Fviii modification trend site

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns area of molecular biology and biochemistry, and can be used in medicine. There is offered mutein conjugate of the blood coagulation factor VIII (FVIII) wherein a residue not being cysteine in position 41, 129, 377, 388, 468, 491, 556, 1804, 1808, 1810, 1812, 1813, 1815 and/or 2118 is substituted by a cysteine residue with polyethylene glycol (PEG) where a PEG molecule is bound with a polypeptide in a mutant cysteine residue.

EFFECT: improved pharmacokinetic properties of the FVIII as an ingredient of the conjugate under the invention with preserved a procoagulant factor activity allows presenting new FVIII PEG-muteins for producing of a pharmaceutical compositions for treating hemophilia.

12 cl, 38 dwg, 8 tbl, 1 ex

 

Cross-reference

This application has the benefit of priority of application No. 60/627277 on U.S. patent, filed November 12, 2004, which is fully incorporated herein by reference.

The scope of the invention

This invention relates to muteena factor VIII (FVIII), which have the ability to communicate in a pre-specified site with one or more biocompatible polymers, such as polyethylene glycol. Also available based on these formulations, dosages, and methods of their administration for therapeutic purposes. These modified FVIII variants and compositions and methods suitable for the treatment of persons affected by hemophilia And in which the frequency of administration of the drug and reduced immunogenic response is reduced.

The level of technology

Hemophilia a is the most common hereditary bleeding disorder with an assessment of occurrence of one in 5000 men. It is caused by deficiency or structural defects of FVIII, the main component of the natural way of blood coagulation. Modern treatment of hemophilia And includes intravenous administration of human FVIII. Human FVIII produced by recombinant method in the form of single-stranded molecules with a size of approximately 300 kDa. It consists of structural domains A1-A2-B-A3-C1-C2 (Thompson, 2003, Semin. Hematol. 29, pp.11-22). The product-precursor pre is brasaetsa in two polypeptide chains size of 200 kDa (heavy) and 80 kDa (light) in the Golgi apparatus, when two chains are held together by metal ions (Kaufman et al., 1988, J. Biol. Chem. 263, p.6352; Andersson et al., 1986, Proc. Natl. Acad. Sci. 83, p.2979).

Domain mutein FVIII seems to be optional, since FVIII with deletionism domain In (BDD, A1-A2 heavy chain 90 kDa plus light chain 80 kDa) also is effective in substitution therapy of hemophilia A. the Sequence of FVIII with deletionism In-domain contains a deletion of all but 14 amino acids In the domain.

Patients with hemophilia And currently treated with intravenous FVIII by necessity or by preventive therapy prescribed several times a week. When prophylactic treatment is given 15-25 IU of factor VIII per 1 kg of body weight three times a week. It constantly requires the patient. Due to the short time half-life in humans, FVIII should be entered frequently. Despite the large size of more than 300 kDa protein of full length FVIII has in humans, the half-life of only about 11 hours (Ewenstein et al., 2004, Semin. Hematol. 41, pp.1-16). The need for frequent intravenous creates huge barriers in keeping sick mode and regimens. For patients, it would be more convenient if there was developed FVIII product, which has a longer half-life and therefore requires less frequent administration. In addition, if the period provived the deposits was increased, might be reduced cost of treatment, because it might take fewer doses.

An additional disadvantage of the existing therapy is that approximately 25-30% of patients develop antibodies that inhibit the activity of FVIII (Georges tarbouriech et al., 2002, Haemophilia 8, pp.1-11). Major epitopes of inhibitory antibodies localized within the A2 domain at residues 484-508, domain A3 on the remains 1811-1818 and C2 domain. Antibody prevents the use of FVIII in substitution therapy, making this group of patients to seek more expensive treatment with vysokopetrovskogo recombinant factor VIII immune resistance and therapy.

In subsequent studies have identified FVIII-inhibitory epitopes of antibodies. In the study 25 inhibitory plasma samples, it was found that 11 of them are connected with a fragment of ASS caused by thrombin and having a light chain size 73 kDa, 4 are connected with the domain A2, and 10 with both fragments (Fulcher, C. et al., 1985, Proc. Natl. Acad. Sci. 2(22), pp.7728-32). In another study, six of the eight inhibitors domain A2 patients were neutralized recombinant A2-polypeptide (Scandella, D. et al., 1993, Blood 82(6), pp.1767-75). The epitopes for six of the nine inhibitors patients noted on the remains 379-538 domain A2 (Scandella, D. et al., 1988, Proc. Natl. Acad. Sci. 85(16), pp.6152-6). The epitope for 18 inhibitor heavy chain is Yu was determined in the same N-terminal region of $ 18.3 kDa domain A2 (Scandella, D. et al., 1989, Blood 74(5), pp.1618-26).

Active recombinant hybrid molecule man/pig mutein FVIII obtained by substitution of residues 387-604 of the A2 domain of human homologous sequence pigs, showed resistance to the inhibitor A2 patients (Lubin, I. et al., 1994, J. Biol. Chem. 269(12), pp.8639-41) and resistance to murine monoclonal antibody MAB 413 IgG, which competes with inhibitors A2 patient in binding to A2 (Scandella, D. et al., 1992, Thromb Haemost. 67(6), RR-71). In the future, this epitope domain A2 was detected in the residue 484-508 domain A2, when the result of the experiments it was found that the MAV 413 IgG and four inhibitor of the patient does not inhibit hybrid human/pig mutein FVIII, in which residues 484-508 domain A2 were replaced with the corresponding residues of the A2 domain pigs (Healey, J. et al., 1995, J. Biol. Chem. 270(24), pp.14505-9). This hybrid FVIII was also more stable, at least half of the 23 patients who were studied plasma (Barrow, R. et al., 2000, Blood 95(2), pp.564-8). When alanine-scanning mutagenesis was identified residue 487 as main to link with all five tested inhibitors patients, despite the fact that all residues 484, 487, 489 and 492 were important for interaction with MAV 413 IgG (Lubin, I., J. Biol. Chem. 272(48), pp.30191-5). The titer of inhibitory antibodies in mice treated with the mutant R484A/R489A/P492, but not mutant R484A/R489A were significantly lower than in mice, p is lucasey control human BDD FVIII (Parker, E. et al., 2004, Blood 104(3), pp.704-10). Thus, the area 484-508 domain A2, apparently, is the binding site for inhibitors to FVIII activity.

Another problem with traditional therapy, in addition to the immune response to FVIII, is that it requires frequent dose due to the short half-life of FVIII in vivo. Investigated the mechanisms of excretion of FVIII from the bloodstream.

Excretion of FVIII from the bloodstream partly due to the specific binding protein related receptor low density lipoprotein (LRP) and liver receptor excretion with broad ligand specificity (Oldenburg et al., 2004, Haemophilia 10 Suppl 4, pp.133-139). Recently it was also shown that the receptor for low density lipoprotein (LDL) plays a role in excretion of FVIII, for example, through joint LRP steps to regulate levels of FVIII in plasma (Bovenschen et al., 2005, Blood 106, pp.906-910). Both interactions are facilitated by binding to heparin sulfate proteoglycans (HSPGs) the cell surface. The half-life from plasma of a mouse can be prolonged up to 3.3 times, when locked LRP, or 5.5 times when blocked as LRP and HSPGs the cell surface (Sarafanov et al., 2001, J. Biol. Chem. 276, pp.11970-11979). It is assumed that HSPGs accumulate FVIII on the cell surface and provide its LRP. The LRP binding sites on FVIII localized to residues 484-509 A2 (Georges tarbouriech et al., 1999, J. Biol. Chm. 274, pp.37685-37692), residues 1811-1818 A3 (Bovenschen et al., 2003, J. Biol. Chem. 278, pp.9370-9377) and the epitope in the C2 domain (Lenting et al., 1999, J. Biol. Chem. 274, pp.23734-23739).

In addition, FVIII derived from the blood by the action of proteases. To understand this effect, it is necessary to understand the mechanism by which FVIII is involved in the clotting of blood. FVIII associated with the factor a background of Villebranda (vWF), circulates, as heterodimer, consisting of heavy and light chains. vWF-binding occurs on the remains 1649-1689 FVIII (Foster et al., 1988, J. Biol. Chem. 263, pp.5230-5234) and part of the C1 domains (Jacquemin et al., 2000, Blood 96, pp.958-965) and C2 (Spiegel, P. et al., 2004, J. Biol. Chem. 279(51), pp.53691-8). FVIII is activated by thrombin, which cleaves the peptide bond after residues 372, 740 and 1689, producing heterotrimer of domains A1, A2 and A3-C1-C2 (Pittman et al., 2001, Proc. Natl. Acad. Sci. 276, pp.12434-12439). Upon completion of the activation of FVIII dissociates from vWF and accumulates on the cell surface of platelets by binding to phospholipid. Phospholipid binding involves residues 2199, 2200, 2251 and 2252 FVIII (Gilbert et al., 2002, J. Biol. Chem. 277, pp.6374-6381). There it binds to coagulation factor IX (FIX) through interaction with residues 558-565 (Fay et al., 1994, J. Biol. Chem. 269, pp.20522-20527), and the remnants 1811-1818 FVIII (Lenting et al., 1996, J. Biol. Chem. 271, pp.1935-1940) and coagulation factor X (FX) through interaction with the remnants 349-372 FVIII (Nogami et al., 2004, J. Biol. Chem. 279, pp.15763-15771) and acts as a cofactor for FIX activation of FX, a significant component of nature the underwater path of blood clotting. Activated FVIII (F Villa) partially inactivated by the protease, activated protein C (APC) via cleavage of FVIII after residues 336 and 562 (Regan et al., 1996, J. Biol. Chem. 271, pp.3982-3987). However, the alleged main factor inactivation is the dissociation of the A2 domain from domains A1 and A3-C1-C2 (Fay et al., 1991, J. Biol. Chem. 266, pp.8957-8962).

One method, which, as has been demonstrated, increases the in vivo half-life of the protein, is PEG-iliriana. PEG-iliriana is a covalent joining of long chain molecules of polyethylene glycol (PEG) to a protein or other molecule. The PEG can have a linear form or a branched form, to get a variety of molecules with different properties. In addition to increasing the half-life of peptides or proteins, PEG-iliriana used to reduce the formation of antibodies that protect the protein from proteases splitting and preservation of material outside the renal filtrate (Hams et al., 2001, Clinical Pharmacokinetics 40, pp.539-51). In addition, PEG-iliriana may also increase the overall stability and solubility of the protein. Finally, supported the concentration of PEG-Yerevani proteins in plasma may reduce the degree of side effects by reducing failures in peak levels of the drug, eliminating thus the need to introduce surficial the practical protein levels at early time periods.

With varying degrees of success were tested random modification of FVIII obtained by directional impact of primary amines (N-terminal and lisini) large polymers, such as PEG and dextran (WO 94/15625, patent US 4970300, patent US 6048720). The most striking improvement, published in a patent application in 1994 (WO 94/15625), showed a 4-fold increase in half-life, however, due to the 2-fold loss of activity after the reaction FVIII full length with a 50-fold excess PEG. In WO 2004/075923 disclosed conjugates FVIII and polyethylene glycol, which are formed through random modification. In the past accidentally PEG-lirovannye proteins, such as interferon-alpha (Kozlowski et al., 2001, BioDrugs 15, pp.419-429), was approved as therapeutic agents.

However, this approach, based on random modifications, is more problematic regarding heterodimeric FVIII. FVIII has hundreds of potential sites PEG-helirovanie, including 158 lysine, two N-end and many histidinol, Surinov, threonines and tyrosines, each of which could potentially be PEG-Yerevani reagents, initially focused on primary amines. For example, it was shown that the major positional isomer for PEG-lirovannomu interferon Alfa-2b is histidine (Wang et al., 2000, Biochemistry 39, pp.10634-10640). In addition, the heterogeneous processing panoras the cluster FVIII may be a mixture of starting material, which further makes the PEG-lirovannye products more complex. An additional disadvantage of nekontroliruemyj customers PEG-helirovanie on FVIII is the potential loss in activity, in cases when the PEG was supposed to join on or near the main active sites, especially in cases where more than one PEG or single PEG large size conjugates with FVIII. Because of the occasional PEG-iliriana constantly produces a large number of PEG-Yerevani products, cleaning with the aim of obtaining nanopeg-Yerevani products will lead to a drastic reduction of the yield of these products. Finally, a great heterogeneity profile of the product will be almost impossible to do continuous synthesis and study of each series. Because commodity production requires a constant, well-researched product, the heterogeneity of the product is a barrier to commercialization. Given all these factors, desired a more specific method, PEG-helirovanie FVIII.

In a recent review (Kochendoerfer, G., Curr. Opin. Chem. Biol. 2005, available online 15 Oct. 2005, direct subject identifier doi: 10.1016/j.cbpa.2005.10.007) were obobschenny different strategies site-directed PEG-helirovanie proteins. One approach includes the introduction of unnatural amino acids into proteins by chemical synthesis or recombinant expre the FIC, followed by adding a derivative of the PEG that is specific to react with unnatural amino acid. For example, unnatural amino acid may be one which contains ketogroup, which is not found in native proteins. However, chemical synthesis of such large proteins, such as FVIII, impossible. Modern limit peptide synthesis is about 50 residues. For large parts of the polypeptide can be ligitamate several peptides, but to get at least FVIII with deletionism In the domain that would require more than 20 legirovanii, resulting in the product yield would be less than 1% even under ideal reaction conditions. Recombinant expression of proteins with unnatural amino acids is limited, mainly, the expression systems of memleketim. I believe that this approach is problematic for large and complex protein, such as FVIII, the expression of which is necessary in systems of mammals.

