysine cytidine monophosphate (GSC) (20 mg; 32 μmol) in pH 7.0. Mg; 2.5 μmol). The mixture was gently rotated until all of the HEP-benzaldehyde was dissolved. During the next 2 hours, a 1 M solution of sodium cyanoborohydride in MilliQ water (5 x 50 μl) was added portionwise to reach a final concentration of 48 mM. The reaction mixture was allowed to stand overnight at room temperature. Excess GSC was then removed by dialysis as follows: The total reaction volume (5250 [mu]l) was transferred to a dialysis card (Slide-A-Lyzer dialysis card, Thermo Scientific product number 66810 with a cutoff of 10 kDa, capacity: 3-12 mL). The solution was dialyzed against 2000 ml of 25 mM Hepes buffer (pH 7.2) for 2 hours and dialyzed for another 17 hours with respect to 2000 ml of 25 mM Hepes buffer (pH 7.2). HPLC verification on a Waters X-Bridge phenyl column (4.6 mm x 250 mm, 5 μm) using a GSC as a reference and a water acetonitrile system (linear gradient of 0-85% acetonitrile over 30 minutes, containing 0.1% phosphoric acid) Excess GSC is completely removed from the internal chamber. The internal chamber material was collected and lyophilized to obtain a 41.5 kDa white powdered HEP-GSC. The HEP-GSC reagent was analyzed by NMR and on SEC chromatography. The HEP-GSC reagent prepared by this procedure contains a HEP polymer attached to sialic acid cytidine monophosphate via a 4-methylbenzhydryl bond.

Step 2: Desialylation of FVIIa L288F T293K K337A:

To a solution of FVIIa L288F T293KK337A (43.5 mg) in 21 ml of 10 mM His, 100 mM NaCl, 60 mM CaCl ₂ , 10 mM PABA, pH 6.7 buffer was added sialidase (U. urealyticum, 9 units/ml). The reaction mixture was incubated at room temperature for 1 hour. The reaction mixture was then cooled on ice and 14 ml of 10 mM His, 100 mM NaCl pH 7.7 was added. 50 ml of 100 mM EDTA solution was then added while maintaining neutral pH. The reaction mixture was then diluted with 50 ml MilliQ water and applied to a 4 x 5 ml interconnected HiTrap Q FF ion exchange distillation column (Amersham Biosciences, GE Healthcare) equilibrated in 50 mM HEPES, 50 mM NaCl, pH 7.0. Non-binding proteins including sialidase were eluted with 5 CV 50 mM HEPES, 150 mM NaCl, pH 7.0. The asialog protein was eluted with 12 CV 50 mM HEPES, 150 mM NaCl, 30 mM CaCl ₂ , pH 7.0. The fractions containing the protein were combined and 0.5 M PABA was added to reach a final concentration of 10 mM. Protein yield was determined by quantifying the FVIIa L288F T293K K337A light chain relative to the FVIIa standard after reduction of gin(2-carboxyethyl)phosphine using reverse phase HPLC as described above. In this manner, 32.5 mg of desialyl FVIIa L288F T293K K337A (2.83 mg/ml) was isolated in 11.5 ml of 50 mM Hepes, 150 mM NaCl, 30 mM CaCl ₂ , 10 mM PABA, pH 7.0.

Addition to 4.2 ml of 50 mM Hepes, 150 mM NaCl, 30 mM CaCl ₂ , 10 mM PABA, pH 7.0 in asialo FVIIa L288F T293K K337A (16.3 mg) in 4.2 ml of 20 mM Hepes, 120 mM NaCl, 50% glycerol, pH 7.0 41.5 kDa HEP-GSC (3 equivalents, 41.5 mg) and rat ST3GalIII enzyme (2.93 mg; 1.1 units/mg). The PABA concentration was then adjusted to 10 mM with 0.5 M aqueous PABA solution and the pH was adjusted to 6.7 with 1 N NaOH. The reaction mixture was incubated overnight at 32 ° C with slow agitation. A solution (356 μl) of 157 mM CMP-NAN in 50 mM Hepes, 150 mM NaCl, 10 mM CaCl ₂ , pH 7.0 was then added, and the reaction was again incubated at 32 ° C for one hour. HPLC analysis showed product distribution with unreacted FVIIa L288F T293K K337A (68%), monoheparin precursor FVIIa (25%) and various polyheparin precursor forms (7%).

