Modified von willebrand factor with prolonged half-cycle in vivo, using it and methods for preparing

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, more specifically to modified von Willebrand factor (VWF), and can be used in medicine. A recombinant method is used to preparing modified VWF fused in C-terminal of its primary translation product with N-terminal of albumin by the linker SSGGSGGSGGSGGSGGSGGSGGSGGSGGSGS. The prepared modified VWF is used as a part of the pharmaceutical composition for treating or preventing coagulation failure.

EFFECT: invention enables preparing the modified VWF which maintains its ability to N-terminal dimerisation and C-terminal multimerisation with a prolonged half-period of functional blood plasma occurrence as compared to the half-period of functional VWF occurrence.

17 cl, 5 dwg, 4 tbl, 11 ex

 

The technical field

The present invention relates to modified sequences of nucleic acids coding for coagulation factor VIII (FVIII) and von Willebrand factor (VWF), as well as their complexes and their derivatives, recombinant expression vectors containing such nucleic acid sequences, cell host transformed with such recombinant expression vectors encoded by such sequences of nucleic acids, recombinant polypeptides and derivatives, which possess biological activity together with extended half-life existence in vivo and/or an increased ratio of the actual maximum concentration in vivo (blood etc) to the expected compared to the unmodified wild-type protein. The present invention also relates to the corresponding FVIII sequences, which result in an increased amount of expression product. The present invention also relates to processes for the production of such recombinant proteins and their derivatives. The present invention also relates to a vector transfer for gene therapy of people, which includes such modified nucleic acid sequences.

Background of invention

There are various on the osenia blood clotting, caused by deficiencies of factors of the blood coagulation system. The most common disorders are hemophilia A and B, which is a consequence of the deficiencies of factor of the blood coagulation system VIII and IX, respectively. Other known bleeding disorder is von Willebrand's disease.

Plasma FVIII exists mainly in the form of ecovalence complex with VWF, and coagulating function is the acceleration-dependent factor IXa conversion of factor X to Xa. Due to the formation of the complex of FVIII and VWF in a long time believed that the function of FVIII and VWF are two functions of the same molecule. Only in the seventies it became clear that FVIII and VWF are separate molecules, which form a complex under physiological conditions. Then in the eighties was determined the dissociation constant of approximately 0.2 nmol/l (Leyte et al., Biochem j 1989, 257: 679-683), and were studied DNA sequences of the two molecules.

Classic hemophilia or hemophilia is A hereditary bleeding disorders. It is a consequence of sex-linked X chromosome FVIII deficiency of the blood clotting system and affects almost exclusively males with a frequency component of one or two individuals per 10,000. The defective X-chromosome is transmitted by female carriers, to the which do not hemophilia. Clinical manifestation of hemophilia A is an increased tendency to bleeding. Before the introduction of treatment with FVIII concentrates the average life expectancy of a person with severe hemophilia were less than 20 years. The use of concentrates of FVIII from plasma has significantly improved the situation for those suffering from hemophilia A patients, much increasing life expectancy, giving most of them the opportunity to live more or less normal life. However, there were some problems with plasma-derived concentrates and their use, the most serious of which was the transfer of viruses. While seriously impressed population of viruses that cause hepatitis B, hepatitis neither-A nor-B and AIDS. Since then there has been recently developed different methods of inactivation of viruses and new highly purified concentrates of FVIII, which set a very high standard of safety for plasma-derived FVIII.

Cloning of cDNA for FVIII (Wood et al. 1984. Nature 312: 330-336; Vehar et al. 1984. Nature 312: 337-342) made possible the expression of FVIII recombinate that led to the development of several products in the form of recombinant FVII, which were permitted by the regulatory authorities between 1992 and 2003. The fact that the Central B-domain of FVIII polypeptide chain between the amino acids Arg-740 and Glu-1649, apparently, is not needed for what Olney biological activity, also led to the development of FVIII with a deletion In the domain.

Mature FVIII molecule consists of 2332 amino acids, which can be grouped into three homologous to A domain, two homologous C-domain B-domain, which are in the order A1-A2-B-A3-C1-C2. The complete amino acid sequence of Mature human FVIII is presented in SEQ ID NO: 15. During secretion into the plasma FVIII processed intracellularly in a number of related metal ion of heterodimers, when single-stranded FVIII is cleaved at the border B-A3 and in different sites within the B-domain. This processing leads to the formation of heterogeneous molecules of the heavy chains consisting of A1, A2 and different parts of the B-domain, which have a molecular weight in the range from 90 kDa to 200 kDa. The heavy chain is linked by means of a metal ion with light chains, which consist of A3, C1 and C2-domain (Georges tarbouriech et al. 2002. Vox Sang. 83: 89-96). In plasma, this heterodimeric FVIII binds with high affinity with von Willebrand factor (VWF), which protects it from premature catabolism. The period of existence is not activated FVIII bound to VWF, is approximately 12 hours in plasma.

The coagulation factor FVIII is activated by proteolytic cleavage by thrombin and FXa in the provisions of amino acids Arg372 and Arg740 within the heavy chain and Arg1689 in the light chain, which leads to the release facto is and Willebrand's disease and the formation of activated heterotrimer FVIII, which will form tenany complex on phospholipid surfaces with FIXa and FX subject to the presence of Ca2+. Heterotrimer consists of the A1-domain fragment mass of 50 kDa, A2-domain fragment mass of 43 kDa and a light chain (A3-C1-C2), fragment mass of 73 kDa. Thus, the active form FVIII (FVIIIa) consists of the A1-subunit, connected via formed by the divalent metal ion communication with split thrombin light chain A3-C1-C2, and free A2-subunit, is relatively weakly associated with A1 and A3 domain.

Avoid excessive coagulation FVIIIa must be inactivated shortly after activation. I believe that the FVIIIa inactivation by activated protein C (APC) by cleavage at Arg336 and Arg562 is not the main speed stage. Rather, it is the dissociation ecovalence attached A2-subunit from heterotrimer, which is believed to determining the speed stage in the inactivation of FVIIIa upon activation by thrombin (Fay et al. 1991. J. Biol. Chem. 266: 8957, Fay & Smudzin 1992. J. Biol. Chem. 267: 13246-50). It is a rapid process, which explains the short half-period of the existence of FVIIIa in the plasma, which is only 2.1 minutes (Georges tarbouriech et al. 2002. Vox Sang. 83: 89-96).

Patients with severe hemophilia A, subjected to preventive treatment, FVIII should be administered intravenously approximately 3 times per week due to short the second half-period of the existence of FVIII in plasma, approximately 12-14 hours. Each intravenous administration is time-consuming, accompanied by pain and entails the risk of infection, especially when this is done mainly at home by patients or parents of children diagnosed with hemophilia A.

It would therefore be highly desirable that a FVIII with increased half-life functional existence, making possible the production of containing FVIII pharmaceutical compositions, which shall be administered less frequently.

Several attempts were made to extend the period of existence is not activated FVIII or by reducing its interaction with cellular receptors (WO 03/093313A2, WO 02/060951A2), by covalent attachment of polymers to FVIII (WO 94/15625, WO 97/11957 and US 4970300), through encapsulation FVIII (WO 99/55306), through the introduction of new binding sites of metal (WO 97/03193), by covalent joining A2-A3 domain to domain using either peptide (WO 97/40145 and WO 03/087355)or disulfide bond (WO 02/103024A2), or through covalent joining A1-A2 domain to domain (WO2006/108590).

Another approach to increase the bottom half of the functional existence of FVIII or VWF is implemented by tahilramani FVIII (WO 2007/126808, WO 2006/053299, WO 2004/075923) or by tahilramani VWF (WO 2006/07101), which, with the result tahilramani increased half-life existence, could indirectly increase the half-period of the existence of FVIII present in the plasma.

Since none of the above approaches have not led to the resolution of the medicinal product FVIII and since the introduction of mutations in the FVIII sequence of the wild type or the introduction of chemical modifications entail at least a theoretical risk of creating immunogenic variants FVIII, there is a continuing need for the development of modified molecules of factor VIII coagulation, which show longer half-life existence.

Because of the potential risk of blood clots more desirable is the lengthening of the period of existence is not activated form of FVIII than FVIIIa.

VWF, which is missing, functionally defective or available only in small quantities in various forms of von Willebrand disease (VWD)is a multimeric adhesive glycoprotein present in the plasma of mammals that has many physiological functions. During primary hemostasis, VWF acts as a mediator between specific receptors on the surface of the platelet and components of the extracellular matrix such as collagen. In addition to the, VWF serves as a carrier and a stabilizing protein to proco-agulant FVIII. VWF is synthesized in endothelial cells and megakaryocytes in the form of molecules of the precursor of 2813 amino acids. Amino acid sequence and cDNA sequence of wild-type VWF reported in Collins et al. 1987, Proc Natl. Acad. Sci. USA 84: 4393-4397. Polypeptide-predecessor, shall-VWF consists of a signal peptide with a length of 22 balance, propeptide length 741 residue polypeptide with a length 2050 residues detected in Mature plasma VWF (Fischer et al., FEBS Lett. 351: 345-348, 1994). After removal of the signal peptide in the endoplasmic reticulum between the two monomers VWF forms a C-terminal disulfide bridge. During further movement through the secretory path added 12 N-sochlenennykh and 10 O-sochlenennykh carbohydrate side chains. Importantly, VWF dimers are multimerization through the N-terminal disulfide bridges and propeptide length of 741 amino acids is cleaved by the enzyme PACE (splitting of paired basic amino acid enzyme)/forenom in the late Golgi apparatus. Propeptide, as well as high-molecular multimeric VWF (VWF-HMWM) are stored in the cells of Babele-Pallada endothelial cells or in the [alpha]-granules of platelets.

After secretion into the plasma protease ADAMTS 13 cleaves VWF within the A1 domain of VWF. Therefore, VWF in the plasma consists of a whole number is of alltimelow, extending from the isolated dimers with Mm 500 kDa to multimers, consisting of up to more than 20 dimers with a molecular weight in excess of 10,000 kDa. This VWF-HMWM have stronger hemostatic activity, which can be defined through ristocetin-cofactor activity (VWF:RCo). The greater the ratio of VWF:RCo/VWF antigen, the greater the relative amount of high-molecular-weight multimers.

Defects in VWF are the cause of von Willebrand disease (VWD), which is characterized by a more or less distinct phenotype of blood clotting disorders. VWD type 3 is the most severe form, in which VWF is completely absent, VWD type 1 is associated with the quantitative loss of VWF and its phenotype may be very weak. VWD type 2 is associated with qualitative defects of VWF and can be as heavy as VWD type 3. VWD type 2 has many podfarm, while some of them are connected with the loss or reduction of high-molecular-weight multimers. VWD type 2a is characterized by the loss of both medium and large multimers. VWD type 2B is characterized by loss of multimers with the highest molecular weight.

VWD is the most common bleeding disorder in humans and can be treated with replacement therapy with the use of concentrates containing VWF plasma or recombinant who CSOs origin. VWF can be prepared from human plasma, as described in EP 05503991. In EP 0784632 describes how the selection of recombinant VWF.

Plasma FVIII binds with high affinity to VWF, which protects it from premature catabolism and therefore, plays, in addition to its role in primary hemostasis, the major role in regulating levels of FVIII in the plasma and, consequently, is a Central factor for the control of secondary hemostasis. The period of existence is not activated FVIII bound to VWF, is approximately 12-14 hours in plasma. In von Willebrand disease type 3, in which VWF is not or almost not present, the period of existence of FVIII is only about 6 hours, leading to symptoms of hemophilia a small to moderate severity in these patients due to reduced concentrations of FVIII. The stabilizing effect of VWF to FVIII was also used for the promotion of recombinant FVIII expression in CHO cells (Kaufman et al. 1989, Mol. Cell Biol.).

Up to the present time the standard treatment of hemophilia A and VWD includes frequent intravenous infusion of drugs FVIII and VWF concentrates or infusion concentrates comprising a complex of FVIII and VWF obtained from plasmas people-donors, or in the case of FVIII infusion of pharmaceutical substances on the basis of recombinant FVIII. Although these substitution therapy is usually effective, for example, for patients with severe hemophilia A, subjected to preventive treatment, FVIII should be administered intravenously approximately 3 times a week due to a short half-period of the existence of FVIII in plasma of approximately 12 hours. Already at levels exceeding 1% of the FVIII activity in non-hemophilia patients, for example, with increasing levels of FVIII 0.01 u/ml, severe haemophilia A is transformed into hemophilia A moderate. When preventive therapy dosing schedules develop so that minimum levels of FVIII activity falling below the levels that make up 2-3% of the FVIII activity in non-hemophilia patients. Each intravenous administration is time-consuming, accompanied by pain and entails the risk of infection, especially when it's mostly done with treatment at home by patients or parents of children diagnosed with hemophilia A. in Addition, frequent intravenous injections inevitably lead to scarring, preventing further infusions. Because prophylactic treatment in severe haemophilia begin early in life, at this age children often are less than 2 years, the introduction of FVIII three times a week in Vienna such young patients is even more difficult. For a limited period of time, the implantation of the infusion system is we can be an alternative. Despite the fact that there may be multiple infection and infusion system can cause discomfort during physical activity, they, nevertheless, are generally considered to be preferable compared to intravenous injection.

The half-life in vivo existence of human VWF in blood is approximately 12 to 20 hours. Prophylactic treatment of VWD, for example, type 3 could also be highly desirable to find ways to extend the half-life of the functional existence of VWF.

Another approach to increase the half-life of the functional existence of VWF is tahilramani VWF (WO 2006/071801), which, with the result tahilramani increased half-life existence, could indirectly increase the half-period of the existence of FVIII present in the plasma.

However, the chemical conjugation of PEG or other molecules with therapeutic proteins always entails the risk of decrease in specific activity due to the screening of important sites of interaction with other proteins, chemical conjugation adds an additional stage in the production of such proteins, reducing the final outputs of the product and making production more expensive. Also not known long-term effects on human health, as is known in the us is Aasee time pegylated therapeutic proteins do not need to type throughout life, as this could be the case for VWF, administered for the prevention of Willebrand's disease, or for FVIII entered in hemophilia A.

