FVIII fusion protein conjugate with extended half-life and its applications

Abstract

The present invention relates to a blood coagulation factor FVIII-fusion protein conjugated to polyethylene glycol having an extended circulating half-life, a method for preparing the same, and its applications.

Description

Technical Field

[0001] Cross-reference This application claims priority based on a Chinese patent application with application number 202210577314.2, filed with the China National Intellectual Property Administration on May 25, 2022, and the entire content thereof is incorporated herein by reference.

[0002] The present invention relates to the field of biopharmaceuticals, and particularly to a recombinant human blood coagulation factor VIII fusion protein conjugate with an extended half-life, a method for producing the same, and its applications.

Background Art

[0003] Hemophilia A is a hereditary blood coagulation disorder caused by a decrease in the activity or functional deficiency of blood coagulation factor VIII (FVIII). Replenishing high-activity FVIII preparations is an effective treatment method for hemophilia A. The FVIII molecule is one of the longest gene fragments that can be cloned to date and is a protein pharmaceutical with the largest molecular weight used clinically. Since the FVIII preparation has a relatively short half-life in plasma, only about 8 to 12 hours, intravenous administration of the FVIII preparation is required about three times a week for prophylactic treatment of severe hemophilia A patients.

[0004] The monomer-dimer hybrid recombinant FVIII-Fc fusion protein (Eloctate) developed by Bioverativ was approved and launched by the US Food and Drug Administration (FDA) in June 2014. According to clinical data, its half-life in the human body is 18.8 hours, only 1.5 - 1.7 times longer than that of the natural protein (Dumont J A et al., Blood, 2012, 119:3024 - 3030; Powell JS et al., Blood, 2012, 119:3031 - 3037), indicating that injections once every 3 - 5 days are still required. Also, in the expression products of HEK-293 cells transfected with the rFVIII Fc and Fc dual-expression vectors constructed by Bioverativ, the expected fusion protein with the rFVIIIFc homodimer structure was not detected, and only the monomer-dimer hybrid rFVIIIFc fusion protein and Fc dimer were expressed.

[0005] To prepare a long-acting formulation of a protein-based drug, it is common to chemically modify the surface of the protein-based drug using a highly soluble polymer such as polyethylene glycol (PEG). Generally, the higher the modification rate, the more significant the reduction in the antigenicity of the protein, and the greater the loss of activity. Currently, it has been reported that Novo Nordisk (N8-GP), Bayer (BAY94-9027), and Baxter (Bax 855) have extended the half-life of FVIII with polyethylene glycol (PEG) and succeeded in developing a long-acting PEGylated FVIII formulation and initiated clinical studies. However, according to pharmacokinetic research data, it was shown that the half-life of the PEGylated FVIII formulation was not significantly extended (Tiede A et al., J Thromb Haemost. 2013;11:670 - 678), (Coyle T et al., Haemophilia. 2012;18(Suppl 3):22), (Turecek PL et al., Hamostaseologie, 2012, 32 Suppl 1:S29 - 38).

[0006] Hemophilia patients need to receive lifelong injections of blood coagulation factors for hemostasis or bleeding prevention. Therefore, in order to reduce the number of administrations, it is required to develop blood coagulation factor preparations with an extended half-life. Also, how to maintain good biological activity while extending the half-life is an issue to be solved.

Summary of the Invention

Problems to be Solved by the Invention

[0007] WO 2019 / 219049 discloses an FVIII-Fc fusion protein modified with PEG that can reduce the severe bleeding rate and 12-hour rebleeding rate of model animals and increase the 48-hour survival rate of model animals in a hemophilia A mouse tail vein transection model.

[0008] The inventors further conducted intensive research and accidentally discovered that arbitrary Fc regions and arbitrary numbers of PEG modifications do not have the same effect of extending the half-life of the FVIII-Fc fusion protein. Instead, for a specific FVIII fragment, an Fc region derived from IgG and a specific number of PEG modifications can bring about an unexpected effect of significantly extending the half-life. According to clinical data, the PEGylated fusion protein according to the present invention has a half-life of 30.87 to 31.91 hours, which is 2.10 to 2.25 times better than Advate and 2.61 to 2.80 times longer than the native protein. Therefore, a dosing frequency of once a week is achieved, and the dosing frequency of the therapeutic agent is significantly reduced. Thus, for patients who require long-term administration, there is no problem taking the medicine on weekends and they can work or study normally on weekdays. The compliance of the patients is significantly improved, and the quality of life of the patients is remarkably enhanced. The present invention has been made in view of this.

Means for Solving the Problems

[0009] According to the present invention, the following technical methods are provided. According to a first aspect of the present invention, there is provided a conjugate of a blood coagulation factor VIII-Fc fusion protein comprising an active portion (FVIII) of blood coagulation factor VIII and an Fc fragment, and polyethylene glycol (PEG), wherein the active portion (FVIII) of the blood coagulation factor VIII and the Fc fragment are directly linked or indirectly linked via a linker so as to form the fusion protein, and the average number of PEG modifications in the conjugate is 3 to 8, 3 to 7, 3 to 6, 4 to 8, 4 to 7, 4 to 6 or 4.2 to 5.1, preferably 4 to 6, more preferably 4.2 to 5.1. As used herein, the average number of PEG modifications of the conjugate is the molar ratio of the PEG to the fusion protein, and may be understood as the average value of the number of PEGs possessed by each modified fusion protein in a sample (where the number of fusion proteins contained therein is one or more). Since the number of PEG molecules possessed by each modified fusion protein may be different, the average value may be an integer or a non-integer.

[0010] In some embodiments, the modifying agent for PEG modification has a structure represented by the following formula (2). Formula (2)

Chemical formula

[0011] In some embodiments, the active portion of the blood coagulation factor VIII is a full-length or truncated human blood coagulation factor VIII, preferably human blood coagulation factor VIII with the B domain truncated, more preferably a peptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a polypeptide having at least 90%, 95% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and still having FVIII activity.

