Isolated molecule of nucleic acid, which codes fused polypeptide that is able to bind vessel endotheliocytes growth factor (vegf), fused polypeptide, copied expressive vector, method for production of fused polypeptide, trap of vegf, pharmaceutical composition, method for treatment and set for treatment of vegf-mediated disease or condition

FIELD: medicine.

SUBSTANCE: invention is related to nucleic acids and multidomain proteins, which are able to bind vessel endotheliocyte growth factor (VEGF), and may be used in medicine. Recombinant method is used to produce polypeptide, which consists of component (R1R2)X and, unnecessarily, multidomain component (MC), which represents aminoacid sequence with length from 1 to 200 of amino acids, having at least one remainder of cysteine, where X≥1, R1 means antibody-like (Ig) domain 2 of VEGF receptor Llt-1, and R2 means Ig-domain 3 of VEGF receptor Flk-1. Produced fused polypeptide does not contain multidomain component in case, when X=2, and in case when X=1, multidomain component represents aminoacid sequence with length from 1 to 15 amino acids. Produced polypeptide is used in composition of pharmaceutical compound for VEGF-mediated disease or condition.

EFFECT: invention makes it possible to produce highly efficient trap of VEGF, special structure of which is suitable for local introduction into specific organs, tissues or cells.

16 cl, 3 tbl, 7 ex

 

The LEVEL of TECHNOLOGY

The technical field to which the invention relates

The invention relates to fused to polypeptides capable of binding growth factor endothelial cells of blood vessels (VEGF), representatives of the family of VEGF and splicing variants with specific desired characteristics, and to therapeutic methods of use.

The INVENTION

In the first aspect, the hallmark of the invention is a nucleic acid molecule encoding a fused polypeptide containing components receptors (R1R2)Xand/or (R1R3)Ywhere R1 means a component of a growth factor receptor on endothelial cells of blood vessels (VEGF) in the form of Ig domain 2 of Flt-1 (Flt1D2), R2 means a component of the VEGF receptor in the form of Ig domain 3 of Flk-1 (Flk1D3) and R3 means a component of the VEGF receptor in the form of Ig-domain 3 Flt-4 (Flt1D3 or R3) and where X≥1 and Y≥1.

In a related second aspect, the hallmark of the invention is a Monomeric VEGF trap or fused polypeptide containing components of the VEGF receptor (R1R2)Xand/or (R1R3)Ywhere X≥1, Y≥1 and R1, R2 and R3 have the meanings defined above. Components of the VEGF receptor R1, R2 and R3 may be directly connected to each other or connected by one or more spacer elements of the sequences. In one specific embodiment, the Monomeric VEGF trap is a (R1R2)Xwhere X=2. More is oncletom version Monomeric VEGF trap is SEQ ID NO: 24 or a functionally equivalent amino acid variant. The invention relates to Monomeric VEGF trap, mainly consisting of the components of the VEGF receptor (R1R2)Xand/or (R1R3)Yand their functionally equivalent amino acid variants.

In the third aspect distinguishing feature of the invention is an isolated nucleic acid molecule encoding a fused polypeptide containing components of the VEGF receptor (R1R2)Xand/or (R1R3)Yand the component, which is a partner in the merger (FP)selected from the group consisting of multimeasure component (MC), whey protein or molecule capable of binding a serum protein. In a preferred embodiment, the FP is multimerization component (MC)capable of interacting with multimerization component in another merged the polypeptide with the formation of a multimeric structure, such as a dimer or trimer. Most preferably, MC is selected from the group consisting of (i) multimeasure component containing fissionable region (C-region), (ii) shortened multimeasure component, (iii) amino acid sequence ranging in length from 1 to about 200 amino acids that has at least one cysteine residue, (iv) latinboy zipper, (v) motif of the spiral loop, (vi) a coil-coil motif and (vii) the domain of immunoglobulin. In addition, there are fused polypeptides, essentially consisting of (R1R2)Xor (R1R3) Yand FP. In a preferred embodiment, the slit polypeptide essentially consists of

(R1R2)Xand MC.

In the fourth aspect distinguishing feature of the invention is fused polypeptide containing components of the VEGF receptor (R1R2)Xand/or (R1R3)Yand FP, which are described above. Components of the receptor can be arranged in a different order, for example (R1R2)X-FP; (R1R2)X-FP-(R1R2)X; FP-(R2R1)Xetc. Components of the fused polypeptide can be directly connected to each other or connected by means of spacer elements of the sequence.