Therefore would be highly desirable to obtain long-lived VWF, which is not chemically modified.

In the prior art have been described merging of clotting factors albumin (WO 01/79271), alpha-fetoprotein (WO 2005/024044) and immunoglobulin (WO 2004/101740) as increasing the period of existence of the polypeptides. It was stated that they are attached to the carboxyl end or amino end or to both ends of the corresponding therapeutic protein component, sometimes linked by peptide linkers, preferably using linkers consisting of glycine and serine.

Ballance and others (WO 01/79271) described polypeptides from a variety of different therapeutic polypeptides, fused at its N - or C-terminal parts with serum albumin human. Presents a long list of possible partners to merge without any messages regarding almost any of these proteins experimental data concerning whether the corresponding fused to albumin proteins biological activity and whether they have improved properties. In the specified list of therapeutic polypeptides also mentioned FVIII and VWF.

It is incredible that the C-terminal fusion seriously R who was smaterials qualified in this field specialist because the C2 domain of FVIII in the very C-terminal part of the FVIII between amino acids 2303 and 2332 FVIII includes the binding site with the membranes of platelets, which is very important for the functioning of FVIII. That is why there are many known amino acid mutations in this region that cause hemophilia A. Therefore surprising that relatively large heterologous polypeptide such as albumin, can be merged with the C-terminal part of FVIII without impeding the functioning of FVIII by preventing its binding to platelets. In addition, the C2 domain also contains a binding site with VWF. This website together with the amino acid sequence 1649-1689 responsible for FVIII binding with high affinity to VWF. So skilled in this technical field specialist could not expect FVIII, fused at its C-terminal part with albumin, will retain its binding to VWF.

Surprisingly it was found that in contrast to the prediction of Ballance and other albumin fusion with an N-end FVIII is not secreted into the culture medium. Therefore, for the reasons detailed above, with even more surprisingly now been found that FVIII, fused at its C-terminal part with albumin secreted into the culture medium and retains its biological function, including binding to membranes of activated clot is Titov and VWF.

Surprisingly it was also found that the modified FVIII of the present invention demonstrates the increase of the ratio of the actual maximum concentration to the expected in vivo by approximately 20% compared to wild-type FVIII.

Qualified in this field specialist also did not consider the fusion of human albumin N - or C-end of VWF. When N-terminal merging albumen part useplease least during the processing of propeptide. Or, if propeptide was not included, could not be multimerization. As discussed above, the C-end of VWF is very important for the initial dimerization and secretion, as demonstrated Schneppenheim and others (Schneppenheim R. et al. 1996. Defective dimerization of VWF subunits due to a Cys to Arg mutation in VWD type MD. Proc. Natl. Acad. Sci. USA 93: 3581-3586; Schneppenheim R. et al. 2001. Expression and characterization of VWF dimerization defects in different types of VWD. Blood 97: 2059-2066.), Baronciani et al. (Baronciani L. et al. 2000. Molecular characterization of a multiethnic group of 21 patients with VWD type 3. Thromb. Haemost 84: 536-540), Enayat et al. (MS Enayat et al. 2001. By aberrant dimerization of VWF as the result of mutations in the carboxy-terminal region: identification of 3 mutations in members of 3 different families with type 2A (phenotype MD) VWD. Blood 98: 674-680; Tjernberg et al. 2006. Homozygous C2362F VWF dosage intracellular retention of mutant VWF resulting in autosomal recessive severe VWD. Br J Haematol. 133: 409-418). So qualified in this field specialist would not even look at the merging of a large protein, such as human albumin, C - or N-end of VWF, as it would be expected that normal Dima who isace or multimerization VWF can be broken. As multimeric VWF with higher molecular weight are the most active in primary hemostasis, qualified in this field specialist'd look for other ways to extend the half-life of the functional existence of VWF.

Surprisingly now been found that the fusion of heterologous polypeptides such as albumin, C-terminal part VWF1 not only allows expression and secretion of recombinant VWF protein from mammalian cells, but also results in a modified VWF molecules, which retain significant activity of VWF to form high-molecular multimer. In addition, such modified VWF molecules exhibit long half-period of the existence and/or increased the ratio of the actual maximum concentration in vivo to expect.

Summary of the invention

The purpose of this invention is the provision of a modified FVIII or modified VWF, and also complexes of modified FVIII with unmodified VWF, complexes of non-modified FVIII with modified VWF, and also complexes of modified FVIII with modified VWF with extended half-life existence in vivo.

In used in the present invention the meaning of the term "modified FVIII or modified VWF" means polypeptides FVIII or VWF, which the slit is to increase the period of existence of the polypeptide, covering natural alleles, variants, deletions and insertions FVIII or VWF.

Another object of this invention is the provision of modified FVIII or modified VWF, and also complexes of modified FVIII with unmodified VWF, complexes of non-modified FVIII with modified VWF, and also complexes of modified FVIII with modified VWF with an increased ratio of the actual maximum concentration in vivo to expect.

Another objective of the present invention is the ability of expression of the modified FVIII or modified VWF, and also complexes of modified FVIII with unmodified VWF, complexes of non-modified FVIII with modified VWF, and also complexes of modified FVIII with modified VWF mammalian cells and the retention of their respective biological activities.

So, the modified FVIII or modified VWF, and also complexes of modified FVIII with unmodified VWF, complexes of non-modified FVIII with modified VWF, and also complexes of modified FVIII with modified VWF of the present invention have, surprisingly, retained biological activity, increased half-life existence in vivo and increased the ratio of the actual maximum concentration in vivo to expect.

To Amiternum potential advantage of those embodiments of the present invention, in which FVIII is modified and in which the A2-domain remains attached to the A3 domain exclusively ecovalence after activation, is that it only increases the half-period of the existence of a non-activated form of FVIII, while the half-period of the existence of an activated form of FVIII remains essentially the same, which could lead to reduced risk of blood clots compared to FVIII variants that cause the stabilization of the activated form of FVIII.

The modified FVIII or modified VWF, and also complexes of modified FVIII with unmodified VWF, complexes of non-modified FVIII with modified VWF, and also complexes of modified FVIII with modified VWF of the present invention can be created by merging component in the form of increasing the period of existence of protein (HLEP) with C-terminal part of FVIII or C-terminal part of VWF.

HLEP in used in the present invention the meaning selected from the group consisting of family members albumin, which includes albumin, afamin, alpha-fetoprotein and communicating with vitamin D protein, as well as portions of constant region of immunoglobulin and polypeptides capable of binding under physiological conditions with family members albumin, and parts of the constant region of the immunoglobulin. Most repectfully HLEP is albumin human.

Therefore, the present invention relates to a modified FVIII or modified VWF, and also complexes of modified FVIII with unmodified VWF, complexes of non-modified FVIII with modified VWF, and also complexes of modified FVIII with modified VWF with(them) in the C-terminal part of the modified FVIII and/or VWF fusion with HLEP characterized(do) the fact that the modified FVIII or modified VWF, and also a complex of modified FVIII with unmodified VWF complex non-modified FVIII with modified VWF or a complex of modified FVIII with modified VWF has a longer half-life functional existence than half the functional existence of FVIII wild type or wild-type VWF or VWF complex of wild-type FVIII wild-type.

The present invention also relates to a C-terminal fusions with more than one HLEP, and HLEP, which is subjected to fusion a few times, can be the same HLEP or can be a combination of different HLEP.

The present invention also relates to the modified FVIII with the C-terminal part of the merger with the HLEP, characterized in that the modified FVIII or modified VWF or a complex of modified FVIII with unmodified VWF complex non-modified FVIII with modified yserowany VWF or a complex of modified FVIII with modified VWF has increased the ratio of the actual maximum concentration in vivo to the expected compared to the ratio of the actual maximum concentration in vivo expected to FVIII wild type or wild-type VWF or VWF complex of wild-type FVIII wild-type.

Another embodiment of the present invention are modified FVIII polypeptide with C-terminal part of the merger with the HLEP, characterized in that the modified FVIII is secreted into the medium for fermentation in greater numbers than wild-type FVIII.

Another aspect of the present invention are polynucleotide or combination of polynucleotides encoding a modified FVIII and/or modified VWF.

The present invention also relates to plasmids or vectors comprising the described polynucleotide, the cells of the host, including this polynucleotide, or a plasmid or vector.

Another aspect of the present invention is a method of producing a modified FVIII or modified VWF or a complex of modified FVIII with unmodified VWF complex non-modified FVIII with modified VWF or a complex of modified FVIII with modified VWF, including:

(a) culturing host cells of the present invention under such conditions that expressed a modified coagulation factor; and

(b) optional selection of the modified coagulation factor from the host cells or from the culture medium.

the Present invention, furthermore relates to pharmaceutical compositions comprising modified FVIII or modified VWF or a complex of modified FVIII with unmodified VWF or a complex non-modified FVIII with modified VWF or a complex of modified FVIII with modified VWF, polynucleotide or a plasmid or vector described herein.

Another aspect of the present invention is the use of the modified FVIII or modified VWF or a complex of modified FVIII with unmodified VWF or a complex non-modified FVIII with modified VWF or a complex of modified FVIII with modified VWF, one or more polynucleotide or one or more plasmids or vectors or the use of host cells in accordance with this invention for the manufacture of a medicinal product for the treatment or prevention of blood clotting disorders.

Detailed description of the invention

The present invention relates to a complex comprising FVIII and VWF, or one of its individual polypeptide components, and at least one polypeptide component of the specified complex fused to C-terminal part of its primary product broadcast with the N-terminal part of increasing the period of existence of the polypeptide (HLEP).

Infusion is her invention also relates to the modified FVIII or modified VWF, or complex comprising modified FVIII and modified VWF or a complex comprising modified FVIII and modified VWF or a complex comprising modified FVIII and modified VWF, and modified FVIII merged in the C-terminal part of the primary product of translation of FVIII with the N-terminal part HLEP, or modified VWF merged in the C-terminal part of the primary product of translation of VWF with the N-terminal part of the HLEP.

In preferred embodiments implementing the present invention relates to a modified FVIII or modified VWF or a complex comprising modified FVIII and modified VWF or a complex comprising modified FVIII and modified VWF or a complex comprising modified FVIII and modified VWF, and

a) a modified FVIII has a longer half-life functional existence than half of the functional existence FVIII wild-type, or

b) the modified VWF has a longer half-life functional existence than half of the functional existence of VWF wild-type, or

c) a complex comprising modified FVIII and modified VWF has a longer half-life functional existence compared to the functional half), the tation of the corresponding complex including wild-type FVIII and VWF wild-type, or

d) a complex comprising modified FVIII and modified VWF has a longer half-life functional existence than half of the functional existence of the corresponding complex with wild-type FVIII and VWF wild-type, or

e) a complex of modified FVIII with modified VWF has a longer half-life functional existence than half of the functional existence of the corresponding complex with wild-type FVIII and VWF wild-type.

The preferred embodiment of the present invention is a modified polypeptide or complex, including the specified modified polypeptide or complex, including these modified polypeptides described above, and the modified polypeptide has a half-period of the functional existence, is increased by at least 25% compared with the half-period of the functional existence of the corresponding wild-type polypeptide, or a complex that includes the specified modified polypeptide or complex, including these modified polypeptides, has a half-period of the functional existence, is increased by at least 25% compared with a half-life functional the existence of the corresponding complex of wild-type FVIII with VWF wild-type.

Another embodiment of the present invention is a modified FVIII or modified VWF or a complex comprising modified FVIII and modified VWF or a complex comprising modified FVIII and modified VWF or a complex comprising modified FVIII and modified VWF, and

a) a modified FVIII has a longer half-life existence as antigen compared to the half-period of existence as FVIII antigen of a wild type, or

b) the modified VWF has a longer half-life existence as antigen compared to the half-period of existence as antigen VWF wild-type, or

c) a complex comprising modified FVIII and remodifications VWF has a longer half-life existence as antigen compared to the half-period of existence as the corresponding antigen complex comprising wild-type FVIII and VWF wild-type, or

d) a complex comprising modified FVIII and modified VWF has a longer half-life existence as antigen compared to the half-period of existence as an antigen corresponding complex of wild-type FVIII with VWF wild-type, or

e) a complex comprising modified FVIII and modified VWF has an elongated p is superiod existence as antigen compared to the half-period of existence as an antigen corresponding complex of wild-type FVIII with VWF wild-type.

The preferred embodiment of the present invention is a modified polypeptide or complex, including the specified modified polypeptide or complex, including these modified polypeptides described above, and the modified polypeptide has a half-period of existence as an antigen, increased by at least 25% compared with the half-period of existence as an antigen corresponding wild-type polypeptide, or a complex that includes the specified modified polypeptide or complex, including these modified polypeptides, has a half-period of existence as an antigen, increased by at least 25% compared with the half-period of existence as an antigen corresponding complex of wild-type FVIII with VWF wild-type.

Another embodiment the present invention is a modified FVIII or modified VWF or a complex comprising modified FVIII and modified VWF or a complex comprising modified FVIII and modified VWF or a complex comprising modified FVIII and modified VWF, and

a) a modified FVIII has increased the ratio of the actual maximum concentration in vivo to the expected compared with the ratio of the true is th maximum concentration in vivo to be expected for wild-type FVIII, or

b) the modified VWF has increased the ratio of the actual maximum concentration in vivo to the expected compared to the ratio of the actual maximum concentration in vivo to be expected for wild-type VWF, or

c) a complex comprising modified FVIII and modified VWF has increased the ratio of the actual maximum concentration in vivo to the expected compared to the ratio of the actual maximum concentration in vivo to the expected for the corresponding complex with wild-type FVIII and VWF wild-type, or

d) a complex comprising modified FVIII and modified VWF has increased the ratio of the actual maximum concentration in vivo to the expected compared to the ratio of the actual maximum concentration in vivo to the expected for the corresponding complex with wild-type FVIII and VWF wild-type, or

e) a complex comprising modified FVIII and modified VWF has increased the ratio of the actual maximum concentration in vivo to the expected compared to the ratio of the actual maximum concentration in vivo to the expected for the corresponding complex with wild-type FVIII and VWF wild-type.