The entire reaction mixture was then applied to a FVIIa-specific affinity column (CV = 24 mL) modified with a Gla-domain specific antibody and first with 2 column volumes of buffer A (50 mM Hepes, 100 mM NaCl, 10 mM CaCl _{2 )} , pH 7.4), followed by stage elution with 2 column volumes of Buffer B (50 mM Hepes, 100 mM NaCl, 10 mM EDTA, pH 7.4). This method essentially follows the principles described by Thim, L et al. Biochemistry (1988) 27, 7785-779.

The product with the unfolded Gla-domain was collected and applied directly to a 3 x 5 ml interconnected HiTrap Q FF ion exchange distillation column (Amersham Biosciences, GE Healthcare) equilibrated with a buffer containing 10 mM His, 100 mM NaCl, pH 6.0. The column was washed with 4 column volumes of 10 mM His, 100 mM NaCl, pH 6.0. With 12CV 10mM His, 100mM NaCl, 10mM CaCl ₂ , pH6.0 (extraction buffer A). The unmodified FVIIa L288F T293K K337A was eluted. 41.5 kDa-HEP-[N]-FVIIa L288F T293K K337A was subsequently eluted with 15 CV 10 mM His, 325 mM NaCl, 10 mM CaCl ₂ , pH 6.0. The purely washed fractions were combined and the protein concentration was determined by the above HPLC quantitative method. 3.42 mg (21%) of pure 41.5 kDa-HEP-[N]-FVIIa L288F T293K K337A was isolated.

Finally, a combined wash containing 41.5 kDa-HEP-[N]-FVIIa L288F T293K K337A was dialyzed against a 10 mM His, 100 mM NaCl, 10 mM CaCl ₂ , pH 6.0 using a Slide-A-Lyzer card (Thermo Scientific) with a cutoff of 10 kD. The fraction was adjusted and the concentration was adjusted to (0.40 mg/ml) by adding a dialysis buffer.

Example 11 - Preparation of 41.5 kDa HEP-[N]-FVIIa W201 T293K

This material was prepared essentially as described in Example 10. FVIIa W201R T293K (40 mg) was initially desialic acid and the asialo FVIIa W201R T293K (27.2 mg) was isolated by Gla-specific ion exchange. The asialog analog was then incubated with 41.5 kDa HEP-GSC (produced as described in Example 10) and ST3GalIII. The conjugated product was subsequently separated by ion exchange chromatography. Final buffer exchange gave 2.9 mg (7.5%) of 41.5 kDa HEP-[N]-FVIIa W201 T293K in 10 mM His, 100 mM NaCl, 10 mM CaCl ₂ , pH 6.0.

Example 12 - Preparation of 41.5 kDa HEP-[N]-FVIIa L288Y T293K

This material was prepared essentially as described in Example 10. FVIIa L288Y T293K (19.9 mg) was initially desialic acid and the asialo FVIIa L288Y T293K (16.9 mg) was isolated by Gla-specific ion exchange. The asialog analog was then incubated with 41.5 kDa HEP-GSC (produced as described in Example 10) and ST3GalIII. The conjugated product was subsequently separated by ion exchange chromatography. Final buffer exchange gave 1.95 mg (11.5%) of 41.5 kDa HEP-[N]-FVIIa L288Y T293K in 10 mM His, 100 mM NaCl, 10 mM CaCl ₂ , pH 6.0.