In the fifth aspect distinguishing feature of the invention is to VEGF trap containing multimer of two or more fused polypeptide consisting of the components of the VEGF receptor (R1R2)Xand/or (R1R3)Yand FP, where component FP is multimerization component (MC), containing the C-region. C-region may be natural or artificial and can be located at any point in multimerization component and functions, providing a splitting of the original MC shortened to MC. The VEGF trap, consisting of two or more fused polypeptides having at least one shortened MC, called "short minilovely".

C-region may be created in MC by insertions, deletions or mutations so that was generated by enzymatic or Henichesk the split site. C-region may be created in any MC in any position in MC; preferably C-region create a full Fc domain or fragment or CH3 domain. C-region may be the site cleaved by an enzyme such as thrombin, fitsin, pepsin, matrilysin or prolidase, or cleaved chemically, for example, formic acid or CuCl2.

In a sixth related aspect distinguishing feature of the invention is shortened minilogue VEGF, which is a multimeric protein containing two or more fused polypeptide consisting of (R1R2)Xand/or (R1R3)Yand multimeasure component, which is shortened by splitting the original MC containing the C-region (tMC).

In the seventh aspect, the hallmark of the invention is fused to the polypeptide consisting of the components of the VEGF receptor (R1R2)Xand/or (R1R3)Yand MC, where MC represents the amino acid sequence ranging in length from 1 to about 200 amino acids, containing at least one cysteine residue, where at least one cysteine residue capable of forming a disulfide bond with a cysteine residue present in another MC fused polypeptide (cMC). In a preferred embodiment, the cMC is an amino acid sequence with a length of 1-50 amino acids, containing at least one cysteine residue. More pre is reverent version of the cMC is the amino acid sequence of length 1-15 amino acids, containing at least one cysteine residue. In an even more preferred embodiment, the cMC is an amino acid sequence with a length of 1-10 amino acids, containing 1-2 cysteine residue. An illustration of this variant of the invention shown in SEQ ID NO: 27 having a signal sequence (1-26), followed by the components R1 (27-129) and R2 (130-231), and follows a sequence of nine amino acids, ending with a cysteine residue. In another embodiment, shown in SEQ ID NO:28, for the signal sequence (1-26) followed by components R1 (27-129) and R2 (130-231), followed by a sequence of six amino acids, ending with a cysteine residue.

In the eighth aspect, the hallmark of the invention is to minilogue VEGF containing multimer of two or more fused polypeptides consisting of (R1R2)Xand/or (R1R3)Yand the cMC. In a more specific embodiment, minilogue is a dimer. An illustration of this option minilovely according to the invention is a dimer fused polypeptide shown in SEQ ID NO: 2, in which each fused polypeptide (R1R2-cMC) has a molecular weight 23,0 kDa and pI which 9.22.

In another embodiment, the cMC has 4 amino acids in length and includes two cysteine residue, for example XCXC (SEQ ID NO: 3). In one illustrative example of this variant of the invention minilogue consists of to the components of the VEGF receptor according to the invention and the cMC consists of ACGC (SEQ ID NO: 4). One illustrative example of this variant of minilovely according to the invention is a dimer fused polypeptide shown in SEQ ID NO: 5, in which each monomer has a molecular mass of 23.2 kDa and pI which 9.22. Another illustrative example of this variant of the invention shown in SEQ ID NO: 26 having a signal sequence (1-26), followed by the components R1 (27-129) and R2 (130-231) followed by a sequence of nine amino acids, ending CPPC.

In all variants of VEGF trap according to the invention (including shortened minicourse VEGF, minilovely VEGF and Monomeric minilovely VEGF) signal sequence (S) can be included at the beginning (or at the N-end) fused polypeptide according to the invention. The signal sequence may be native to the cell, recombinant or synthetic. If the signal sequence linked to the N-end of the first receptor component, the fused polypeptide can be labeled, such as S-(R1R2)X.

The components of the fused polypeptide can be directly connected to each other or may be connected by spacers. In specific embodiments, one or more receptor components and/or components that are partners in the merger, the merged polypeptide directly linked to each other without spacers. In other variations of the one or more receptor components and/or components, partners in the merger, connected by spacers.

The invention relates to vectors containing the nucleic acid molecules according to the invention, including expressing vectors, containing a nucleic acid molecule, functionally associated with a sequence expression regulation. The invention, furthermore, relates to systems host-vector to obtain the fused polypeptide, which contains expressing the vector in a suitable cell host; systems host-vector, in which a suitable cell host is a bacterial, yeast cell, insect cell, mammalian cell; the cell is E. coli or COS cell or CHO. In addition, there are traps VEGF according to the invention, modified by acetylation or pegylation. How acetylation or paglierani protein are well known in this field.

In the linked ninth aspect distinguishing feature of the invention is a method for VEGF trap according to the invention, comprising culturing the host cell transtitional vector containing the nucleic acid sequence according to the invention, under conditions suitable for expression of the protein of the host-cell, and removing the thus obtained fused polypeptide.