Another preferred embodiment of the present invention is a modified polypeptide or complex, kiuchumi specified a modified polypeptide, or complex comprising these modified polypeptides described above, and the modified polypeptide has a ratio of the actual maximum concentration in vivo to expect increased by at least 10% compared to the ratio of the actual maximum concentration to the expected in vivo to the corresponding wild-type polypeptide, or a complex that includes the specified modified polypeptide or complex, including these modified polypeptides, is the ratio of the actual maximum concentration in vivo to expect increased by at least 10% compared to the ratio of the actual maximum concentration in vivo to the expected for the corresponding complex of wild-type FVIII with VWF wild-type.

Another preferred embodiment of the present invention is

a) a modified polypeptide or complex, including the specified modified polypeptide or complex, including these modified polypeptides described above, and at least one polypeptide component of the specified complex fused to C-terminal amino acid of its primary product broadcast with the N-terminal part HLEP, or

b) a modified polypeptide or complex, including the specified modified polypeptide or complex including at Azania modified polypeptides, described above, and at least one polypeptide component of the specified complex fused to C-terminal part of its primary product broadcast with N-terminal amino acid HLEP, or

c) a modified polypeptide or complex, including the specified modified polypeptide or complex, including these modified polypeptides described above, and at least one polypeptide component of the specified complex fused to C-terminal amino acid of its primary product broadcast with N-terminal amino acid HLEP.

Another preferred embodiment of the present invention is a modified polypeptide or complex, including the specified modified polypeptide or complex, including these modified polypeptides described above, and the modified polypeptide has at least 10% of the biological activity of the wild-type polypeptide, or a complex comprising the modified polypeptide or complex, including these modified polypeptides has at least 10% of the biological activity of the corresponding complex of wild-type FVIII with VWF wild-type.

In the present invention also includes a method of preparation of the modified FVIII or modified VWF, with increased half the fun is inogo existence, including the merger of the N-terminal part of increasing the period of existence of the polypeptide with C-terminal part of the primary product of translation of FVIII or C-terminal part of the primary product of translation of VWF, as well as the method of preparation of a complex comprising modified FVIII and modified VWF or a complex comprising modified FVIII and modified VWF or a complex comprising modified FVIII and modified VWF, by mixing the modified FVIII prepared in the manner described above, with wild-type VWF or by mixing wild-type FVIII with modified VWF, prepared as described above, or by mixing the modified FVIII and modified VWF prepared by the method described above.

In the present invention also includes the use of

a) the modified FVIII prepared in the manner described above, and VWF wild-type, or

(b) wild-type FVIII and modified VWF, prepared as described above, or

c) modified FVIII prepared in the manner described above, and modified VWF, prepared as described above

for the production of a combined pharmaceutical preparation for simultaneous, separate or sequential use in the treatment of disorders of the Holy is rivamonti blood, preferably for the treatment of hemophilia A and/or von Willebrand disease.

"Half of the functional existence" in accordance with the present invention is a half-period of the existence of the biological activity of the modified FVIII or modified VWF or a complex of modified FVIII with unmodified VWF or a complex non-modified FVIII with modified VWF or a complex of modified FVIII with modified VWF after its introduction to the mammal and can be measured in vitro in the blood samples taken at different intervals of time at a specified mammal after administration of the modified FVIII or modified VWF or a complex of modified FVIII with unmodified VWF or a complex non-modified FVIII with modified VWF or a complex of modified FVIII with modified VWF.

The expression "merge" or "merged" refers to the addition of amino acids to the C-terminal part of FVIII and/or C-terminal part of VWF. When the reference here to "merge with the C-terminal amino acid FVIII" or "merge with C-terminal VWF amino acid" refers to a fusion with the C-terminal amino acid FVIII in position 2332 Mature wild-type FVIII or just with the C-terminal amino acid of VWF in position 2050 Mature wild-type VWF. Mature FVIII or Mature VWF means corresponding polypeptides off propeptide. However, the present invention also covers the merge with the C-terminal part of FVIII or merge with the C-terminal part of VWF". These expressions are used in the present invention sense may also include a fusion molecule with FVIII and/or VWF, respectively, from which demeterova one or more amino acids, up to n amino acids from the C-terminal amino acids FVIII and/or VWF. The number n is an integer, which must not exceed 5%, preferably 1% of the total number of amino acids FVIII and/or VWF. Usually n is equal to 20, preferably 15, more preferably 10, more preferred 5 or less (for example, 1, 2, 3, 4 or 5).

In one embodiment, the modified FVIII has the following structure:

N-FVIII-C-L1-H,[formula 1]

where

N represents the N-terminal part of FVIII,

L1 represents a chemical bond or a linker sequence,

H represents the HLEP and

C represents the C-terminal part of FVIII.

In another embodiment, the modified VWF has the following structure:

N-VWF-C-L1-H,[formula 2]

where

N represents the N-terminal part of VWF,

L1 represents a chemical bond or a linker sequence,

H represents the HLEP and

C represents the C-terminal part of VWF.

L1 may be a chemical bond or a linker sequence consisting of one or more amino acids, for example, 1-20, 1-15, 1-10, 1-5 or 1-3 (for example, 1, 2 or 3) amino acids, which may be the same or different from each other. Typically, the linker sequences are not present in the corresponding position in the coagulation factor wild type. Examples of suitable amino acids present in L1, include Gly, and Ser.

The preferred sequence HLEP below. The present invention covers merge with "N-terminal amino acid corresponding HLEP or merging with the N-terminal part of the corresponding HLEP, which includes the N-terminal deletions of one or several amino acids HLEP.

The modified FVIII or modified VWF or a complex of modified FVIII with unmodified VWF complex non-modified FVIII with modified VWF or a complex of modified FVIII with modified VWF of the present invention may include more than one sequence HLEP, for example two or three sequences HLEP. These multiple sequence HLEP can be merged with the C-terminal part of FVIII and/or C-terminal part of VWF in tandem, for example, in the form of successive repetitions.

FVIII can processionals PR is Teoreticheskie at different stages. For example, as noted above, during its secretion into the plasma-chain FVIII is cleaved intracellular border B-A3 and in different sites within the B-domain. A heavy chain linked by a metal ion with a light chain having a hierarchy of domains A3-C1-C2. FVIII is activated by proteolytic cleavage at positions of the amino acids Arg372 and Arg740 within the heavy chain and Arg1689 in the light chain, creating heterotrimer activated FVIII, consisting of the A1-domain A2 domain and the light chain (A3-C1-C2), fragment mass of 73 kDa. Thus, the active form FVIII (FVIIIa) consists of the A1-subunit, connected via formed by the divalent metal ion communication with split thrombin light chain A3-C1-C2, and free A2-subunit, is relatively weakly associated with A1 and A3 domain.

Accordingly, the present invention is also covered by the modified FVIII, which is presented not in the form of a single-chain polypeptide, and consists of several polypeptides (e.g., one or two, or three), which are connected to each other through nequality links.

Preferably N-FVIII-C includes the full sequence of FVIII. Also included N-terminal, C-terminal or internal deletions FVIII provided that the biological activity of FVIII is saved. In used in the present invention the biological sense is aktivnosti saved if FVIII with deletions retains at least 10%, preferably at least 25%, more preferably at least 50%, most preferably at least 75% of the biological activity of wild-type FVIII. The biological activity of FVIII may be determined by the specialist, as described below.

Appropriate analysis to determine the biological activity of FVIII is, for example, a single-stage or two-stage analysis of coagulation (Rizza et al. 1982. Coagulation assay of FVIII:C and FIXa in Bloom ed. The Hemophilias. NY Churchchill Livingston 1992) or the analysis of FVIII:C by using a chromogenic substrate (S. Rosen, 1984. Scand J Haematol 33: 139-145, suppl.). The contents of these links are included here by reference.

The cDNA sequence and amino acid sequence of the Mature form of wild-type FVIII coagulation of the person represented in SEQ ID NO: 14 and SEQ ID NO: 15, respectively. Reference to the position of the specific amino acids of the sequence indicates the position of this amino acid in the protein FVIII wild-type and does not exclude the presence of mutations, such as deletions, insertions and/or substitutions in other positions in the sequence, to which reference is made. For example, a mutation in the "Glu2004" with reference to SEQ ID NO: 15 does not exclude that in the modified homologue lacks one or more amino acids in positions 1 through 2332 SEQ ID NO: 15.

The term "factor VIII roll is her blood system", "factor VIII" and "FVIII" are used here interchangeably. "Factor VIII blood clotting system includes factor VIII wild-type blood coagulation system, as well as derivatives of factor VIII wild-type blood coagulation system with proco-agulant activity of factor VIII wild-type blood coagulation system. Derivatives may have deletions, insertions and/or additions compared to the amino acid sequence of wild-type FVIII. The term "FVIII" includes proteoliticeski versions of the forms FVIII, for example the form to activation, comprising a heavy chain and light chain.

The term "FVIII" includes any variants or mutant FVIII having at least 25%, more preferably at least 50%, most preferably at least 75% of the biological activity of factor VIII wild-type.

As non-limiting examples of FVIII molecules include FVIII mutants that prevent or reduce splitting under the action of APC (Amano 1998. Thromb. Haemost. 79: 557-563), FVIII mutants, additional stabilizing A2 domain (WO 97/40145), FVIII mutants, leading to increased expression (Swaroop et al. 1997. JBC 272: 24121-24124), FVIII mutants that reduce its immunogenicity (Lollar 1999. Thromb. Haemost. 82: 505-508), FVIII, reconstructed from separate expression of heavy and light chains (Oh et al. 1999. Exp. Mol. Med. 31: 95-100), FVIII mutants that reduce causes catabolism of FVIII light is ivanie with receptors, like HSPG (heparin-sulfate-proteoglycans) and/or LRP (protein related receptor low-density lipoprotein) (Ananyeva et al. 2001. TCM, 11: 251-257), FVIII variants stabilized by a disulfide bond (Gale et al., 2006. J. Thromb. Hemost. 4: 1315-1322), FVIII mutants with improved characteristics secretion (Miao et al., 2004. Blood 103: 3412-3419), FVIII mutants with increased cofactors specific activity (Wakabayashi et al., 2005. Biochemistry 44: 10298-10304), FVIII mutants with enhanced biosynthesis and secretion, reduced interaction with chaperones of the endoplasmic reticulum, improved transfer from the endoplasmic reticulum to the Golgi apparatus, increased activation or resistance to inactivation and increased half-life existence (summarized Pipe 2004. Sem. Thromb. Hemost. 30: 227-237). All these mutants and variants FVIII included here generally by reference.

VWF can processionals proteoliticeski at different stages. For example, as noted above, the protease ADAMTS13 cleaves VWF within the A2 domain of VWF. Accordingly, the present invention is also covered by the modified VWF, which was proteoliticeski split, e.g., ADAMTS13. This splitting could lead to a multimeric chains VWF, which include at their ends, at least one or at most two VWF monomer, which has tsebelis under the action of ADAMTS 13.

Preferably N-VWF-C R is t full sequence of VWF. Also included N-terminal, C-terminal or internal deletions VWF, provided that the biological activity of VWF is stored. In used in the present invention sense the biological activity is retained if VWF with deletions retains at least 10%, preferably at least 25%, more preferably at least 50%, most preferably at least 75% of the biological activity of wild-type VWF. The biological activity of wild-type VWF may be determined by the specialist, using methods to determine ristocetin-cofactors activity (Federici AB et al. 2004. Haematologica 89: 77-85), VWF binding to glycoprotein Ibα glycoprotein complex Ib-V-IX platelets (Sucker et al. 2006. Clin Appl Thromb Hemost. 12: 305-310), or the analysis of binding with collagen (Kallas & Talpsep. 2001. Annals of Hematology 80: 466-471).

"FVIII and/or VWF" in the above definition also includes natural allelic variations that may exist or occur during the transition from one individual to another. "FVIII and/or VWF" in the above definition also includes variants of FVIII and/or VWF. These options differ by one or more amino acid residues from the sequence of the wild type. Examples of such differences can include conservative amino acid substitutions, i.e. substitutions within groups of amino acids with similar characteristics who, for example, (1) small amino acids, (2) acidic amino acids, (3) polar amino acids, (4) basic amino acids, (5) hydrophobic amino acids and (6) aromatic amino acids. Examples of such conservative substitutions are shown in the following table.

Table 1
(1)AlanineGlycine
(2)Aspartic acidGlutamic acid
(3)AsparagineGlutamineSerineThreonine
(4)ArginineHistidineLysine
(5)IsoleucineLeucineMethionineValine
(6)PhenylalanineTyrosine Tryptophan

One or more HLEP can be merged with the C-terminal part of FVIII preferably so that there have been difficulties in the ability of FVIII to bind, for example, VWF, platelet or FIX.

One or more HLEP can be merged with the C-terminal part of VWF preferably so that there have been difficulties in the ability of VWF to bind, for example, FVIII, platelets, heparin or collagen.

After endogenous FVIII activation during coagulation in vivo preservation of the increased half-life functional existence now activated FVIII can no longer be desirable, because it could lead to thrombotic complications, that is already the case for such activated factor wild type coagulation, as FVIIa (Aledort 2004. J Thromb Haemost 2: 1700-1708), and what could be more relevant, if activated factor had an increased half-life functional existence. Therefore, another purpose of this invention is the provision of long-lived FVIII molecules, after which endogenous activation in vivo or with availability of the cofactor really have the bottom half of the functional existence, comparable to those remodifications FVIII. As a non-limiting example of this can be achieved by introducing a cleavage site, for example, for the factor to which gulali between the C-terminal part of FVIII and HLEP. When using such connecting FVIII-HLEP sequences of activation of the chimeric protein FVIII of the present invention will cause a concomitant complete separation of FVIIIa from the component in the form HLEP. Accordingly, in one embodiment, the half-period of the functional existence of endogenous activated modified FVIII is essentially the same with the bottom half of the functional existence of activated FVIII wild type (for example, ±15%, preferably ±10%).