[0012] In some other embodiments, the Fc fragment is an Fc fragment derived from IgG, preferably an Fc fragment derived from IgG1, IgG2 or IgG4, more preferably an Fc fragment derived from IgG2.

[0013] In some other embodiments, the amino acid sequence of the Fc fragment is (i) the amino acid sequence shown in SEQ ID NO: 3, (ii) the amino acid sequence shown in SEQ ID NO: 4, and (iii) a sequence selected from the amino acid sequence shown in SEQ ID NO: 5.

[0014] In some other embodiments, the conjugation of the fusion protein and PEG is random conjugation or site-specific conjugation. The conjugation site is a free amino group, thiol group, glycosyl group and / or carboxyl group, preferably a free amino group.

[0015] In some other embodiments, the linker contains a flexible unit and a rigid unit.

[0016] In some other embodiments, the flexible unit contains two or more amino acid residues selected from glycine, serine, alanine and threonine. Preferably, the flexible peptide linker has the following general formula: (GS) a (GGS) b (GGGS) c(GGGGS) d It has an array represented by (where a, b, c, and d are integers of 0 or more, and satisfy the relationship of a + b + c + d ≥ 1).

[0017] Preferably, the flexibility unit is (i) GSGGGSGGGGSGGGGS (SEQ ID NO: 6), (ii) GSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 7), (iii) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 8), (iv) GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 9), and (v) includes an amino acid sequence selected from the group consisting of GGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 10).

[0018] In some other embodiments, the rigidity unit is the carboxy-terminal peptide of human chorionic gonadotropin β subunit, or has 70%, 80%, 90%, 95% or more identity with the amino acid sequence of the carboxy-terminal peptide of human chorionic gonadotropin β subunit. Further, the rigidity unit may contain one, two or more glycosylation sites.

[0019] Preferably, the rigidity unit is (i) PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 11), (ii) SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 12), (iii) SSSSKAPPPS (SEQ ID NO: 13), (iv) SRLPGPSDTPILPQ (SEQ ID NO: 14), and (v) includes an amino acid sequence selected from the group consisting of GSGGGGSGGGGSGGGGSGGGGSGGGGSSSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 15).

[0020] In some other embodiments, the blood coagulation factor VIII-Fc fusion protein sequentially comprises, from the N-terminus to the C-terminus, a B-domain-deleted human blood coagulation factor VIII, a flexibility unit, a rigidity unit, and an Fc fragment. The B-domain-deleted human blood coagulation factor VIII comprises the amino acid sequence shown in SEQ ID NO: 2, the flexibility unit comprises the amino acid sequence shown in SEQ ID NO: 7, the rigidity unit comprises the amino acid sequence shown in SEQ ID NO: 12, and the Fc fragment comprises the amino acid sequence shown in SEQ ID NO: 4. The B-domain-deleted human blood coagulation factor VIII comprises the amino acid sequence shown in SEQ ID NO: 2, the flexibility unit comprises the amino acid sequence shown in SEQ ID NO: 6, the rigidity unit comprises the amino acid sequence shown in SEQ ID NO: 11, and the Fc fragment comprises the amino acid sequence shown in SEQ ID NO: 4. The B-domain-deleted human blood coagulation factor VIII comprises the amino acid sequence shown in SEQ ID NO: 2, the flexibility unit comprises the amino acid sequence shown in SEQ ID NO: 7, the rigidity unit comprises the amino acid sequence shown in SEQ ID NO: 12, and the Fc fragment comprises the amino acid sequence shown in SEQ ID NO: 5. Or The B-domain-deleted human blood coagulation factor VIII comprises the amino acid sequence shown in SEQ ID NO: 2, the flexibility unit comprises the amino acid sequence shown in SEQ ID NO: 6, the rigidity unit comprises the amino acid sequence shown in SEQ ID NO: 11, and the Fc fragment comprises the amino acid sequence shown in SEQ ID NO: 5.

[0021] In some embodiments, the PEG molecule is linked to the primary amine (-NH2) group on the lysine residue in the blood coagulation factor VIII-Fc fusion protein via a succinimidyl carboxymethyl ester (SCM) which is an active linker. Preferably, the modifier for PEG modification has a structure represented by the following formula (2). Formula (2)

Chemical formula

[0022] According to a second aspect of the present invention, there is provided a pharmaceutical composition comprising the conjugate and a pharmaceutically acceptable carrier.

[0023] According to a third aspect of the present invention, there is provided the use of the conjugate in the manufacture of a medicament for preventing and / or treating a bleeding disorder. Preferably, the bleeding disorder is a bleeding disorder in a patient with congenital or acquired FVIII deficiency, or spontaneous or surgical bleeding in a patient with hemophilia A.

[0024] According to a fourth aspect of the present invention, there is provided a method for preventing and / or treating a bleeding disorder, comprising administering the conjugate to a subject in need thereof. Preferably, the bleeding disorder is selected from a bleeding disorder in a patient with congenital or acquired FVIII deficiency, and spontaneous or surgical bleeding in a patient with hemophilia A.

[0025] According to the present invention, there is provided a method for producing the conjugate according to the first aspect, step 1 of producing the blood coagulation factor VIII-Fc fusion protein, step 2 of reacting the fusion protein obtained in step 1 with PEG (where the molar ratio of PEG to the fusion protein is (50 to 120):1, preferably 100:1, and PEG is a branched PEG having a molecular weight of 30 to 50 kD, preferably 40 kD), and step 3 of purifying the conjugate obtained in step 2.

[0026] In some embodiments, the reaction conditions include reacting at 20°C ± 5°C for 1 to 3 hours, preferably for 2 hours. To more clearly illustrate the embodiments of this application and the technical methods of the prior art, the following briefly introduces the drawings required for the embodiments and the prior art. Obviously, the drawings described below are only some embodiments of the present invention, and those skilled in the art may obtain other embodiments from these drawings without creative labor.