Another approach to site-directed PEG-helirovanie protein is targeted impact amine N-Terminus of the main chain PEG-aldehydes. This process requires a low pH to achieve specificity in relation to other amine groups, which, however, is incompatible with a narrow interval near neutral pH, necessary for the stability of FVII (Wang et al., 2003, International J. Pharmaceutics 259, pp.1-15). Moreover, N-terminal PEG-iliriana FVIII may not lead to improved half-life from plasma, if this region is not involved in the excretion of plasma. In fact, the N-terminal region of the light chain of FVIII involved in binding to vWF, protein carrier, which is essential for the preservation of FVIII in the circulation. For N-terminal modification factor FVIII most important connection with vWF can be destroyed or weakened. Thus, the N-terminal PEG-iliriana FVIII can have the opposite effect of reducing the half-life of FVIII from plasma.

WO 90/12874 reveals site-specific modification of human IL-3, colony-stimulating factor granulocyte and eritropoetinov polypeptides by introducing cysteine or replacing them with another amino acid followed by the addition of the ligand, which contains a sulfhydryl reactive group. The ligand selectively attached to cysteine residues. Modification of FVIII or any of its variants is not disclosed.

For the reasons set forth above, there is a need for an improved version of FVIII, which has a greater duration of action in vivo and reduced immunogenicity, preserving, at the same time, the functional activity. In addition, it is desirable that this protein was produced as homogeneous PR is the product of a reliable method.

A brief summary of the invention

The purpose of this invention is the provision conjugated to a biocompatible polymer functional FVIII polypeptide having improved pharmacokinetic and therapeutic properties.

Another purpose of this invention is the provision conjugated to a biocompatible polymer protein FVIII with deletionism In the domain that have improved pharmacokinetic properties.

Another objective of the invention is the provision conjugated to a biocompatible polymer functional FVIII polypeptide having weakened binding protein-related receptor low density lipoprotein (LRP)receptor low density lipoprotein (LDL), heparin-sulfate proteoglycans (HSPGs) and/or inhibitory antibodies against FVIII.

Another objective of the present invention is the provision of an improved variant FVIII, which has a greater duration of action in vivo and reduced immunogenicity and which may be produced as a homogeneous product in a safe manner.

In one aspect of the invention provides a conjugate having procoagulant activity of factor FVIII, including functional polypeptide factor FVIII, covalently coupled to one or more of their pre-defined sites with one or more the biocompatible polymers, where the predefined site is not N-terminal amine. The invention also includes a method of obtaining this conjugate, providing matirovanie nucleotide sequence that encodes a functional polypeptide factor FVIII, with the aim of substitution in the pre-site sequence that encodes a cysteine residue; the expression of the mutated nucleotide sequence to obtain a modified cysteine mutein; cleaning mutein; responding mutein with a biocompatible polymer that is activated to react with polypeptides essentially only on the introduced cysteine residues so as to form a conjugate; and purification of the conjugate. The invention is also directed to pharmaceutical compositions comprising the conjugate and a pharmaceutically acceptable adjuvant, and for the treatment of hemophilia by introducing a therapeutically effective quantities of these pharmaceutical compositions to a mammal that needs them.

The invention also relates to a method of site-directed PEG-helirovanie mutein factor FVIII, comprising (a) expression of site-directed mutein factor FVIII, where mutein has a cysteine substitution at amino acid residue on the outer surface mutein factor FVIII and this cysteine Kairouan; (b) contacting the t is steinbaugh mutein with a reducing agent under conditions in which cysteine mutein gently recovers and separates the cap; (C) remove cap and reductant from cysteine mutein; and (d) processing at least after approximately 5 minutes after removal of the reducing agent, cysteine mutein using PEG-containing, sulfhydryl-binding fragment under conditions in which the formed PEG-pilirovanny mutein factor FVIII.

Brief description of drawings

Figure 1. Maps vectors and mutagenesis strategy for PEG-Malinov.

Figure 2. The profile of the UV absorption at 280 nm versus time for PEG-2, a protein purified through a chromatographic column with monoclonal antibodies to FVIII. Chromatography was performed using chromatographic system ACT® Explorer 100 from Amersham Bioscience.

Figure 3. Three-stage method of site-directed PEG-helirovanie. PEG is a cysteine-reactive PEG, such as PEG-maleimide. Closed bars indicate disulfide education, and open - restored cysteine.

Figure 4. Site-directed PEG-iliriana PAG.

Figure 5. Site-directed PEG-iliriana PAG.

Figa. Site-directed PEG-iliriana BDD, PEG, 4, 5 and 6. The top panel were stained with the antibody heavy (H) chain, and the bottom panel is an antibody to the light (L) chains. "U" - raw material containing both N and L.

Fig.6b. PEG-Yerevan the e PEG and PEG with PEG and PEG as a control. First, purified PEG-mutiny (S) restored using Tris(2-carboxyethyl)phosphine (TSER) and after removal of the reducing agent (R) PEG-jiroveci using PEG 12 kDa ("12") or 22 kDa ("22"). Samples were applied to a 6%Tris-glycine SDS PAGE and stained with the antibody to the heavy chain (HC) in the left pane or the antibody to the light chain (LC) in the right pane. "U" - raw material containing both NA and LC. PEG-lirovannye strips of colored dots.

Figs. PEG-iliriana PEG+6 PEG and PEG as a control. PEG, PEG or PEG+6 restored using TSER and after removal of the reducing agent (R) PEG-jiroveci with PEG 5 kDa ("5") or 43 kDa ("43"), PEG+6 PEG-jiroveci using PEG 12, 22 and 33 kDa. Samples were applied to a 6%Tris-glycine SDS PAGE and stained Kumasi left for proteins or antibody to the heavy chain (H) or light chain (L). "U" - raw material containing both N and L. PEG-lirovannye strips of colored dots.

Fig.6d. PEG-iliriana FVIII wild-type full length (KG-2) with PEG as a control. Left gel were stained with Kumasi for proteins, and the right gel - iodine for PEG. "BDD U" - raw BDD-material containing both N and L. PEG-lirovannye strips of colored dots.

7. Cleavage by thrombin PEG-lirovannomu PEG. N-terminal half of the A2 domain is colored in blue and the C-concava the half - in green, the epitope antibodies R8B12 tinted dark green (right sample FVIII). PEG (lane 1) and 22 kDa-PEG-pilirovanny PEG (lane 2) were treated with thrombin (lanes 3 and 4, respectively) and then sprayed on a 7%Tris-acetate gel (Invitrogen) and stained with the antibody R8B12. Each track contains 50 ng FVIII.

Fig. Cleavage by thrombin PEG-lirovannomu FVIII wild-type full length (KG-2). "S" = the source material KG-2. "R" = restored KG-2, the reducing agent is removed. "R" = "R", PEG-pilirovanny PEG 43 kDa. "Net" = "R", cleared of excess PEG. L = light chain. PEG-lirovannye strips of colored dots.

Fig.9. Staining with iodine PEG-lirovannomu PEG. 22 or 43 kDa-PEG-pilirovanny PEG inflicted on 6%Tris-glycine gel and stained with antibody R8B12 to FVIII (lanes 1 and 2) or iodine (lanes 3 and 4). Two spots were aligned according to their marker lanes molecular weight. Lanes 1 and 2 contain about 30 ng FVIII, while lanes 3 and 4 contain about 2 mg.

Figure 10. MALDI-mass spectrometry analysis of PEG-lirovannomu, napag-lirovannomu PEG. MALDI-mass spectrometry was performed on PEG (Figa) or 22 kDa-PEG-iliriana PEG (Fig.10b). After PEG-helirovanie peak heavy (H) chain PEG significantly reduced and a new peak (N+PEG) with center at 111 kDa (PEG 22 kDa + heavy chain 89 kDa). The expected peak PEG-iliovasilema (L) chain with center at 100 kDa (PEG 22 kDa + light chain 83 kDa) was not detected.

11. MALDI-mass spectrometry PEG-lirovannomu, napag-lirovannomu PEG after cleavage by thrombin.

Fig. MALDI-mass spectrometry analysis of PEG-lirovannomu PEG before and after cleavage by thrombin.

Fig. The profile of the UV absorption at 280 nm PEG-lirovannomu PEG, purified on column used in gel chromatography.

Fig. The profile of the UV absorption at 280 nm PEG-lirovannomu, napag-lirovannomu PEG, purified on a cation-exchange column.

Fig. The profile of the UV absorption at 280 nm PEG-lirovannomu, napag-lirovannomu PEG on column used in gel chromatography.

Fig. Comparison of the activity of PEG-lirovannomu protein with the activity of NAAG-lirovannomu protein, measured by chromogenic analysis and coagulation analysis. Purified FVIII full length represented as KG-2. The resulting percent activity was determined by dividing the value of the sample treated with PEG after recovery and removal of the reducing agent, the amount of sample treated with buffer control, whereas the PEG-helirovanie.

Fig. Pharmacokinetic (PK) study of rabbit relative to PEG-lirovannomu PEG in comparison with PEG.

Fig. RK-study rabbit relative to PEG-lirovannomu PEG compared to BDD and PEG. P-values are comparisons between PEG-Yerevani PEG and BDD.

Phi is .19. RK-study rabbit relative to PEG-lirovannomu PEG compared to BDD and PEG.

Fig. RK-study rabbit relative to PEG-lirovannomu FVIII wild-type full length ("fl") in comparison with remodification FVIII fl.

Fig. RK-study hemophilic mouse relative to PEG-lirovannomu PEG compared to BDD and PEG.

Fig. RK-the study of normal mouse relative to the 22 and 43 kDa-PEG-lirovannomu PEG compared to BDD.

Fig. RK-study 22 normal mouse relative to PEG-lirovannomu PEG size of 22 kDa in comparison with BDD during the whole time.

Fig. The histogram collection Hemophilic Mice (BDD) Factor VIII, showing the pharmacokinetic evaluation of half-life BDD Factor VIII in two species in the analysis hemophilic mouse.

Fig. Study break kidney hemophilic mouse relative to PEG-lirovannomu PEG size of 22 kDa compared to BDD. As a result of processing a media mouse had blood loss of 25 μl/g body weight.

Fig. Chromogenic activity of PEG-lirovannomu PEG and BDD in the presence of increasing amounts of antibodies to FVIII. Epitope antibodies is indicated in parentheses.

Fig. Chromogenic activity of PEG-lirovannomu PEG in the presence of increasing amounts of antibodies mAB 413 to FVIII.

Fig. Chromogenic activity BDD, 43 kDa-PEG-lirovannomu PEG, 33 kDa-PEG-lirovannomu PEG and 3 kDa-djpeg-lirovannomu PEG+6 in the presence of human plasma, derived from patients who have developed inhibitors to FVIII. Inhibitor titer and data collected blood was noted above. The top two panels include data of plasma collected from patients, diluted in 5-405 time. The bottom left panel displays a dilution of 1:15 for patients with plasma HRF-828. The bottom right panel confirms that 0,064 IU/ml was used for each sample FVIII in the upper two panels are not dose.

Fig. Method of screening and confirmation PEG-helirovanie. The top panel shows a schematic screening PEG-helirovanie transient expressed PEG-Malinov. The bottom panel shows the Western analysis of PEG-Yerevani products using specific antibodies to heavy ("H") circuit (left) or specific antibodies to light (L) chains (right). PEG-lirovannye strips of colored dots. "U" - raw material containing both N and L.

Fig. Screening PEG-helirovanie PEG-17. Western analysis of PEG-Yerevani products using specific antibodies to the heavy ("H") circuit (R8B12 and 58.12) or specific antibodies to the light (L) chains of antibodies (C7F7 and GM). All three mutein selected heavy chain, with a relative efficiency of PEG-helirovanie PAG~PAP>PAG. PEG-lirovannye strips of colored dots. "U" - raw material containing both N and L.

Figure 1. Gel showing PEG-iliriana PEG+14 as a function of the concentration of the reducing agent. PEG+14 was treated 67-670 μm TSAR for 30 min at 4°C. the Reducing agent was removed rotating column, followed by PEG-iliriana using PEG 12 kDa. Heavy and light chain of FVIII colored "H" and "L" respectively. Two points indicate heavy and light PEG-lirovannye chain.

Fig. Expanded mass spectra PEG+14, processed 67-670 μm TSAR with the remote then reducing agent.

Detailed description of the invention

The present invention is based on the discovery that polypeptides having the activity of FVIII, can be covalently linked to a previously defined site, which is not the N-terminal amine with a biocompatible polymer such polypeptides substantially retain its coagulation activity. In addition, these polypeptide conjugates have improved circulation time in the blood and reduced antigenicity. The conjugates according to the invention have the advantage over the conjugates known in the prior art, which have random attaching a polymer to FVIII or are joining the N-end. Site-directed connection allows you to design modifications which do not affect the area required for biological activity and, thereby substantially retain FVIII. It also allows you to attach polymers to block the binding sites involved in the excretion of FVIII. Site-directed connection also allows to obtain a more homogeneous product than heterogeneous conjugates obtained in the prior art by random joining of polymers. Due to the fact that the accession to the N-terminal amine is not performed, the conjugates according to the present invention does not lose activity when attaching a ligand to the active site of FVIII polypeptide. It is assumed that the N-terminal region of the light chain is involved in the binding of factor vWF to FVIII, which is a stabilizing binding in the blood stream.