In yet another embodiment of the present invention, however, one or more sites of proteolytic cleavage, preferably the sites of cleavage by thrombin in Arg740 and/or Arg372, motivovany or dellarovere in order to prevent splitting and to have the insertion protein, which demonstrates the superior properties like increased half-cycle of the functional existence even in the form of an activated molecule.

In another embodiment, the present invention FVIII proteins of the present invention can be expressed in the form of two separate chains (see below).

Modified FVIII in accordance with this invention can be a single-stranded polypeptide, or it may be composed of two or three polypeptide chains that are linked via non-covalent tie the th, due to proteolytic processing.

In another embodiment, the present invention motivovany or deleterow amino acids in the cleavage site PACE/farinon (Arg1648) or near this site to prevent splitting PACE/Purina. I believe that this is a consequence of the single-stranded merged FVIII/HLEP molecule with increased half-life existence.

In one embodiment of the present invention, the modified FVIII of the present invention shows an increased half-life functional existence in comparison with the appropriate form of FVIII, not containing attached HLEP, and/or form of wild-type FVIII. Half of the functional existence is possible, for example, to determine in vivo in models of hemophilia And animals, such as mice with off FVIII in which one would expect longer haemostatic effect of the modified FVIII compared to wild-type FVIII. Hemostatic effect can be verified, for example, by defining a period of time to stop the bleeding after clipping of the tail.

In one embodiment of the present invention the bottom half of the functional existence is the half-period of the existence of the biological activity of FVIII after its introduction to the mammal and is measured in vitro. Half the fun is the national existence of the modified FVIII in accordance with the present invention, more than half of the functional existence of FVIII, with no modification, when testing the same species. Half of the functional existence is increased preferably by at least 10%, preferably at least 25%, more preferably at least 50% and even more preferably by at least 100% compared to the form of FVIII wild-type.

Half of the functional existence of the modified FVIII, comprising a modification in the form HLEP, can be determined by introducing the corresponding modified FVIII (and in comparison with wild-type FVIII) rats, rabbits or other species of experimental animals intravenously or subcutaneously and tracking elimination of biological activity of the specified modified or unmodified coagulation factor in the blood samples taken at appropriate intervals after application. Suitable test methods are described here for the analysis activity.

The bottom half of the functional existence in accordance with another embodiment of the present invention is a half-period of the existence of the biological activity of VWF after its introduction to the mammal and is measured in vitro. Half of the functional existence of the modified VWF in accordance with the present invention more functional half su is estovakia VWF, with no modification, when testing the same species. Half of the functional existence increased by at least 10%, preferably at least 25%, more preferably at least 50% and even more preferably by at least 100% compared with VWF, with no modification, and/or form of VWF wild-type.

Half of the functional existence of the modified VWF, comprising a modification in the form HLEP, can be determined by introducing the corresponding modified VWF (and in comparison with that of non-modified VWF) rats, rabbits or other species of experimental animals intravenously or subcutaneously and tracking elimination of biological activity of the specified modified or non-modified VWF in the blood samples taken at appropriate intervals after application. Suitable test methods are described here for the analysis activity.

As a surrogate marker for a half period of the existence of biological activity can also be measured levels of antigen in the form of the modified FVIII or FVIII wild-type or levels of antigen in the form of a modified VWF or VWF wild type. Thus, the present invention also covered by the modified molecules is FVIII and/or VWF, with the C-terminal part of FVIII and/or VWF merge with the HLEP, characterized in that the modified FVIII or modified VWF or a complex of modified FVIII with unmodified VWF or a complex non-modified FVIII with modified VWF or a complex of modified FVIII with modified VWF has a longer half-period of the existence of FVIII and/or VWF antigen compared to the half-period of the existence of FVIII and/or VWF antigen, in which there is no specified insertion. "The half-period of the existence of FVIII antigen in accordance with the present invention is a half-period of the existence of the antigen in the form of FVIII after its introduction to the mammal and is measured in vitro. "The half-period of the existence of VWF antigen in accordance with the present invention is a half-period of the existence of the antigen in the form of VWF after its introduction to the mammal and is measured in vitro. How antigenic analysis based on the specific antibodies in the format immunoassay known to the expert and commercially available (e.g., Dade Behring, Instrumentation Laboratory, Abbott Laboratories, Diagnostica Stago). Half of the functional existence and a half of existence as an antigen can be calculated from the time the beta phase of elimination by the formula t1/2=ln2/k, given that k is the angle of the slope of the regression line.

In another embodiment, the half-period of the functional existence of endogenous activated modified FVIII is lengthened compared with that of activated wild-type FVIII. The increase may be greater than 15%, e.g. at least 20% or at least 50%. Again the values of such half-cycle of the functional existence can be calculated as described above for the half-periods of the functional existence. Increased half-periods of the existence of endogenous activated modified FVIII molecules can be useful in situations in which achievable only very low levels of FVIII, which therefore are not tromboobrazovanie. Such situations can arise, for example, by treatment in the form of gene therapy, which often can be achieved only low speed expression. Therefore, such stable FVIII molecule could be useful, for example, in gene therapy despite the risk of blood clots associated with such FVIII molecules, if they were introduced in the form of proteins in high or physiological doses.

In another embodiment of the present invention, the modified FVIII of the present invention demonstrates increased the ratio of the actual maximum concentration to the expected in vivo as compared to that of wild-type FVIII and options is qualified VWF of the present invention demonstrates increased the ratio of the actual maximum concentration to the expected in vivo as compared to that of wild-type VWF. The ratio of the actual maximum concentration to the expected in vivo can be determined in vivo, for example, in normal animals or in models of hemophilia A in animals, such as mice with off FVIII, or in models VWD, like mice with off VWF, which would be expected that increased the percentage of modified FVIII or VWF of the present invention will be detected using assays of antigen or activity in the bloodstream soon (from 5 to 10 min) after intravenous administration compared with the corresponding wild-type FVIII or VWF wild-type.

Preferably the ratio of the actual maximum concentration in vivo to the expected increased by at least 10%, more preferably at least 20% and even more preferably by at least 40% compared with the form of the wild-type FVIII or VWF wild-type.

In yet another embodiment of the present invention the constant region of immunoglobulins or parts thereof are used as HLEP. Preferably use the Fc-region composed of CH2 and CH3 domains and the hinge region of IgG, more preferably IgG1, or its fragments or variants with mutations that enhance binding to the neonatal receptor for Fc fragment (FcRn).

Another purpose of this invention is the provision of long-lived FVIII molecules, which after proteolytic PR is testirovanie in vivo really have the bottom half of the functional existence, comparable to those of unmodified FVIII. This can be achieved by saving specific sites of cleavage in a modified FVIII, resulting in proteolytic cleavage, for example, when the contact activated coagulation factors, which leads to the separation of FVIII from HLEP. Accordingly, in one embodiment, the half-period of the functional existence proteoliticeski processioning modified FVIII is essentially identical with that of unmodified FVIII, with no modification, and/or it is essentially identical to that of wild-type FVIII (for example, ±15%, preferably ±10%).

Another embodiment of the present invention are modified FVIII polypeptide, which merged with the HLEP, for example, albumin, C-terminal part of the FVIII molecule that actually have a reduced binding to VWF or not associated with VWF at all.

Another purpose of this invention is the provision of long-VWF molecules, which after proteolytic processing in vivo indeed have functional properties comparable to those of non-modified VWF. This can be achieved by using the save or insert specific cleavage sites in a modified(th), VWF (see below), leading to proteolytic who have cleavage for example, when the contact activated coagulation factors, which leads to the separation of VWF from HLEP. Accordingly, in one embodiment, the half-period of the functional existence proteoliticeski processioning modified VWF is essentially identical with that of non-modified VWF, with no modification, and/or it is essentially identical to that of wild-type VWF (for example, ±15%, preferably ±10%).

Another preferred embodiment of the present invention is coexpressed VWF wild-type and modified VWF in accordance with the present invention, with the consequence of multimeric VWF, comprising unmodified and modified VWF monomers.

The linker sequence

In accordance with this invention component in the form of therapeutic polypeptide can be connected with component in the form HLEP using a peptide linker. The linker should be non-immunogenic and can be necessasary or biodegradable linker.

Darassalam linkers may be composed of alternating residues of glycine and serine, as shown in the example in WO2007/090584.

In another embodiment of the present invention the peptide linker between the component in the form of FVIII and/or VWF and component as albumin consists of peptide sequences which serve as natural interdomain linkers in proteins person. Preferably such peptide sequences in their natural environment are localized close to the surface of the protein and achievable for the immune system, so we can assume the presence of natural tolerance to this sequence. Examples are given in WO2007/090584.

Biodegradable linkers should be sufficiently flexible to allow cleavage by proteases. In a preferred embodiment, cleavage of the linker occurs relatively quickly as the activation of FVIII within the fused protein, if the protein is modified FVIII.

Preferably biodegradable linker includes a sequence derived from

a) enter by itself therapeutic polypeptide if it contains the sites of proteolytic cleavage, which proteoliticeski split during activation of therapeutic polypeptide,

b) which is a substrate of the polypeptide that is cleaved by the protease, which is activated or formed with the participation of therapeutic polypeptide,

c) a polypeptide involved in coagulation or fibrinolysis.

In a more preferred embodiment, the linker includes a sequence of FVIII and/or VWF, which should lead to decrease nomu risk neoantigenic properties expressed fused protein. Also in the case where therapeutic protein is FVIII, which must be proteoliticeski activated, the kinetics of cleavage of the peptide linker will accurately reflect the kinetics associated with coagulation activation Imogene.

In a preferred embodiment, therapeutic polypeptide is Imogen FVIII and HLEP is albumin. In this case, the linker sequence or derived from sequences of the regions activated FVIII, from the region of the cleavage of any substrate FIX, such as FX or FVII or from the region of the cleavage which any substrate polypeptide, which is cleaved by the protease, the activation of which involved FIXa.

In the preferred highly embodiment, the linker peptide is of the FVIII and composed of sequences covering the sites of cleavage by thrombin in the provisions of amino acids 372, 740 and 1689 SEQ ID NO: 15, respectively. In another preferred embodiment, the linker peptide is from FX, FIX, FVII or FXI.

Preferably the linker peptides are cleaved by proteases of the blood coagulation system, for example, FIIa, FIXa, FXa, FXIa, FXIIa and FVIIa.

These linker sequences can also be used in a modified VWF of the present invention.

Given as examples of the combination of therapeuti the definition of the polypeptide, biodegradable linker and HLEP include design, listed in WO2007/090584 (for example, in table 2 and Fig. 4) and in WO2007/144173 (for example, in tables 3a and 3b), but are not limited to.

Increasing the period of existence of the polypeptide (HLEP)

As used here, "increasing the half-period of the existence of a polypeptide" is chosen from the group consisting of albumin, family member albumin, a constant region of immunoglobulin G and its fragments and polypeptides capable of binding under physiological conditions with albumin, family members albumin, and parts of the constant region of the immunoglobulin. It can be full size increases the half-period of the existence of a protein described herein (e.g., albumin, a member of the family albumin, or a constant region of immunoglobulin G) or one or more of its fragments, which are capable of stabilizing or lengthening of time of therapeutic activity or biological activity of the coagulation factor. The length of such segments can be 10 or more amino acids, or it may include at least about 15, at least about 20, at least about 25, at least about 30, at least about 50, at least about 100 or more consecutive amino is the slot of the sequence HLEP or may include part or all of the specific domains corresponding to HLEP provided that fragment HLEP provides prolonged functional half existence, constituting at least 25%, compared with wild-type FVIII or VWF wild-type.

Part of the HLEP of the proposed structures with the inclusion of coagulation factor of the present invention may be a variant of the normal HLEP. The term "variants" includes insertions, deletions and substitutions, either conservative or non-conservative, and such changes do not alter significantly the active site or active domain, which provides the biological activity of the modified FVIII or modified VWF.

In particular, the proposed design merge with FVIII HLEP or VWF with the HLEP of the present invention may include naturally occurring polymorphic variants HLEP and fragments HLEP. HLEP can be from any vertebrate, in particular any mammal, such as human, monkey, cow, sheep or pig. HLEP not from mammals include, but without limitation, HLEP chicken and salmon.

Albumin as HLEP

The terms "serum albumin man" (HSA) and human albumin (HA) and "albumin" (ALB) are used in this application interchangeably. The term "albumin" and "serum albumin" broader and include serum albumin human (and fragments and variants), and albumin from other species (and fragments and variants).

As used here, "albumin" refers collectively to the albumin polypeptide or amino acid sequence of albumin, or fragment or variant of albumin, having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or fragments, especially the Mature form of human albumin, here presented in SEQ ID NO: 16, or albumin from other vertebrates or its fragments, or analogs or variants of these molecules or their fragments.

In particular, the proposed design merge with FVIII and/or VWF of the present invention may include naturally occurring polymorphic variants of human albumin and fragments of human albumin. Generally speaking, the length of the fragment or variant of albumin will be at least 10, preferably at least 40, more preferably more than 70 amino acids. Preferably the variant of albumin may consist of or alternatively include at least one whole domain of albumin or fragments of these domains, for example, domain 1 (amino acids 1-194 SEQ ID NO: 16), 2 (amino acids 195-387 SEQ ID NO: 16), 3 (amino acids 388-585 SEQ ID NO: 16), 1 + 2 (amino acids 1-387 SEQ ID NO: 16), 2 + 3 (amino acids 195-585 SEQ ID NO: 16) or 1 + 3 (amino acids 1-194 SEQ ID NO: 16 + amino acids 388-585 SEQ ID NO: 16). Each domain is itself composed of two homologous subdomains, namely, 1-105, 120-194, 195-291, 316-387, 388-491 and 512-585, with a flexible linker regions between subdomains comprising residues with Lys106 to Glu119, with Glu292 to Val315 and Glu492 to Ala511.