Brief Description of the Drawings

[0027]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Modes for Carrying Out the Invention

[0028] Hereinafter, to make the objectives, technical solutions, and advantages of the present invention more clear, the present invention will be described in more detail with reference to the drawings and embodiments. Obviously, the embodiments described herein are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative labor are also included within the protection scope of the present invention.

[0029] The term "Factor VIII" is also called Factor VIII or FVIII, and is a large and complex glycoprotein mainly produced by hepatocytes. The term "active portion of Factor VIII" refers to the portion showing FVIII activation of the fusion protein of the present invention. Native human FVIII consists of 2351 amino acids, including a signal peptide, and contains a plurality of different domains limited by homology, and has three A domains, one specific B domain, and two C domains. These domains are arranged in the order of NH2-A1-A2-B-A3-C1-C2-COOH. FVIII circulates in plasma as two chains separated at the B-A3 boundary. These two chains are linked by divalent metal ion bonds. Here, the A1-A2-B chain is called the heavy chain (HC), and A3-C1-C2 is called the light chain (LC).

[0030] Endogenous Factor VIII molecules circulate in vivo as a molecular library having B domains of different sizes. In vivo, sequential enzymatic cleavage of the B domain occurs, producing a molecular library having B domains of different sizes. It is generally considered that the occurrence of cleavage at position 740 (whereby the last part of the B domain is cleaved) is related to thrombin activation.

[0031] In the present invention, "Factor VIII" includes its naturally occurring wild-type sequence (for example, SEQ ID NO: 1), and variants such as mutant proteins obtained by substitution, deletion or addition of one or more amino acids and simultaneously maintaining the activation of Factor VIII.

[0032] In one embodiment, the blood coagulation factor VIII is a molecule with the B domain cleaved, and the remaining domains basically correspond to the sequences shown by amino acid numbers 1 to 745 and 1640 to 2332 shown in SEQ ID NO: 1. Further, the B domain-cleaved molecule of the present invention may slightly differ from the sequence shown by SEQ ID NO: 2, that is, the remaining domains (i.e., three A domains and two C domains) have one or more amino acid substitutions, additions or deletions with respect to the amino acid sequence shown by SEQ ID NO: 2. For example, there are differences of one, two, three, four, five, six, seven, eight, nine, ten or more amino acids, or differences of about 1%, 2%, 3%, 4% or 5%. Such differences modify the binding ability of factor VIII to various other components (e.g., LRP, multiple receptors, other coagulation factors, cell surface) while maintaining the basic activity of factor VIII, and glycosylation sites and the like are introduced and / or removed.

[0033] For the Fc fragment, modifications can be made to improve other functions such as binding to complement and / or binding to specific Fc receptors. Mutations at positions 234, 235, and 237 of the IgG Fc domain can usually lead to a decrease in binding to the FcγRI receptor and further a decrease in binding to the FcγRIIa and FcγRIII receptors. These mutations do not change the binding to the FcRn receptor and promote the extension of the circulating half-life through the endocytosis recycling pathway. The modified IgG Fc domain of the fusion protein of the present invention preferably contains one or more of the mutations (L234A, L235E, and G237A) that result in a decrease in affinity for a specific Fc receptor and the mutations (A330S and P331S) that result in a reduction in complement binding via C1q.

[0034] The polyalkylene glycol (PEG) in the present invention may be linear or branched. The main chain of the branched polymer is well-known in the art. Generally, a branched polymer has a central branched core portion and one or more linear polymer chains linked to this central branched core. In the present invention, it is preferable to use PEG in a branched form. In one embodiment, the branched polyethylene glycol is represented by the general formula R(-PEG-OH)m (wherein R represents a core portion such as glycerol or pentaerythritol, and m represents the number of arms).

[0035] In one embodiment, the branched PEG (also referred to as mPEG, methoxy-PEG for example) has 2 branches, and in that case, it is also referred to as "Y-shaped" PEG (mPEG for example), that is, a branched PEG containing 2 PEGs or linear methoxy PEGs.

[0036] In certain embodiments of the present invention, PEG modification (i.e., conjugation) is used, and more preferably, mPEG modification is used. Here, the modification is a random modification or a site-specific modification. The sites for the modification include free amino groups, thiol groups, glycosyl groups and / or carboxyl groups, preferably free amino groups.

[0037] In this specification, the PEG molecule for cross-linking reaction with a protein is also called a modifier. Generally, it is an activated polyethylene glycol (also called a polyethylene glycol modifier or a PEG modifier), that is, a polyethylene glycol having a functional group such as an active linker. In this specification, terms such as "modifier", "modifier for PEG modification", "polyethylene glycol modifier" and "PEG modifier" can be used interchangeably herein.

[0038] In a specific embodiment of the present invention, the modifier used for the random modification of the free amino group may be one selected from mPEG-SS (methoxypolyethylene glycol-succinimidyl succinate), mPEG-SC (methoxypolyethylene glycol-succinimidyl carbonate), mPEG-SPA (methoxypolyethylene glycol-succinimidyl propionate), mPEG-SG (methoxypolyethylene glycol-succinimidyl glutarate), and the like. The modifier for the N-terminus is one selected from mPEG-ALD (methoxypolyethylene glycol-acetaldehyde), mPEG-pALD (methoxypolyethylene glycol-propionaldehyde), mPEG-bALD (methoxypolyethylene glycol-butylaldehyde), and the like. The shapes of the modifiers mPEG-SS, mPEG-SC, mPEG-SPA, mPEG-SG, mPEG-ALD, mPEG-pALD, and mPEG-bALD are linear or branched.

[0039] In a specific embodiment of the present invention, the modifier used for the random modification of the free thiol group is one selected from mPEG-mal (methoxypolyethylene glycol-maleimide), mPEG-OPSS (methoxypolyethylene glycol-orthopyridyl disulfide), mPEG-Vinylsulfone (methoxypolyethylene glycol-vinyl sulfone), mPEG-Thiol (methoxypolyethylene glycol-thiol), and the like.