Definition

Biocompatible polymer. Biocompatible polymer includes polyalkylene oxides, such as polyethylene glycol (PEG), dextrans, Kolomenskoye acid or other polymers based on hydrocarbons, polymers of amino acids, derivatives of Biotin, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, a copolymer of ethylene and maleic acid anhydride, copolymer of styrene with maleic acid anhydride, polyoxazoles, polyacrylonitril, heparin, albumin, cellulose, hydrolysates of chitosan, starches such as hydroxyethyl-starch and hydroxypropyl-starches, glycogen, agarose and derivatives thereof, guar gum, pullulan, inu is in, xanthan gum, carrageenan, pectin, alginic acid hydrolysates, other biopolymers, and any cash equivalents. Preferred is polyethylene glycol, and even more preferred is methoxypolyethyleneglycol (mPEG). Other useful polyalkyleneglycols compounds are polypropyleneglycol (PPG), polietilenglikoli (PBG), PEG-glycidyl esters (EpoxPEG), PEG-externaleditor (CDI-PEG), branched polyethylene glycols, linear glycols, fork glycols and multibeam or "hyperbranched" glycols (star-PEG).

Polyethylene glycol (PEG). "PEG" and "polyethylene glycol" are used herein interchangeably and include any water-soluble poly(ethylene oxide). In a typical case, Page for use in accordance with the invention include the following structures: "--(OCH2CH2)n--"where (n) is 2 to 4000. Used herein, the PEG also includes "--CH2CH2-O(CH2CH2O)n--CH2CH2--" and "--(och2CH2)nO--", depending on whether substituted or not substituted terminal oxygens. Throughout the description and points of claim, remember that the term "PEG" includes patterns having different terminal and terminal kiperousa" groups, including, without limitation, as hydroxyl or C1-20-alkoxy gr is the PAP. The term "PEG" means a polymer that contains a majority, i.e. more than 50%, of the repeating subunits --OCH2CH2--. With regard to specific forms, the PEG can have any number of varieties of molecular weights and structures or geometries, such as branched, linear, forked and multi-functional.

PEG-iliriana. PEG-iliriana is the process by which polyethylene glycol (PEG) covalently attached to a molecule such as a protein.

Activated or active functional group. When a functional group, such as a biocompatible polymer, referred to as an activated functional group readily reacts with the electrophile or nucleophile on another molecule.

FVIII with deletionism In-domain (BDD). The term BDD used here is characterized by the presence of an amino acid sequence that has a deletion of all but the 14 amino acids of the b-domain of FVIII. The first 4 amino acids of the b-domain ((SFSQ, SEQ ID NO:1) linked to the last 10 residues In domain (NPPVLKRHQR, SEQ ID NO:2). (Lind, P. et al., 1995, Eur. J. Biochem. 232, pp.19-27). Used here BDD has the amino acid sequence of SEQ ID NO:3.

FVIII. Factor VIII (FVIII) blood coagulation is a glycoprotein synthesized and secreted into the bloodstream by the liver. When blood circulation, it binds to factor von Willebrand is a (vWF, also known as related to Factor VIII antigen) with the formation of a stable complex. Upon activation by thrombin he dissociates from the complex and interacts with other blood clotting factors in the coagulation cascade, which eventually leads to the formation of blood clots. Human FVIII full length has the amino acid sequence of SEQ ID NO:4, although possible allelic variants.

The functional factor VIII polypeptide. The term "functional factor VIII polypeptide"used herein means a functional polypeptide or combination of polypeptides, which are capable, in vivo or in vitro, to correct a deficiency of factor VIII in humans, characterized by, for example, hemophilia A. In natural condition factor VIII has numerous degradation and versions of the forms. As shown here, they happen proteoliticeski from its predecessor, the single-chain protein. Such single-chain protein and various degradation products, which have biological activity correction of the deficiency of factor VIII in humans, will be the functional factor VIII polypeptide. There are probably allelic variants. Functional factor VIII polypeptides will be all such allelic variations, glycosylated variants, modifications and fragments, leading to about wodnym factor VIII to the extent that as far as they contain the functional segment of the human factor VIII, and the special characteristic of human factor VIII functional activity remains essentially unaffected. These derived factor VIII having the desired functional activity can be easily identified described here is simple in vitro tests. In addition, the functional factor VIII polypeptide capable of catalyzing the conversion of factor X to XA in the presence of factor IXa, calcium and phospholipid, and to correct the coagulation defect in plasma derived from affected by hemophilia And individuals. Disclosed here is the sequence of human factor VIII amino acid sequences and their functional area make obvious to a person skilled in the art fragments that can be separated by cutting with restriction enzymes DNA or proteolytic or other degradation of the protein human factor VIII.

The term "FIX", as used here, means Coagulation Factor IX, which is also known as Human Factor GC Coagulation or Thromboplastin Component Plasma.

Terim "FX", as used here, means Coagulation Factor X, which is also known under the name of Human Factor X Coagulation and under the eponym factor Stewart-Great the EPA.

The pharmacokinetics. "Pharmacokinetics" ("RK") is a term used to describe the properties of absorption, distribution, metabolism and elimination of drugs in the body. Improving the pharmacokinetics of drugs means improving those characteristics that make the drug more effective in vivo as a therapeutic agent, especially the duration of its useful effect in the body.

Mutein. Mutein is a derived through genetic engineering protein arising as a result of mutation of the protein or polypeptide induced in the laboratory.

The terms "protein" and "polypeptide"as used herein are synonymous.

The term "receptor excretion of FVIII"used here means a receptor region on the functional FVIII polypeptide which is associated or connected with one or more other molecules, which leads to the excretion of FVIII from the bloodstream. Receptors removal of factor VIII include, in particular, the region of the FVIII molecules that bind to LRP, LDL receptor and/or HSPG.

Discussion

It seems that any functional polypeptide factor FVIII can be mutated in a pre-specified site and then covalently linked in this site with biocompatible polymer in accordance with the methods according of the bretania. Suitable polypeptides include, in particular, full-size factor FVIII with the amino acid sequence shown in SEQ ID NO:4, and BDD FVIII having the amino acid sequence shown in SEQ ID NO:3. BDD FVIII is preferred.

Biocompatible polymer used in the conjugates according to the invention, can be any of the polymers mentioned above. Biocompatible polymer is selected to achieve the desired improvements in pharmacokinetics. For example, the identity, size and structure of the polymer is selected such as to increase the half-life of the polypeptide having the activity of FVIII, from blood flow or to reduce the antigenicity of the polypeptide without unwanted decrease of its activity. Preferably, the polymer comprises PEG and even more preferably is at least 50% of the molecular weight of PEG. In one embodiment, the execution of the polymer is a polyethylene glycol, a terminal kopirovany limit capitalsim fragment, such as hydroxyl, alkoxy, substituted alkoxy, alkenone, replaced alkenone, alkyloxy, replaced alkyloxy, aryloxy and replaced aryloxy. Even more preferred are polymers comprising methoxypolyethyleneglycol. And even more preferred are polymers comprising methoxypolyethyleneglycol in the region have the size in the range from 3 kDa to 100 kDa, more preferably from 5 kDa to 64 kDa or 5 kDa to 43 kDa.

Preferably, the polymer contains reactive fragment. For example, in one embodiment, the polymer contains a sulfhydryl reactive fragment that can react with cysteine functional polypeptide factor FVIII with the formation of covalent bonds. Such sulfhydryl reactive fragments comprise thiol, triflate, TResult, aziridine, oxirane, S-pyridyl or maleimide fragments. It is preferable maleimides fragment. In one implementation, the polymer is linear and has on one end cap, which is not very reactive towards sulfhydryl (such as methoxycoumarin), and the other end is a sulfhydryl reactive fragment. In the preferred implementation of the conjugate comprises a PEG-maleimide and has a size in the range of from 5 kDa to 64 kDa.

In the following examples provide guidance on the selection of suitable biocompatible polymers.

Site-directed mutation of the nucleotide sequence that encodes a polypeptide having the activity of FVIII, may be carried out by any method known in the art. Preferred methods include mutagenesis with the aim of introducing cysteine codon in the a site selected for covalent attachment of the polymer. This is can be done using a commercially available kit for site-directed mutagenesis, such as a set for the site-directed mutagenesis, Stratagene cQuickChange™ II kit for site-directed mutagenesis No. K1600-1 Clontech Transformer, the system for site-directed mutagenesis No. 12397014 Invitrogen GenTaylor, set system for site-directed mutagenesis in vitro No. Q6210 Promega Altered Sites II or kit for PCR mutagenesis No. so RR016 Takara cool simple point pointers rubber Bio LA.

The conjugates according to the invention can be prepared by first codon substitution of one or more amino acids on the surface of the functional FVIII polypeptide with cysteine codon for further obtaining cysteine mutein in a recombinant expression system, by conducting the reaction mutein with the cysteine-specific polymeric reagent and cleaning mutein.

In this way the addition of polymer to the site cysteine can be performed by maleimide, functionally active polymer. Examples of this technology are given below. The number of sulfhydryl reactive polymer should be at least equimolar to the molar quantity cysteine, which must be produced, and is preferably present in excess. Preferably, the sulfhydryl reactive polymer is used at least in 5-fold molar excess of and even more preferably used, p is at least in a ten-fold excess. Other conditions for the covalent joining, are included in the scope of specialist knowledge in this field of technology.

In the examples which follow, mutiny are conventional in the art name. Full amino acid sequence of the Mature factor VIII, which is presented in SEQ ID NO:4 is the basis for giving the names of the mutants. Being secretively protein FVIII contains a signal sequence that proteoliticeski cleaved during translation process. After removal of the signal sequence of 19 amino acids of the first amino acid secreted FVIII product is alanine.

When speaking about the mutated amino acids in BDD-FVIII, the mutated amino acids are indicated in this document traditional way, their position in the sequence of full length FVIII. For example, consider below mutein PEG indicated CS because it lysine (K) at the same positions in 1808 sequence of full length, replaced by a cysteine (C).

Predefined site for covalent binding of the polymer it is best to choose from sites located on the surface of the polypeptide, which do not affect the activity of FVIII or other mechanisms that stabilize FVIII in vivo, such as wezwanie with vWF. In addition, it is better to choose such sites are known in the mechanisms through which FVIII is deactivated or removed from the bloodstream. Detail the choice of these sites is discussed below. Preferred sites encompass amino acid residue at or near the binding site (a) protein related receptor low density lipoprotein, (b) heparansulfate of proteoglycan, (C) receptor low density lipoprotein and/or (d) antibody inhibiting factor FVIII. The expression "on or near the binding site" refers to the residue, which is close enough to the binding site, so that covalent joining of biocompatible polymer to this site would lead to steric damage to the binding site. It is assumed that such a site, for example, is within a 20 Å from the binding site.

In one embodiment, a biocompatible polymer is covalently attached to the functional factor VIII polypeptide at amino acid residue at or near (a) receptor removal of factor VIII, as defined above, (b) binding site of the protease that can degrade factor VIII and/or (C) the binding site of inhibitory antibodies to factor VIII. Proteases can be activated protein C (APC). In another embodiment, a biocompatible polymer is covalently attached functionalname the factor VIII polypeptide in a pre-specified site so binding protein related receptor low-density lipoprotein, the polypeptide is weaker than the same polypeptide, when he deconjugation, and preferably weaker by more than two times. In one embodiment, a biocompatible polymer is covalently attached to the functional factor VIII polypeptide in a pre-specified site so that binding of heparansulfate proteoglycans with polypeptide is weaker than the same polypeptide, when he deconjugation, and preferably weaker by more than two times. In the following embodiment, a biocompatible polymer is covalently attached to the functional factor VIII polypeptide in a pre-specified site so that binding of inhibitory antibodies to factor VIII polypeptide with is weaker than the same polypeptide, when he deconjugation, preferably weaker, more than twice than the binding with the polypeptide, when he deconjugation. In another implementation biocompatible polymer is covalently attached to the functional factor VIII polypeptide in the pre-site, so that receptor binding of low density lipoprotein with polypeptide is weaker than the same polypeptide, when he deconjugation, preferably weaker bol is e than twice. In another embodiment, a biocompatible polymer is covalently attached to the functional factor VIII polypeptide in the pre-site, so that the plasma protease degrades the polypeptide is weaker than when the polypeptide deconjugation. In the following embodiment, a degradation of the polypeptide by the protease plasma is more than two times weaker than the degradation of the polypeptide, when he deconjugation, under these same conditions for the same period of time.

The affinity of binding of LRP, LDL and HSPG with FVIII can be determined using the technique of surface plasmon resonance (Biacore). For example, FVIII may be caused directly or indirectly through FVIII-antibody on a Biacore™chip, and, passing through the chip, various concentrations LRP can be measured as the rate of Association and dissociation rate (Bovenschen, N. et al., 2003, J. Biol. Chem. 278(11), pp.9370-7). The ratio of the two velocities gives the value of affinity. When PEG-Yerevani it would be desirable two, preferably five times, more preferably ten-fold and even more preferably 30-fold lower affinity.

