TWI535454B - Conjugated factor viii molecules - Google Patents

Description

Conjugated factor VIII molecule

The present invention relates to conjugated Factor VIII molecules. In particular, the invention relates to conjugated Factor VIII molecules having an improved circulating half-life.

Hemophilia A is an inherited bleeding disorder caused by deficiency or dysfunction of factor VIII (FVIII) activity. The clinical manifestation is not that the first stage of hemostasis - the formation of blood clots occurs normally - but that the clot is unstable due to the lack of stage II thrombin formation. The disease is treated by intravenous injection of blood or recombinantly produced factor FVIII.

Current treatment recommendations are shifting from traditional on-demand treatment to prevention. The circulating half-life of endogenous FVIII is 12-14 hours and thus prophylactic treatment requires several times a week to allow the patient to obtain a substantially asymptomatic life. Intravenous (IV) administration causes significant inconvenience and/or pain for many people, especially children and minors. Thus, there is a need in the art for a novel Factor VIII product having Factor VIII activity which is preferably structurally homogeneous, preferably safe, and preferably has a significantly extended circulating half-life to reduce the number of weekly doses of Factor VIII. In addition, there is a need in the industry for a relatively simple method for obtaining and producing such molecules.

It is known in the art to PEGylate Factor VIII to extend the circulating half-life. However, obtaining a safe product with a homogenous structure and a significantly improved cyclic half-life is still an obstacle. Methods that can be used to generate conjugated Factor VIII molecules are generally laborious and/or often result in lower yields and/or product structure identities. WO2008011633 proposes the use of artificially constructed O-linked glycosylation sites to obtain treatments with prolonged therapeutic protein circulating half-life Sexual proteins, however, do not reveal conjugated factor VIII molecules.

In a first aspect, the invention relates to a B domain truncated Factor VIII molecule having an improved circulating half-life, the molecule covalently covalently with a hydrophilic polymer via an O-linked oligosaccharide in a truncated B domain Conjugation in which Factor VIII activation results in the removal of covalently conjugated side groups.

In other aspects, the invention is further directed to methods of obtaining such molecules, uses of such molecules, and pharmaceutical compositions comprising such molecules.

Accordingly, provided herein are conjugated Factor VIII molecules having an improved circulating half-life in which a conjugated pendant group (eg, a hydrophilic polymer) is removed upon activation. The molecules of the invention preferably have a homogenous structure - at least for the position of the hydrophilic polymer in the truncated B domain - and preferably have advantageous security properties. In addition, a relatively simple method of obtaining such molecules is further provided herein. Preferably, the activated Factor VIII molecule of the invention is similar to endogenously activated Factor VIII.

In the figure, the size of the conjugated group is sometimes referred to as "K", which has the same meaning as KDa (kiloton) herein.

Figure 1: Schematic representation of the PEGylation process of O-linked oligosaccharides. This figure does not represent an exhaustive list of possible ways of achieving the products obtained in the examples.

Figure 2: Ion exchange chromatography (A) of the reaction mixture on Source 15Q. SDS-PAGE (B) of the molecular marker (left) of the collected fractions.

Figure 3: Purification of the capped product on a superdex 200 size exclusion chromatography.

Figure 4: Coagulation activity of O-glycol PEGylated rFVIII using various aPTT reagents. (A) shows the ratio of coagulation activity to chromogenic activity. (B) shows specific clotting activity.

Figure 5: In vivo effect (blocking time) of 40K-PEG-[O]-N8 in FVIII KO mice.

Figure 6: A flow chart showing the process steps involved in the production of the sugar PEGylation factor FVIII in accordance with the present invention.

Figure 7: Schematic representation of the Factor VIII molecule of the invention produced in the Examples.

Figure 8: shows a representative branched PEG polymer useful in the examples of the invention, which is referred to herein as "SA-glycerol-PEG."

definition:

Factor VIII Molecule: FVIII/Factor VIII is a large complex glycoprotein produced primarily by hepatocytes. FVIII consists of 2351 amino acids, including signal peptides, and is defined by homology to contain several different domains. There are three A domains, one B domain, and two C domains. The domain sequence can be listed as NH2-A1-A2-B-A3-C1-C2-COOH. FVIII circulates in plasma in the form of two strands separated at the B-A3 border. The chains are linked by a divalent metal ion. The A1-A2-B chain is referred to as the heavy chain (HC), and the A3-C1-C2 is referred to as the light chain (LC).

The endogenous Factor VIII molecule circulates in vivo in the form of an aggregate of molecules having different size B domains. Progressive enzymatic removal of the B domain is likely to occur in vivo, resulting in aggregates of molecules with different size B domains. It is generally believed that the occurrence of cleavage at position 740 (by which the last portion of the B domain is removed) is associated with thrombin activation. However, this does not exclude that, for example, a Factor VIII variant that has been disrupted by the cleavage site at position 740 may be active.

As used herein, "Factor VIII" or "FVIII" refers to a human plasma glycoprotein that is a member of the endogenous coagulation pathway and is essential for coagulation. "Native FVIII" is a full length human FVIII molecule shown in SEQ ID NO. 1 (amino acid 1-2332). The B domain spans the amino acid 741-1648 in SEQ ID NO 1.

SEQ ID NO 1:

The Factor VIII molecule of the invention is a B domain truncated factor FVIII molecule, wherein the remaining domains correspond to amino acids 1-740 and 1649-2332 of SEQ ID NO. The sequence shown. Thus, the molecules of the invention are recombinant molecules produced in a transformed host cell, preferably derived from a mammal. However, the remaining domains (ie, the three A domains and the two C domains) may differ slightly from the amino acid sequences (amino acids 1-740 and 1649-2332) shown in SEQ ID NO: 1, for example, 1%, 2%, 3%, 4% or 5%. Specifically, amino acid modifications (substitutions, deletions, etc.) are introduced in the remaining domains to improve, for example, the binding of Factor VIII to various other components such as vW factors, LPR, multiple receptors, other coagulation factors, cell surfaces, and the like. The ability seems to be feasible. Furthermore, it may also be feasible for the Factor VIII molecules of the invention to include other post-translational modifications in, for example, a truncated B domain and/or one or more other domains of such molecules. Such other post-translational modifications may be in a variety of molecular forms that are conjugated to the Factor VIII molecules of the invention, such as polymeric compounds, peptide compounds, fatty acid-derived compounds, and the like.

The Factor VIII molecule of the present invention has Factor VIII activity whether modified in the B domain or whether it has other post-translational modifications, meaning that it can function in a similar or equivalent manner to FVIII function in the coagulation cascade. Interaction with FIXa on activated platelets induces the formation of FXa and supports the formation of blood clots. This activity can be assessed in vitro by techniques well known in the art, such as clot analysis, endogenous thrombin potential analysis, and the like. The Factor VIII molecule of the invention has at least about 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least FVIII activity of native human FVIII. 90%, and 100% or even 100% or more.

B domain: The B domain in Factor VIII spans the amino acid 741-1648 in SEQ ID NO 1. The B domain is cleaved at several different sites, resulting in greater heterogeneity of circulating plasma FVIII molecules. The exact function of the highly glycosylated B domain is not known. It is known that this domain is not essential for FVIII activity in the coagulation cascade. The following facts support this apparent lack of function: B domain deleted/truncated FVIII appears to have the same in vivo characteristics as seen for full length native FVIII. Despite this, there are indications that the B domain can reduce the attachment to the cell membrane, at least in serum-free conditions.