[0040] In a specific embodiment of the present invention, the modifier used for the random modification of the glycosyl group and / or carboxyl group is mPEG-ZH (methoxypolyethylene glycol-hydrazide).

[0041] In a specific embodiment of the present invention, the structure of the modifier for the mPEG modification is represented by the following formula (1). Formula (1)

Chemical formula

[0042] In certain embodiments of the present invention, the structure of the modifier for the mPEG modification is represented by the following formula (2). Formula (2)

Chemical formula

[0043] The size of the polymer main chain can be changed, but typical ranges for polymers (e.g., PEG, mPEG, PPG, or mPPG) are from about 0.5 KD to about 160 KD, for example, from about 1 KD to about 100 KD. More specifically, the size of each conjugated hydrophilic polymer of the present invention mainly varies within the ranges of about 1 KD to about 80 KD, about 2 KD to about 70 KD, about 5 KD to about 70 KD, about 10 KD to about 60 KD, about 20 KD to about 50 KD, about 30 KD to about 50 KD, or about 30 KD to 40 KD. It should be understood that these sizes are not exact measured values but represent approximate values. This is a common practice in the art.

[0044] In one specific embodiment, the size of the PEG or mPEG used in the present invention is preferably 35 KD or more (i.e., not less than 35 KD), 40 KD or more, 45 KD or more, 50 KD or more, 55 KD or more, 60 KD or more, 65 KD or more, or 70 KD or more. For example, specifically, the molecular weight is 40 KD, 50 KD, 60 KD, 70 KD, 80 KD, 90 KD, 100 KD, 110 KD, 120 KD, 130 KD, 140 KD, 150 KD, or 160 KD.

[0045] In one specific embodiment, the blood coagulation factor VIII-Fc fusion protein sequentially includes, from the N-terminus to the C-terminus, a B-domain-deleted human blood coagulation factor VIII, a flexibility unit, a rigidity unit, and an Fc fragment. Here, the B-domain-deleted human blood coagulation factor VIII includes the amino acid sequence shown in SEQ ID NO: 2, the flexibility unit includes the amino acid sequence shown in SEQ ID NO: 7, the rigidity unit includes the amino acid sequence shown in SEQ ID NO: 12, and the Fc fragment includes the amino acid sequence shown in SEQ ID NO: 4 (FL1G2-0). The B-domain-deleted human blood coagulation factor VIII includes the amino acid sequence shown in SEQ ID NO: 2, the flexibility unit includes the amino acid sequence shown in SEQ ID NO: 6, the rigidity unit includes the amino acid sequence shown in SEQ ID NO: 11, and the Fc fragment includes the amino acid sequence shown in SEQ ID NO: 4 (FL2G2-0). The B-domain-deleted human blood coagulation factor VIII includes the amino acid sequence shown in SEQ ID NO: 2, the flexibility unit includes the amino acid sequence shown in SEQ ID NO: 7, the rigidity unit includes the amino acid sequence shown in SEQ ID NO: 12, and the Fc fragment includes the amino acid sequence shown in SEQ ID NO: 5 (FL1G4-0), or alternatively the B-domain-deleted human blood coagulation factor VIII includes the amino acid sequence shown in SEQ ID NO: 2, the flexibility unit includes the amino acid sequence shown in SEQ ID NO: 6, the rigidity unit includes the amino acid sequence shown in SEQ ID NO: 11, and the Fc fragment includes the amino acid sequence shown in SEQ ID NO: 5 (FL2G4-0).

[0046] In another specific embodiment, the conjugate is FL 1G2-0, FL2G2-0, FL1G4-0 or FL2G4-0 randomly modified with PEG.

[0047] In another specific embodiment, the random modification includes that the PEG molecule is linked to the primary amine (-NH2) group on the lysine residue in the fusion protein via a succinimidyl carboxymethyl ester (SCM) which is an active linker.

[0048] In another specific embodiment, the PEG is a branched PEG (Y-shaped PEG) containing two PEGs or linear methoxy PEG.

[0049] In another specific embodiment, the PEG has a structural formula represented by formula (2). In formula (2), m2 is 2, m3 is 1, and the PEG preferably has a molecular weight of 40 kD.

[0050] In another specific embodiment, the PEG modifier represented by formula (2) forms an amide bond with the primary amine on the lysine residue (shown by the following formula (4)), and the PEG molecule is linked to the fusion protein molecule. Formula (4)

Chemical formula

[0051] In another specific embodiment, the PEG in the conjugate has a structure represented by the following formula (5). Formula (5)

Chemical formula

[0052] The term "improved circulating half-life" means that the molecule of the present invention has a modified circulating half-life, preferably an extended circulating half-life, compared to the wild-type Factor VIII molecule. The circulating half-life is preferably at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 100%, more preferably at least 125%, more preferably at least 150%, more preferably at least 175%, more preferably at least 200%, and most preferably at least 250% or 300% extended. Even more preferably, the circulating half-life of said molecule is extended by at least 400%, 500%, 600%, or even 700%.

[0053] The term "pharmaceutically acceptable carrier" includes, but is not limited to, saline, buffers, glucose, water, glycerol, alcohol and combinations thereof. Usually, the pharmaceutical preparation should be prepared according to the administration mode. The pharmaceutical composition of the present invention is prepared in the form of an injection, and is prepared by a general method using, for example, an aqueous solution containing physiological saline or glucose and other adjuvants. It is preferable to prepare the said pharmaceutical composition under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount. Also, the pharmaceutical preparation of the present invention may be prepared in the form of a sustained-release preparation.

Example

[0054] Example 1: Preparation and purification of hFVIII fusion protein A series of hFVIII fusion protein expression plasmids were constructed according to molecular cloning techniques well-known to those skilled in the art, and each expression plasmid was transfected into DHFR-deficient CHO cells (see U.S. Patent US4,818,679), and each hFVIII fusion protein was expressed (Table 1). For the specific preparation and purification procedures of the fusion proteins, see Chinese Patent ZL201610692838.0 and International Publication No. 19 / 219049. They are hereby incorporated by reference into this specification.