Degradation of FVIII protease ARS can be measured by any means known to specialists in this field of technology.

In one embodiment, a biocompatible polymer is covalently attached to p is dipeptide in one or more positions of the amino acids 81, 129, 377, 378, 468, 487, 491, 504, 556, 570, 711, 1648, 1795, 1796, 1803, 1804, 1808, 1810, 1864, 1903, 1911, 2091, 2118 and 2284 factor VIII. In another embodiment, a biocompatible polymer is covalently attached to the polypeptide at one or more positions of the amino acids 377, 378, 468, 491, 504, 556, 1795, 1796, 1803, 1804, 1808, 1810, 1864, 1903, 1911 and 2284 factor VIII, and (1) binding of the conjugate with protein related receptor low-density lipoprotein, is less than the binding of the unconjugated polypeptide to a protein related receptor low-density lipoprotein; (2) binding of the conjugate to a receptor low-density lipoprotein less than the binding of the unconjugated polypeptide to the receptor lipoprotein low density; or (3) binding of the conjugate with protein-related receptor low density lipoprotein and lipoprotein receptor low density less than the binding of the unconjugated polypeptide to a protein related receptor low-density lipoprotein, and lipoprotein receptor low density.

In another embodiment, a biocompatible polymer is covalently attached to the polypeptide at one or more positions of the amino acids 377, 378, 468, 491, 504, 556 and 711 of factor VIII, and the binding of the conjugate with heparansulfate the proteoglycan is less than the binding of the unconjugated polypeptide with heparansulfate Proteas what Econom. In the following embodiment, a biocompatible polymer is covalently attached to the polypeptide at one or more positions of the amino acids 81, 129, 377, 378, 468, 487, 491, 504, 556, 570, 711, 1648, 1795, 1796, 1803, 1804, 1808, 1810, 1864, 1903, 1911, 2091, 2118 and 2284 factor VIII, and the conjugate has a smaller binding inhibitory antibodies to factor VIII than the unconjugated polypeptide. In a further embodiment, the biocompatible polymer is covalently attached to the polypeptide at one or more positions of the amino acids 81, 129, 377, 378, 468, 487, 491, 504, 556, 570, 711, 1648, 1795, 1796, 1803, 1804, 1808, 1810, 1864, 1903, 1911, 2091, 2118 and 2284, and preferably one or more of the provisions of 377, 378, 468, 491, 504, 556, and 711 of factor VIII, and the conjugate undergoes less degradation by plasma protease that can degrade factor VIII than the unconjugated polypeptide. More preferably, the protease plasma was activated protein C.

In the following embodiment, a biocompatible polymer is covalently attached to the factor VIII deletionism In-domain positions of the amino acids 129, 491, 1804 and/or 1808, more preferably in the provisions 491 or 1808. In a further embodiment, the biocompatible polymer is attached to the factor VIII polypeptide at amino acid position 1804 and includes polyethylene glycol. Preferably, one or more pre for the data sites for attaching a biocompatible polymer are controlled by site-specific mutation of cysteine.

Predefined sites for attachment of a biopolymer can be one or more sites, preferably one or two customers, on the functional factor VIII polypeptide. In particular executions of the polypeptide is nanopeg-Yerevani or djpeg-Yerevani.

The invention also relates to a method for producing a conjugate, providing matirovanie nucleotide sequence that encodes a functional polypeptide factor VIII, a substitution of the sequence cysteine codon in the pre-site; the expression of the mutated nucleotide sequence to produce a modified cysteine mutein; cleaning mutein; responding mutein with a biocompatible polymer that is activated for reaction with a polypeptide essentially only restored cysteine residues, which form a conjugate; and purification of the conjugate. Another variant implementation of the invention is a method of site-directed PEG-helirovanie mutein factor VIII, comprising: (a) the expression of site-directed mutein factor VIII, where mutein has a cysteine substitution of amino acid residue on the outer surface mutein factor VIII and this cysteine kaparot; (b) contacting the cysteine mutein with a reducing agent under conditions in which cysteine mutein gently restore vlivaetsa and separates the cap; (C) removing the cap and reductant from cysteine mutein; and (d) after at least about 5 minutes, preferably at least 15 minutes, even more preferably at least 30 minutes after removal of the reducing agent is treated with cysteine mutein Peg, including sulfhydryl-binding fragment, under such conditions to form a PEG-pilirovanny mutein factor VIII. Sulfhydryl-binding fragment PEG selected from the group consisting of Colnago, triflates, cresylate, aziridines, oxiranes, S-peredelnogo and maleimides fragments, preferably of maleimide.

The invention also relates to pharmaceutical compositions for parenteral administration comprising a therapeutically effective amount of the conjugates of the present invention and a pharmaceutically acceptable adjuvant. Pharmaceutically acceptable adjuvants are substances that can be added to the active ingredient, to help formulate or stabilize the drug and not to cause any significant adverse Toxicological effects to the patient. Examples of such adjuvants are well known to experts in the art and include water, sugars such as maltose or sucrose, albumin, salt, etc. Other adjuvants are described, for example, E.W.Martin in Remington''s Pharmaceutical Sciences. So the e compositions will contain an effective amount of the conjugate described herein together with a suitable amount of carrier in order to prepare pharmaceutically acceptable compositions suitable for effective administration host. For example, the conjugate may be parenterally administered to the persons suffering from hemophilia A, at a dose, which may vary depending on the severity of injury bleeding. The average dose administered intravenously, are within 40 units per kilogram on preoperative indications, 15-20 units per kilogram in a small bleeding and 20-40 units / kg, administered over an 8-hour period as a maintenance dose.

In one embodiment, the method comprises substitution of one or more located on the surface of the BDD of the amino acids cysteine, getting cysteine mutein in the expression system mammalian recovery of cysteine, which was Kairouan during the expression of the cysteine from the medium of cultivation, removal of the reducing agent to give the opportunity BDD-disulfides to restore the structure, and reaction with the reagent cysteine-specific biocompatible polymer, such as PEG-maleimide. Examples of such reagents are added to maleimide sizes PEG 5,22 or 43 kDa, available from Nektar Therapeutics (San Carlos, the Caliph.) under catalog numbers Nektar 2D2M0H01 mPEG-MAL, MW 5000 Da, 2D2M0P01 mPEG-MAL, MW 20 kDa, 2D3X0P01 mPEG2-MAL MW 40 kDa, respectively, or 12, or 33 kDa, available from NOF Corporation (Tokyo, Japan) is od catalog numbers NOF Sunbright ME-120MA and Sunbright ME-300MA respectively. PEG-pilirovanny the product was then purified using ion exchange chromatography to remove unreacted PEG and gel chromatography to remove unreacted BDD. This method can be used to identify and election protection from any unwanted interactions with FVIII, such as receptor-mediated elimination from the bloodstream, binding inhibitory antibodies and degradation by proteolytic enzymes. We observed that the PEG-reagent supplied by Nektar or NOF as having a size of 5 kDa, tested in our laboratory, was having a size of 6 kDa, and, similarly, the PEG-reagent supplied as a linear 20 kDa, testing was having a size of 22 kDa; supplied as a 40 kDa testing had a 43 kDa; and supplied as a 60 kDa was tested as having a 64 kDa in our laboratory. To avoid confusion, we here discuss the use of molecular weight, as he was identified in our laboratory, with the exception of PEG 5 kDa, about which we speak of as having a size of 5 kDa, as he is identified by the manufacturer.

In addition to cysteine mutations at positions 491 and 1808 in BDD (disclosed above) were implemented cysteine mutations at positions 487, 496, 504, 468, 1810, 1812, 1813, 1815, 1795, 1796, 1803 and 1804, to potentially block LRP-binding after PEG-helirovanie. In addition to after PEG-the modeling block LRP and HSPG-binding, were made to cysteine mutation at positions 377, 378 and 556. Provisions 81, 129, 422, 523, 570, 1864, 1911, 2091 and 2284 were selected as evenly spaced on BDD to site-directed PEG-iliriana Pagami large (>40 kDa) in these regulations, together with PEG-Yerevanian the native glycosylation sites (41, 239 and 2118) and the websites of the LRP-binding completely covered the surface of BDD and so we can identify a new mechanism for removal from the bloodstream for BDD.

In one embodiment, a medium for culturing cells contains cysteine that "kaparot" cysteine residues in mateine through the formation of disulfide bonds. In the preparation of the cysteine conjugate mutein produced in a recombinant system, kaparot the cysteine from the medium of cultivation, and this cap is removed by a mild recovery, which frees the cap before adding cysteine-specific polymeric reagent. Other methods known in the field for site-specific mutations in FVIII, can also be used, as they are obvious to a person skilled in this field.

Examples

ANALYSIS OF ACTIVITY OF THE STRUCTURAL RELATIONS OF FVIII. FVIII and BDD-FVIII are very large, complex molecules with many different sites involved in biological reactions. Previous attempts covalently modifier is to them, to improve pharmacokinetic properties, had conflicting results. What molecules could specifically mutate and then a site-specific way to add the polymer was unexpected. In addition, there were also unexpected results in improved pharmacokinetic properties and preservation activity, whereas, previously known polymeric conjugates were inherent problems of occurrence of non-specific increase or decrease activity.

In one embodiment, the invention provides for site-directed mutagenesis using the cysteine-specific ligands, such as PEG-maleimide. Neutropenia BDD does not have any cysteine available for reaction with PEG-maleimide, so that only the mutated position of cysteine would be the site of PEG-helirovanie. More specifically, the BDD-FVIII contains 19 cysteines, 16 of which form disulfides and the remaining 3 of which are free cysteine (McMullen et al., 1995, Protein Sci. 4, pp.740-746). Structural model of BDD assumes that all 3 free cysteine within the structure (Stoliova-McPhie et al., 2002, Blood 99, pp.1215-1223). Because okislennye cysteine cannot be PEG-Yerevani PEG-maleimide, 16 cysteines that form in BDD disulfides may not be PEG-Yerevani, while the first will not be restored. On the basis of the structures of the first BDD model 3 free cysteine in BDD cannot be PEG-Yerevani without prior denaturation of the protein, to these cysteine became available PEG-reagent. Thus, it is not obvious it is possible to achieve specific PEG-helirovanie BDD PEG-Yerevanian on native cysteine residues without serious changes in the structure of the BDD that will very likely lead to the violation of its functions.

The redox state 4 cysteines in-domain full length FVIII unknown. PEG-iliriana 4 cysteines in-domain can be possible if they do not form disulfides and are located on the surface of the structure. However, since full-FVIII and BDD have similar pharmacokinetic (PK) profile and a similar elimination half-life in vivo (Gruppo et al., 2003, Haemophilia 9, pp.251-260), it is unlikely that PEG-iliriana In-domain will result in improved half-life from plasma, unless it appears that the PEG also protects areas that are not related to In-domain.

To pre-determine the site of the polypeptide having the activity of FVIII, to attach the polymer, which will retain the activity of factor VIII and improve the pharmacokinetics, the following recommendations are based on BDD-FVIII. Modifications should be directed to mechanisms for the removal, inactivation and immunogenic mechanisms, such as LRP, HSPG, ARS, and the binding sites of inhibitory antibodies. The structure of the BDD shown in Stoilova-McPhie. S. et al., 2002, Blood 99(4), pp.1215-23. For example, Strait to herawati half-life, you can enter a single PEG in a specific site on or near sites LRP-binding in the range of residues 484-509 of the A2 domain and in the range of residues 1811-1818 domain A3. The introduction of bulk PEG in these sites should interrupt the ability of FVIII to contact LRP and reduce the excretion of FVIII from the bloodstream. It is also assumed that for prolonging the half-life period without significant effect on the activity of the PEG can be entered on the balance of 1648, which is located at the junction of the In-domain and A3-domain of the molecule in the full length, and 14-amino acid linker I BDD between domains A2 and A3.

The specificity of PEG-helirovanie can be achieved through the introduction of a single cysteine residue in domain A2 or A3, using methods of mutagenesis using recombinant DNA, using the site-specific PEG-iliriana introduced cysteine by cysteine-specific PEG-reagent, such as PEG-maleimide. Another advantage of PEG-helirovanie on residues 484-509 and 1811-1818 is that these two epitopes are two of the three main classes of inhibitor antigenic sites in patients. To achieve the maximum effect of improving the half-life from blood flow and reducing the immunogenic response can PEG to reroute as A2, A3 sites LRP-binding to get dipag-pilirovanny product. It should be noted that PEG-iliriana HV the three areas 1811-1818 can lead to a significant loss of activity, because this region is also involved in the binding of FIX. Site-directed PEG-iliriana inside the area 558-565 should resolve HSPG binding, but may also reduce the activity, since this area is also associated with the FIX.

To identify a new mechanism for the excretion of FVIII, you can also PEG-reroute additional sites on its surface. PEG-iliriana domain A2 can give the additional advantage that the A2 domain dissociates from FVIII after activation and, presumably, is removed from the bloodstream faster than the rest of the FVIII molecule, because of its smaller size. On the other hand, PEG-pilirovanny A2 may not be large enough to avoid elimination by the kidneys, and to have a half-life from plasma is comparable to that for the remainder of FVIII, and, thus, it is possible to restore activated FVIII in vivo.