The VEGF trap according to the invention therapeutically p is iminime for the treatment of any disease or condition which improves becomes weakened or depressed during removal, inhibition or reduction of VEGF. A partial list of specific conditions that improve when the inhibition or reduction of VEGF, includes an example of undesirable leakage of plasma or vascular permeability, unwanted growth of blood vessels, such as in tumors, edema associated with inflammatory diseases such as psoriasis or arthritis, including rheumatoid arthritis; asthma; generalized edema associated with burns; ascites and pleural effusion associated with tumors, inflammation or trauma; chronic airway inflammation; asthma; syndrome capillary leak; sepsis; kidney disease associated with increased leakage of protein; adenocarcinoma of the pancreatic ducts (PDAC), and eye diseases such as age related macular degeneration and diabetic retinopathy. Minilogue VEGF, in particular, applicable to the treatment of eye diseases and as an aid in eye surgery, including surgery for glaucoma; and treatment of intraocular tumors, such as uveal melanoma, retinoblastoma, by delivery to the vitreous body.

Accordingly, in the tenth aspect, the hallmark of the invention is therapeutic the manual treatment is associated with VEGF disease or condition, including the introduction of VEGF trap according to the invention to a subject suffering from associated with VEGF disease or condition. Although any mammal can be treated with therapeutic methods according to the invention, the subject preferably is a sick person suffering from or at risk of developing a condition or disease that can be improved, weakened, ingibirovalo or treated with VEGF trap.

In the eleventh aspect, the hallmark of the invention are methods for diagnosing and forecasting, as well as kits for the identification, quantitative analysis and/or tracking of VEGF using minilogue according to the invention.

In the twelfth aspect, the hallmark of the invention are pharmaceutical compositions containing the VEGF trap according to the invention with a pharmaceutically acceptable carrier. Such pharmaceutical compositions may contain a trap of dimer fused polypeptide or nucleic acid encoding the fused polypeptide. Minilovely according to the invention find particular application when the conditions under which you want the VEGF trap with reduced half-life in serum (e.g., more rapid clearance and/or increased penetration in the tissue due to the smaller size. Specific applications of minilovely VEGF VK is ucaut, for example, diseases in which it is desirable local introduction in a particular tissue or cell. Examples of this condition are ophthalmic eye disease.

Other objects and advantages will become apparent upon reading the following detailed description.

DETAILED description of the INVENTION

Before you find a description of the proposed methods, it should be understood that this invention is not limited to the described specific ways and experimental conditions, as such methods and conditions may vary. Also it should be understood that as used in this description, the terminology is used only to describe specific options and is not intended to be limiting, as the scope of the present invention will be limited only by the attached claims.

In used in this description and the attached claims the sense of the form of the singular include references to the plural, unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods and/or the steps specified in the description of the type and/or those that will become obvious to specialists in this field when reading the description, etc.

Unless otherwise noted, all technical and scientific terms used in the present description, have the same meaning, which is usually understood as a specialist in the field to which this invention relates. Although in practice or testing of the present invention can be used any methods and materials similar or equivalent to the substances listed in this description describes the preferred methods and substances. All publications referred to herein, are incorporated into it by reference to describe the methods and/or agents, in connection with which the cited publication.

General description

The invention relates to a VEGF trap, is able to bind and inhibit the activity of VEGF, which is a monomer or multibeam one or more fused polypeptides. Molecules according to the invention bind and inhibit the biological activity of VEGF and/or physiological reaction or response. Description based on receptor antagonistic VEGF trap VEGF Flt1D2.Flk1D3.FcΔC1(a) (SEQ ID NO: 7-8) and VEGFR1R2-FcΔC1(a) (SEQ ID NO: 9-10), see PCT WO/0075319, the contents of which are incorporated in this description by reference in full.

Minilogue according to the invention is smaller than full-sized trap, approximately 50-60 kDa compared to the 120 kDa original traps, and includes Monomeric trap, consisting mainly of domains of VEGF receptors (R1R2)X, (R1R3)Yor combinations thereof, traps, educated otwal is part of the original multimeric traps having component, which is a partner in the merger, which represents multimediali component (MC), containing the region of the cleavage (C-region); or binding cysteine residue or amino acid sequence that contains one or more cysteine residues, with domains of the receptor component or between domains of the receptor component. In specific embodiments, minilogue according to the invention has a molecular mass of less than 60 kDa as measured by SDS-page analysis; more preferably about 50 kDa; even more preferably about 20-30 kDa, or about 25 kDa and is able to bind VEGF with an affinity comparable to the full-size original trap, described in PCT/US00/14142.