B-domain truncated/deleted factor VIII molecule: The endogenous full-length FVIII synthesized is in the form of a single-stranded precursor molecule. Prior to secretion, the precursor is cleaved into heavy and light chains. Recombinant B domain deleted FVIII can be produced by two different strategies. Synthesis of a B-domain-free heavy and light chain (double-stranded strategy) in the form of two different polypeptide chains, or a B-domain deleted FVIII in the form of a single precursor polypeptide chain, respectively, with a full-length FVIII precursor In the same way, it is cleaved into heavy and light chains (single chain strategy).

In a B domain deleted FVIII precursor polypeptide, the heavy and light chain portions are typically separated by a linker. To minimize the risk of introducing an immunogenic epitope in a B domain deleted FVIII, the linker sequence is preferably derived from the FVIII B domain. The linker must comprise a protease recognition site that separates the B domain deleted FVIII precursor polypeptide into a heavy chain and a light chain. Amino acids 1644-1648 constitute this recognition site in the B domain of full length FVIII. The thrombin site that results in the removal of the linker upon activation of the B domain deleted FVIII is located in the heavy chain. Thus, the size of the linker and the amino acid sequence are unlikely to affect its removal from the remaining FVIII molecules by thrombin activation. Deletion of the B domain facilitates the production of FVIII. Nonetheless, the B domain portion can be included in the connector without reducing productivity. The negative effect of the B domain on productivity is not due to the B domain of any particular size or sequence.

The truncated B domain may contain several O-glycosylation sites. However, according to a preferred embodiment, the molecule comprises only one, or two, three or four O-linked oligosaccharides in the truncated B domain.

According to a preferred embodiment, the truncated B domain comprises only one potential O-glycosylation site and the hydrophilic polymer is covalently conjugated to the O-glycosylation site.

The O-linked oligosaccharide in the B domain truncated molecule of the invention can be attached to an O artificially produced by recombinant means and/or by exposing a "hidden" O-glycosylation site by truncating the B domain. - glycosylation site. In both cases, the molecules can be produced by designing a B domain truncated Factor VIII amino acid sequence and subsequently subjecting the amino acid sequence to in silico analysis to predict truncation Probability of O-glycosylation sites in the B domain. Can be suitable Molecules having such glycosylation sites are synthesized in host cells with relatively high probability, and then the glycosylation pattern is analyzed and subsequently the molecules having O-linked glycosylation in the truncated B domain are selected. Suitable host cells for the production of recombinant Factor VIII proteins are preferably derived from mammals to ensure that the molecule is glycosylated. In the practice of the invention, cell line mammalian cells, more preferably established mammalian cell lines, including but not limited to CHO (eg, ATCC CCL 61), COS-1 (eg, ATCC CRL 1650), baby hamster kidney (BHK), and HEK293 (eg, ATCC CRL 1573; Graham et al, J. Gen. Virol. 36: 59-72, 1977) cell lines. A preferred BHK cell line is the tk-ts13 BHK cell line (Waechter and Baserga, Proc. Natl. Acad. Sci. USA 79: 1106-1110, 1982), hereinafter referred to as BHK 570 cells. The BHK 570 cell line is available under the ATCC Accession No. CRL 10314 from the American Type Culture Collection, 12301 Parklawn Dr., Rockville, MD 20852. The tk-ts13 BHK cell line is also available from ATCC under accession number CRL 1632. A preferred CHO cell line is the CHO K1 cell line derived from CCC61 from ATCC and the cell lines CHO-DXB11 and CHO-DG44.

Other suitable cell lines include, but are not limited to, rat Hep I (rat hepatoma; ATCC CRL 1600), rat Hep II (rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), human lung ( ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1); DUKX cells (CHO cell line) (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77: 4216-4220, 1980) (DUKX cells are also known as DXB11 cells) ), and DG44 (CHO cell line) (Cell, 33: 405, 1983; and Somatic Cell and Molecular Genetics 12: 555, 1986). 3T3 cells, Namalwa cells, myeloma, and fusions of myeloma with other cells can also be used. In some embodiments, the cell can be a mutant or recombinant cell, eg, a zymogram line of a catalytic protein post-translational modification that exhibits a qualitative or quantitative difference in performance compared to the cell type from which it is derived (eg, such as a glycosyltransferase and/or Or a cell such as a glycosylase such as a glycosidase or a processing enzyme such as a propeptide. DUKX fine Cells (CHO cell lines) are particularly preferred.

The currently preferred cell lines are HEK293, COS, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) and myeloma cells, and Chinese hamster ovary (CHO) cells are particularly preferred.

Thus, the inventors of the present invention have shown that a "hidden" O-glycosylation site in the Factor VIII B domain can be activated by truncating the B domain. Although not wishing to be bound by any theory, this phenomenon can be attributed to the tertiary structure of the molecule in the altered truncated B domain. This allows the "hidden" O-glycosylation site to be glycosylated in the truncated B domain. One advantage of this approach is to provide recombinant molecules with advantageous safety properties (e.g., in terms of allergenicity). Another advantage may be that it represents a relatively simple method of obtaining a B domain truncation variant with an O-linked oligosaccharide in the B domain, since the glycosylation site in the B domain is inherently sufficient, previously confirmed in It is difficult to construct artificial O-glycosylation sites in recombinant proteins.

The length of the B domain in the wt FVIII molecule is about 907 amino acids. The length of the truncated B domain of the molecules of the invention may vary from about 10 amino acids to about 700 amino acids, such as from about 12 to about 500 amino acids, from 12 to 400 amino acids, 12- 300 amino acids, 12-200 amino acids, 15-100 amino acids, 15-75 amino acids, 15-50 amino acids, 15-45 amino acids, 20-45 Amino acid, 20-40 amino acids, or 20-30 amino acids. The truncated B domain may comprise a heavy chain fragment and/or a light chain fragment and/or an artificially introduced sequence not found in the wt FVIII molecule. The terms "B domain truncation" and "B domain deletion" are used interchangeably herein.

Improved circulating half-life: The molecules of the invention have an improved circulating half-life, preferably a prolonged circulating half-life, compared to wild-type Factor VIII molecules. Preferably, the cycle half-life is extended by 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%, and 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%, more preferably 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% . Even more preferably, the circulating half-life of the molecules is extended by at least 400%, 500%, 600%, or even 700% relative to the circulating half-life of wild-type FVIII.

Hydrophilic polymer: The modifying group/hydrophilic polymer of the present invention is preferably non-naturally occurring. In one example, a "non-naturally occurring modifying group" is a polymeric modifying group in which at least one polymeric moiety is not naturally occurring. In another example, the non-naturally occurring modifying group is a modified carbohydrate. The position at which the modifying group is functionalized is selected so as not to prevent the "modified sugar" from being added to the polypeptide in an enzymatic manner. "Modified sugar" also refers to any glycosyl analog moiety that is functionalized with a modifying group and is a substrate of a native or modified enzyme (eg, a glycosyltransferase).

The polymeric modifying group added to the polypeptide can alter the properties of the polypeptide, for example, its bioavailability, biological activity, or its in vivo half-life. Examples of polymers of the present invention include water soluble polymers which may be linear or branched and which may include one or more independently selected polymeric moieties such as polyalkylene glycols and derivatives thereof. The polymer modifying groups of the present invention may include water soluble polymers such as polyethylene glycol and its derivatives (PEG, m-PEG), polypropylene glycol and its derivatives (PPG, m-PPG), and the like.