IDENTIFICATION of SITES of PEG-helirovanie IN the AREAS A2 and A3. Five positions (Y487, L491, K, L504 and Q468, the relevant provisions of PEG1-5) in or near the intended LRP-binding A2 were selected as examples of site-directed PEG-helirovanie based on the surface location and direction in the external side of the path α-β. In addition, these balances are approximately equidistant from each other in the spatial structure of molecules, so together they can represent a whole about the art. Eight of the provisions(1808, 1810, 1812, 1813, 1815, 1795, 1796, 1803, 1804, relevant PEG6-14) in or near the intended LRP-binding A3 were selected as examples for carrying out site-directed PEG-helirovanie. PEG (C) is bordered 1811-1818 and natural N-linked glycosylation site in position 1810. PEG-iliriana in position 1810 (PEG) leads to the substitution of sugar on the PEG. The mutation at position T 1812 PEG also eliminates the glycosylation site. Although, as predicted, PEG-position (K1813) is directed inward structure, it was selected for the case, if the model structure is incorrect. PEG 10 (Y1815) is a three-dimensional hydrophobic amino acids within loop LRP-binding and may be the primary balance of interaction, because the hydrophobic amino acids are usually found in the center of the protein-protein interactions. Because the area 1811-1818, as stated, is involved in the binding of both LRP and FEX, an assumption was made that the PEG-iliriana inside this loop can lead to reduced activity. In this regard, near loop 1811-1818, but not inside it, were designed PEG-PEG(1795, 1796, 1803, 1804), that became possible dissociation of the binding of LRP and FIX with PEG of different sizes.

To simultaneously block both customers LRP-binding, can be made double PEG-iliriana, for example, the state is x PEG and PEG.

Since the area 558-565, has been shown to be associated with HSPG and FIX inside this field sites were not created. Instead, between the areas of LRP and HSPG-binding A2 were designed PEG-PEG (377, 378 and 556), attached to the PEG could hinder interactions, and to interrupt the possible interaction between them. Additional websites which are located on the surface and directed outward, could be selected within or near the areas of LRP and HSPG-binding. To identify new mechanisms of excretion, can be systematically added to reroute FVIII. In addition to PEG-17 as tie-points for PEG-helirovanie you can use three other natural site of glycosylation, namely N14, N239 and N2118 corresponding PEG 18-20, since they must be located on the surface. On the BDD model in addition to the functional interaction sites for vWF, FIX, FX, phospholipid and thrombin were mapped surface area within a radius of 20 angstroms from β atoms PEG, PEG and four glycosylation site.

Then, based on their ability to cover almost completely the remaining BDD surface with a radius of 20 angstroms from each of them β atoms were selected PEG-29 corresponding Y81, F129, K, K, K, N1864, T, Q2091 and Q2284. Because these provisions are completely on the surface, are what nesne directed and are far from natural cysteine, they were also selected to minimize possible incorrect formation of disulfides. The radius of 20 angstroms was chosen because it is assumed that the big PEG, such as branched PEG 64 kDa, has the ability to cover a sphere with a radius of about 20 angstroms. PEG-iliriana PEG-29 together with PEG and PEG and sites of glycosylation PEG 18, 19 and 20 will probably protect almost completely non-functional surface of FVIII.

The provisions of PEG-helirovanie that lead to improved properties such as improved RK-profile, higher stability or reduced immunogenicity, can be combined to obtain multipage-pilirovanny product with the most improved properties. PEG and PEG were constructed by removing disulfides, located on the surface of A2 - and A3-domains, respectively. For PEG-helirovanie PEG, or SA must be freed from its disulfide partner S. Also, PEG, SO should allow S to be PEG-Yerevani.

MUTAGENESIS. Substrates for site-directed PEG-helirovanie FVIII can be created by introduction of a cysteine codon in the a site selected for PEG-helirovanie. For all PEG-mutants used kit for site-directed mutagenesis, Stratagene cQuickChange™ II kit Stratagene 200523 from Stratagene Corporation, La Yola, the Caliph.). Method with it-directed mutagenesis cQuickChange™ was carried out, using PfuTurbo® DNA polymerase and thermal cycler. Two complementary oligonucleotide primers containing the desired mutation were bongiovani using PfuTurbo, which does not displace the primers. As the matrix was used Dunaeva DNA, containing the gene for wild-type FVIII. After many cycles of elongation product was digested with endonuclease DpnI, which is specific for methylated DNA. Newly synthesized DNA containing the mutation was not methylated, while the parent wild type DNA was methylated. Cleaved DNA was then used for transformation supercompetent XL-1 Blue cells.

The efficiency of mutagenesis was almost 80%. The reaction mutagenesis was carried out either in pSK207+BDD C2.6 or pSK207+BDD (Figure 1). Successful mutagenesis was confirmed by DNA sequencing, and the appropriate fragments containing the mutation was included in the major site of expression vector mammalian pSS207+BDD encoding FVIII. After doing all mutations were again confirmed by sequencing. For A3-Malinov PEG 6, 7, 8, 9 and 10 mutagenesis was performed in the vector pSK207+BDD C2.6. After confirmation by sequencing of the mutant fragment Cloned/Pme was subcloned into pSK207+BDD. BDD-mutein then was subcloned into expressing vector pSS207+BDD. For A3-Malinov PEG 11, 12, 13, 14 mutagenesis was carried out directly in the vector pSK207+BDD and confirmed seque what funding mutant BDD then subclavian in pSS207+BDD. For A2-Malinov PEG 1, 2, 3, 4 and 5 mutagenesis was performed in the vector pSK207+BDD C2.6. Confirmed by sequencing the mutant was subcloned into pSK207+BDD, and then in pSS207+BDD.

PRIMERS (ONLY SEMANTIC THREAD), USED FOR MUTAGENESIS are LISTED FOR EACH REACTION:

EXPRESSION MUTEIN. After introduction into the vector that confers resistance to Hygromycin (GIHR), PEG-mutiny were transfected into NCV cells (U.S. Patent 6136599), mixed with a Reagent for Transfection Of 293 Pectin (Invitrogen Corp. cat. No. 12347-019) according to the manufacturer's instructions. The expression of FVIII in a three-day posttransfection period was assessed by chromogenic analysis Coatest (Chromogenix Corp. cat. No. 821033, see Example 12, a chromogenic assay) (table 1). Then the transfected cells were placed under selective influence of 50 µg/ml Giger In growth medium, supplemented with 5% fetal bovine serum (FBS). After the emergence of resistant to Gigr In the colonies they were collected and were skanirovali on the expression of FVIII by chromogenic analysis Latest. Then stable expressing FVIII cells were introduced into the medium containing the additive HSPG. Cells were grown and sown at a concentration of 1 × 106cells/ml in Catalogne flask with fresh medium. The culture fluid (TCF), collected after 3 days was used for the purification of FVIII BDD-Malinov. The FVIII activity of TCF were analyzed using Coatest (table 1). (N/A = analysis not conducted).

Table 1
The level of expression of PEG-Malinov in transient and stable transfected
Total credits PEG-Malinov
Titer (IU/ml)
MutationID MuteinThe transient.The camera. cell
Y487CPEG0,07N/A
L491CPEG0,601,96
K496CPEG0,45N/A
L504CPEG0,38to 5.57
Q468CPEG0,698,14
K1808C PEG0,542,73
N1810CPEG0,210,5
TSPEG0,16N/A
KSPEG0,357,74
Y1815CPEG0,09N/A
D1795CPEG0,27N/A
Q1796CPEG0,29N/A
R1803CPEG0,11N/A
K1804CPEG0,181,14
L491C/K1808CPEG+60,112,48
L491C/K1804CPEG+140,1 7,19
K377CPEG0,1112,58
NSPEG0,150,97
K556CPEG0,090,15
N41CPEG0,05N/A
N239CPEG0,16N/A
N2118CPEG0,13N/A
Y81CPEG0,36N/A
F129CPEG0,252,55
K422CPEG0,28N/A
K523CPEG<0,05N/A
K570CPEG<0,05N/A
N1864CPEG0,15N/A
TSPEG0,28N/A
Q2091CPEG0,20N/A
Q2284CPEG0,17N/A
SAPEG<0,050,20
SAPEG0,301,80

CLEANING MALINOV. After collecting the cell culture supernatant containing secreted malinowy the FVIII protein, the supernatant was filtered through an 0.2 micron membrane filter to remove any remaining cells. Then the supernatant was concentrated either by ultrafiltration or by using anion exchange. Then he was sent to immunoaffinity column where the components of the environment of the cultivation of cells and the majority in the forest proteins from the host cells were removed. Then the eluate with immunoaffinity column has buhariwala diafiltrate in the buffer composition containing sucrose, and frozen. Exit and collect the protein through the column with monoclonal to FVIII antibodies were evaluated by chromogenic assay. Samples loaded flowing through the column, different buervenich fractions, stripes and diafiltration of the eluate chromatographic studies were analyzed for FVIII activity (table 2). Table 2 shows the collection PAG-mutein with a column with monoclonal antibodies. Antibodies are antibodies C7F7. The percentage of collection in Table 2 is determined using a chromogenic assay. The final yield was 73%. The Figure 2 shows a graph of UV-absorption at 280 nm versus time for PEG-protein, purified through a chromatographic column with monoclonal antibodies to FVIII. Chromatography was performed using chromatographic system ACT® Explorer 100 from Amersham Bioscience. This system uses multi-wave UV-Visible monitor and throughput cell 2 mm PEG-mutein blueraven with a column in the presence of high salt, and peak elution is indicated by absorption at 280 nm, so the analysis of FVIII activity.

Table 2
Collection PAG-muteena column with the monoclonal antibodies to FVIII
Stage% recovery
C7F7 loaded100
C7F7 in the stream1,1
C7F7-washed0,2
C7F7 the eluate86
C7F7 band0,0
C7F7 UF/DF73

PEGYLATION. Native FVIII full length or BDD cannot be PEG-Yerevan cysteine-specific PEG without recovery and denaturation at more than 100-fold excess of PEG: protein ratio (data not shown) confirms the hypothesis, based on the model structure BDD that all native cysteine form disulfides or inside FVIII. Cysteine FVIII-mutiny, expressed and purified using the above standard protocols could not be PEG-Yerevani cysteine-specific PEG-maleimide reagent, presumably because they are FVIII-cysteine Kairouan through reaction with sulfhydryl groups, such as cysteine and β-mercaptoethanol present in the growth medium of the cells. This issue could potentially be solved eliminare what W cysteine and β-mercaptoethanol from the environment of the cultivation, but this can lead to decrease in FVIII products, and would not impede sulfhydryl released by cells, block entered FVIII-cysteine.

In another aspect of the invention has been developed a three-stage method, enabling site-specific PEG-helirovanie FVIII (Figure 3). In stage 1 purified cysteine FVIII-lutein in an amount of about 1 micron gently restored by reducing agents, such as approximately 0.7 mm Tris(2-carboxyethyl)phosphine (TSER) or 0.07 mm dithiothreitol (DTT)for 30 minutes at 4°C to separate the cap. In stage 2, the reducing agent was removed together with the cap by means of the method gel chromatography (SEC), such as the passage of a sample through a rotating column (BioRad®)to enable disulfides FVIII reform, leaving the introduced cysteine free and restored. At stage 3 after at least 30 minutes after removal of the reductant released cysteine FVIII-mutein worked at least 10-fold molar excess of PEG-maleimide sizes in the range from 5 to 64 kDa (Nektar Therapeutics and N.O.F. Corporation) for at least 1 hour at 4°C. This method gives the profile of the product, in good agreement with existing data for many reactions, repeated by different researchers.

Due to the fact that the method of rotating the column to remove TSER not the two which is a large-scale, was selected by gel-filtration chromatography desalting. However, after validation of this method using a sample labeled TSAR, it was shown that TSAR was elyuirovaniya in measured quantities into the pores of the column and not directly in the salt fraction, as would be expected from a molecule with a low molecular weight. Western blot analysis showed a significant background PEG-helirovanie, probably due to incomplete removal TSAR. In a separate intermediate experiments it was shown that purified C7F7-material could be substantially purified further from other impurity proteins by using anion-exchange chromatography combined with a salt gradient. Then it was decided to restore C7F7-material using TSER, as described above, and then to process the material through the anion exchange column. Due to differences in charge, FVIII protein was able to help it, while TSAR flowed through the column and was gone. At the same time during elution salt gradient FVIII protein was purified from the majority of the remaining impurity proteins. This means that later the resulting PEG-iliriana it would be theoretically more homogeneous with a cleaner source material. However, after testing by sample labeled TSAR, it was shown that measurable levels TSAR discovered luorophenyl in the gradient with FVIII. Therefore, it was decided after anion-exchange chromatography to apply gel-filtration chromatography with desalting to these two stages, successively used, led to the complete destruction of TSER and exclude nonspecific PEG-helirovanie.