Example 13 - Preparation of 41.5 kDa HEP-[N]-FVIIa L288Y T293R

This material was prepared essentially as described in Example 10. After asiatic acid, desialyl FVIIa L288Y T293R (30 mg) was reacted with 41.5 kDa HEP-GSC (produced as described in Example 10) and ST3GalIII. The conjugated product was subsequently separated by ion exchange chromatography. The final buffer exchange to give 4.33mg (14.4%) 41.5kDa HEP- [ N] -FVIIa L288Y T293R, which is obtained in 10mM His, 100mM NaCl, 10mM CaCl 2, pH 6.0 in.

Example 14 - Preparation of 41.5 kDa HEP-[N]-FVIIa T293K K337A

This material was prepared essentially as described in Example 10. After asiatic acid, desialyl FVIIa L288Y T293R (8 mg) was reacted with 41.5 kDa HEP-GSC (produced as described in Example 10) and ST3GalIII. The conjugated product was separated by ion exchange chromatography. The final buffer exchange yielded 1.72 mg (15%) of 41.5 kDa HEP-[N]-FVIIa T293K in 10 mM His, 100 mM NaCl, 10 mM CaCl ₂ , pH 6.0. K337A.

Example 15 - Functional Properties of Modified Combination FVIIa Variants

The proteolytic activity and antithrombin reactivity of the FVIIa combination variant conjugated to PEG or heparin precursor (HEP) (as described in Examples 9-14) were characterized as described in Example 5. The results are summarized in Table 7. These data show that in the case of PEG or HEP, chemical modification of FVIIa reduces the proteolytic activity of the FVIIa variant, but for some variants, it is allowed to retain higher proteolytic activity than wild-type FVIIa and additionally allow retention of antithrombin resistance Sex.

Example 16 - Evaluation of hemophilia A-like whole blood thromboelastography

A thromboelastography (TEG) analysis was selected to assess the activity of FVIIa variants in whole blood thrombus A-like whole blood by comparison with FVIIa. TEG analysis provides the characteristics of the entire coagulation process - initiation, propagation, and final clot strength measurements. In addition to the possible effects of shear forces in flowing blood and vascular structures, TEG analysis simulates the viscoelastic properties of clot whole blood in this method. In vivo conditions of pseudocoagulation (Viuff, E. et al., Thrombosis Research, (2010), Vol. 126, pp. 144-149). Each TEG analysis was initiated by the use of kaolin and the TEG parameter clot time (R) and maximum thrombus generation rate (MTG) were recorded and reported in Table 8. The clot time (R) represents the latency (2 mm amplitude) from the placement of blood in the sample cup until the formation of a clot; and the maximum thrombus formation (MTG) indicates the rate of clot formation. The clot time (R) in seconds was determined using standard TEG software; and the MTG was calculated as the TEG trajectory multiplied by a one-step derivative of 100 (100 x mm/sec).

Blood samples were obtained from normal, healthy donors who are members of the Danish National Corps of Voluntary Blood Donors and meet blood donation criteria. Blood samples were taken in a 3.2% citrate blood collection tube (Vacuette reference 455322, Greiner bio-one, lot A020601 2007-02) and analyzed within 60 minutes. By adding anti-human FVIII (sheep anti-human FVIII, batch AA11-01, Haematologic Technologies, VT, USA) antibody to a final concentration of 10 Bethesda units (BU) / ml (final 0.1 mg / ml) from normal human whole blood Hemophilia A-like blood was prepared and gently spun at 2 rpm/min for 30 minutes at room temperature. Test compounds were added at final concentrations of 0.1, 1, 10 and 25 nM in addition to FVIIa L288Y T293K tested at 0.069, 0.69, 6.9, 17.3 nM and FVIIa W201R T293K tested at 0.076, 0.76, 7.6, 19.1 nM.