The term "water soluble" refers to a moiety that has a certain detectable solubility in water. Methods for detecting and/or quantifying water solubility are well known in the art. Exemplary water soluble polymers of the invention include peptides, saccharides, polyethers, polyamines, polycarboxylic acids, and the like. The peptide may have a mixed sequence and may be composed of a single amino acid, for example, a polylysine. An example of a polysaccharide is polysialic acid. An example of a polyether is polyethylene glycol, for example, m-PEG. An example of a polyamine is polyethyleneimine, and a polyacrylic acid is a representative polycarboxylic acid.

The polymer backbone of the water soluble polymer of the present invention may be polyethylene glycol (i.e., PEG). The term PEG in connection with the present invention includes polyethylene glycol in any form, including alkoxy PEG, difunctional PEG, multi-arm PEG, forked PEG, branched PEG, pendant PEG (ie having one or a plurality of PEG or related polymers flanked by functional groups of the polymer backbone, or a PEG having a degradable bond.

The polymer backbone can be linear or branched. Branched polymer backbones are well known in the art. Typically, a branched polymer has a central branched core portion and a plurality of polymeric linear chains attached to the central branched core. PEG is often used in a branched form, which can be prepared by adding ethylene oxide to a polyol such as glycerin, pentaerythritol, and sorbitol. The central branch portion can also be derived from several amino acids such as lysine or cysteine. In one example, a branched polyethylene glycol can be represented by the formula R(-PEG-OH)m, wherein R represents a core moiety, such as glycerol or pentaerythritol, and m represents the number of arms. Multi-arm PEG molecules, such as those described in U.S. Pat.

Figure 8 shows a representative branched PEG polymer that can be used in embodiments of the invention, which is referred to herein as "SA-glycerol-PEG." Figure 8A shows an exemplary SA-glycerol-PEG component of CMP-SA-glycerol-PEG or SA-glycerol-PEG linked to the amino acid of a glycan or polypeptide. Figure 8B shows the SA-glycerol-PEG moiety attached to a glycan or polypeptide via a Gal residue. Figure 8C shows the SA-glycerol-PEG moiety attached to a glycan or polypeptide via a Gal-GalNAc residue. Figure 8D shows the SA-glycerol-PEG moiety attached to the amino acid of the polypeptide through the Gal-GalNAc moiety. In various embodiments, the AA is threonine or serine. In an exemplary embodiment, AA is converted to an O-linked glycosylation site by deletion of the B domain of the FVIII polypeptide. The discussion of polymer molecular weight in the following paragraphs is generally applicable to the branched PEG shown in Figure 8. In Figure 8, the subscript "n" indicates any integer that provides a linear (and therefore branched) m-PEG having the desired molecular weight as discussed in the paragraph. In various embodiments, "n" is selected such that the linear m-PEG moiety is from about 20 KDa to about 40 KDa, such as about 20 KDa, about 30 KDa, or about 40 KDa. An integer corresponding to the molecular weight of the m-PEGs corresponds to from about 400 (e.g., from about 455) to about 900 (e.g., about 910). Thus, "n" is selected to provide a branched PEG of from about 40 KDa to about 80 KDa (eg, about 40 KDa, about 50 KDa, about 60 KDa, about 70 KDa, or about 80 KDa).

Many other polymers are also suitable for use in the present invention. Non-peptide and water soluble polymer backbones are especially useful in the present invention. Examples of suitable polymers include, but are not limited to, other polyalkylene glycols such as polypropylene glycol ("PPG"), copolymers of ethylene glycol and propylene glycol, and the like, poly(oxyethylated polyols), poly(enols) ), poly(vinylpyrrolidone), poly(hydroxypropylmethacrylamide), poly([α]-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, poly(N) - propylene decyl morpholine (for example, as described in U.S. Patent No. 5,629,384, the disclosure of which is incorporated herein in its entirety by reference in its entirety in its entirety)

While the molecular weight of each chain of the polymer backbone may vary, it is typically in the range of from about 100 Da to about 160,000 Da, for example, from about 5,000 Da to about 100,000 Da. More specifically, the conjugated hydrophilic polymers of the present invention may range in size from about 500 Da to about 80,000 Da, such as from about 1000 Da to about 80,000 Da; from about 2000 Da to about 70,000 Da; from about 5,000 to about 70,000 Da; 5000 to about 60,000 Da; about 10,000 to about 70,000 Da; about 20,000 to about 60,000 Da; about 30,000 to about 60,000 Da; about 30,000 to about 50,000 Da; or about 30,000 to about 40,000 Da. It should be understood that these dimensions represent estimates rather than accurate measurements. According to a preferred embodiment, the molecules of the invention are conjugated to a heterogeneous population of hydrophilic polymers, for example, having a size of, for example, 10,000, 40,000, or 80,000 Da +/- about 5,000, about 4,000, about 3,000, about 2,000, or about 1000 Da. PEG.

O-linked oligosaccharides: Both N-glycans and O-glycans can be attached to proteins by the production of proteins. When the nascent protein translocates from the ribosome to the endoplasmic reticulum, the cellular N-glycosylation device recognizes and phosphorylates the N-glycosylation signal (NXS/T motif) in the amino acid chain (Kiely et al., 1976; Glabe et al., 1980).

Similarly, O-glycans can be attached to specific O-glycosylation sites in the amino acid chain, but the motif that triggers O-glycosylation is more heterogeneous than the N-glycosylation signal, and The ability to predict O-glycosylation sites in amino acid sequences is not sufficient (Julenius et al., 2004). because Thus, the construction of artificial O-glycosylation sites has some uncertainty. It is generally assumed that the native FVIII molecule does not contain any O-glycosylation sites, and those skilled in the art are therefore expected to construct at least one artificial O-glycosylation site and insert into the B domain associated with the practice of the invention.

Thus, an O-linked oligosaccharide in the truncated Factor VIII B domain is covalently linked to a naturally occurring O-linked glycosylation sequence or an O-linked glycosylation sequence artificially constructed by recombinant techniques.

According to a preferred embodiment of the invention, the O-linked oligosaccharide is linked to a naturally occurring O-linked glycosylation sequence which is not subjected to glycosylation in the wild-type Factor VIII molecule, However, since the B domain is truncated, it becomes O-glycosylated. Examples thereof are shown in the examples and in SEQ ID NO 2 (the truncated B domain corresponds to the amino acid 742-763). It may seem possible that even if the B domain is truncated at a slightly different place, the truncated B domain is slightly shorter than SEQ ID NO 2 (eg shorter than SEQ ID NO 2 1, 2, 3, 4) Or 5 amino acids) or longer (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50) The amino acid, the "hidden" O-glycosylation site in SEQ ID NO 2, also becomes glycosylated. The method activates a "hidden" O-glycosylation site by truncating the B domain rather than creating an artificial O-glycosylation site, which has the advantage of producing favorable safety properties (ie, reduced allergenicity, etc.) The advantages of molecules. Other O-glycosylation sites in the Factor VIII B domain can also be activated by truncating the molecule in different ways.