ANALYSIS OF PAGLIERANI BY ELECTROPHORESIS IN POLYACRYLAMIDE GEL WITH SODIUM DODECYL SULFATE (SDS-PAGE) AND WESTERN BLOTTING. PEG-pilirovanny product can be analyzed using electrophoresis on pampering 6% Trisglycine SDS-polyacrylamide gel (Invitrogen). If you continue electrophoresis gel can be painted, Kumasi Blue (Coomassie Blue) to identify all proteins or be subjected to standard Western blot Protocol for the identification of the figure of the PEG-helirovanie in various areas of FVIII. Staining of the blot mouse monoclonal antibody R8B12 or C7F7 created for the C-terminal region of the heavy chain of FVIII or N-terminal region of the light chain of FVIII, respectively, must identify the PEG-iliriana respective circuits. Staining with 413-antibodies to the field 484-509 FVIII must determine whether PEG-iliriana really site-specific or not, for Malinov, such as PEG-4. Similarly, staining with antibody CLB-CAg A, which recognizes the region 1801-1823 FVIII must determine whether PEG-iliriana really the AIT-specific or not, for Malinov, such as PEG-10.

It was shown that PEG-iliriana PEG (L491C) is selective for the heavy chain relative to the light chain and especially the election for the region 484-509 (Figure 4), while PEG (K1808C), as was shown to be an election for relatively light chain heavy chain (Figure 5).

For the study, depicted in Figure 4, PEG-mutein (tracks 1 and 8) were reconstructed using TSER, then TSAR removed (tracks 2 and 9) and treated with 5-, 12-, 22-, 33 - or 43-kDa PEG-maleimide (tracks 3-7 and 10-14). Napag-pilirovanny FVIII were investigated in raw form (H+L) and treated strips of heavy (H) and light (L) chains. All three bands are detected on the gel, colored Kumasi Blue (bottom right), while Western-staining specific to the chain antibodies showed only unreacted and the corresponding circuit. When painting R8B12 (top left) lane of the heavy chain (H) is strongly decreased in intensity when PEG was treated with PEG-maleimide, and created a new band, which was higher than the parent H-band, in proportion to the size of the PEG. When painting C7F7 (bottom left) strip light chain (L) (multiple bands due to heterogeneous glycosylation) did not change in intensity. Raw H+L band in both colourings shifted, poskoljko-chain is part of the raw FVIII. Staining of Kumasi also confirms much more PEG-iliriana heavy chain, i.e. the decrease of intensity of the H-band than the light chain. Finally, PEG-lirovannye bands lose relatively more in the intensity of staining 413-antibody (top right), than when painting with R8B12, depending on the size of the PEG, presumably due to site-specific PEG-helirovanie in 491, which blocks the binding 413-antibodies with 484-509. The amount of FVIII deposited on the track, approximately 30 ng for two left gels, about 1000 ng to the upper right of the gel and about 2000 ng to the bottom right of the gel.

Recovery, followed by removal of the reducing agent, does not alter the migration of FVIII (lane 1 compared to lane 2 and lane 8 compared with lane 9). The addition of 22 kDa-PEG to PEG blocks the binding 413-antibodies, which is consistent with a specific PEG-Yerevanian in position 491 (Figure 4, upper right gel). It also suggests that PEG-pilirovanny PEG will have lower immunogenicity in humans, because 413-antibody, as shown, has the same epitope as inhibitory antibodies to A2 person (Scandella et al., 1992, Thromb. Haemost. 67, pp.665-71).

For the study, depicted in Figure 5, PEG-mutein restored using TSER, then TSAR removed (tracks 1 and 6) and were processed through the 5-, 12-, 22 -, or 33 kDa-PEG-maleimide (lanes 2-5 and 7-10). Napag-pilirovanny FVIII were investigated in raw form (H+L) and treated strips of heavy (H) and light (L) chains. Due to the mutated residues PEG (K1808) on the light chain of the PEG-iliriana was detected only in the light chain, but not heavy. The amount of FVIII deposited on the track is about 100 ng to the left of the gel and about 30 ng to the right of the gel.

BDD, which was tested as a control, did not show any significant PEG-helirovanie after processing more than 100-fold molar excess of PEG-maleimide, even after the above procedure, recovery and removal of the reducing agent (Figure 6A). This same method was applied to PEG and PEG (Figure 6A). When compared with PEG, these mutiny were not PEG-Yerevani as effectively, but they were selective for the heavy chain, like PEG (L491C). The effectiveness of PEG-helirovanie PEG (CS) is relatively low, probably because it is very close to the site of N-linked glycosylation on N1810, which can block PEG-iliriana in position 1808. Therefore, we have designed PEG (N1810C)to delete the native glycosylation in 1810. PEG shows superior efficacy of PEG-helirovanie compared to PEG, if you compare them head to head" (Figure 6b). Similarly, PEG shows a few who Uchuu the effectiveness of PEG-helirovanie, than PEG.

PEG+6, the double mutant BDD may be PEG-Yerevan as heavy and light chain, because PEG is a cysteine mutation of the heavy chain, while PEG is a mutation of the light chain (Figure 6C). This method was applied also to FVIII wild-type full length (Figure 6d). PEG-iliriana was detected for the largest fragment of the heavy chain, which includes A1, A2, and most of the In-domain. Figure PEG-helirovanie suggests nanopeg-iliriana, and that there is only one PEG-pilirovanny cysteine.

ANALYSIS OF PAGLIERANI THROUGH CLEAVAGE BY THROMBIN AND WESTERN BLOTTING. PEG-pilirovanny product can be treated with thrombin (40 IU/μg FVIII) at 37°C for 30 minutes. Used thrombin also contains ARS as an impurity. Cleavage by thrombin leads to 50 kDa-A1 - 43 kDa-A2-domain of the heavy chain, whereas the cleavage of APC leads to the splitting of the A2-domain fragments 21 and 22 kDa (Figure 7). Staining with R8B12-antibody, which recognizes the C-end of the heavy chain, will identify only intact A2 domain and 21 kDa, C-terminal fragment (FVIII 562-740). Thus, if the PEG-iliriana PEG would be specific to the position 491, 43 kDa-A2-domain would have to be PEG-Yerevani, except for the 21 kDa C-terminal fragment. It was an action is positive confirmed by Western blotting for the 22 kDa-PEG-lirovannomu PEG, shown in Figure 7. Thus, by elimination of PEG-iliriana PEG was localized to the N-terminal 22 kDa fragment (FVIII 373-561) A2 domain. Because PEG-maleimide is completely selective for zisteinom at pH 6.8 and the only native FVIII-cysteine in the field 373-561 come from hidden disulfide between 528 and 554, it is highly likely that PEG PEG-Yerevan on the introduced cysteine at position 491. Western-staining treated with thrombin PEG-lirovannomu PEG using N-terminal part of the antibody heavy chain FVIII showed PEG-helirovanie A1-domain (data not shown). Election PEG-iliriana PEG using the method thrombin cleavage was also confirmed for PEG 5, 12, 33, and 43 kDa (data not shown). Cleavage by thrombin PEG-lirovannomu FVIII wild-type full length shows that PEG-Yerevan only In-domain (Figure 8).

ANALYSIS OF PAGLIERANI BY STAINING WITH IODINE. To confirm that the emerging new bands detected by staining Kumasi Blue and Western, are actually strips, showing the PEG-Yerevani was applied staining with barium iodide, which is specific to the PEG (Figure 9). PEG-pilirovanny PEG analysed on 6%Trisglycine gel (Invitrogen) and stained R812-antibody heavy chain or a solution of iodide of barium (Lee e al., Pharm Dev Technol. 1999 4:269-275). PEG-lirovannye bands were found between the two spots, using the molecular weight marker to align them, confirming, thus, the PEG-iliriana heavy chain of FVIII.

ANALYSIS OF PAGLIERANI USING MALDI-MACC-SPECTROSCOPY. To confirm PEG-helirovanie A2 domain in the heavy chain sample rFVIII before and after PEG-helirovanie analyzed by mass spectrometry using laser desorption/ionization in the presence of matrix (MALDI). The samples were mixed and were led on misheneva MALDI-tablet matrix from sinadinovic acid in 30% acetonitrile, 0.1% of TFA. Then they were analyzed in the spectrometer Voyager DE-PRO in positive linear mode. The results presented in Figure 10, showed that light chain PEG has a centre of 83 kDa, and a heavy chain (HC) - 89 kDa. Obtained for PEG-lirovannomu sample spectrum showed a decrease in NA-peak and the formation of a new peak centered at 111 kDa. This confirms the PEG-iliriana heavy chain. No PEG-helirovanie light chain (105 kDa) above the detection limit were observed.

Then the samples were subjected to cleavage by thrombin at 20 units of thrombin/mg of FVIII at 37°C for 30 minutes and determined the concentration of FVIII amino acid analysis (Commonwealth Biotechnologies, Inc.). A heavy chain was split at 48 kDa (A1) N-terminal fraction and 43 kDa (A2) faction is. The obtained MALDI spectrum for PEG-lirovannomu sample (Figure 11) shows the loss peak of 43 kDa and the appearance of a new peak of 65 kDa, due to PEG-lirovannomu A2 domain. PEG-iliriana light chain above the detection limit again was not observed. These results reaffirm the PEG-iliriana A2 domain of FVIII. The same analysis was applied to PEG-lirovannomu PEG and confirmed PEG-iliriana ASS-fragment light chain (Figure 12).

Measurement of the ACTIVITY

COAGULATION ANALYSIS. Test method coagulation FVIII:C is a single-stage analysis based on the activated partial thromboplastin time (artt). In the presence of Factor IXa, calcium and phospholipid FVIII acts as a cofactor in enzymatic conversion of Factor X to XA. In this analysis of the diluted test samples incubated at 37°C with a mixture of substrate from FVIII-deficient plasma and art-reagent. To the incubated mixture, add calcium chloride, and coagulation begins. Between the time (in seconds)that is required for the formation of a clot, and the logarithm of the concentration of FVIII:C there is an inverse ratio. The activity levels for unknown samples interpolate by comparing the clotting time of various dilutions of the test material with the curve constructed from a series of dilutions of the standard material with a known activity, averageat in International Units per ml (IU/ml).

CHROMOGENIC ANALYSIS. Method chromogenic analysis consists of two consecutive stages, where the color intensity is proportional to the activity of FVIII. In the first stage, the activate Factor X to FXa FIX with its cofactor, FVIIIa in the presence of optimal amounts of calcium ions and phospholipids. Excessive amounts of Factor X such that the rate of activation of Factor X depends only on the number of FVIII. In the second stage, the Factor Ha hydrolyzes the chromogenic substrate, giving the chromophore, and the color intensity is read photometrically at 405 nm. Calculates the value of the unknown, and the validity of the analysis is checked using a statistical method, the tangent angle of the curve. Activity is expressed in International Units per ml (IU/ml).

Loop 1811-1818 involved in binding to FIX, but the importance of individual provisions within this loop has not been determined. Mutiny PEG-10 find almost identical specific chromogenic activity relative to the native FVIII (table 3). Table 3 shows the percent specific activity (SA) PEG-maleinos and PEG-lirovannomu PEG or PEG relatively BDD. SA was determined by dividing the chromogenic, coagulation activity or activity of the vWF-binding total antigen ELISA value (TAE). Then SA PEG-Yerevani of Malinov divided into SA BDD (8 IU/μg chromium is genetic, 5 IU/μg of coagulation and 1 vWF/TAE) and multiplied by 100 to obtain the percentage of SA is given in Table 3 under the headings "chromogenic", "coagulation" and "vWF/TAE".

Table 3
The percentage of specific activity (SA) PEG-maleinos and PEG-Yerevani PEG and PEG relatively BDD
MutationChromogenicCoagulationvWF/TAE
BDD100100100
PEGY487C
PEGL491C125130138
BAG redL491C13714198
PEG-5 kDaL491C124 93125
PEG
PEG-12 kDaL491C1182571
PEG
PEG-22 kDaL491C1031387
PEG
PEG-33 kDaL491C1301759
PEG
PEG-43 kDaL491C91957
PEG
PEGK496C
PEGL504C
PAQ468C92
PEGK1808C8360100
PEG-33 kDaK1808C42690
PEG
PEGN1810C100
PEGTS100
PEGK1813C83
PEGY1815C75
PEGD1795C
PEGQ1796C
PEGR1803C
PEGK1804C
PEG+6S/S
PEGK377C82
PEGNS126the
PEGK556C43
PEGN41C80
PEGN239C
PEGN2118C127
PEGY81C
PEGF129C83
PEGK422C
PEGK523C
PEGK570C
PEGN1864C
PEGTS
PEGQ2091C
PEGQ2284C

As shown in Table 3, "PEG red" is PEG-lutein, which has been treated with a reducing agent, followed by removal of the latter. This recovery procedure is slightly modifies three functional activity of FVIII. PEG-mutein conjugated with PEG, having a size in the range of from 5 kDa (PEG-kDa) to 43 kDa (PEG-da), does not lose a significant amount of chromogenic activity, but has a much lower coagulation activity, when the size of the PEG increases beyond the 5 kDa. It is also possible moderate decrease in vWF-binding to PEG-lirovannomu PAG larger size.

TOTAL ANTIGEN ELISA (TAE). FVIII PE UNOSAT on a microtiter plate, which are coated with a polyclonal antibody to FVIII. The FVIII binding is detected using a biotinylated polyclonal antibodies to rFVIII and conjugate streptavidin-horseradish peroxidase (PSC). Peroxidase-streptavidin complex gives a color reaction after adding tetramethylbenzidine (TMB) substrate. Concentration of samples interpolating from a standard curve using a model with four appropriate parameters. The results of FVIII expressed in µg/ml.