Data from kaolin-induced TEG showed that all compounds dose-dependently reduced clot time (R-time) and increased maximal thrombus formation (MTG) in hemophilia A-like blood (Table 8). All 40k-HEP-[N]-FVIIa-variants showed shorter or similar clot times compared to FVIIa when evaluated at the highest test concentration. In addition, the maximum thrombus formation of the variant is similar to or relative to FVIIa. In addition, the data show that 40k-heparin pro-formation of FVIIa variants reduces 40k-heparin pro-formation when compared to the corresponding FVIIa variant (no 40k-heparin precursor) The activity of the compound. Table 8 shows the R-time (clot time) and MTG (maximal thrombus formation) of the test compound in the kaolin-induced TEG in hemophilia A-like whole blood. The highest concentration of test compound was 25 nM divided by FVIIa L288Y T293Kthat tested at 17.3 nM and FVIIa W201R T293K tested at 19.1 nM. FVIIa, 40k-PEG-[N]-FVIIa and 40k-HEP-[N]-FVIIa were tested in four individual donors (n=4) and the remaining compounds were tested in two individual donors (n=2) . The data in square brackets indicates the range of parameters from the four individual donors.

Operating parameters.

Example 17 - Evaluation of PK in rats

Pharmacokinetic analysis of FVIIa variants in unmodified form or conjugated to PEG or heparin precursor (HEP) sugars was performed in rats to assess their effect on in vivo survival of FVIIa. Spirogoli rats (three/group) were administered intravenously. Collected at the appropriate time to completely form wherein Stabylite ^TM (TriniLize Stabylite tube; Tcoag Ireland Ltd., Ireland) stabilized plasma samples and frozen until further analysis. The clot activity of the plasma samples was analyzed (as described in Example 7) and the FVIIa-antithrombin complex was quantified by ELISA. Pharmacokinetic analysis was performed by using a non-compartmental method of Phoenix WinNonlin 6.0 (Pharsight). The following parameters were evaluated: Cmax (maximum concentration) of FVIIa-antithrombin complex, T1⁄2 (functional terminal half-life) of clot activity, and MRT (functional mean residence time).

Briefly, FVIIa-antithrombin complexes were measured by using enzyme immunoassay (EIA). A single anti-FVIIa antibody that binds to the N-terminus of the EGF-domain and does not block antithrombin binding is used to capture the complex (Dako Denmark A/S, Glostrup; product code O9572). Multiple anti-human AT antibody peroxidase conjugates were used for detection (Siemens Healthcare Diagnostics ApS, Ballerup/Denmark; product code OWMG 15). A preformed purification complex of human wild-type or variant FVIIa and plasma-derived human antithrombin was used as a standard for constructing an EIA calibration curve. Plasma samples were diluted and analyzed and the average concentration of repeated measurements was calculated. The analytical accuracy within the EIA is between 1% and 8%.

The pharmacokinetic evaluation parameters are listed in Table 9. The test variant exhibited a smaller accumulation of the FVIIa-antithrombin complex (rat AT complex) relative to wild-type FVIIa, with the plasma level approaching the detection level. In addition, 40k-HEP-[N]-FVIIa L288F T293K was observed (in 18.4 hours in rats) significantly extended functional half-life compared to 40k-PEG-[N]-FVIIa (7.4 hours in rats).

In conclusion, Lys present at position 293 increases T1⁄2 in rats and reduces FVIIa-antithrombin complex formation. In addition, the introduction of a glycoconjugated heparin precursor substantially improved the T1⁄2 of the rat.

Example 18 - Liquid formulation of FVIIa L288Y T293K stabilized via active site

The stability of FVIIa in solution is limited by various modifications to the polypeptide chain due to autolysis, oxidation, deamidation, isomerization, and the like. The foregoing studies have identified three sites on the heavy chain that are susceptible to self-protein attack; these sites are Arg290-Gly291, Arg315-Lys316, and Lys316-Val317 (Nicolaisen et al, FEBS, 1993, 317:245-249). The calcium free conditions further promote proteolytic release of the first 38 residues of the light chain comprising the gamma-carboxy glutamic acid (Gla) domain.