[0055] Table 1: Composition and Sequence of Fusion Proteins

Table 1

[0056] The hFVIII-L1-G1 and hFVIII-L2-G1 fusion proteins are disclosed in International Publication No. 19 / 2219049 and are used as controls in this application, corresponding to hFVIII-L1-Fc (FL1F-0) and hFVIII-L2-Fc (FL2F-0), respectively.

[0057] Example 2: Preparation and Purification of hFVIII Fusion Proteins Modified with mPEG According to the description in International Publication No. 19 / 2219049, FL1G1-40Y modified with 40 kD PEG (40 kD, Y-shaped PEG-modified FL1G1-0, and the subsequent naming rules are the same) was prepared and purified.

[0058] In this example, the hFVIII fusion protein FL1G2-40Y modified with 40 kD PEG was prepared and purified according to the following method.

[0059] A Y-shaped Y-SCM-40K PEG with a molecular weight of 40 kD and an active linker of SCM (the molecular formula is represented by Formula (2). However, m2 is 2 and m3 is 1. Manufactured by Beijing KeyGen Biotech Co., Ltd., Y-NHS-40K) and an hFVIII fusion protein were used for a cross-linking reaction. The active linker of PEG is succinimidyl carboxymethyl ester, which can easily react with the primary amine (-NH2) group on the lysine residue in the protein to form a stable amide bond, so a protein-PEG cross-linked product can be obtained. Y-SCM-40K PEG and the hFVIII fusion protein FL1G2-0 after filtration and concentration were weighed, and conjugates were prepared under the condition that the molar ratio of PEG:fusion protein was, for example, 30:1, 50:1, 100:1, 120:1. Here, the raw materials were charged at a molar ratio of PEG:protein = 100:1 (mass ratio is 10.26:1), reacted at 20°C ± 5°C for 2 hours, filtered through a 0.2 μm filter membrane after cross-linking, and the sample was stored at 2 - 8°C for a while and then further purified. The conjugate prepared in this way (the conjugate of this example) showed an excellent half-life extension effect in subsequent detections.

[0060] Purification: First, it was isolated by S200 (manufactured by GE Healthcare) molecular sieve chromatography. Using the binding buffer (binding buffer: 20 mM His-HCl, 0.1 M NaCl, 5 mM CaCl2, 0.02% Tween 80, pH 6.8 - 7.2), the chromatography column was equilibrated with a linear flow rate of 150 cm / h and a liquid feed volume of 3 - 5 column volumes (CV). The sample was injected into the column at a linear flow rate of 150 cm / h. After the completion of sample injection, using the equilibration buffer (20 mM His-HCl, 200 mM NaCl, 5 mM CaCl2, 0.02% Tween 80, pH: 6.8 - 7.2), the chromatography column was washed with a linear flow rate of 150 cm / h and a liquid feed volume of 3 - 5 column volumes (CV) until the pH and electrical conductivity matched the buffer. It was eluted using the buffer (20 mM His-HCl, 0.1 M NaCl, 5 mM CaCl2, 0.02% Tween 80, pH 6.8 - 7.2), and the sample at a peak where A280 / 260 was greater than 1.8 was collected. In step 2, it was isolated using a Source 15Q (manufactured by GE Healthcare) anion chromatography column. Using the binding buffer (binding buffer: 20 mM His-HCl, 0.1 M NaCl, 5 mM CaCl2, 0.02% Tween 80, pH 6.8 - 7.2), the chromatography column was equilibrated with a linear flow rate of 150 cm / h and a liquid feed volume of 3 - 5 column volumes (CV). The sample isolated by the molecular sieve chromatography in step 1 was injected into the column at a linear flow rate of 150 cm / h. After the completion of sample injection, using the equilibration buffer (20 mM His-HCl, 0.1 M NaCl, 5 mM CaCl2, 0.02% Tween 80, pH 6.8 - 7.2), the chromatography column was washed with a linear flow rate of 150 cm / h and a liquid feed volume of 3 - 5 column volumes (CV) until the pH and electrical conductivity matched the buffer.Elution was carried out at a linear velocity of 100 cm / h from 0 to 100% using elution buffer (20 mM His-HCl, 2 M NaCl, 5 mM CaCl2, 0.02% Tween 80, pH 6.8 - 7.2). Samples at elution peaks with A280 / 260 greater than 1.8 were collected in separate test tubes, and purity detection and the average number of PEG modifications were confirmed respectively.

[0061] Purity measurement of FL1G2-40Y by SEC-HPLC method Measurement was performed using a TSKgel UltraSW Aggregate chromatographic column (7.8 mm × 300 mm, 3 μm). The mobile phase was (0.3 mol / L arginine, 0.2 mol / L sodium chloride, 0.01 mol / L calcium chloride anhydrous, 0.02 mol / L histidine, 0.02% poloxamer 188, pH 7.0)-10% acetonitrile. Isocratic elution was carried out with a flow rate of 0.5 mL / min and an ultraviolet detection wavelength of 280 nm. The test sample was diluted to a concentration of about 0.30 mg / mL using a dilution buffer. 100 μL of the test solution was taken and injected into the liquid chromatography. The purity of the test sample was calculated by the area percentage method. As shown in Figure 1, the purity of the test sample was 99.5%.

[0062] Measurement of the average number of PEG modifications of FL1G2-40Y by the combined method of SEC-HPLC-UV-RID Measurement was performed using a TSKgel UltraSW Aggregate chromatographic column (7.8 mm × 300 mm, 3 μm), with the mobile phase being (0.3 mol / L arginine, 0.2 mol / L sodium chloride, 0.01 mol / L calcium chloride anhydrous, 0.02 mol / L histidine, 0.02% poloxamer 188, pH 7.0)-10% acetonitrile. Isocratic elution was carried out at a flow rate of 0.5 mL / min, with an ultraviolet detection wavelength of 280 nm, a column temperature of 25 °C, and a differential refractive index detector temperature of 30 °C. The UV peak area and RI peak area of the hFVIII fusion protein, the UV peak area and RI peak area of the purified conjugate solution, and the RI peak area of the PEG standard substance solution were measured and recorded. Standard curves were created using the hFVIII fusion protein and the PEG standard substance respectively. FL1G2-40Y was diluted with a dilution buffer to a concentration of about 0.20 mg / mL, 100 μL was taken and injected into liquid chromatography, and the average modification number of the test sample PEG was calculated by the external standard method.