ELISA vWF BINDING. FVIII was allowed to contact with vWF much hemophilic plasma in solution. Then the complex of FVIII-vWF were transferred to microtiter plate coated with vWF-specific monoclonal antibody. The binding of FVIII to vWF detected using biotinylated polyclonal antibodies to rFVIII and conjugate streptavidin-horseradish peroxidase (PSC). Peroxidase-streptavidin complex gives a color reaction after addition of the substrate. Concentration of samples interpolating from a standard curve using a model with four appropriate parameters. The results of the FVIII-binding expressed in µg/ml was found Not to have any significant impact on any of the activities after PEG-helirovanie, which would be consistent with the PEG-Yerevanian-domain.

Table 4
Specific activity (SA) FVIII wild-type full length (KG-2) before and after PEG-helirovanie through the PEG of different size
SampleTai, ug/mlCoagulation analysisChromogenic analysisvWF ELISA
IU/mlIU/ug% Ref.IU/mlIU/μg% Ref.mg/mlvWF/TAE% Ref.
KG-2 source1,314,83,6100the ceiling of 5.604,31000,420,32100
Only restore.0,933,13,4934,084,4103
KG-2-kDa PEG0,712,53,5963,094,3102
KG-2-12 kDa PEG0,592,3a 3.91072,995,0118
KG-2-22 kDa PEG0,632,5a 3.91083,064,81130,190,3094
KG-2-30 kDa PEG0,592,54,11143,015,1119 0,190,32100
KG-2-43 kDa PEG0,522,44,61282,865,5129

PURIFICATION of PEG-lirovannomu FVIII ION exchange CHROMATOGRAPHY. PEG-pilirovanny FVIII is passed through the anion-exchange or cation-exchange column, where the protein binds to the column, while the excess of free PEG-reagent not bound and removed, passing through the column. Then PEG-mutein elute from the column using a gradient of sodium chloride. To ensure that erwerbende with column fractions containing PEG-pilirovanny mutein, used painted iodide barium 4-12% Bis-Tris gel loaded, passing through the column and processed in the gradient fractions.

PURIFICATION of PEG-lirovannomu FVIII GEL CHROMATOGRAPHY Anion-exchange fraction, containing most of PAG-muteena are pooled and concentrated using ultrafiltration, which is then injected into the column used in gel chromatography. Then the column elute using a buffer composition. Due to differences in size and shape about Aina, depending on, does the PEG with protein, this column separates the PEG-pilirovanny PEG-mutein from any of the other PEG that do not PEG-iliriana. Fractions of PEG-lirovannomu FVIII-muteena unite on the basis of considerable activity of FVIII, then frozen for subsequent animal studies and molecular studies. Figure 13 compares the elution of Nepal-lirovannomu PEG-muteena with elution 43 kDa-PEG-lirovannomu PEG-muteena. PEG-pilirovanny PEG aluinum much earlier, which indicates that the increase in its size and shape from covalently attached PEG.

With regard to such Malinov as PEG, which show lower efficacy of PEG-helirovanie, i.e. less than 50%, then the most efficient purification scheme, which gives high-purity, Tonopah-pilirovanny product is using a combination of cation-exchange chromatography followed by gel-chromatography. For example, with regard to PEG, cation-exchange chromatography purifies PEG-pilirovanny PEG (earlier eluting fraction, Figure 14) from the greater part of Nepal-lirovannomu PEG (later eluting fraction, Figure 15). Then using gel chromatography PEG-pilirovanny protein (earlier eluting fraction, Figure 15) is purged from the rest of Nepal-lirovannomu protein (later eluting fraction, Figure 5).

THE INFLUENCE OF THE SIZE OF THE PEG ON THE ACTIVITY. To check whether the size of the PEG on coagulation and chromogenic FVIII activity after PEG-helirovanie, purified FVIII full length, PEG, PEG and PEG-14 was restored using TSER with the subsequent removal of the reducing agent and reaction with buffer control or PEG, having a size in the range from 6 kDa to 64 kDa. The resulting PEG-pilirovanny FVIII were analyzed directly without remove excess PEG or napag-lirovannomu FVIII. Control experiments showed that the excess PEG does not affect the activity of FVIII.

The Figure 16 shows the results of this study. Purified FVIII full length presented in Figure 16 as KG-2. The percentage of activity shown in Figure 16, was determined by dividing the value of the sample treated with PEG after recovery and removal of the reducing agent, the amount of sample treated with buffer control, whereas the PEG-helirovanie. The resulting PEG-helirovanie compared with all the PEG for any given FVIII-construct. He is about 80% for KG-2, PEG and PEG and about 40% for PEG. For example, the processed buffer control PEG has coagulation activity of 6.8 IU/ml in contrast to the 3.2 IU/ml for 12 kDa-PEG-lirovannomu sample PAG. However, the effectiveness of PEG-helirovanie was about 80%, and a value of 3.2 IU/ml is Agregat the th activity of about 80% PEG-lirovannomu and 20% napag-lirovannomu. Believing that napag-pilirovanny sample has the same activity as the processed buffer control PEG, the percentage of the activity of NAAG-lirovannomu to PEG-iliriana PEG is 34%=(3,2-6.8 times 20%)/(6.8 times 80%).

PEG-iliriana inside A2 or A3 domain in position PEG, PEG and PEG BDD leads to considerable loss of coagulation activity, when the size of the PEG increases beyond 6 kDa. However, PEG-iliriana inside In-domain cysteine domain of native FVIII full length does not affect coagulation activity. Interestingly, the chromogenic activity of all PEG-Yerevani constructs does not change. This may be due to differences in the analyses. Perhaps the small substrate chromogenic peptide has easier access to the PEG-iliriana complex of FVIII/FIX/FX than the substrate larger protein used in coagulation analysis. Alternatively, the PEG may affect the activation mutein. It would be easier to detect using one-stage coagulation analysis than the two-stage chromogenic assay.

To confirm the observed PEG-effects on coagulation activity PEG, 6 and 14 several PEG-Yerevani constructs were purified from excess PEG and napag-lirovannomu material. Because the PEG does not affect chromogenic activity, the ratio of the chromogenic activity of coagulation is all right is estimated on the relative influence of PEG on coagulation activity (table 5). Larger PEG in this position, such as PEG and more Pegov, as in this case with the construct PEG+6, cause a higher loss of coagulation activity.

Table 5
The ratio of chromogenic and coagulation activity for the purified PEG-lirovannomu BDD
PEG-pilirovanny BDDChromogenic IU/ml/Coagulation IU/ml
ID-samplePEGRaw attitudeAttitude regarding BDD
BDDwithout PEG1,71
PEG (pool 2)22 kDa 49195
PEG43 kDa* 4912515
PEG12 kDa 180853
PEG (continue) 33 kDa 1808137
PEG (new)33 kDa 180885
PEG+6 (LSP25)33 kDa in 491, Mono106
PEG+6 (LSP22)33 kDa in 491/1808, Dee2414
PEG+6 (ESP)33 kDa in 491/1808/A3, Three6035
PEG64 kDa*129148
PEG12 kDa 18043,21,9
PEG20 kDa*18044,22,5
PEG33 kDa 180452,9
PEG+14 (ESP19)33 kDa in 491/1804, Dee2112
* branched PEG

RK STUDY OF THE RABBIT. In order to understand the influence of PEG-helirovanie on the pharmacokinetics (PK) FVIII, RK-studies were performed on several species. For research were used NZW SPF rabbits: 10 females, 5 rabbits per group, 2 groups (PAG FVIII and 22 kDa-PEG-pilirovanny PEG). Samples were diluted in sterile PBS to a final concentration of 100 IU/ml (chromogenic units). Each rabbit was injected at the ear vein dose of 1 ml/kg (100 IU/kg) diluted test or control substances. In various after the introduction of the time took blood samples (1 ml) in a 1 ml syringe (filled with 100 μl of 3.8%Na-citrate) from the Central ear artery in specified periods of time after administration of the dose. Plasma samples were incubated R8B12-antibody to the heavy chain, placed in a 96-well plate, for a specific input capture human FVIII. Activity captured FVIII was determined by chromogenic assay (Figure 17). PEG-pilirovanny PEG and PEG-pilirovanny PEG were compared with BDD (Figures 18 and 19), while the number of allocated PEG-Yerevani of Malinov from plasma was higher compared with the number of BDD. PEG-pilirovanny FVIII wild-type full length explicitly did not show such properties (Figure 20).

RK STUDY OF THE MOUSE. As the second species in the ROK studies were used ICR-normal or hemophilic, FVIII-Def is citye mice (Taconic, Hudson, New Th.). Normal mice were used for the study; 5 mice in the group at one time. The test materials were dissolved in the composite buffer to the nominal final concentration of 25 IU/ml Each mouse was injected 4 ml/kg (~0.1 ml of total volume) diluted test material via the tail vein. Blood samples (or 0.3 to 0.45 ml for the study of normal or hemophilic mice, respectively) was collected in a 1 ml syringe (filled with 50 or 30 μl of 3.8%Na-citrate for studies of normal or hemophilic mice, respectively) of the inferior Vena cava in a given period of time (one animal per sample). Plasma samples were analyzed for the concentration of FVIII using the method described above chromogenic assay. A greater number of PEG-lirovannomu PEG stands out from the plasma compared to BDD or PEG (Figure 21). A greater number of PEG-lirovannomu PEG stands out from the plasma compared to BDD (Figures 22 and 23).

5.3
Table 6
The final results RK-study of PEG-lirovannomu FVIII, showing the half-life from plasma in hours. *Initial preparation 33 kDa-PEG-lirovannomu PEG with a half-life of 9.6 h in rabbits was not as clean as the next drug that gave Perry the d half-life of 17.4 h
ConstructHalf-life, hTypes
BDD6.6Normal rabbit
PEG4.8Normal rabbit
PEG-22 kDa PEG7.5Normal rabbit
PEG-43 kDa PEG8.0Normal rabbit
PEG-12 kDa PEG8.2Normal rabbit
PEG-33 kDa PEG*9.6Normal rabbit
PEG-33 kDa PEG17.4Normal rabbit
BDD4.5Normal mouse
PEG-22 kDa PEG7.3Normal mouse
PEG-12 kDaNormal mouse
PEG-33 kDa PEG7.3Normal mouse
PEG-12 kDa PEG5.5Normal mouse
PEG-64 kDa9.2Normal mouse

Table 7
The collection of PEG-Yerevani PEG-Malinov from plasma in hemophilic mice. Shows a quantitative improvement in the collection of the plasma during the postinjection period of 16 hours compared to BDD-control, carried out under the same conditions
MuteinPEGThe number of times
PEG 612 kDa2.9
PEG 633 kDa2.9
PEG 2+633 kDa3.3
PEG 1433 kDa2.5
The EH 2+6 33 kDa4.4
PEG 2+1433 kDa2.1
PEG 2264 kDa3.2

COLLECTION (BDD) OF FACTOR VIII IN HEMOPHILIC MICE. The histogram collection (BDD) of Factor VIII in hemophilic mice, are shown in Figure 24, illustrates the pharmacokinetic (PK) evaluation of the half-life of two types of BDD Factor VIII in the analysis of hemophilic mice. This analysis was conducted to measure the concentration in the plasma as BDD Factor VIII (marked on Figure 24 as "wt" or BDD Factor VIII wild-type)and PEG 2+6 double PEG-lirovannomu variant of BDD Factor VIII (identified here as L491C, K1808 dual variant of BDD Factor VIII) at three time points after intravenous injection in a mouse model. While time 0.8 and 4 hours were comparable, 16-hour score deserves special attention. As for 16 hours, then after 16 hours after administration in the rat plasma was about four times (400%) more option double PEG-lirovannomu BDD Factor VIII (PEG 2+6) compared to napag-pilirovanny molecule.

THE MODEL OF RUPTURE OF THE KIDNEY. To determine whether PEG-lirovannye FVIII-mutiny effective to stop bleeding in hemofil the th mouse we used the model of rupture of the kidney. Hemophilic mice (C57/BL6 disturbed FVIII gene) was anestesiologi under isofluorane and weighed. Expose the inferior caval vein and injected 100 μl of either saline or FVIII using gauge needle 31st size. The needle was carefully removed and applied pressure to the injection site for 30-45 seconds to prevent bleeding. Two minutes later right kidney was exposed and held between the clamps along the vertical axis. Using a scalpel No. 15, the kidney is cut horizontally at a depth of 3 mm To ensure uniform depth of damage to the kidney is lightly held in the middle part that was exposed to the same fabric on each side of the clamp. The bare surface of the kidney was cut to the depth of the clip. Blood loss was calculated as described above. To determine the correlation between the dose and response FVIII renal hemorrhage in mice was tested various doses of FVIII. PEG-pilirovanny PAGE shows comparable with BDD activity reduce blood loss after injury of the mouse kidney (Figure 25). Thus, although the coagulation activity of PEG-lirovannomu PEG lower than the BDD, this model of rupture of the kidney shows that the efficiency of in vivo PEG-lirovannomu PEG was not significantly reduced compared to BDD, which is consistent with the data chromogenic analysis.