Here, we use the small molecule PCI-27483-S (2-{2-[5-(6-methylindol-1H-benzimidazol-2-yl)-6,2'-dihydroxy-5'- Amidoxime-biphenyl-3-yl]-acetamido}-succinic acid, which stabilizes the active site of FVIIa via non-covalent interactions and prevents heavy chains in liquid formulations Solubilization (see WO 2014/057069 for additional details on PCI-27483-S).

Quantification of heavy chain cleavage has been assessed by analysis of reduced FVIIa L288Y T293K by reverse phase HPLC (RP-HPLC). The assay solution was reduced in 127 mM dithiothreitol (DTT) and 3 M guanidine hydrochloride, and the solutions were incubated at 60 ° C for 15 minutes, followed by the addition of 1 μL of concentrated acetic acid (/50 μL of the initial analysis solution) and cooled to 25 °C. 25 μg of reduced FVIIa L288Y T293K was then injected onto an ACE 3 μM C4 column (300 Å, 4.6 x 100 mm; Advanced Chromatography Technologies, Inc., Scotland) equilibrated at 40 °C. The protein fragment was isolated as a linear gradient with Action Phase A consisting of 0.05% trifluoroacetic acid (TFA) in water and 35%-80% Action Phase B consisting of 0.045% TFA in 80% acetonitrile. The gradient time was 30 minutes, the flow rate was 0.7 mL/min, and the elution peak was detected at an absorbance of 215 nm.

By 1.47 mg/mL CaCl ₂ , 7.5 mg/mL NaCl, 1.55 mg/mL L-histamine, 1.32 mg/mL glycine glycine, 0.5 mg/mL L-methionine, 0.07 mg/ Formulation of FVIIa L288Y T293K with mL polysorbate 80, 0.021 mg/mL PCI-27483-S, protein concentration of 1 mg/mL (ie, 1:1.75 protein inhibitor molar ratio) and final pH of 6.8 . The solution was incubated at 30 ° C for 1 month under static conditions and away from light. As can be seen in Table 10, the presence of PCI-27483-S resulted in near complete inhibition of heavy chain cleavage of FVIIa L288Y T293K; whereas the addition of PCI-27483-S resulted in a significant increase in cleavage at positions 315-316 and 290-291. .

Table 10 Heavy chain on day 0 corresponding to two different cleavage sites as determined in RP-HPLC chromatograms after 28 days of incubation with and without PCI-27483-S inhibitor The percentage of the peak area of the fragment increases.

Example 19 - Computer Simulation Evaluation of Immunogenic Risk

Computer simulation studies to investigate whether a novel peptide sequence produced by a protein engineered to produce a FVIIa analog can produce a class II major histocompatibility complex (MHC-II) (also known as HLA-II) that binds to the human body. Peptide sequence. This binding is essential for the presence of T cell epitopes. The peptide/HLA-II binding prediction soft system used in this study is based on two algorithms, NetHHCIIpan 2.1 for HLA-DR prediction (Nielsen et al. 2010) and NetMHCII 2.2 for HLA-DP/DQ prediction (Nielsen et al. 2009). ).

The immunogenicity risk score (IRS) was calculated to enable comparison of different FVIIa analogs in terms of immunogenic risk potential. Calculations were performed as follows: FVIIa wild type was used as a reference and only predicted 15mers in the reference (FVIIa wild type) with a predicted rank of 10 or less were included in the analysis. The HLA-II dual genes are classified into three categories: a first type having a weight of 2 (rank number <=1), a second type having a weight of 0.5 (1>rank number <=3), and a weight having a weight of 0.2. Class 3 (3>rank number <=10). The class weight (2.5 or 0.2) is multiplied by the frequency of the dual gene (for each population) to obtain the IRS. Calculate the sum of IRS for each population and each HLA-II (DRB1, DP, and DQ).