O-linked oligosaccharide sugar PEGylation: The biosynthesis of O-glycans can be altered and terminated by the addition of sialic acid residues at a relatively early stage of biosynthesis. Certain sialyltransferases are capable of acting on GalNAcα-Ser/Thr, or early O-glycan core subtypes (after core 1 GalT action). The term T antigen is associated with the presence of Galβ1-3GalNAcα-Ser/Thr disaccharide. The production of such structures involves competition between the glycosyltransferases for the same substrate and thus the actual extent of glycosyltransferase activity in the Golgi and the subcellular distribution that determines the structural results of O-glycan biosynthesis and diversification. As shown in Figure 1, only Galβ1-3GalNAcα-Ser/Thr disaccharide is suitable for glycoPEGylation.

However, the availability of this structure can be greatly increased by treating the protein with sialidase or core 1 GalT or a combination thereof. As a result of the PEGylation process of the sugar, sialic acid PEG was added to the native structure by the Galβ1-3GalNAcα-Ser/Thr disaccharide bonded to the target protein by α3 (Fig. 1).

Other hydrophilic polymers can also be attached to the O-linked oligosaccharides. The basic requirement for enzymatically conjugate other hydrophilic polymers to FVIII via O-glycans can be coupled to the glycidinyl-sialic acid derivative via a free amine group as disclosed in WO03031464. This can be achieved by a variety of coupling chemistries known to those skilled in the art. Examples of active biocompatible polymers include polyalkylene oxides such as, but not limited to, polyethylene glycol (PEG), 2-(methacryloxy)ethylphosphonium choline (mPC) polymers (eg, WO03062290) Said), dextran, polyacetamide, or other carbohydrate-based polymers, amino acids or polymers of specific peptide sequences, biotin derivatives, polyvinyl alcohol (PVA), polycarboxylates Acid ester, polyvinylpyrrolidone, polyethylene-co-maleic anhydride, polystyrene-co-malic anhydride, polyoxazoline, polypropylene decylmorpholine, heparin, albumin, cellulose, chitosan Hydrolysate, starch such as hydroxyethyl starch and hydroxypropyl starch, glycogen, agarose and its derivatives, guar gum, amylopectin, inulin, xanthan gum, carrageenan, pectin, Alginate hydrolysate, other biopolymers, and any equivalents thereof.

Pharmaceutical Compositions: Pharmaceutical Compositions It is preferred herein to encompass compositions suitable for parenteral administration comprising a Factor VIIII molecule of the invention, such as a ready-to-use sterile aqueous composition or may be, for example, in water or an aqueous buffer. Reconstituted dry sterile composition. The compositions of the present invention may comprise a variety of pharmaceutically acceptable excipients, stabilizers and the like.

Additional ingredients in such compositions may include wetting agents, emulsifying agents, antioxidants, accumulating agents, tonicity adjusting agents, chelating agents, metal ions, oily vehicles, proteins (eg, For example, human serum albumin, gelatin or protein) and zwitterions (eg, amino acids such as betaines, taurine, arginine, glycine, lysine, and histidine). Of course, such additional ingredients should not adversely affect the overall stability of the pharmaceutical formulations of the present invention. Parenteral administration can be carried out by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe (optional pen-type syringe). Alternatively, parenteral administration can be carried out by means of an infusion pump. Another selection system composition can be a solution or suspension suitable for administration of the FVIII compound in the form of a nasal or pulmonary spray. As a further alternative, a pharmaceutical composition comprising a compound of the invention FVIII may also be suitable for transdermal administration (for example by needle-free injection or patch, optionally as an iontophoresis patch), or via a mucosa (for example, containing a garment). Cast.

Thus, in a first aspect, the invention relates to a B domain truncated Factor VIII molecule having an improved circulating half-life via an O-linked oligosaccharide and a hydrophilic polymer in a truncated B domain Covalent conjugation in which factor VIII activation (molecular activation) results in the removal of a covalently conjugated hydrophilic polymer.

According to one embodiment, the hydrophilic polymer is PEG. The size of the PEG polymer can vary from 10,000 to about 160,000 Da; for example, 10,000 to 80,000 Da, such as about 10,000; 15,000; 20,000; 25,000; 30,000; 35,000; 40,000; 45,000; 50,000; 55,000; 60,000; 65,000; 70,000; Or 80,000 Da. Preferably, the O-linked oligosaccharide is attached to the O-glycosylation site produced by truncating the B domain rather than by insertion of an artificial O-glycosylation site not found in the wt FVIII molecule. Point.

According to a particularly preferred embodiment, the molecule of the invention comprises an amino acid sequence as set forth in SEQ ID NO 2. These molecules have the same characteristics that the activated FVIII molecule is identical to the naturally active FVIII molecule. This feature seems to have advantageous characteristics in the safety evaluation.

The invention also relates to pharmaceutical compositions comprising the molecules of the invention.

The invention further relates to a method of obtaining a molecule of the invention, wherein the method comprises passing a B domain truncated Factor VIII molecule to a hydrophilic polymer such as a PEG group via The O-linked oligosaccharide is conjugated in the truncated B domain. Accordingly, the invention also relates to molecules obtained by or by such methods.

In another aspect, the invention relates to a method of treating a hemophilia disorder comprising administering to a patient in need thereof a therapeutically effective amount of a molecule of the invention.

The term "treatment" as used herein refers to the medical treatment of any human or other animal individual in need thereof. It is expected that the individual has performed a physical examination by a medical practitioner who gives a tentative or definitive diagnosis indicating that the use of the particular treatment is beneficial to the health of the human or other animal individual. The timing and purpose of the treatment may vary from person to person depending on the current state of the individual. Thus, the treatment can be prophylactic, palliative, symptomatic, and/or curative.

In still another aspect, the invention relates to the use of a molecule of the invention as a medicament and to the use of a molecule of the invention for the manufacture of a medicament for the treatment of hemophilia.

In a final aspect, the invention relates to a method of constructing a B domain truncated Factor VIII molecule of the invention, the method comprising: (i) truncating the B domain and optionally the truncated Factor VIII molecule Analysis of the amino acid sequence to identify potential O-linked glycosylation sites; (ii) production of the molecule in a suitable host cell; and (iii) selection of O-linked glycans in the truncated B domain The molecule.

Instance Example 1 Production of recombinant B domain truncated O-glycosylation factor VIII

An example of an amino acid sequence of a B domain deleted Factor VIII molecule is given in SEQ ID NO 2. This polypeptide may also be referred to as "N8". This molecule contains the 21 amino acid residue linker sequence (SFSQNSRHP S QNPPVLKRHQR - underlined S- line serine residue having the PEGylated O-glycan in Example 2).

The Factor VIII molecule of the present invention may be mentioned in various ways in the examples - but all references to the Factor VIII molecule refer to the Factor VIII molecule of the present invention, or alternatively selected to be converted into A Factor VIII molecule in the process of the Factor VIII molecule of the invention.

SEQ ID NO 2:

Cell line and culture method:

A mammalian expression plasmid encoding a B domain deleted type Factor VIII having the amino acid sequence as shown in SEQ ID NO 2 was constructed using Factor VIII cDNA. This plasmid encodes a Factor VIII heavy chain comprising amino acid 1-740 of full length human Factor VIII and a Factor VIII light chain comprising amino acid 1649-2332 of full length human Factor VIII. The heavy and light chain sequences are ligated to the amino acid 741-750 and 1638-1648 sequences of full length human Factor VIII by a 21 amino acid linker. Chinese hamster ovary (CHO) cells were transfected with the BDD Factor VIII-encoding plasmid and selected using the dihydrofolate reductase system to finally produce pure suspension production cells cultured in animal-free medium.