[0063] The results of the modification number were calculated as follows. 1) The PEG moiety in the conjugate has no UV absorption peak at a wavelength of 280 nm, and the UV absorption value of the conjugate with the same protein content is the same as that of the fusion protein before modification. Therefore, the content of the fusion protein moiety in the purified conjugate was calculated by the standard curve method. 2) Since the RI absorption value of the fusion protein solution and the corresponding component concentration show a linear relationship, the RI absorption value of the fusion protein moiety in the conjugate was determined by the standard curve method. 3) Since the RI absorption value of the conjugate is the sum of the RI absorption value of the PEG moiety in the conjugate and the RI absorption value of the fusion protein moiety, the RI absorption value of the PEG moiety in the conjugate was determined. 4) Since the RI absorption value of PEG and the corresponding component concentration show a linear relationship, the content of the PEG moiety in the conjugate was calculated by the standard curve method. 5) The ratio of the number of moles of the PEG moiety to the number of moles of the fusion protein moiety in the conjugate was the modification number of PEG in the single protein.

[0064] For exemplary RI chromatography and UV chromatography, please refer to FIGS. 2-6. The measurement data are shown in Tables 2-4 below. The linear equations showing the relationship between the RI peak area and the component concentration of the hFVIII fusion protein and the PEG standard substance are Y = 2E+06X - 197.15 (R 2 = 0.9998), Y = 1E+06X + 1960.1 (R 2 = 0.9998), respectively. The linear equation showing the relationship between the UV peak area and the component concentration of the rhFVIII fusion protein is Y = 17590X - 13.542 (R 2 = 0.9999). The average values of the corresponding RI peak areas and UV peak areas of the conjugates after purification at 0.20 mg / mL from different lots were 579850.509 and 3483.775, respectively. Thus, the calculated result of the average number of PEG modifications in the test conjugate was approximately 4.6.

[0065] Table 2: Linear relationship between RI peak area and concentration of hFVIII fusion protein

Table 2

[0066] Table 3: Linear relationship between RI peak area and concentration of PEG standard substance solution

Table 3

[0067] Table 4: Linear relationship between UV peak area and concentration of hFVIII fusion protein

Table 4

[0068] As a result of detecting the average number of PEG modifications in FL1G2-40Y prepared from different lots, similar results of 4.2 - 5.1 were obtained, as shown in Table 5 below.

[0069] Table 5: Detection Results of Average Number of PEG Modifications in FL1G2-40Y in Different Lots

Table 5

[0070] Example 3: Direct Measurement of Biological Activity of Fusion Protein by One-Step Coagulation Method The method for measuring the titer of human blood coagulation Factor VIII used in this example is also called the one-step coagulation method. For the specific procedure, please refer to the general rules in the third part of the Chinese Pharmacopoeia 2020 edition. The biological activity of FVIII by the one-step coagulation method is measured by the ability to correct the prolonged clotting time of plasma due to the deficiency of Factor VIII. A kit Coagulation Factor VIII Deficient Plasma (Cat.No.OTXW 17) manufactured by German Siemens was used. The test method included diluting the WHO FVIII activity reference substance to 1 IU / ml with 5% FVIII-deficient plasma, and then diluting the above solution 10-fold, 20-fold, 40-fold, and 80-fold with 5% FVIII-deficient plasma respectively, and measuring the clotting time for each. The logarithm of the activity of the known reference substance and the measured clotting time were obtained respectively, and linear fitting was performed to plot the standard curve. The test sample was diluted to about 1 IU / ml with 5% FVIII-deficient plasma, and then diluted 10-fold and 20-fold with 5% FVIII-deficient plasma respectively, and the clotting time was measured in the same way. After substituting into the standard curve to obtain the numerical value of the titer of the test sample FVIII, the specific activity (unit: IU / mg) of the test sample FVIII was calculated. As a result of the test, the corresponding specific activities of FL1G1-40Y and FL1G2-40Y were 1210 IU / mg and 1300 IU / mg respectively, indicating that the specific activity of FL1G2-40Y was stronger than that of FL1G1-40Y.

[0071] Example 4: Pharmacokinetic Study of hFVIII Fusion Protein Modified with PEG in HA Mice Eighteen HA mice were randomly divided into three groups: the FL1G1-40Y group, the FL1G2-40Y group, and the recombinant factor VIII (Xyntha formulation) control group (6 males / group), and each group was injected intravenously once at 200 IU / kg. Blood samples (about 0.12 mL) were collected from the non-administered site of the subcutaneous vein in the hind limbs of the animals into 1.5 mL sodium citrate blood collection tubes (1:9). The blood collection times were 0 hours before administration, 10 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 32 hours, 48 hours, 72 hours, and 96 hours after the start of administration for each group of animals. Centrifugation was performed at 2-8°C within 30 minutes after blood collection (about 5000 rpm, 5 minutes). After separating the plasma samples, they were dispensed into two test tubes and stored in an ultra-low temperature freezer (-70°C to -90°C). It was specified that the process from blood collection of the plasma samples to centrifugation should be completed within 2 hours. The drug concentration in the plasma was measured and analyzed using a validated ELISA method, and the pharmacokinetic parameters of each administration group were calculated using the compartment model analysis method of Phoenix WinNonlin software (Certara L.P., version 8.2). The pharmacokinetic parameter results of each group are shown in Table 6 below. As a result of the test, when administered at the same dose, the corresponding half-lives of the control drug Xyntha, FL1G1-40Y, and FL1G2-40Y were 9.46 hours, 15.53 hours, and 20 hours, respectively. From this result, after modification with PEG, FL1G2-40Y was shown to have a 2.11-fold and 1.29-fold improvement in half-life compared to Xyntha and FL1G1-40Y, respectively, and obtained a significantly extended half-life, which has not been reported in the prior art.