The calculated risk scores for the selected single and combined variants are provided in Table 11. Particularly advantageous combinations include L288F/T293K, L288F/T293K/K337A, L288Y/T293K, and L288Y/T293K/K337A, which simultaneously exhibit high proteolytic activity and less susceptibility to inhibition by antithrombin.

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<211> 254

<212> PRT

<213> Dog

<400> 4

<210> 5

<211> 255

<212> PRT

<213> Pigs

<400> 5

<210> 6

<211> 255

<212> PRT

<213> Cattle

<400> 6

<210> 7

<211> 253

<212> PRT

<213> mouse

<400> 7

<210> 8

<211> 253

<212> PRT

<213> Rat

<400> 8

<210> 9

<211> 253

<212> PRT

<213> Rabbit

<400> 9

Claims (15)

A Factor VII polypeptide comprising two or more substitutions relative to the amino acid sequence of human Factor VII (SEQ ID NO: 1), wherein T293 is by Lys (K), Arg (R), Tyr (Y) Or Phe(F) substitution; and L288 is replaced by Phe(F), Tyr(Y), Asn(N), Ala(A) or TrpW and/or W201 by Arg(R), Met(M) or Lys (K) substitution and / or K337 is replaced by Ala (A) or Gly (G). The Factor VII polypeptide of claim 1, wherein T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F) and L288 is by Phe (F), Tyr (Y), Asn (N), Ala (A) or Trp (W) substitution. A Factor VII polypeptide according to claim 1 or 2, wherein T293 is replaced by Lys (K) and L288 is replaced by Phe (F). A Factor VII polypeptide according to claim 1 or 2, wherein T293 is replaced by Lys (K) and L288 is replaced by Tyr (Y). A Factor VII polypeptide according to claim 1 or 2, wherein T293 is replaced by Arg (R) and L288 is replaced by Phe (F). A Factor VII polypeptide according to claim 1 or 2, wherein T293 is replaced by Arg (R) and L288 is replaced by Tyr (Y). The Factor VII polypeptide of any one of claims 1 to 6, wherein K337 is substituted by Ala (A). A Factor VII polypeptide according to claim 1, wherein T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F) and W201 is by Arg (R), Met (M) or Lys (K) replacement. a Factor VII polypeptide according to claim 8 wherein T293 is replaced by Lys (K) and W201 is replaced by Arg(R). A Factor VII polypeptide according to any one of the preceding claims, wherein the Factor VII polypeptide is coupled to at least one half-life extending moiety. The Factor VII polypeptide of claim 10, wherein the half-life extending moiety is selected from the group consisting of biocompatible fatty acids and derivatives thereof, such as hydroxyethyl starch (HES) hydroxyalkyl starch (HAS), polyethylene. Alcohol (PEG), poly(Glyx-Sery)n (HAP), hyaluronic acid (HA), heparosan polymer (HEP), phosphocholine-based polymer (PC polymer), Fleximer, polydextrose , polysialic acid (PSA), Fc domain, transferrin, albumin, elastin-like peptide (ELP), XTEN polymer, PAS polymer, PA polymer, albumin binding peptide, CTP peptide, FcRn binding peptide and Any combination of them. The Factor VII polypeptide of claim 11, wherein the half-life extending moiety is a heparin precursor polymer. The FVII polypeptide according to any one of claims 1 to 12, wherein the proteolytic activity is wild-type human factor VIIa as measured by in-tube proteolytic analysis in the absence of soluble tissue factor. At least 110% of the proteolytic activity of SEQ ID NO: 1); and as measured by antithrombin inhibition assay in the presence of low molecular weight heparin and in the absence of soluble tissue factor, compared to wild-type human Factor VIIa , its antithrombin reactivity is less than 20%. A Factor VII polypeptide for use as a pharmaceutical product according to any one of claims 1 to 13. A factor VII polypeptide as claimed in claim 14 for use as a medicament for the treatment of a coagulopathy.