The first step of the method is to inoculate a cell vial from a working cell bank vial to a chemically defined and animal free growth medium. After thawing, the cells were first incubated in a T-flask. One day or two after thawing, the cells were transferred to shake flasks and expanded by serial dilution to make the volume of the culture cell density was maintained between 0.2-3.0 × 10 ⁶ cells / ml. The next step is to transfer the shake flask culture to the seed bioreactor. At this point the culture volume is expanded again and then finally transferred to the production bioreactor. The same chemically defined and animal-free medium was used for all inoculum expansion steps. After being transferred to the production bioreactor, the medium is supplemented with a component capable of increasing the concentration of the product. In a production bioreactor, cells were cultured in a three-day cycle in a repeated batch process. At harvest, 80-90% of the culture volume was transferred to the harvesting tank. The remaining medium is then diluted with fresh medium to achieve initial cell density and then a new growth phase begins.

The harvest batch is purified by centrifugation and filtration and transferred to a storage tank, after which the purification process begins. The buffer is added to the free cell harvest in the storage tank to stabilize the pH.

At the end of the production run, the cells are collected and frozen to end the production cell bank. The cell bank was tested for mycoplasma, sterility and virus contamination.

Purification: Separation of B domain-deficient Factor VIII from cell culture media using a four-step purification procedure consisting of a concentration step on a Capto MMC column, an immunoadsorption chromatography step, an anion exchange chromatography, and a final gel filtration step . The following procedure is typically used: 11 liters of sterile filter medium is pumped onto a Capto MMC column (GE Healthcare, Sweden) (1.6 x 12 cm) which has been subjected to a flow rate of 15 ml/min of buffer A (20 mM imidazole, 10 mM CaCl). ₂ , 50 mM NaCl, 0.02% Tween 80, pH = 7.5) equilibrated. The column was washed with 75 ml of buffer A, followed by washing with 75 ml of buffer A containing 1.5 M NaCl. The protein was eluted with 20 mM imidazole, 10 mM CaCl ₂ , 0.02% Tween 80, 2.5 M NaCl, 8 M ethylene glycol (pH = 7.5) at a flow rate of 1 ml/min. 8 ml of the fraction was collected and analyzed for factor VIII activity (CoA-test). Concentrations containing Factor VIII are concentrated and typically achieve an aggregate volume of approximately 50 ml.

A monoclonal antibody against Factor VIII has been developed (Kjalke Eur J Biochem 234 773). A map analysis of the epitope (not shown) has been found to recognize that the antibody F25 recognizes the far C-terminal sequence of the heavy chain from amino acid residues 725 to 740. This F25 antibody was coupled to NHS activated agarose 4FF (GE Healthcare, Bio-Sciences AB, Uppsala, Sweden) at a density of 2.4 mg/ml gel essentially as described by the manufacturer. The pool from the previous step was diluted 10-fold with 20 mM imidazole, 10 mM CaCl ₂ , 0.02% Tween 80 (pH = 7.3) and applied to a F25 agarose column (1.6 x 9.5 cm) which had been used with 0.5 ml. The /min flow rate was equilibrated with 20 mM imidazole, 10 mM CaCl ₂ , 150 mM NaCl, 0.02% Tween 80, 1 M glycerol (pH = 7.3). The column was washed with equilibration buffer until the UV signal was stable, and then the column was washed with 20 mM imidazole, 10 mM CaCl ₂ , 0.65 M NaCl (pH = 7.3) until the UV signal stabilized again. Factor VIII was eluted with 20 mM imidazole, 10 mM CaCl ₂ , 0.02% Tween 80, 2.5 M NaCl, 50% ethylene glycol (pH = 7.3) at a flow rate of 1 ml/min. 1 ml of the fraction was collected and analyzed for factor VIII activity (CoA-test). Concentrations containing Factor VIII are concentrated and typically achieve an aggregate volume of about 25 ml.

Prepare buffer A for the ion exchange step (20 mM imidazole, 10 mM CaCl ₂ , 0.02% Tween 80, 1 M glycerol, pH = 7.3) and buffer B (20 mM imidazole, 10 mM CaCl ₂ , 0.02% Tween 80, 1 M glycerol, 1 M NaCl, pH = 7.3). The Macro-Prep 25Q Support column (1 x 10 cm) (Bio-Rad Laboratories, Hercules, CA, USA) was equilibrated with 85% buffer A/15% buffer B at a flow rate of 2 ml/min. The pool from the previous step was diluted 10-fold with buffer A and pumped onto the column at a flow rate of 2 ml/min. The column was washed with 85% buffer A/15% buffer B at a flow rate of 2 ml/min and factor VIII was eluted with 120 ml at a flow rate of 2 ml/min using a linear gradient from 15% buffer B to 70% buffer B. 2 ml of the fraction was collected and analyzed for Factor VIII activity (CoA-test). Concentrations containing Factor VIII are concentrated and typically a volume of pool of approximately 36 ml is obtained.

The pool from the previous step was applied to a Superdex 200 preparative (GE Healthcare, Bio-Sciences AB, Uppsala, Sweden) column (2.6 x 60 cm) with 20 mM imidazole, 10 mM CaCl ₂ , 0.02% Tween 80, 1 M glycerol. 150 mM NaCl (pH = 7.3) was equilibrated and dissolved at 1 ml/min. 3 ml of the fraction was collected and analyzed for Factor VIII activity (CoA-test). Concentrations containing Factor VIII are concentrated and typically a volume of pool of approximately 57 ml is obtained. The pool containing Factor VIII was stored at -80 °C.

The overall yield of about 15% was obtained using the four-step purification procedure described above as judged by CoA activity and ELISA measurements.

Cell line used to make N8 recombinant Chinese hamster ovary (CHO) cell line The expression plasmid #814 F8-500 in pTSV7 consisting of the pTSV7 expression vector and the insert containing the cDNA encoding the F8-500 protein was stably transfected. "N8" is intended herein to correspond to a protein having an amino acid sequence as set forth in SEQ ID NO 2. Starting from the N-terminus, the F8-500 protein (N8) consists of the FVIII signal peptide (amino acid-19 to -1), followed by the B-free FVIII heavy chain (amino acid 1-740), 21 amino group. The acid linker (SFSQNSRHPSQNPPVLKRHQR) and the FVIII light chain (amino acid 1649-2332 of wild type human FVIII) are composed. The 21 amino acid linker sequence is derived from the FVIII B domain and consists of the amino acids 741-750 and 1638-1648 of full length FVIII.

CHO cells were transfected with 814 F8-500 in pTSV7 and selected using a dihydrofolate reductase system to ultimately produce pure suspension production cells cultured in animal-free medium. Production runs are initiated by thawing the working cell bank vials and expanding the cells prior to transfer to the production bioreactor. The same chemically defined and animal-free medium was used for all inoculum expansion steps. After being transferred to the production bioreactor, the medium is supplemented with a component capable of increasing the concentration of the product. In a production bioreactor, cells were cultured in a three-day cycle in a repeated batch process. At harvest, 80-90% of the culture volume was transferred to the harvesting tank. The remaining medium is then diluted with fresh medium to achieve initial cell density and then a new growth phase begins. The harvest batch is purified by centrifugation and filtration and transferred to a storage tank, after which the purification process begins. The buffer is added to the free cell harvest in the storage tank to stabilize the pH.