[0072] Table 6: PK-related parameters in HA mice

Table 6

[0073] Example 5: Pharmacokinetic study of PEG-modified hFVIII fusion protein in cynomolgus monkeys Twenty-four cynomolgus monkeys were randomly divided into four groups (half males and half females, 6 monkeys / group). Groups 1-3 were each intravenously injected with FL1G2-40Y at 50, 125, and 300 IU / kg, respectively, and Group 4 was administered Xyntha at 125 IU / kg. Blood samples (about 1.8 mL) were collected from the non-injected site of the subcutaneous vein in the hindlimb of the animals into 2 mL sodium citrate blood collection tubes (1:9). The blood collection times were 0 hours before administration, 10 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 32 hours, 48 hours, 72 hours, 96 hours, 120 hours, and 168 hours after the start of administration for each group of animals. Centrifugation was performed at 2-8°C within 30 minutes after blood collection (about 4000 rpm, 5 minutes). After separating the plasma samples, they were dispensed into two test tubes and stored in an ultra-low temperature freezer (-70°C to -90°C). It was specified that the process from blood plasma sample collection to centrifugation should be completed within 2 hours. The drug concentration in the plasma was measured and analyzed using a validated ELISA method, and the pharmacokinetic parameters of each administration group were calculated using the compartment model analysis method of Phoenix WinNonlin software (Certara L.P., version 8.2). The pharmacokinetic parameter results of each group are shown in Table 7 below. As a result of the test, when administered at the same dose of 125 IU / kg, the corresponding half-lives of the control drug Xyntha and FL1G2-40Y were 7.57 hours and 27.8 hours, respectively. It was shown that FL1G2-40Y had a 3.67-fold improvement in half-life compared to Xyntha, which has not been reported in the prior art.

[0074] Table 7: PK-related parameters in cynomolgus monkeys

Table 7

[0075] Example 6: Pharmacokinetic study of hFVIII fusion protein modified with PEG in patients with severe hemophilia A Selection Criteria: The conditions that the subjects must meet are as follows. 1) 12 years old ≤ age < 60 years old, male; 2) Clinically diagnosed as severe hemophilia A patient (Factor VIII coagulation < 1%), and it has been confirmed by past medical records that the subject has received Factor VIII coagulation agent treatment (EDs ≥ 150); 4) Non-acute bleeding state; 5) Negative for past Factor VIII coagulation inhibitors (< 0.6 BU) and no inhibitor family history; 6) Platelet count > 100,000 cells / μL; 7) Prothrombin time is normal or INR < 1.3; 8) Thrombin time (TT) is normal; 9) Past test results of vWF-related tests are normal; 10) Lupus anticoagulant factor is negative.

[0076] Clinical Design: In the trial, two dosing groups were set up, with the dosages being 25 IU / kg and 50 IU / kg respectively, and the number of effective cases in each group was set to be 6 or more. First, the subjects were administered Advate (recombinant human Factor VIII coagulation, ADVATE) as a control drug once, and then the test drug was administered once. The subjects in each group were admitted to the clinical trial center on the day before dosing (-1 day), and were intravenously administered on an empty stomach in the morning on the dosing day.

[0077] The plasma drug concentration was measured and analyzed using a validated ELISA method, and the kinetic parameters of each dosing group were calculated using the compartment model analysis method of Phoenix WinNonlin software (Certara L.P., version 8.2). The results of the pharmacokinetic parameters of each group are shown in Table 8 below.

[0078] Table 8: PK-related Parameters in Severe Hemophilia A Patients

Table 8

[0079] As a result, when administered at doses of 25 IU / kg and 50 IU / kg, the half-lives of Advate were 14.18 hours and 14.68 hours, respectively. When FL1G2-40Y was administered at doses of 25 IU / kg and 50 IU / kg, the half-lives were 31.91 hours and 30.87 hours, respectively. Compared with Advate, the half-lives were improved by 2.25 times (25 IU / kg) and 2.10 times (50 IU / kg), respectively. The marketed drug Xyntha had a half-life of 13.76 hours in the body of patients aged 12 and above when administered at a dose of 50 IU / kg, which is equivalent to that of Advate administered at the same dose. Therefore, compared with Xyntha, FL1G2-40Y was reasonably predicted to have a significantly longer half-life in the body and a significantly extended half-life.

[0080] It should be noted that the above description is only the optimal embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and principle of the present invention are all included in the protection scope of the present invention.

Claims

1. A conjugate of a blood coagulation factor VIII-Fc fusion protein containing the active portion (FVIII) of blood coagulation factor VIII and an Fc fragment, and polyethylene glycol (PEG), wherein the active portion (FVIII) of the blood coagulation factor VIII and the Fc fragment are directly linked or indirectly linked via a linker so as to form the fusion protein, the average number of PEG modifications in the conjugate is 3 to 8, 3 to 7, 3 to 6, 4 to 8, 4 to 7, 4 to 6, or 4.2 to 5.1, preferably 4.2 to 5.1, provided that the average number of modifications is the molar ratio of the PEG to the fusion protein, the conjugate.

2. The modifying agent for PEG modification is represented by the following formula (2): Formula (2) 【Chemical 1】 (However, m ２ is in the range of 0 ≤ m ２ ≤ 6, preferably 2, and m ３ is in the range of 0 ≤ m ３ ≤ 6, preferably 1, and mPEG- represents a polyethylene glycol group having one end capped with a methoxy group) and has a structure represented by Preferably, the molecular weight of the PEG is 30 kD to 50 kD, preferably 40 kD, the conjugate according to claim 1.