Part of the albumin proposed structures merge with FVIII and/or merge with VWF of the present invention may include at least one subdomain or domain HA or conservative modifications.

Afamin, alpha-fetoprotein and communicating with vitamin D protein as HLEP

In addition to albumin, alpha-fetoprotein, another member of the family albumin allegedly increases the half-period of the existence of attached therapeutic polypeptide in vivo (WO 2005/024044). The albumin family of proteins, evolutionary related serum protein vectors, consists of albumin, alpha-fetoprotein (AFP; Beattie & Dugaiczyk 1982, Gene 20: 415-422), afamin (AFM; Lichenstein et al. 1994. J. Biol. Chem. 269: 18149-18154) and bind to the vitamin D protein (DBP; Cooke & David 1985, J. Clin. Invest. 76: 2420-2424). Their genes are a cluster of many genes with structural and functional similarities, CharterWay in the same chromosomal region in humans, mice and rats. The structural similarity of family members albumin says about their suitability as HLEP. Therefore, another aim of the present invention is the use of family members albumin, fragments and variants as HLEP. The term "variations is you" includes insert, deletions and substitutions, either conservative or non-conservative, provided that the required functionality is still there.

Family members albumin may include the full length of the corresponding protein AFP, AFM and DBP, or may include one or more of its fragments, which are capable of stabilizing or lengthening of time of therapeutic activity. The length of such segments can be 10 or more amino acids, or they may include about 15, 20, 25, 30, 50 or more consecutive amino acids from the corresponding protein sequence, or may include part or all of the specific domains of the corresponding protein, provided that the fragments HLEP provide prolonged functional half existence, constituting at least 25%. Family members albumin insertion of proteins of the present invention may include naturally occurring polymorphic variants of AFP, AFM and DBP.

Immunoglobulins as HLEP

In the art it is known that the constant region (Fc) of an immunoglobulin G (IgG) increases the half-period of the existence of therapeutic proteins (Dumont JA et al. 2006. BioDrugs 20: 151-160). The constant region of the heavy chain of IgG consists of 3 domains (CH1-CH3) and the hinge region. The immunoglobulin sequence may be from any mammal or from subclass the s IgG1, IgG2, IgG3 or IgG4, respectively. As HLEP can also be used IgG and IgG fragments without antigennegative domain. Part of therapeutic polypeptide is combined with IgG or IgG fragments, preferably by means of the hinge region of the antibody or peptide linker, which may be an even split. In several patents and patent applications describe the fusion of therapeutic proteins with a constant regions of immunoglobulin to increase the half-periods of the existence of in vivo therapeutic proteins. In the US 2004/0087778 and WO 2005/001025 describes fusion proteins of biologically active peptides with Fc-domains, or at least parts of the constant regions of immunoglobulins, which increase the half-period of the existence of the peptide, which otherwise would quickly would be eliminated in vivo. Were described fused proteins Fc-IFN-β, which has gained increased biological activity, long half-life existence in blood flow and a greater solubility (WO 2006/000448). Were described proteins Fc-EPO with a longer half-life existence in serum and increased in vivo activity (WO 2005/063808), as well as the merger of Fc with G-CSF (WO 2003/076567), glucagon-like peptide-1 (WO 2005/000892), factors of the blood coagulation system (WO 2004/101740) and interleukin-10 (US 6,403,077), all of them had the property of increasing the half-period of existence.

Poly is ucleotide

The present invention also relates to polynucleotide, codereuse modified coagulation factor, preferably, a modified version of FVIII and/or modified variant of VWF described in this application. The term "polynucleotide(s)" generally refers to any polyribonucleotide or polymethacrylamide, which may be unmodified RNA or DNA or modified RNA or DNA. Polynucleotide may be single - or double-stranded DNA, single - or double-stranded RNA. Used herein, the term "polynucleotide(s)" also includes DNAs or RNAS that include one or more modified bases and/or unusual bases such as inosine. It will be clear that DNA and RNA can enter the number of modifications that serve many useful purposes known to skilled in the art specialists. The term "polynucleotide(s)"as it is used here, covers such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA-the characteristics of viruses and cells, including, for example, simple and complex cells.

A qualified specialist will be clear that due to the degeneracy of the genetic code specific polypeptide can be encoded by different polynucleotide. These options is covered by this invention.

Preferably polynucleotide of the present invention is selected polynucleotides. The term "isolated" polynucleotide refers to polynucleotide that is essentially free from other nucleic acids sequences, for example and without limitation, other chromosomal and extrachromosomal DNA and RNA. Selected polynucleotide can be purified from the host cell. To get the selected polynucleotides can be used conventional methods of purification of nucleic acids, known qualified specialists. The term also includes recombinant polynucleotide and chemically synthesized polynucleotide.

The present invention also refers to a group of polynucleotides, which together encode a modified FVIII and/or modified VWF of the present invention. First polynucleotide in this group can encode the N-terminal part of the modified FVIII and/or modified VWF, and the second polynucleotide can encode the C-terminal part of the modified FVIII and/or modified VWF.

Another aspect of the present invention is a plasmid or vector comprising polynucleotide in accordance with the present invention. Preferably a plasmid or vector is an expression vector. In a specific embodiment, the vector is the I vector transfer for gene therapy of people.

The present invention also refers to a group of plasmids or vectors, which include the above group polynucleotides. The first plasmid or vector may contain the first polynucleotide, and the other plasmid or vector may contain the specified second polynucleotide. As an example, and with reference to the factor VIII coagulation in the first expression vector can be cloned sequence encoding the signal peptide, A1 - and A2-domains, the remainder of the sequence B-domain and HLEP, and the second expression vector can be cloned sequence encoding the A3, C1 and C2 with the proper sequence of the signal peptide. Both expression vector cotransfected in a suitable cell host, which would cause the expression of light and heavy chains of FVIII molecule of the present invention and the formation of functional protein.

Alternatively, the first expression vector clone sequence encoding the signal peptide FVIII, A1 - and A2-domains, and the second expression vector clone sequence encoding HLEP, A3, C1 and C2 FVIII with the proper sequence of the signal peptide. Both expression vector cotransfected in a suitable cell host, which would cause the expression of light and heavy chains of FVIII molecule of the present invention and the formation of the functionality of the nogo protein.

Alternatively, both coding sequences clone in one expression vector, using either two separate promoter sequence, or one promoter element as part of the internal landing ribosomes (IRES) to control the expression of both chains of FVIII.

Still another aspect of the present invention is a host cell comprising polynucleotide, plasmid or vector of the present invention, or a group of polynucleotides, or group of plasmids or vectors described herein.

Cells are the masters of the present invention can be used in the method of producing a modified coagulation factor, preferably a modified FVIII molecule, which is part of this invention. This method includes:

(a) culturing host cells of the present invention under such conditions that Express the desired protein insertion; and

(b) optional selection of the desired insertion of the protein from the host cells or from the culture medium.

Preferred is the treatment of the modified FVIII and/or modified VWF of the present invention to >80% purity, more preferably >95% purity, and particularly preferred is a pharmaceutically pure state, in which the purity with respect to contaminating macromolecules, particularly other protein and nucleic acids, over 99.9% and no infectious and pyrogenic agents. Preferably selected or purified modified FVIII and/or modified VWF of the present invention essentially contains no other, unrelated polypeptides.

Different products of the present invention is applicable as medicines. Accordingly, the present invention relates to pharmaceutical compositions comprising modified FVIII and/or modified VWF as described here, polynucleotide of the present invention, or a plasmid or vector of the present invention.

The present invention also relates to a method of treatment of an individual suffering from bleeding disorders such as hemophilia A or B. the Method comprises the introduction of a specified individual an effective amount of FVIII and/or modified VWF or a modified VWF or a complex of modified FVIII with unmodified VWF or a complex non-modified FVIII with modified VWF or a complex of modified FVIII with modified VWF, which are described here. In another embodiment, the method includes the administration to an individual an effective amount of polynucleotide of the present invention, or a plasmid or vector of the present invention. Alternatively, the method may include the introduction of individual EF the objective number of host cells of the present invention, are described here.

Expression of the mutant

For production of recombinant mutant proteins at high levels in specific cells-the owners need the Assembly of the above-mentioned modified cDNA in efficient transcription units together with suitable regulatory elements in the recombinant expression vector, which can be reproduced in various expression systems, in accordance with methods known to skilled in the art specialists. Effective regulatory transcription elements could occur from viruses with cells of animals as their natural hosts, or from chromosomal DNA of animal cells. Preferably, you can use the combination of the promoter-enhancer derived from simian vacuolating virus 40, adenovirus, polyomavirus BK, human cytomegalovirus, or the long terminal repeat of rous sarcoma virus, or a combination of the promoter-enhancer, including genes, highly constitutively transcribed in the cells of animals, such as beta-actin or GRP78. To achieve sustained high levels of mRNA transcribed from cDNA, a transcription unit must contain at their 3'-proximal part of the DNA region that encodes a polyadenylation sequence, which is the terminator Tr is scriptie. Preferably this sequence is from the area of early transcription simian vacuolating virus 40, a gene beta-globulin rabbit or gene activator tissue plasminogen person.

cDNA are then integrated into the genome of a suitable cell line host for expression of the modified protein FVIII and/or VWF. Preferably, this cell line should be a line of cells of vertebrates to ensure proper installation, the formation of disulfide bonds, glycosylation at asparagine and other post-translational modification, and secretion into the culture medium. Examples of other posttranslational modifications are O-sulfation of tyrosine and proteolytic processing of the growing polypeptide chain. Examples of cell lines that can be used are cells COS monkey, L-cells, mouse C127 cells, mouse cells, BHK-21 hamster cells, kidney 293 human embryo cells and CHO hamster.

A recombinant expression vector comprising the cDNA can be inserted in the line of animal cells in several different ways. For example, recombinant expression vectors can be created from vectors on the basis of various animal viruses. Their examples are based vectors baculovirus, vaccinia virus, adenovirus, and preferably bullish popes is mamavirus.

Transcription unit comprising the DNA, you can also enter in the cells of animals together with other recombinant gene which may function as a dominant breeding marker in these cells, to facilitate the allocation of specific t-cell clones, whose genome was integrated recombinant DNA. Examples of this type of genes dominant breeding markers are aminoglycoside-phosphotransferase Tn5, which imparts resistance to geneticin (G418), hygromycin-phosphotransferase, giving resistance to hygromycin, and puromycin-acetyltransferase, giving resistance to puromycin. The recombinant expression vector encoding such a breeding marker may either be the same vector as the vector comprising cDNA of the desired protein, or it can be a single vector, which is introduced simultaneously and integrates into the genome of the host cell, which is often a consequence of a strong physical connection between the different transcriptional units.

Other types of genes breeding markers that can be used with cDNA of the desired protein, based on different transcriptional units encoding dihydrotetrazolo (dhfr). After the introduction of this type of the gene into cells, which lack endogenous dhfr activity, preferably kletchino (DUKX-B11, DG-44), it will make possible the growth of these cells in media lacking nucleosides. An example of such environment is F12 ham without gipoksantina, thymidine and glycine. These genes dhfr can be entered together with including FVIII cDNA transcription units in CHO cells the above-mentioned type, or United in one and the same vector or different vectors, thereby creating a cell line dhfr+producing recombinant protein.

When growing the above cell lines in the presence of cytotoxic dhfr inhibitor methotrexate will see new lines of cells resistant to methotrexate. These cell lines can produce recombinant protein with increased speed due to the increased number of dhfr and transcriptional units for the desired protein. When the reproduction of these cell lines to increasing concentrations of methotrexate (1-10000 nm), you can get new lines of cells that produce the desired protein with a very high speed.

The above cell line producing the desired protein can be grown on a large scale, either in suspension culture or on various solid substrates. Examples of these substrates are micropolicy based dextranomer or collagen matrices or solid substrate in the form of hollow fibers or various ceramic materials. When is Yerevani in suspension cell culture or microbology cultivation of the above cell lines can be carried out either in the form of cultivation in closed volume, or perfusion cultivation with a continuous flow air-conditioned environment for long periods of time. Thus, in accordance with the present invention, the above cell lines are fully suitable for the development of industrial process for the production of the desired recombinant mutant proteins.

Cleaning and preparation

Recombinant modified FVIII protein and/or recombinant modified VWF protein that accumulates in the environment secreting cells of the above types, you can concentrate and purify by using a number of biochemical and chromatographic methods, including methods that use differences in size, charge, hydrophobicity, solubility, specific affinity, etc. between the desired protein and other substances in the medium for cell culture.

An example of such purification is the adsorption of the recombinant mutant protein monoclonal antibody directed against, for example, HLEP, preferably albumin human, or against the corresponding coagulation factor, which is immobilized on a solid substrate. After adsorption of the modified FVIII and/or modified VWF substrate, washing and desorption of the protein can be further purified by a series of chromatographic methods based on azannyh above properties. The order of the stages of purification choose, for example, in accordance with the bandwidth and selectivity stages, the stability of the substrate or other aspects. For example, the preferred stages of purification are, but without limitation, the stage ion-exchange chromatography, stage immunoaffinity chromatography stage affinity chromatography, phase chromatography based on hydrophobic interactions, phase chromatography using dye-phase chromatography on hydroxyapatite, stage multimodal chromatography and stage of gel filtration.

To minimize theoretical risk of viral infections in the process may include additional stages to effectively inactivate or remove viruses. For example, these stages are heat treated in the liquid or solid state, processing solvents and/or detergents, radiation in the visible or ultraviolet region of the spectrum, gamma rays or nanofiltration.

Modified polynucleotide (e.g., DNA) of this invention can also be integrated into the vector transfer for gene therapy of people.

Various embodiments of described herein can be combined with each other. The present invention will be further described in detail in the following examples. This description of a particular VA is Ianto implementation of the present invention will be made in connection with the accompanying figures.