Example 2 PEGylation of recombinant B domain truncated and O-glycosylated factor VIII:

The recombinant Factor VIII molecule obtained in Example 1 was conjugated to polyethylene glycol (PEG) using the following procedure.

In order to efficiently PEGylate the recombinant Factor VIII molecule obtained in Example 1, a concentration of FVIII greater than 5 mg/ml is preferred. Since FVIII is generally insoluble at this concentration, screening is performed on the selected buffer composition (some of these results are shown in Table 1).

Based on these considerations, we have found that a buffer containing 50 mM MES, 50 mM CaCl ₂ , 150 mM NaCl, 20% glycerol (pH 6.0) is a suitable reaction buffer.

Recombinant FVIII purified as described above in a reaction buffer by stepwise dissolution on a Poros 50 HQ column, on a Sartorius Vivaspin (PES) filter (with a cutoff value of 10 kDa) or at Amicon 10kDa MWCO PES Ion exchange was performed on the filter to concentrate to a concentration of 6-10 mg/mL. By factor VIII (BDD) (final about 4.7 mg/mL) with sialidase (A. ureafaciens) (159 mU/mL), CMP-SA-glycerol-PEG-40kDa (5 mol. eq) .) and MBP-ST3Gal1 (540 mU) were mixed in reaction buffer (50 mM MES, 50 mM CaCl ₂ , 150 mM NaCl, 20% glycerol, 0.5 mM anti-protease, pH 6.0) to initiate sugar pegylation of FVIII. The reaction mixture was incubated at 32 ° C until the total conversion yield was about 20-30%.

After incubation, the samples were diluted with buffer A (25 mM Tris, 5 mM CaCl ₂ , 20 mM NaCl, 20% glycerol, pH 7.5) and applied to a Source 15Q column (1 cm id x 6 cm, 4.7 mL, 1 mL/min, 280 nm) )on. The binding substance was washed with buffer A and eluted with buffer B (25 mM Tris, 5 mM CaCl ₂ , 1 M NaCl, 20% glycerol, pH 7.5) using a step gradient. The sugar PEGylated Factor VIII-(O)-SA-glycerol-PEG-40kDa was eluted from the column at about 25% buffer B. Figure 2 shows ion exchange chromatography of the reaction mixture on Source 15Q.

For the exposure to the free galactose moiety of the N-glycan during the capsidylase treatment, the pooled fraction of Factor VIII-SA-glycerol-PEG-40kDa (final 1.0 mg/mL) was combined with CMP-SA (2,000 mol) Eq) and MBP-SBD-ST3Gal3 (400 mU/mL) were mixed in reaction buffer (50 mM MES, 20 mM CaCl ₂ , 150 mM NaCl, 10 mM MnCl ₂ , 20% glycerol, pH 6.0) and incubated at 32 ° C for 11 hours.

The resulting blocked glycated PEGylated Factor VIII-SA-glycerol-PEG-40kDa was separated from CMP-SA and ST3GalIII by gel filtration on a Superdex 200 column (10 cm inner diameter x 300 mm; 280 nm). The column has been equilibrated with 50 mM MES, 50 mM CaCl ₂ , 150 mM NaCl, 10% glycerol (pH 6.0) at a flow rate of 0.25 mL/min. The product factor VIII-SA-glycerol-PEG-40kDa eluted at 38 min. Figure 3 shows the purification of the capped product using Superdex 200 size exclusion chromatography. The peak fractions were collected, aliquoted and subsequently analyzed.

The purpose of the capping procedure is to reduce the in vivo clearance of the conjugated Factor VIII molecule.

Example 3 O-glycan PEGylated rFVIII activity in chromogenic FVIII activity assay:

The activity of the O-glycol PEGylated rFVIII obtained in Example 2 was evaluated in the chromogenic FVIII assay using Coatest SP reagent (Chromogenix) as follows: rFVIII sample and calibrator (7th International FVIII standard from NIBSC) Dilute in Coatest assay buffer (50 mM Tris, 150 mM NaCl, 1% BSA, pH 7.3, containing preservative). 50 μl of sample, standards, and buffer negative controls were added in duplicate to 96-well microtiter plates (Nunc). Factor IXa/Factor X reagent, phospholipid reagent and CaCl ₂ from the Coatest SP kit were mixed at 5:1:3 (vol:vol:vol) and 75 μl of this material was added to the wells. After incubation for 15 min at room temperature, 50 μl of Factor Xa substrate S-2765/thrombin inhibitor I-2581 mixture was added and the reaction was incubated for 10 min at room temperature followed by the addition of 25 μl of 1 M citric acid (pH 3). The absorbance at 415 nm was measured on a Spectramax microtiter plate reader (Molecular Devices) using 620 nm as the reference wavelength. Negative control values for all samples were subtracted and a calibration curve was drawn by linear regression of the absorbance values and FVIII concentration plots. The specific activity was calculated by dividing the sample activity by the protein concentration, which was determined by size exclusion HPLC and integrating the light chain peaks in the HPLC chromatogram (ie, excluding the PEG moiety). The data in Table 2 shows that the specific chromogenic activity of the O-glycolized pegylated rFVIII compound is maintained, meaning that Factor VIII activity appears to be retained in the PEGylated variant.

The data indicated in parentheses are the average deviation and standard deviation of the values measured separately.

Example 4 Activity of O-glycan PEGylated rFVIII in FVIII coagulation activity assay

The activity of O-glycol PEGylated rFVIII was further evaluated in the FVIII coagulation assay. The rFVIII sample was diluted in HBS/BSA (20 mM hepes, 150 mM NaCl, pH 7.4, containing 1% BSA) to about 10 U/ml, followed by 10-fold dilution in FVIII-deficient plasma containing VWF (Dade Behring). Samples and calibration plasma standards (HemosIL calibrated plasma from Instrumentation Laboratory) were then diluted in HBS/BSA to produce four (samples) or six (calibrators) different concentrations. A single factor program is used to measure the clotting time on an ACL 9000 instrument (Instrumentation laboratory), where The sample/standard was mixed with an equal volume of FVIII-deficient plasma containing VWF (Dade Behring), calcium and aPTT reagent, and the clotting time was measured. The following reagents were used: Synthasil (HemosIL, Instrumentation Laboratory), actin FS (Activated PTT reagent, Dade Behring), Stago (STA® PTT-A, Stago), and dAPPTin (DAPPTIN) ® TC, Technoclone). Sample activity is calculated based on a half log plot of clotting time versus calibrant concentration.

The coagulation activity (Fig. 4) of the O-sugar PEGylated rFVIII compounds (control, 10, 40 and 80 kDA PEG, respectively) was reduced to varying degrees depending on the size of the PEG and the aPTT reagent used. The use of Synthasil or dAPPTin as an aPTT reagent resulted in a gradual decrease in coagulation activity with PEG size. Using Stago's aPTT reagent, a 50% reduction in coagulation activity was observed for all three O-glycolized PEGylated N8 compounds evaluated. When actin FS was used as the aPTT reagent, the specific clotting activity was maintained at about 10,000 IU/mg. The data indicates that the aPTT assay is affected by the presence of the PEG moiety, however, the specific clotting activity of rFVIII is not attenuated when PEGylated with the selected aPTT reagent (eg, actin FS) O-glycan.