3. The active portion of the blood coagulation factor VIII is a full-length or truncated human blood coagulation factor VIII, preferably a B-domain truncated human blood coagulation factor VIII, more preferably a peptide containing the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a polypeptide having at least 90%, 95% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and still having FVIII activity, the conjugate according to claim 1 or 2.

4. The Fc fragment is an Fc fragment derived from IgG, preferably an Fc fragment derived from IgG1, IgG2 or IgG4, more preferably an Fc fragment derived from IgG2, the conjugate according to any one of claims 1 to 3.

5. The amino acid sequence of the Fc fragment is (i) the amino acid sequence shown in SEQ ID NO: 3, (ii) the amino acid sequence shown in SEQ ID NO: 4, and (iii) an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 5, the conjugate according to claim 4.

6. The linker contains a flexible unit and a rigid unit, the conjugate according to any one of claims 1 to 5.

7. wherein the flexibility unit has an array represented by the following general formula: (GS) ａ (GGS) ｂ (GGGS) ｃ (GGGGS) ｄ (wherein a, b, c and d are integers of 0 or more, and satisfy the relationship of a + b + c + d ≧ 1). Preferably, the flexible unit is (i) GSGGGGGSGGGGGSGGGGG (SEQ ID NO: 6), (ii) an amino acid sequence selected from the group consisting of GSGGGGGSGGGGGSGGGGGSGGGGGSGGGGGS (SEQ ID NO: 7), (iii) GGGGGGSGGGGGSGGGGGSGGGGGS (SEQ ID NO: 8), (iv) GSGGGGGSGGGGGSGGGGGSGGGGGSGGGGGSGGGGGSGGGGGSGGGGGS (SEQ ID NO: 9), and (v) an amino acid sequence selected from the group consisting of GGGGSGGGGGSGGGGGSGGGGGS (SEQ ID NO: 10), the conjugate according to claim 6.

8. The rigid unit contains the carboxyl-terminal peptide of human chorionic gonadotropin β subunit, Preferably, the rigid unit is (i) PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 11), (ii) SSSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 12), (iii) SSSSSKAPPPS (SEQ ID NO: 13), (iv) SRLPGPSDTPILPQ (SEQ ID NO: 14), and (v) an amino acid sequence selected from the group consisting of GSGGGGGSGGGGGSGGGGGSGGGGGSGGGGGSSSSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 15), the conjugate according to claim 6.

9. The conjugate in which the blood coagulation factor VIII-Fc fusion protein sequentially contains a B domain-deleted human blood coagulation factor VIII from the N-terminus to the C-terminus, a flexibility unit, a rigidity unit, and an Fc fragment, the B domain-deleted human blood coagulation factor VIII contains the amino acid sequence shown in SEQ ID NO: 2, the flexibility unit contains the amino acid sequence shown in SEQ ID NO: 7, the rigidity unit contains the amino acid sequence shown in SEQ ID NO: 12, and the Fc fragment contains the amino acid sequence shown in SEQ ID NO: 4, the B domain-deleted human blood coagulation factor VIII contains the amino acid sequence shown in SEQ ID NO: 2, the flexibility unit contains the amino acid sequence shown in SEQ ID NO: 6, the rigidity unit contains the amino acid sequence shown in SEQ ID NO: 11, and the Fc fragment contains the amino acid sequence shown in SEQ ID NO: 4, the B domain-deleted human blood coagulation factor VIII contains the amino acid sequence shown in SEQ ID NO: 2, the flexibility unit contains the amino acid sequence shown in SEQ ID NO: 7, the rigidity unit contains the amino acid sequence shown in SEQ ID NO: 12, and the Fc fragment contains the amino acid sequence shown in SEQ ID NO: 5, or or The conjugate according to any one of claims 1 to 8, wherein the B-domain-deleted human blood coagulation factor VIII contains the amino acid sequence represented by SEQ ID NO: 2, the flexible unit contains the amino acid sequence represented by SEQ ID NO: 6, the rigid unit contains the amino acid sequence represented by SEQ ID NO: 11, and the Fc fragment contains the amino acid sequence represented by SEQ ID NO:

5.

10. The B-domain-deleted human blood coagulation factor VIII contains the amino acid sequence represented by SEQ ID NO: 2, the flexible unit contains the amino acid sequence represented by SEQ ID NO: 7, the rigid unit contains the amino acid sequence represented by SEQ ID NO: 12, and the Fc fragment contains the amino acid sequence represented by SEQ ID NO: 4, The PEG molecule is linked via a succinimidyl carboxymethyl ester (SCM), which is an active linker, to the primary amine (—NH ２ 2) group on a lysine residue in the blood coagulation factor VIII-Fc fusion protein, The modifier for PEG modification is represented by the following formula (2): Formula (2) [Chemical 2] (However, m ２ is 2, and m ３ is 1), and has a structure represented by the above, and the molecular weight of the PEG is 40 kD. The conjugate according to claim 9.

11. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

12. Use of the conjugate according to any one of claims 1 to 10 in the manufacture of a medicament for preventing and / or treating hemorrhagic diseases.

13. The use according to claim 12, wherein the hemorrhagic disease is a hemorrhagic disease in a patient with congenital or acquired FVIII deficiency or spontaneous or surgical bleeding in a patient with hemophilia A.

14. A method for preventing and / or treating hemorrhagic diseases, the method comprising administering the conjugate according to any one of claims 1 to 10 to a subject in need thereof, Preferably, the hemorrhagic disease is selected from hemorrhagic diseases in patients with congenital or acquired FVIII deficiency and spontaneous or surgical bleeding in patients with hemophilia A.

15. A method for producing the conjugate according to any one of claims 1 to 10, 1) a step of producing the blood coagulation factor VIII-Fc fusion protein; 2) a step of reacting the fusion protein obtained in step 1) with PEG (wherein the molar ratio of PEG to the fusion protein is (50 to 120):1, preferably 100:1, and PEG is a branched PEG having a molecular weight of 30 to 50 kDa, preferably 40 kDa); 3) a step of purifying the conjugate obtained in step 2).

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