Modified FVIII and/or modified VWF as described in this invention, can be composed into pharmaceutical preparations for therapeutic use. Purified protein can be dissolved in conventional, physiologically compatible aqueous buffer solutions to which can be added optional pharmaceutical excipients to provide pharmaceutical preparations.

Such pharmaceutical carriers and excipients, as well as suitable pharmaceutical preparations are well known in the art (see, for example, "Pharmaceutical Formulation Development of Peptides and Proteins", Frokjaer et al., Taylor & Francis (2000) or the "Handbook of Pharmaceutical Excipients", 3rdedition, Kibbe et al., Pharmaceutical Press (2000)). In particular, the pharmaceutical composition comprising the variant polypeptide of the present invention can be prepared in lyophilized or stable liquid form. Variant polypeptide can be liofilizirovanny by using a number of well-known in the art procedures. Lyophilized preparations recreate before use by adding one or more pharmaceutically acceptable diluents, such as sterile water for injection or sterile physiological saline solution.

Drugs compositions give the individual any pharmaceutically appropriate way of introduction. Various systems Dostuk is known and can be used for injection of the composition using any suitable way. Preferably the compositions of the present invention is administered systemically. For systemic use insertion proteins of the present invention are for parenteral (e.g. intravenous, subcutaneous, intramuscular, intraperitoneal, intracerebral, intra-lungs, intranasal, or transdermal) or enteral (e.g. oral, vaginal or rectal) delivery according to conventional methods. The most preferred routes of administration are intravenous and subcutaneous administration. Drugs can enter non-stop with the help of infusion with bolus injections. Some drugs include system slow release.

Insertion proteins of the present invention is administered to patients in therapeutically effective dose means the dose that is sufficient to invoke the desired effects, prevention or reduction of the severity or spread of the disease or symptom being treated, without reaching dose that causes unacceptable adverse side effects. The exact dose depends on many factors, such as symptom, medication, route of administration, and must be determined in preclinical and clinical trials for each of the corresponding symptom.

The pharmaceutical compositions of the present invention can enter the ü separately or in combination with other therapeutic agents. These funds may be included as part of the same medicines. One example of such means is a combination of modified FVIII with unmodified VWF or a combination of non-modified FVIII with modified VWF or a combination of modified FVIII with modified VWF.

Brief description of drawings

Fig. 1: Levels of antigen in the form of wild-type FVIII and polypeptide C-terminal merge with FVIII albumin and the levels of their activity.

Fig. 2: Comparison of the pharmacokinetics of FVIII:Ag (antigen) of a person in mice, which is off VWF, after intravenous injection of 100 u (FVIII:Ag)/kg FVIII wild-type and merged with FVIII protein 1656 (mean; n=4/time point).

Fig. 3: Relationship VWF:RCo/VWF:Ag for supernatant cell cultures containing recombinant VWF wild-type (1570/1212), recombinant fused c VWF protein (1572/1212), contains at its C-end linked albumin, or a cell culture for the combined expression containing a mixture of recombinant VWF wild-type (1570/1212) and recombinant fused with VWF protein (1572/1212), transfected at a ratio of 5:1. In each case, were obtained the values that make up approximately 0.8, which is close to 1, which is theoretical ratio of NHP in accordance with the definitions of units.

Fig. 4: Electrophoresis in SDS-agarose gels of recombinant VWF dick is type (1570/1212), expressed in HEK cells, (B) and recombinant fused with VWF protein (1572/1212), also expressed in HEK cells (A). Bands were revealed using either antibodies against VWF, or antibodies against albumin (HSA).

Fig. 5: Comparison of the pharmacokinetics of recombinant human VWF wild-type and recombinant fused with VWF protein after intravenous injection of 100 IU VWF:Ag rats (mean; n=2-3/time).

Examples

Example 1: Creating vectors for the expression of molecules C-terminal fusion with FVIII albumin

Expression plasmid based on the pIRESpuro3 (BD Biosciences)containing the cDNA sequence of the full-size FVIII in its multiple cloning site (pF8-FL) was used first to create the FVIII with a deletion In the domain. This used the oligonucleotides F8-1 F8-2 (SEQ ID NO: 1 and 2) in the experiment site-directed mutagenesis according to standard protocols (using a kit for site-directed mutagenesis QuickChange XL, Stratagene, La Jolla, CA, USA)using pF8-FL as a matrix for dellarovere B-domain. In the second stage was built in sequence, encoding amino acid sequence RRGR to connect R740 A2-domain R1648 A3-domain. This was done in another multi site-directed mutagenesis using the primers F8-3 F8-4 (SEQ ID NO 3: and 4). The resulting plasmid was named pF8-457.

Design to merge with FVIII albumin was created in stages. First, the cleavage site PinAI was inserted into the 3'-end of FVIII. This was created using the pF8-457 as a matrix, using the primers for PCR We2827 and We2828 (SEQ ID NO: 5 and 6), the PCR fragment, which was subsequently purified from the gel, cut with restriction endonucleases BspE1 and Notl and ligated with pF8-457, pre-split by enzymes BspE1 and Notl. Then the resulting plasmid (pF8-1433) cut by enzymes PinAI and Notl and embed the fragment obtained by PCR to contain the cDNA of human albumin plasmid using the primers We 2829 and We 2830 (SEQ ID NO: 7 and 8), and subsequently cleaved by enzymes PinAI and Notl. The resulting expression plasmid (pF8-1434) contained a sequence encoding a FVIII with a deletion of the B-domain, followed by site for PinAI to include linkers (encoding amino acid sequence ThrGly) and the sequence encoding the albumin human. Amino acid sequence encoded pF8-1434, presented as SEQ ID NO: 9.

The linker sequence separating FVIII and albumin as components, can be easily incorporated into the newly created site PinAI described above. Embedding two linker sequences described next. In addition, on the basis of pF8-1434, on top could be deleteroute linker TG, and m is tenderly to enter even deletions within the C-end of FVIII or N-Terminus of albumin, using site-directed mutagenesis.

The incorporation of degradable linker originating from the site of cleavage by thrombin FVIII: First I created a PCR fragment containing the sequence encoding the site of cleavage by thrombin in position 372, by PCR using the primers We2979 and We2980 (SEQ ID NO: 10 and 11) and pF8-457 as a matrix. This fragment was purified, digested with enzyme PinAI and ligated with the cleaved by the enzyme PinAI pF8-1434. Sequencing confirmed the integration of the fragment in the correct orientation, the resulting plasmid was named pF8-1563.

Embedding a flexible linker of glycinol/Surinov: PCR fragment containing the sequence encoding the linker of 31 amino acids - glycinol/Surinov, amplified by PCR with pFVII-937 as described in WO2007/090584 using primers We2991 and We2992 (SEQ ID NO: 12 and 13). This fragment was then purified, digested with restriction endonuclease PinAI and ligated with split PinAI pF8-1434. Sequencing confirmed the integration of the fragment in the correct orientation, the resulting plasmid was named pF8-1568.

Using protocols and plasmids described above, and applying the techniques of molecular biology known skilled in the art specialists (and are described, for example, in Current Protocols in Molecular Biology, Ausubel FM et al. (eds.) John Wiley & Sons, Inc.;http://www.currentprotocols.com/WileyCDA/), a specialist can create the th other designs to replace albumin other HLEP or embed any linker described in the website for PinAI. The transfer of the cDNA for FVIII/albumin into suitable vectors, such pIRESneo3 (Invitrogen) and pEE12.4 (Lonza), allowed us to Express the corresponding protein merge with FVIII albumin in CHO cells and selected expressing his clones.

Example 2: Transfection and protein expression FVIII and VWF

Expression plasmids were reproduce inE. coliTOP10 (Invitrogen, Carlsbad, CA, USA) and purified using standard protocols (Qiagen, Hilden, Germany). Cells HEK-293 (Invitrogen) was transferrable using the reagent lipofectamine 2000 (Invitrogen), and were grown in serum-free medium (Invitrogen 293 Express) in the presence of 4 μg/ml puromycin and optional 0.5 IU/ml VWF. The CHO cells (CHO-S, Invitrogen; CHOK1SV, Lonza) were transferrable using the reagent lipofectamine 2000 (Invitrogen), and were grown in serum-free medium (Invitrogen CD CHO, 6 mm glutamine in the case of CHO-S and CD-CHO in the case of CHOK1SV) in the presence of 500-1000 µg/ml geneticin (only in the case of CHO-S). For the expression of FVIII optional was added 0.5 IU/ml VWF. For the expression of VWF was cetrespectively expression plasmid encoding a PACE/furin, (pFu-797), described in WO2007/144173. In another experiment two plasmids encoding wild-type VWF and VWF, fused at the C-end with albumin, was cotranslationally with pFu-797, which resulted in multimeric VWF, comprising the monomers in the form of VWF wild-type and the monomers in the form merged with VWF albumin (see Fig. 3). Populations of transfected cells were sown from T-FL is contained in a rotating vials or small fermenters, of which supernatant collected for purification.

Table 2 lists the data for expression in HEK-293 when using the constructions described in example 1.

Table 2
DesignActivity [u/ml]
pF8-4571,54
pF8-457+0,5 u/ml VWF1,66
pF8-14341,59
pF8-1434+0,5 u/ml VWF1,82
pF8-1563+0,5 u/ml VWF2,04
pF8-1568+0,5 u/ml VWF1,21

Example 3: speed Increased protein expression of FVIII fusion with albumin

In Fig. 1 summarizes the results of studies of the expression of the fusion protein with FVIII albumin in serum-free cell culture. Cells HEK-293 were transferrable in three repetitions using pF8-1434 (C-terminal fusion with FVIII albumin) and pF8-457 (FVIII wild-type), respectively, were seeded in T80-vials equal number of cells were grown in the absence of stabilizing VWF. Supernatant cultures were then harvested at 96, 120 and 144 hours and tested on the act shall want to make FVIII.

The results showed the effect of increasing expression caused by component in the form of albumin in her presence as an integral part of the FVIII molecule in cell culture. Therefore, productivity was undoubtedly increased in the case of a fused protein compared to wild-type FVIII (Fig. 1).

Example 4: Purification of FVIII proteins

It contains the FVIII molecule the supernatant from expressing culture was added number immunoaffinity resin, sufficient for almost complete binding of FVIII activity. Immunoaffinity resin was prepared by covalent binding of a suitable Mat against FVIII resin Sephacryl S1000, used as a carrier. After washing the resin it filled the chromatographic column, and again carried out washing. Elution was carried out using a buffer containing 250 mm CaCl2and 50% of ethylene glycol.

Obtained immunoaffinity chromatography (IAC) the fractions containing the activity of FVIII:C were pooled, dialyzed against buffer preparation (including excipients: sodium chloride, sucrose, histidine, calcium chloride and Tween 80) and concentrated. Samples were either stored frozen or liofilizirovanny, using a suitable lyophilization cycle.

Alternative containing FVIII supernatant from a cell culture concentrate/purify first use IO is obmennoi chromatography and then with further purification, using immunoaffinity chromatography (IAC). In this case, the eluate obtained by ion-exchange chromatography, loaded onto a column for IAC, using the above-mentioned resin.

Example 5: analysis of the activity of FVIII and FVIII antigen

For determining the activity of FVIII:C in vitro used or the analysis of coagulation (e.g., using reagent Pathromtin SL and plasma FVIII deficiency supplied by Dade Behring, Germany), or analysis using a chromogenic substrate (e.g., analysis of FVIII:C Coamatic created Haemochrom). These analyses were performed in accordance with the manufacturers ' instructions.

FVIII antigen (FVIII:Ag) were determined using ELISA, the performance of which is known to the skilled in the art specialists. Briefly, microplates were incubated with 100 µl per well of antibody for capture (sheep IgG against human FVIII, Cedarlane CL20035K-C, diluted 1:200 in buffer A [Sigma C3041]) for 2 hours at ambient temperature. After washing tablets three times with buffer B (Sigma P3563) serial dilutions of tested sample in the buffer for dilution of the sample (Cedarlane), and serial dilutions of the drug FVIII (CSL Behring; 200 - 2 IU/ml) in buffer for dilution of the sample (in volume per well: 100 μl) were incubated for two hours at ambient temperature. After three with ADI washing with buffer B to each well was added 100 μl of the antibody for detection (sheep IgG against human FVIII, Cedarlane CL20035K-D, peroxidase labeled) at a dilution of 1:2 in buffer, and incubated for a further one hour at ambient temperature. After three stages of washing with buffer B to each well was added 100 μl of substrate solution (1:10 (volume ratio) TMB OUVF:buffer TMB OUVG, Dade Behring), and incubated for 30 minutes at ambient temperature in the dark. Add 100 μl of stop reagent (Dade Behring, a) did the samples ready to read in the appropriate microplate reader device at a wavelength of 450 nm. Then the expected concentration in the tested samples, using the standard calibration curve, constructed using the drug FVIII as a reference.

Example 6: Evaluation of pharmacokinetics merged with FVIII protein in mice, which is off VWF, after a single intravenous injection

To compare the pharmacokinetics of FVIII wild-type (DNA 457) and protein fusion with the C-end of FVIII (DNA 1656) both FVIII were injected into mice intravenously. Mouse line, which is off VWF (Denis C. et al, Proc. Natl. Acad. Sci. USA, 1998, Vol. 95, 9524-9529), was selected because, among other functions VWF serves as a carrier and a stabilizing protein for FVIII, protecting, thus, FVIII from premature decomposition, for example, proteases and from premature elimination from the bloodstream. For unmodified FVIII is very important n the disturbed interaction with VWF, as illustrated by the cases of hemophilia a, caused by a mutation in the C-terminal region, resulting in loss of binding to VWF. In the case of the modified FVIII this coupling may, however, be even undesirable, for research or achieve improved pharmacokinetics. Accordingly, both the product was administered intravenously at a dose of 100 u (FVIII:Ag)/kg bolus two groups of mice (table 3). Blood samples were collected retroorbital at appropriate intervals beginning 5 minutes after the application of tested substances and up to 24 hours. One sample of blood/mouse were collected, transformed into the plasma and stored frozen at -20°C until analysis. The concentration of FVIII:Ag man was determined using an ELISA specific for human FVIII, or complex ELISA specific for human albumin and FVIII, respectively. For calculation of pharmacokinetic parameters used average concentration in plasma samples for each time group. Half life was calculated using the moments of the beta phase of elimination by the formula t1/2=ln2/k, given that k is the angle of the slope of the regression line. The result is shown in Fig. 2. Surprisingly, merged with FVIII protein 1656 (t1/2=3,06 h, between 5 and 960 min) was approximately 3-4 times longer end of the half-period of existence sravnenie is with FVIII wild-type (t 1/2=0.8 h, between 5 and 240 min). In addition, the ratio of the actual maximum concentration in vivo to expect for merged with FVIII protein 1656 was increased by approximately 20% compared to wild-type FVIII (table 4).