Example 5: Effect of O-linked PEGylation of rFVIII on Cofactor Activity and FVIII Activation Rate

Incorporation of activated FVIII into the FIXa-FVIIIa complex increases the catalytic efficiency of FIXa-catalyzed FX activation by five orders of magnitude (van Dieijen et al., (1981) J Biol Chem 256:3433) and FIXa-FVIIIa complex assembly and Characterization of FX activation kinetics is a sensitive measure of the functional integrity of the FVIIIa molecule. The cofactor activity of thrombin-activated rFVIII or PEG-rFVIII was characterized by measuring the kinetic parameters of FIXa-catalyzed FX activation in the presence of phospholipid and thrombin-activated rFVIII or PEG-rFVIII. Using FVIIIa activity assay (FIXa-cofactor activity assay), FIXa and FVIIIa were titrated to each other at a fixed concentration (0.1 nM) of rFVIIIa or FIXa to obtain the apparent affinity constant (K _{1⁄2 FIXa} ) and functional FVIIIa concentration of FIXa for rFVIIIa. . The Michaelis constant (k _m ) and the number of conversions (k _cat ) of FX activation were obtained by titrating FX against a fixed concentration of FIXa-FVIIIa complex.

FIXa-cofactor activity assay was performed as follows: Fresh coagulation for each test was prepared by incubating rFVIII (usually 0.7 nM, 1 U/mL) with 5 nM human alpha-thrombin for exactly 30 seconds at 37 °C. Enzyme activated rFVIII and PEG-rFVIII variants. Subsequently, preparation was carried out by subsampling the above activated reactants to FIXa, phospholipid vesicles (phospholipid TGT, from Rossix [Mölndal, Sweden]), hirudin, broad-spectrum protease inhibitors (Pefabloc) Xa and CaCl ₂ . The FX activation rate was quantified in the mixture; FX activation was initiated by adding FX and allowing 30 or 60 seconds at 37 °C. Activation was terminated by diluting the FX activating reaction into ice cold buffer containing EDTA. FXa concentration was quantified using FXa-specific chromogenic receptors by reading the absorbance at 405 nM in an ELISA reader. The absorbance was converted to FXa concentration using a reference curve prepared using purified FXa. The turnover number of the FIXa-rFVIIIa complex assembled using self-activated rFVIII or PEG-rFVIII variants converts the FX activation rate to rFVIIIa concentration.

The thrombin-catalyzed rate of rFVIII activation was measured by quantifying the initial (0-3 min) formation of rFVIIIa in a mixture containing 0.7 nM rFVIII or PEG-rFVIII and 0.13 nM human alpha-thrombin. The formation of FVIIIa is linear with time. The FVIIIa activation rate is expressed as the number of moles of rFVIIIa (v/[rFVIII] ₀ ) formed per minute of rFVIII originally present per mole.

O- linked glycosylation of rFVIII PEGylated rFVIII does not affect the presence of thrombin catalyzed activation rate of the catalytic FIXa or FX activation k _m or k _cat (Table 3) activation of rFVIII. Furthermore, O- linked glycosylation pegylation does not affect the apparent _{K d} (K ½FIXa) Interaction of rFVIIIa-FIXa.

Figure 4 shows the clotting activity of O-glycol PEGylated rFVIII using various aPTT reagents. The data is shown as the ratio of coagulation activity to chromogenic activity (A) or to specific coagulation activity (B). The mean and standard deviation of the values from the three independent experiments are shown.

Table 3: Kinetic constants of rFVIII activation rate and FX activation by FIXa

The data is the average and standard deviation of 3-6 measurements.

Example 6 Pharmacokinetics of glycoPEGylated B domain deleted (BDD)-FVIII in FVIII KO mice and vWF KO mice

The pharmacokinetics of PEGylated BDD-FVIII with various PEG sizes were performed after intravenous administration of 280 IU/kg to FVIII KO mice.

The following compounds were studied: BDD-FVIII, BDD-FVIII-10K PEG (O-glycan, 0129-0000-1005), BDD-FVIII-40K PEG (O-glycan, 0129-0000-1003), BDD-FVIII- 2x40K PEG (O and N-glycan 0129-0000-1008-1A), BDD-FVIII-80K PEG (N-glycan, 0129-0000-1012, O-glycan 0129-0000-1009).

Animal Research Design: Based on the background of C57B1/6 exon 16 gene knockout, Taconic M&B was reared to Factor VIII gene knockout (FVIII KO) mice. Male and female mice weighing approximately 25 g and 19-26 weeks of age were used at a ratio of approximately 1:1. The mice did not return completely. No FVIII was detected in this mouse strain.

Mice were given a single intravenous injection of 280 IU/kg of the compounds listed above in the tail vein. If the mouse is administered intravenously, the mouse is replaced with another mouse. After administration, blood samples of the ocular plexus were collected from uncoated glass capillaries from the time of administration until 64 hours after administration. Three samples were taken from each mouse and 2, 3 or 4 samples were taken at each time point. The blood was stabilized in sodium citrate (9:1) and diluted in FVIII COA SP buffer (1:4), followed by centrifugation at 4000 g for 5 minutes. The plasma obtained from the diluted blood is frozen in dry ice at -80 ° C, followed by FVIII chromogenic activity and / or FVIII Antigen analysis was performed quantitatively.

Quantitative plasma analysis:

FVIII chromogenic activity was determined by using reagents from the Coatest SP kit (Chromogenix). Diluted plasma samples, calibrators (ILS calibrated plasma) stored in Coatest SP buffer, and buffer negative controls (50 [mu]l) were added in duplicate to 96-well microtiter plates (Nunc). Factor IXa/Factor X reagent, phospholipid reagent and CaCl ₂ from the Coatest SP kit were mixed at 5:1:3 (vol:vol:vol) and 75 μl of this material was added to the wells. After incubation for 15 min at room temperature, 50 μl of Factor Xa receptor S-2765/thrombin inhibitor I-2581 mixture was added and the reaction was incubated for 10 min at room temperature followed by the addition of 25 μl of 2% citric acid. Absorbance at 405 nm was measured on a Spectramax microtiter plate reader (Molecular Devices). The FVIII activity in the plasma samples was calculated from calibration curves prepared by calibrating dilutions with International Plasma Standards (ILS).

The FVIII antigen assay was an ELISA kit purchased from Diagnostica Stago (Asserachrom VIII: CAg) using two monoclonal antibodies directed against the light chain of human FVIII. The calibrant (diluted compound) or plasma sample was diluted at least 50-fold in the coatest SP dilution buffer provided by the kit, applied to the pre-coated wells and the ELISA was performed according to the manufacturer's instructions. The values used to report pharmacokinetic studies are based on standard curves prepared from the compounds themselves.

Estimation of pharmacokinetic parameters:

Pharmacokinetic analysis was performed by non-domain method (NCA) of data using ILS as a calibrator (data based on chromogenic activity), using the compound itself as a calibrator (data based on ELISA). The following parameters were estimated from the data: Cmax (the maximum concentration after intravenous administration at the first sampling time point), Tmax (the maximum concentration time after intravenous administration at the first time point), AUC0-∞ (from time 0 to time) The area under the infinite curve), T1⁄2 (terminal half-life), CL (clearance rate), and Vss (steady-state distribution volume). All calculations were performed using WinNonlin Pro version 4.1.