ANALYSIS OF INGIBIROVANY THE ANTIBODIES. Adding polymer with high molecular weight such as polyethylene glycol (PEG), specific to the position 491 (i.e. PEG) should reduce binding and sensitivity to MAV 413, greatly increasing the fraction of inhibitory antibodies in patients because many patients develop inhibitory antibodies to the same epitope of the MAB 413. To test this, an increasing number MAV 413 incubated with ninasimone quantities (0,003 IU/ml) BDD or 43 kDa-PEG-lirovannomu PEG and tested for functional activity chromogenic assay (Figure 26). As control was used R8B12, reinhibition antibody, and ESH4, inhibitory antibody aimed at the C2-domain. PEG-pilirovanny PAG indeed more resistant to inhibition of mAB 413 than BDD, and shows a similar pattern of inhibition in the presence of a control antibody that does not bind near position 491. In addition, the protective effect of PEG against the inhibition of mAB 413 depends on the size of the PEG, while the larger PEG, the stronger the effect (Figure 27). To check out whether the PEG-pilirovanny more resistant FVIII inhibitory antibodies in patients chromogenic activity was measured in the presence of a number of selected plasma samples originating from patients with hemophilia And who have developed inhibitors to FVIII. 8 tested clicks scah plasma of patients 43 kDa-PEG-pilirovanny PEG was more resistant to inhibition of plasma of patients than BDD 4 plasma samples of patients. For example, PEG-pilirovanny PEG, PEG or PEG+6 showed higher residual activity than BDD in the plasma of one patient compared with other plasma (Figure 28). Dipag-pilirovanny PEG+6, apparently, is more stable than nanopeg-pilirovanny PEG or PEG. These results suggest that PEG-lirovannye PEG-mutiny can be more effective in the treatment of patients who develop inhibitors to FVIII.

HIGH-THROUGHPUT SCREENING OF PAGLIERANI. The effectiveness of PEG-helirovanie specific PEG-muteena unpredictable, especially in view of the fact that there is no direct structural information on BDD. For example, based on the structural model of BDD, it is possible to predict that the effectiveness of PEG-helirovanie PEG or PEG must be very high, like activity PEG and PEG, since all three points are on the surface and directed outwards in accordance with the structure. Thus, to use the PEG to search for a new mechanism of excretion via systematic PEG-iliriana will need to skanirovat a large number of Malinov.

To quickly skanirovat a large number of PEG-maleinos, has developed a new high-performance method by which to test the effectiveness of PEG-helirovanie and functional aktivnosta-Yerevani products from transient transfected with Malinov. Only 5-10 ml of the transient downregulation of PEG-Malinov such small chromogenic value FVIII as 0.1 to 0.2 IU/ml, concentrate about 50 times, using the device 30K MWCO Amicon-centra Ultra, so that the concentration of FVIII reaches more than 1 nm, almost approaching the magnitude of the affinity of the antibody to interact with FVIII. Concentrated PEG-mutein (~300 μl) incubated with ~30 µl of polymer C7F7-antibodies to FVIII overnight at 4°C, washed, elute, subjected to dialysis and restore. The reducing agent is removed and the recovered PEG-mutiny PEG-jirout and examined by Western analysis as described above (Figures 29 and 30). The relative effectiveness of PEG-helirovanie transient downregulation of PEG-Malinov corresponds exactly to that of purified PEG-Malinov.

This method can be skanirovat many PEG-Malinov for one or two months. For example, PEG (K1804C BDD) had, at least about 80% PEG-helirovanie in the light chain of 12 kDa-Peg and had no PEG-helirovanie in the heavy chain (data not shown), which is consistent with K1804C-mutation, located on the light chain. Based on the structure of the BDD α-β distance between K1804 and K1808 (PEG-position) is only 8.4 Angstrom, and it can be assumed that the introduction of the 43 kDa-PEG in this situation will lead to the improvement of the RK, similar to 33 kDa-PEG-lirovannomu PEG with a much better result, PEG-helirovanie. About the relative results PEG-helirovanie for all tested PEG-Malinov are summarized in Table 8. PEG-iliriana was highly selective for the specific chain of FVIII, which was introduced cysteine mutation and in which each mutein with cysteine in the heavy chain becomes PEG-Yerevani only on the heavy chain, while each mutein with a cysteine in the light chain becomes PEG-Yerevani only on the light chain. Numbers Malinov 2-31 represent cysteine mutation of substitution of the native amino acids in BDD in the position indicated by cysteine. PEG+6 is the double muteena BDD, where position 491 and 1808 were replaced by cysteine. A1 and A2 (In-domain for KG-2, full length FVIII) belong to the heavy chain, while the A3, C1 and C2 belong to the light chain. The effectiveness of PEG-helirovanie was assessed by the study PEG-Yerevani products on SDS PAGE, comparing the intensity of the PEG-pilirovanny strip with napag-pilirovanny band: +++ ~>80% result PEG-helirovanie, ++ ~30-70% result + ~10-30% result - ~<10% of the result.

491
Table 8
The effectiveness of PEG-helirovanie for different PEG-Yerevani FVIII
PEG-muteinPositionDomainN-PEGL-PEG
2A2+++-
4504A2+-
5468A2+-
61808A3-++
71810A3-++
81812A3--
91815A3--
111795A3-+
121796A3-+
13 1803A3-++
141804A3-+++
15377A2+++-
16378A2+++-
17556A2++-
202118A3-+
2181A1++-
22129A1++-
23422A2--
25 570A2--
261864A3-++
271911A3-+++
282091C1-++
292284C2-+

PEG-muteinPositionDomainn-PEGL-PEG
30711A2+-
311903A3-++
2+6490/1808 A2/A3+++++
2+14490/1804A2/A3++++++
KG-2In+++-

MASS SPECTROMETRIC ANALYSIS OF THE RECOVERED PEG-MALINOV. To determine the identity of the "cap"that prevents direct PEG-iliriana PEG-Malinov or full length FVIII, PEG+14 restored using TSER at concentrations in the range from 67 mm to 670 microns. The resulting PEG-helirovanie improved in proportion to the increase in the number TSER (Figure 31). The same samples were also analyzed by a mass spectroscopy to PEG-helirovanie (Figure 32). In order to obtain a protein domain that could be examined directly, the samples were digested with thrombin at a ratio of 20 units/mg FVIII for 30 minutes at 37°C. Cleavage by thrombin produces A2-fragment, which includes the remains of 372-740 and not busy sites of glycosylation. Split samples were introduced into the system liquid chromatography with reversed phase C4 and with a column eluent was introduced directly into a quadrupole time-of-flight mass spectrometer through RAS is elitely interface. The mass spectrum from the bottom of the chromatographic peak corresponding to the A2 domain, was developed to give the mass of the intact protein. To restore the A2-domain PEG+14 gave a lot of that on 118 daltons larger than predicted theoretically. When the concentration TSAR increases, there is a new peak, which has the exact predicted mass of the A2 domain. The share of this new peak increases with increasing concentration TSAR. The difference in 118 Dalton can be attributed to containerbase on the Cys residue 491 through the formation of disulfide with cysteine (119 Yes) and instrumental error. Thus, this shows that PEG-mutiny keeravani by cysteine, which prevents direct PEG-iliriana.

All disclosed here links included here in their entirety.

1. Conjugate with procoagulant activity of factor VIII, which represents the mutated factor VIII polypeptide with SEQ ID NO:3, which necesairly residue in position 41, 129, 377, 378, 491, 468, 556, 1804, 1808, 1810, 1812, 1813, 1815 and/or 2118 according to the numbering of the residues in SEQ ID NO:4 substituted cysteine residue, where the mutated factor VIII polypeptide covalently linked to polyethylene glycol mutant cysteine residue.

2. The conjugate according to claim 1, where the polyethylene glycol contains methoxypolyethyleneglycol.

3. The conjugate according to claim 2, where methoxytrityl glycol has a size in the range from 5 to 64 kDa.

4. The conjugate according to claim 1, where the binding of inhibitory antibodies to factor VIII with conjugate is less than factor VIII delegated domain.

5. The conjugate according to claim 1, where the polyethylene glycol is attached to the mutated factor VIII polypeptide at amino acid position 1804 according to the numbering of the residues in SEQ ID NO:4.

6. A method of obtaining a conjugate with procoagulant activity of factor VIII, including:
matirovanie nucleotide sequence that encodes a functional factor VIII polypeptide with SEQ ID NO:3, to replace the codon nechitailova amino acid residue in position 41, 129, 377, 378, 491, 468, 556, 1804, 1808, 1810, 1812, 1813, 1815 and/or 2118 according to the numbering of the residues in SEQ ID NO:4 cysteine codon amino acid residue;
the expression of the mutated nucleotide sequence in the cell host, which is cultivated in cell culture medium containing sulfhydryl group;
cleaning mutein;
contacts mutein with a reducing agent under conditions in which mutein gently restored and released sulfhydryl group;
deleting released sulfhydryl groups and reductant from mutein;
response mutein with polyethylene glycol, which is activated for reaction with the restored cysteine residues with sulfhydryl fragment, later, at measures which, about 5 min after removal of the reductant;
purification of the conjugate.

7. The method according to claim 6, in which mutein reacts with maleimide groups on the peg.

8. The method according to claim 6, in which released sulfhydryl group and the reducing agent is removed from mutein by pressure on the size of molecules or ion-exchange chromatography.

9. The method according to claim 6, in which the sulfhydryl fragment PEG selected from the group consisting of Colnago, triflates, cresylate, aziridines, oxiranes, S-peredelnogo and maleimides fragments.

10. The method according to claim 6, in which the polyethylene glycol is a PEG-maleimide and has a size in the range from 5 to 64 kDa.

11. Pharmaceutical composition with procoagulant activity of factor VIII for parenteral administration containing a therapeutically effective amount of the conjugate according to claim 1 and a pharmaceutically acceptable adjuvant.

12. Method for the treatment of hemophilia, providing an introduction to the needy in this patient an effective amount of a composition according to claim 11.



 

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SUBSTANCE: invention refers to producing versions of group I Poaceae (holy grass) allergen, also can be used either for specific immunotherapy (hyposensitisation) of patients with grass pollen allergy, or for preventive immunotherapy of grass pollen allergies. The produced versions are characterised by Cys41 Ser, Cys57Ser, Cys69Ser, Cys72Ser, Cys77Ser, Cys83Ser and Cysl39Ser substitutes in a Phi p1 mature protein sequence. Also, a structure of the allergen versions can be presented with no fragments relevant to amino acid residues 1-6, 1-30, 92-104, 115-119, 175-185 and 213-220 or 1-6, 115-119 and 213-220 as a part of a primary sequence of Phi p1 mature protein.

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FIELD: medicine.

SUBSTANCE: invention can be used for haemopoietic system recovery and treatment of the conditions associated with depressed or disturbed haematopoiesis. A method of preparing a therapeutic composition for short-term haematopoiesis recovery involves cultivation ex vivo of a parent cell population containing CD34+CD90+ haemopoietic stem cells (HSC) from a variety of allogenic donors in a culture medium. The culture medium contains thrombopoietin (TPO) or its analogue which is bound with c-Mpl, and a stem cell factor (SCF) or its analogue which is bound with c-kit. It is followed with resuspension of said expansed myeloid precursor cells in a pharmaceutically acceptable medium applicable for introduction in a host mammal.

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Exenatid analogues // 2422461

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new Exenatid analogues of general formula H-HisGlyGluGlyThrPheThrSerAspLeuSerLysGlnNleGluGluGluAlaValArgLeuPhe-IleGluTrpLeuLysAsnGlyGlyProSerSerGlyXProProProSer-ol where X is selected from L-Ala or D-Ala. The produced analogues show the stability higher than Exenatid, high insulinotropic activity, and also have high activity on renal sodition discharge without potassium ion loss, intensifis calcium and magnesium ion discharge, osmotically free water dishcarge. These properties can be effective for water-salt metabolism rebalancing along with glycemia normalisation.

EFFECT: production of the compositions which can be used for treatment of diabetes, and also for treatment and prevention of diabetic nephropathy and cardiac failure.

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FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology and deals with recombinant plasmid DNA, which contains sequence of gene of mature staphylokinase Staphylococcus aureus with replacement of codons K74, E75 and R77 with triplets, which code Ala, of strain Escherichia coli MZ09 and method of obtaining recombinant protein, which contains sequence of gene of mature staphylokinase with replacement of codons K74, E75 and R77 with Ala-coding triplets. Essence of method includes recombinant plasmid DNA, which codes sequence of gene of mature protein of staphylokinase from Staphylococcus aureus staphylokinase with replacement of codons K74, E75 and R77 with Ala-coding triplets, which has nucleotide sequence, given in dwg.1. Invention also includes strain Esherichia coli MZ09, producent of recombinant staphylokinase, with sequence, given in dwg.1, as well as method of its obtaining.

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FIELD: medicine, pharmaceutics.

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EFFECT: invention ensures extension of spectrum of substances with heparine-like action.

4 dwg

FIELD: medicine, pharmaceutics.

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19 cl, 10 ex

FIELD: medicine.

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EFFECT: method ensures increase of treatment efficiency, reduction of number of obstetrical complications during pregnancy and labour and, thus, prevent possible perinatal loss.

2 tbl

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