Table 3
Exposed treatment groups to compare the pharmacokinetics of FVIII in mice, which is off VWF
TreatmentDose (FVIII:C)/volume/scheme introduction/introduction
Wild-type100 u (FVIII:Ag)/kg24
/0.2 ml/20 g every two weeks/t=0 h/intravenous
Merged with FVIII protein 1656100 u (FVIII:Ag)/kg24
/0.2 ml/20 g every two weeks/t=0 h/intravenous

Table 4
Bioavailability (%) wild-type FVIII and modificirovannogo FVIII, merged with FVIII protein 1656, after intravenous injection to mice, which is off VWF
Treatment Bioavailability (%)
FVIII wild-type100
merged with FVIII protein 1656120,4

Example 7: Creating vectors for the expression of VWF wild-type and fusion proteins with VWF albumin

First I created an expression plasmid containing the cDNA sequence of a full-sizedVWF in its multiple cloning site. This amplified encoding VWF sequence using polymerase chain reaction (PCR)using the primer set VWF+ and VWF- (SEQ ID NO: 17 and 18), under standard conditions known to skilled in the art specialists (and described in Current Protocols in Molecular Biology, Ausubel FM et al. (eds.) John Wiley & Sons, Inc.;http://www.currentprotocols.com/WileyCDA/with a plasmid containing cDNA VWF (commercially available, for example, pMT2-VWF from ATCC, No. 67122). The resulting PCR fragment was digested with restriction endonuclease EcoRI and ligated with expression vector pIRESpuro3 (BD Biosciences, Franklin Lakes, NJ, USA), which was linearized with EcoRI. The resulting expression plasmid containing VWF cDNA wild-type 3' from the CMV promoter, called pVWF-1570.

The PCR fragment containing the sequence encoding the linker of 31 amino acids - glycinol/Surinov, and cDNA albumin human, amplified with pFVII-937, opican the th in WO2007/090584, using primers We2994 and We1335 (SEQ ID NO: 19 and 20). This PCR fragment was then digested with restriction endonuclease Notl and ligated with Notl cleaved pVWF-1570. The resulting plasmid containing the sequence encoding the wild-type VWF, the linker sequence and albumin human, called pVWF-1574.

To achieve the expression of the fused protein was required DeleteMovie several grounds between VWF and the linker sequence. This was performed using site-directed mutagenesis according to standard protocols (using a kit for site-directed mutagenesis QuickChange XL, Stratagene, La Jolla, CA, USA), using the oligonucleotides We2995 and We2996 (SEQ ID NO: 21 and 22). The resulting expression plasmid, named pVWF-1572, contained encoding VWF sequence in reading frame with sequences encoding the linker of 31 amino acids - glycinol/Surinov and human albumin. Amino acid sequence of expressed recombinant fused with VWF protein represented in SEQ ID NO: 25. Amino acid sequence of preprally VWF person presented as SEQ ID NO: 24.

Using protocols and plasmids described above, and applying the techniques of molecular biology known skilled in the art specialists (and are described, for example, in Current Protocols in Molecular Biology, t is m), a specialist can create other designs to replace the sequence of the albumin sequence of another HLEP or sequence of the linker a different linker sequence.

Example 8: Purification of VWF and proteins merge with VWF albumin

Supernatant cell cultures containing recombinant wild-type VWF (rVWF wild-type or recombinant proteins merge with VWF albumin (rVWF-FP), sterilized by filtration through 0.2 μm filter and dialyzed against equilibrium buffer (EB; 10 mm Tris-HCl, 10 mm CaCl2, pH 7.0). This material was then introduced into the column Heparin Fractogel, balanced with buffer EB. The column was washed with buffer EB, and VWF proteins were suirable using 500 mm NaCl in EB. Peak elution was concentrated and dialyzed against buffer FB (3 g/l sodium chloride, 20 g/l glycine, 5.5 g/l tinatawag citrate dihydrate, pH 7.0). Finally, the material was sterilized by filtration and frozen in the aliquot. If necessary, apply additional purification stages, including anyone and/or cation exchange chromatography, HIC and SEC.

Example 9: analysis of the activity of VWF and VWF antigen

The samples were analyzed using immunoturbidimetric determination of VWF:Ag (OPAB03, Siemens Healthcare Diagnostics, Marburg, Germany) and binding to collagen (Technozym VWF:CBA ELISA, ex. No. 5450301 set the calibrator 5450310 and control set 545032, Technoclone, Vienna, Austria), as described by the manufacturers.

Check VWF:RCo was carried out using the reagent BC VWF Siemens Healthcare Diagnostics, Marburg, Germany, in accordance with the description manufacturers. International concentrated standard was used as a standard drug for calibration own standard product for daily use.

Relationship of VWF:RCo to VWF:Ag was calculated for comparison of this parameter for different check designs. As shown in Fig. 3, the ratio of VWF:RCo/VWF:Ag was comparable for rVWF wild-type and recombinant protein C-terminal fusion with VWF albumin.

For pharmacokinetic analysis of VWF antigen were determined using ELISA, the performance of which is known to the skilled in the art specialists. Briefly, microplates were incubated with 100 µl per well of antibody for capture (rabbit IgG against human VWF, Dako A0082 [Dako, Hamburg, Germany) diluted 1:200 in buffer A [Sigma C3041, Sigma-Aldrich, Munich, Germany]) overnight at ambient temperature. After washing tablets three times with buffer B (Sigma P3563) each well was incubated with 200 ál of buffer From Sigma P3688) for 1.5 hours at ambient temperature (for blocking). After three more stages of washing buffer In serial dilutions of tested sample buffer, and is also serial dilutions of standard human plasma (ORKL21; 20-0,2 IU/ml; Siemens Healthcare Diagnostics, Marburg, Germany) in buffer (in volume per well: 100 μl) were incubated for 1.5 hours at ambient temperature. After three stages of washing with buffer B to each well was added 100 μl of the antibody for detection (rabbit IgG against human VWF, Dako P0226 peroxidase labeled) at a dilution of 1:16000 buffer and incubated for 1 hour at ambient temperature. After three stages of washing with buffer B to each well was added 100 μl of substrate solution (OUVF, Siemens Healthcare Diagnostics) and incubated for 30 minutes at ambient temperature in the dark. Add 100 µl of stop solution (a, Siemens Healthcare Diagnostics) was making the samples ready for reading in the appropriate microplate reader device at a wavelength of 450 nm. Then the expected concentration in the tested samples, using the standard calibration curve, constructed using the standard human plasma as a reference.

Example 10: Analysis of multimeric VWF and proteins merge with VWF albumin

Analysis of multimeric VWF was performed using electrophoresis in SDS-agarose gels as previously described (Tatewaki et al., Thromb. Res. 52: 23-32 (1988), and Metzner et al., Haemophilia 4 (Suppl. 3): 25-32 (1998)) with slight modifications. Briefly, after equilibration in the movable buffer ready to use 1% agarose minigel (BioRad) was used for the article is harrisonii way to the maximum extent. Comparable amounts of VWF antigen were subjected to electrophoresis in SDS-agarose gels. After Western blotting of the VWF protein bands were detected using antibodies against VWF (DAKO, No. cont. 0854) or albumin, and then labeled with alkaline phosphatase antibodies against IgG (SIGMA, No. cont. 1305), and color reaction was quantified using densitometry.

Using recombinant VWF wild-type (1570/797) and recombinant fused with VWF protein (1572/797) can be demonstrated using Western blotting and detection with use of antibodies against albumin or VWF that recombinant fused with VWF protein forms a normal distribution of multimers detected using both antibodies against albumin and antibodies against VWF (Fig. 4). This confirms the fact that, although each subunit of a multimeric VWF contains albumin, formed a regular pattern of multimeric VWF. It is obvious that component in the form of albumin did not inhibit any of the N-terminal dimerization nor the C-terminal multimerization VWF molecules.

Example 11: Evaluation of pharmacokinetics of VWF protein and merge with VWF albumin in rats after a single intravenous injection

Recombinant fused with VWF protein and recombinant wild-type VWF was administered intravenously to each of 4 rats CD in General. The dose was 100 E (VWF:Ag)/kg body weight, the amount of injection of 4 ml/kg

Blood samples were taken retroorbital at appropriate intervals beginning 5 minutes after the application of tested substances, using changing patterns in the sampling that resulted in samples from 2 animals/time point (t=0, 5, 30, 90 min, 4 h, 1 day for subset 1 and 0, 15 min, 1, 2, 8 h and 2 days for a subset No. 2). The scheme was designed to minimize the potential effects of sampling on the plasma concentration, which was determined. The blood was turned into plasma and kept deep-frozen until analysis. Subsequently, we determined the levels of VWF:Ag in plasma using ELISA as described in example 9. For calculation of pharmacokinetic parameters used average concentration in plasma. Half life was calculated using the moments of the beta phase of elimination by the formula t1/2=ln2/k, given that k is the angle of the slope of the regression line.

The result is shown in Fig. 5 (n=2/time; the average value). It was estimated that the final half of existence are 32,4 min for recombinant fused with VWF protein and 2.6 min for recombinant wild-type VWF. The ratio of the actual maximum concentration to the expected in vivo was also increased for recombinant fused with VWF protein, accounting for 42.1%, and compared with 16.1 percent for recombinant VWF wild-type.

1. The modified von Willebrand factor (VWF), including VWF, fused at the C-terminal part of its primary product broadcast with N-terminal cha is part of albumin by a linker SSGGSGGSGGSGGSGGSGGSGGSGGSGGSGS, where the modified VWF has a longer half-life functional existence than half of the functional existence of VWF.

2. Modified VWF according to claim 1, where the modified VWF has a half-period of the functional existence, is increased by at least 25% compared with the half-period of the functional existence of VWF.

3. Modified VWF according to claim 1, where the modified VWF has a longer half-life existence as antigen compared to the half-period of existence as antigen VWF.

4. Modified VWF according to claim 3, where the modified VWF has a half-period of existence as an antigen, increased by at least 25% compared with the half-period of existence as antigen VWF.

5. Modified VWF according to claim 1, where the modified VWF has increased the ratio of the actual maximum concentration in vivo to the expected compared to the ratio of the actual maximum concentration in vivo to expect for VWF.

6. Modified VWF according to claim 5, where the modified VWF has a ratio of the actual maximum concentration in vivo to expect increased by at least 10% compared to the ratio of the actual maximum concentration in vivo to expect for VWF.

7. Modified VWF according to claim 1, where the modified VWF has at least 10% bio is logicheskoi activity of VWF.

8. Polynucleotide encoding a modified VWF according to any one of claims 1 to 7.

9. The expression vector containing polynucleotide in item 8.

10. A host cell that can Express the exogenous genetic material containing polynucleotide of claim 8 or a vector according to claim 9.

11. The method of obtaining modified VWF, including:
(a) culturing the host cell of claim 10 under such conditions that the expressed modified VWF; and
(b) the allocation of the modified VWF from the host cell or from the culture medium.

12. Pharmaceutical composition for treatment or prevention of blood clotting disorders, including the modified VWF according to any one of claims 1 to 7, polynucleotide of claim 8 or a vector according to claim 9.

13. The modified VWF according to any one of claims 1 to 7, polynucleotide of claim 8, vector according to claim 9 or host cell of claim 10 for the manufacture of a medicinal product for the treatment or prevention of blood clotting disorders.

14. Use item 13, where the coagulation disorder is a hemophilia A.

15. Use item 13, where the coagulation disorder is a von Willebrand's disease.

16. The use according to any one of p-15, where treatments include gene therapy people.

15. The method of preparation of the modified VWF has increased the half-life of the functional existence, including the surrounding fusion N-terminal albumin with the C-terminal part of the primary product of translation of VWF via a linker SSGGSGGSGGSGGSGGSGGSGGSGGSGGSGS.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biochemistry. Disclosed is L-fucose α1→6 specific lectin, which is extracted from a basidiomycete or an ascomycete or an ascomycete, characterised by peak molecular weight of about 4500 m/z, determined via MALDI-TOF mass spectrometry analysis. The novel L-fucose α1→6 specific lectin has high affinity for a L-fucose α1→6 sugar chain, represented by an association constant of 1.0×104 M-1 or higher (at 25°C), and has an association constant of 1.0×103 M-1 or lower (at 25°C) with high-mannose sugar chains and/or glucolipids which do not contain an L-fucose α1→6 sugar chain. In one version, the disclosed L-fucose α1→6 specific lectin is a protein or a peptide which consists of an amino acid sequence selected from SEQ ID NO:2-6. The L-fucose α1→6 specific lectin is used for specific detection of a L-fucose α1→6 sugar chain and effective purification of the L-fucose α1→6 sugar chain or a sugar chain which does not contain L-fucose α1→6.

EFFECT: obtaining L-fucose α1→6 specific lectin.

16 cl, 38 dwg, 8 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology and biotechnology. There are presented versions of nucleic acids each of which codes a heavy-chain amino acid sequence of immunoglobulin IgG1. The above chain contains glycine-lysine dipeptide coded by ggaaaa, ggcaaa or gggaaa codon at the C terminal of the CH3 domain. There are described: a plasmid coding a heavy chain of immunoglobulin; version cells providing immunoglobulin IgG1 expression; a method for producing immunoglobulin in mammalian cells; a method for improving immunoglobulin expression in the mammalian cells; - using the versions of a nucleic acid.