After intravenous injection of 280IU/Kg BDD-EVIII, BDD-FVIII-10KDa PEG, BDD-FVIII-40KDa PEG, BDD-FVIII-2x40KDa PEG and BDD-FVIII-80KDa PEG into FVIII KO mice, the PEG size increases The half-life was extended to 7.8 h (BDD-FVIII) to 15-16 h (Table 4), which corresponds to a 2-fold increase. Similarly, as PEG size increased, clearance decreased and MRT increased (Table 4).

in conclusion:

After intravenous administration of 280 IU/kg to FVIII KO mice, PEGylation of BDD-FVIII sugars increased T1⁄2 by 1.3-2.1 fold compared to BDD-FVIII. As the size of the PEG group increased in the range of 10 KDa to 80 KDa PEG, we observed an increase in T1⁄2.

Example 7 Prolonged hemostasis of 40K-PEG-[O]-N8 compared with Advate in type A hemophilia mice induced by FeCl ₃ induced injury

Mice Study 40K-PEG- [O] -N8 phase duration to effect recombination of FVIII (Yifei Te) in the hemophilia A (F8-KO) in the FeCl ₃ model of injury induced in it.

Mice were anesthetized and placed on a heating pad (37 ° C) to maintain body temperature. A flow probe (0.5 PSB nano probe) that exposes the carotid artery and measures blood flow by supersonics is placed around the artery. Injury (iron-mediated chemical oxidation) was induced by placing a filter paper (2 x 5 mm) briefly immersed in a 10% FeCl ₃ solution around the exposed carotid artery. Remove the filter paper after 3 minutes. The artery is then washed three times with 0.9% NaCl and finally a surgical surgical lubricant (Surgilube) is applied to exclude air from the flow probe and to ensure that the blood flow is optimized. Blood flow (ml / min) 25min and was determined by the amount of time measured from the blocking saturated filter paper to remove FeCl ₃ until blood flow was 0ml / min of time (min) after removal of the recording paper saturated FeCl _3. If no blockage occurred after 25 min, the block time was reported to be 25 min, even though no blockage occurred during the observation period. Treatment with F8-KO mice (n=6-10) was performed with Effort (280 U/kg), 40K-PEG-[O]-N8 (280 U/kg), or vehicle. The injury was induced with FeCl ₃ 5 min (acute effect) or 24, 48, 60, and 72 hours after administration. Blood flow (ml/min) was recorded for 25 min after removal of FeCl ₃ and the blocking time was subsequently determined.

No occlusion occurred in the vehicle-treated F8-KO mice, but in all mice treated with 40KDa-PEG-[O]-N8 and Effort, the occlusion occurred 5 minutes after the administration (acute effect). The mean occlusion time was 4.3 ± 0.4 min and 5.2 ± 0.7 min, respectively. In F8-KO mice treated with 40KDa-PEG-[O]-N8, the mean occlusion time was extended to 13.8 ± 3.4 min at 72 hours after administration. In contrast, F8-KO mice treated with Effort had a blocking time of 13.0 ± 3.4 min and 15.9 ± 2.9 min after 24 and 48 hours, respectively. Importantly, no obstruction was observed after 60 and 72 hours of Effort administration. In all mice treated with 40KDa-PEG-[O]-N8, occlusion was observed 24 hours after administration, whereas only 67% of Ephel-treated mice developed occlusion. After 72 hours, occlusion was still visible in 63% of 40KDa-PEG-[O]-N8 treated mice, whereas no obstruction was observed at 60 and 72 hours after administration of Effie.

The prolongation effect of 40KDa-PEG-[O]-N8 in F8-KO mice.

The injury was induced with FeCl ₃ at 5 min (acute effect), 24, 48, 60, and 72 hours after administration of 280 IU/kg 40 KDa-PEG-[O]-N8, 280 IU/kg Effort, or vehicle. Blood flow (ml/min) was recorded for 25 min after removal of FeCl ₃ and the blocking time was subsequently determined. At 60 and 72 hours after administration, no blockage occurred in the mice administered with Effie. The mean and SEM of 6-10 mice per group are shown. Kruskal-Wallis test (including Dunn's post test) was used to compare the block time between different groups. *: p <0.05; **: p < 0.01.

In short, compared to the model of FeCl ₃ -induced damage to the F8-KO mice with Yifei Te 40KDa-PEG- [O] -N8 hemostatic effect of significantly extended.

<110> Danish company Novo-Nodisk

<120> Conjugated Factor VIII Molecule

<130> 101961-5191WO

<140> 098106523

<141> 2009-02-27

<150> US 61/032,006

<151> 2008-02-27

<150> US 61/043,354

<151> 2008-04-08

<150> US 61/058,869

<151> 2008-06-04

<160> 2

<170> PatentIn vcrsion 3.5

<210> 1

<211> 2332

<212> PRT

<213> Homo sapiens

<400> 1

<210> 2

<211> 1445

<212> PRT

<213> Artificial sequence

<220>

<223> B-domain truncated human factor VIII

<400> 2

Claims (17)

A B domain truncated Factor VIII molecule having an improved circulating half-life, the molecule being covalently conjugated to a hydrophilic polymer via an O-linked oligosaccharide in the truncated B domain, wherein activation of Factor VIII results in Removal of the covalently conjugated hydrophilic polymer. The molecule of claim 1, wherein the heavy and light chain portions of the Factor VIII precursor polypeptide are separated by a linker, wherein the sequence of the linker is derived from a Factor VIII B domain, and wherein the linker comprises a protease At the recognition site, the protease can separate the B domain deleted FVIII precursor polypeptide into a heavy chain and a light chain. A molecule according to claim 1 or 2, wherein the B domain is 20-30 amino acids in length. The molecule of claim 1 or 2, wherein the B domain is 20 amino acids in length. The molecule of claim 1 or 2, wherein the hydrophilic polymer is a polysaccharide. The molecule of claim 1 or 2, wherein the hydrophilic polymer is PEG. The molecule of claim 6, wherein the PEG is about 40,000 Da in size. The molecule of claim 1 or 2, wherein the molecule comprises an amino acid sequence as set forth in SEQ ID NO 2. A molecule according to claim 1 or 2, wherein the B domain is one amino acid shorter than SEQ ID NO 2. A molecule according to claim 1 or 2, wherein the molecule is produced by a method comprising the steps of: a) transfecting a mammalian host cell with a vector encoding a Factor VIII molecule comprising SEQ ID NO 2; b) being suitable for the host The culture step under conditions in which the factor VIII molecule is expressed in the cell Host cell of a); c) harvesting the Factor VIII molecule from the host cell culture of step b); and d) covalently conjugated the Factor VIII molecule to the hydrophilic polymer via an O-linked oligosaccharide. A molecule according to claim 1 or 2 for use as an agent for the treatment of hemophilia. A molecule according to claim 1 or 2, which is administered by subcutaneous administration to treat hemophilia. The numerator of claim 1 or 2 is for the treatment of hemophilia by intravenous administration. A pharmaceutical composition comprising the molecule of any one of claims 1 to 10. A pharmaceutical composition according to claim 14 which is for use as an agent for the treatment of hemophilia. The pharmaceutical composition of claim 14, which is for treating hemophilia by subcutaneous administration. The pharmaceutical composition of claim 14 which is administered by intravenous administration to treat hemophilia.