EFFECT: using the invention provides preventing the by-product expression of weight 80 kDa that can find application in producing immunoglubulins.

18 cl, 7 dwg, 3 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: application of yeast strain Komagataella pastoris RNCIM Y-727 as the recipient to construction of producers of target protein is characterised, optionally comprising introduction of mutations into it, providing the use of auxotrophic selective markers.

EFFECT: solution can be applied in preparation of recombinant proteins without the use of methanol as inductor of gene expression.

8 dwg, 4 tbl, 10 ex

FIELD: biotechnology.

SUBSTANCE: nucleotide sequences are formed, encoding the hybrid proteins EPO-TR 1.6, EPO-TR 4 and EPO-TR 6. Protein EPO-TR 1.6 is recombinant human erythropoietin fused with a fragment TR 1.6 of the human protein MUC1. Protein EPO-TR 4 is recombinant human erythropoietin fused with a fragment TR 4 of the human protein MUC1. The hybrid protein EPO-TR 6 is recombinant human erythropoietin fused with a fragment TR 6 of the human protein MUC1. Hybrid proteins are produced by the roller cultivation in the suitable conditions the modified mammalian cell line CHO containing a nucleotide sequence encoding the protein with subsequent isolation of the hybrid protein from the culture fluid.

EFFECT: invention enables to produce the hybrid recombinant human erythropoietin having the prolonged action.

4 cl, 4 dwg, 7 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology, namely to muteins of human tear lipocalin, and can be used in medicine. Mutein of human tear lipocalin (hTLc) has identifiable affinity of binding with human receptor Met (c-Met) receptor tyrosine kinase, or its domain, or fragment of human c-Met. Mutein contains from 6 to 18 amino acid substitutions relative to amino acid sequence of mature lipocalin of human tear liquid (SWISSPROT DATABANK ENTRY P31025; SEQ ID NO:36), selected from group, consisting of Arg 26→Thr, Val, Pro, Ser, Gly; Glu 27→Gln, Gly, Val, Ser; Phe 28→Met, Asp; Pro 29→Leu, Ile, Ala, Trp; Glu 30→Leu, Gly, Arg, Phe; Met 31→Ser; Asn 32→Leu, Arg, Val, Gln; Leu 33→Tyr, Val, Ile, Thr, Phe; Glu 34→Val, Arg, Ala; Leu 56→Asn; Ile 57→Gln; Ser 58→Ile, Val; Asp 80→Tyr; Lys 83→Ala; Glu 104→Asp; Leu 105→Thr; His 106→Trp and Lys 108→Gly. Mutein can also additionally contain the following substitutions: Cys 61→Ser; Cys 101→Ser; Cys 153→Ser; Arg 111→Pro; Lys 114→Trp; Thr 37→Ser; Met 39→Ile, Leu; Asn 48→Ser; Lys 52→Thr, Met; Met 55→Leu; Lys 65→Arg, Leu; Ala 79→Leu, Ser; Ala 86→Thr; Ile 89→Ser, Gln, Thr, His; Thr 40→Cys; Glu 73→Cys; Arg 90→Cys; Asp 95→Cys; Lys 121→Cys; Asn 123→Cys and Glu 131→Cys.

EFFECT: invention makes it possible to efficiently treat pathological disorders, which involve pathway HGF/c-Met, as well as to perform identification of human c-Met in sample.

40 cl, 16 dwg, 9 tbl, 25 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to field of microbiology and deals with method of obtaining RTX-toxin ApxI. Claimed method is realised by cultivation of bacteria Actinobacillus pleuropneumoniae in culture medium, which provides growth of bacteria, and said culture medium contains borogluconate in concentration lower than 60 mmol/l in order to form in medium calcium-bologluconate complex.

EFFECT: invention makes it possible to increase output of RTX-toxin ApxI, which can be applied in production of vaccines.

5 cl, 4 tbl

FIELD: biotechnologies.

SUBSTANCE: invention refers to a method for obtaining an antibody, the pharmacokinetic properties of which have been changed at maintaining antigen-binding activity of a variable area, which provides for the following stages: (a) obtaining antibodies in which there has been modified a charge of amino-acid residues chosen from amino-acid residues in positions 31, 61, 62, 64 and 65 of the variable area of a heavy chain and in positions 24, 27, 53, 54 and 55 of the variable area of a light chain in compliance with numbering as per Kabat system, where modification of the charge of amino-acid residues leads to the change of 1.0 or more at a theoretical isoelectric point of the variable area of the antibody, and (b) extracting an antibody with stored antigen-binding activity from antibodies obtained at stage (a).

EFFECT: invention allows effective change in pharmacokinetic properties of an antibody, thus maintaining its antigen-binding activity.

FIELD: biotechnologies.

SUBSTANCE: invention relates to method for obtaining of RTX-toxins Apxl or ApxIII by culturing of Actinobacillus pleuropneumoniae bacteria in liquid culture media. Characterised method consists in the following: during exponential growth phase of bacteria and production of RTX-toxins air passes through the medium, carbon dioxide content in air is above normal atmospheric level and is up to 10 % vol.

EFFECT: invention allows increasing Apxl or ApxIII toxins output, this may be used during vaccines production.

7 cl, 4 tbl

FIELD: biotechnologies.

SUBSTANCE: physiologically active protein or polypeptide are fused with version of alpha-1-antitrypsin, which has at least one mutated aminoacid residue. Mutations are performed in the following positions: asparagine residue instead of proline residue in position 357; or asparagine residue instead of proline residue in position 357 and threonine residue instead of serine in position 359; or asparagine residue instead of proline residue in position 357 and serine residue instead of cysteine in position 232; or asparagine residue instead of proline residue in position 357, threonine residue instead of serine in position 359 and serine residue instead of cysteine in position 232.

EFFECT: invention allows increasing half lifetime of physiologically active protein or polypeptide in vivo by maintaining its stable circulation in blood.

7 cl, 13 dwg, 7 ex

FIELD: biotechnologies.

SUBSTANCE: method involves cultivation in the appropriate conditions of yeast Saccharomyces cerevisiae and release of target protein; besides, release is directed with leader polypeptide, which has amino acid sequence SEQ ID NO1 and representing a variant of a pro-area of leader polypeptide of protein PpPIRl Pichia pastoris.

EFFECT: invention enlarges the range of methods for obtaining target protein in yeast Saccharomyces cerevisiae and increases possibilities for effective synthesis of such proteins.

2 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is an antibody specifically binding to an epitope on CD43 and CEA and modified in a heavy and/or light chain constant region. There are disclosed polynucleotides, vector, host cell and methods for producing the antibody according to the invention, as well as pharmaceutical composition, kit and method of treating non-haemopoietic cancer.

EFFECT: this invention can find further application in therapy and diagnosing of CD43 or CEA mediated diseases.

35 cl, 15 tbl, 5 ex, 4 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a recovered human integrin α5β1 monoclonal antibody. The antibody is characterised by the fact that it contains 6 CDR, 3 CDR from a light chain and 3 CDR from a heavy chain. A nucleic acid (NA) coding the antibody according to the invention, an expression vector containing a NA molecule, a host cell containing the vector, and a method for preparing the antibody on the basis of the cell are described. There are disclosed: a composition and a method for growth inhibition of the tumour cells expressing human integrin α5β1 on the basis of the antibody. What is described is a version of the method for growth inhibition of the tumour cells expressing human integrin α5β1 using the composition.

EFFECT: invention provides the new antibodies with high (approximately nm, as measured by FACS) binding affinity for human integrin α5β1 that can find application in medicine in therapy of the tumours mediated by integrin α5β1 expression.

13 cl, 36 dwg, 3 tbl, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, virology and medicine. The first flaviviral particle comprises a psudoinfecting viral genome coding cis-active promoter elements required for the RNA replication, coat proteins and a complete kit of non-structural proteins of the flavivrus, and not coding capside proteins of the flavivirus. The second flaviviral particle comprises a complementary genome coding the cis-active promoter elements required for the RNA replication, a capside protein and a complete kit of proteins of the flavivrus, and not coding coat proteins. Since the genetic material of the flavivirus is distributed between two genomes, the flavivirus is replication-deficient and is not able to induce a disease, however it is able to induce an immune response. What is also described is a method for preparing this combination, a pharmaceutical composition and a method for using it. The invention can be used in medicine.

EFFECT: what is presented is a combination of the flaviviral particles.

13 cl, 18 dwg, 14 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. What is presented is an isolated monoclonal antibody or its immunoreactive fragment binding to the respiratory syncytial virus (RSV) G protein epitope of the strain A2. There are also described a nucleic acid molecule coding it, a host cell containing this nucleic acid molecule, a method for producing the antibody and a pharmaceutical composition containing this antibody.

EFFECT: presented group of inventions can be used for treating respiratory syncytial virus.

14 cl, 37 dwg, 1 tbl, 9 ex

Antibody to epha2 // 2525133

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of immunology, medicine and biotechnology. Claimed are versions of anti-EPHA2 antibodies. Claimed antibodies are bound with polypeptide, consisting of amino acids 426-534 in SEQ ID NO:8. Also described are hybridomes, which produce such antibodies, and pharmaceutical compositions and methods of application of said antibodies and compositions.

EFFECT: invention can be used in medicine.

74 cl, 14 dwg, 14 ex, 1 tbl

FIELD: biotechnology.

SUBSTANCE: invention relates to an agent for treatment of ischemic lesions of tissues, which is a mixture with the ratio of 1.5-3 of two cultures of mesenchymal stem cells, one of which is modified by the genetic structure based on the viral vector which provides hyperproduction of vascular endothelial growth factor, and the other is modified by the genetic structure based on the viral vector which provides hyperproduction of angiopoietin, and a method of treatment of ischemic lesions of tissues by puncture of ischemic tissue, and can be used in medicine.

EFFECT: invention enables to achieve effective vascularisation and repair of ischemic tissue.

4 cl, 4 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: inventions relate to the field of immunology. Claimed are a single-chain antibody, specific to a carcinoembryonic antigen, a chimeric mononuclear T-cell receptor, a vector, a host cell and a method of diagnostics or treatment of diseases, characterised by the presence of antigens, capable of binding with the chimeric receptor. Described is a genetic construction, coding chimeric monomolecular T-cell receptors, in which an effector fragment of the T-cell receptor is combined with an antigen-recognising part, which represents variable fragments of two different antibodies to the carcinoembryonic antigen (CEA).

EFFECT: claimed inventions can be used in T-cell cancer therapy.

7 cl, 4 dwg, 3 ex, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to chemical-pharmaceutical industry and represents a preparation for involving a mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow, which is introduced into the blood vessel or muscle and which contains any of components: (a) protein HMGB1; (b) HMGB1 protein-secreting cell; (c) a vector, into which HMGB1 protein-coding DNA is inserted; (d) protein HMGB2; (e) HMGB2 protein-secreting cell; (f) a vector, into which HMGB2 protein-coding DNA is inserted; (g) protein HMGB3; (h) HMGB3 protein-secreting cell; and (i) a vector, into which HMGB3 protein-coding DNA is inserted.

EFFECT: elaboration of the preparation for involving the mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow.

3 cl, 6 ex, 1 tbl, 14 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Method includes introduction of RNA molecule into a bird egg. Introduced RNA molecule contains double-stranded region and results in reduction of the level of molecule of RNA and/or protein, included into determination of sex in birds, in the egg. Invention can be used in poultry breeding.

EFFECT: claimed is method of changing sex characteristics in birds.

7 cl, 3 dwg, 6 tbl, 2 ex

FIELD: food industry.

SUBSTANCE: invention refers to the field of biotechnology and food industry. Presented is a barley plant that yields grain and is homozygotic in at least two loci for the genetic variations having been bred, representing: a) allele wherein most of or all the genes coding B-hordein in Locus Hor2 are removed, and b) mutant allele in the barley Locus Lys3 so that the grain contains neither B-, nor C- hordeins, the said genetic variations present in Lines Riso 56 and Riso 1508 barley accordingly; absence of B-hordeins is to be revealed by absence of amplified DNA using primers: 5'B1hor: 5'-CAACAATGAAGACCTTCCTC-3', 3'B1hor: 5'-TCGCAGGATCCTGTACAACG-3', while absence of C-hordeins is to be revealed by absence of the 70 kDa strip during study of the grain alcohol-soluble extract by means of SDS-PAGE. Additionally presented are: barley grain cropped from the said plant; B- and C-hordein-free products produced from the said grain such as flour, malt and beer. Additionally described are methods for production of food products barley (flour, whole-grain flour, starch, malt) and beverages using grain cropped from the barley plant having the above characteristics. Proposed is a method for identification of barley grain suitable for production of a malt-based food product and/or beverage suitable for consumption by a person suffering from gluten-sensitive enteropathy which method includes: a) production of one or more materials: i) sample of a plant capable to yield the said grain, ii) grain, iii) malt produced from the grain, and/or iv) extract of the said grain; b) analysis of Stage a) material for presence of at least one hordein and/or at least one hordein-coding gene with selection of grain having the gene pattern of the above plant.

EFFECT: invention allows to manufacture B- and C-hordein-free malt-based food products or beverages.

27 cl, 14 dwg, 10 tbl, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry, particularly to a recovered polypeptide which is a biological target for methane-producing cell inhibition, as well as to a recovered polynucleotide which codes this polypeptide. There are disclosed expression vector and cloning vector containing this polynucleotide, and host cells containing the above expression vector. There are described conjugated molecules or fused molecule for methane-producing cell inhibition, as well as antibody or its functional fragment which binds to the above polypeptide. The invention also covers a pharmaceutical composition and methods for inhibiting and identifying the methane-producing cell with the use of the above conjugated molecule or fused molecule and the antibody or its fragment.

EFFECT: invention enables inhibiting the methane-producing cell effectively.

19 cl, 9 dwg, 6 ex

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