Structure of nucleic acids and expression

The present invention relates to the construction of nucleic acid molecules encoding a separate fused polypeptide capable of binding VEGF, or a multimeric VEGF trap. The nucleic acid molecules according to the invention can encode components of wild-type receptors R1, R2 and/or R3 or their functionally equivalent variants. Variants of the amino acid sequence of the receptor components R1, R2 and/or R3 traps according to the invention can also be obtained by creating mutations in the coding nucleic acid molecules. So the e options include, for example, deletions, or insertions, or substitutions of amino acid residues in the amino acid sequence R1, R2 and/or R3. Can be made of any combination of deletions, insertions and substitutions to obtain the final construct, provided that the final construct possesses the ability to bind and inhibit VEGF.

These molecules are nucleic acid is inserted into a vector that can Express the fused polypeptide when introduced into a suitable cell host. Appropriate cell hosts include, but are not limited to, cells, bacteria, yeast, insects and mammals. You can apply any of the methods known to the person skilled in the art, for integration of DNA fragments into a vector to construct expressing vectors encoding the fused polypeptide according to the invention, under control of signals regulation of transcription/translation.

The expression of the nucleic acid molecules according to the invention may be regulated by a second nucleic acid sequence so that the molecules expressives in a host transformed with the recombinant DNA molecule. For example, expression can be controlled by any promoter/enhancer element known in this field. Promoters that can be used to regulate expression of a chimeric polypeptide of the molecular is, include, without limitation long terminal repeat (Squinto et al. (1991) Cell 65: 1-20); the area of early SV40 promoter, the CMV promoter, the M-MuLV, the promoter timedancing, the regulatory sequences of the gene of metallothionine; prokaryotic expressing vector, such as the promoter of b-lactamase or the tac promoter (see also Scientific American (1980) 242: 74-94); promoter elements of yeast or other fungi such as the Gal promoter 4, ADH, PGK, alkaline phosphatase promoter, and the field of regulation of tissue-specific transcription derived from such genes as elastase I.

Expressing the vectors that can replicate in a bacterial or eukaryotic host containing the nucleic acid molecules according to the invention, used for transfection of the host and thus to control the expression of such nucleic acids to obtain the fused polypeptide according to the invention, which forms a trap, the ability to communicate with VEGF. Transfetsirovannyh cells can temporarily or preferably a constitutive and constantly Express the VEGF trap according to the invention.

Trap according to the invention can be purified by any method that provides the subsequent formation of a stable biologically active traps. For example, but not to limit, the factors can be extracted from cells or in the form of soluble proteins or in the form of inclusion bodies, of which they can be extracted quantitatively 8M hydrochloride guanidine and dialysis (see, for example, U.S. patent No. 5663304). In order to further purify the factors, you can use an ordinary ion-exchange chromatography, chromatography based on hydrophobic interaction chromatography with reversed-phase or gel-filtering.

Components of the VEGF receptor

Components of the VEGF receptor in miniauskaf VEGF consist of Ig domain 2 of Flt-1 (Flt1D2) (R1), Ig domain 3 of Flk-1 (Flk1D3) (R2) (together, R1R2), and/or R1 and Ig domain 3 of Flt-4 (Flt1D3) (R3) (together R1R3). It is implied that the term "Ig-domain of Flt-1, Flt-4 or Flk-1 not only covers the full domain of the wild type, but also its variants with insertions, deletions and/or substitutions, which essentially retain the functional properties of the intact domain. The person skilled in the art without difficulty be clear, that can be obtained numerous variants of the above Ig-domains, which will retain essentially the same functional properties as the domain of the wild type.

It is implied that the term "functional equivalents" when used with reference to R1, R2 or R3, covers the domain of R1, R2 or R3 is at least one change, for example by deletion, connection and/or replacement, which retains essentially the same functional properties as the domain of R1, R2 or R3 wild type, that is essentially the equivalent binding to VEGF. It will be clear that can be made by different amino acid substitutions at R1, R2 or R3, without departing from the essence of the invention in relation to the ability of these receptor components to bind and inactivate VEGF. Functional properties of traps according to the invention can be defined by any suitable screening analysis, known in the field to measure the desired characteristics. Examples of such assays are described in the experimental section below, are used to determine the binding properties of traps for VEGF (Kd), as well as their half-life in the case of dissociation of the complex trap-ligand (T1/2). Other tests, such as changing abilities specific to contact VEGF, can be measured in the analysis of binding VEGF competitive type. Modify the properties of the protein, such as thermal stability, hydrophobicity, susceptibility to proteolytic degradation or the tendency to aggregation can be measured by methods known to experts in this field.

The components of the fused polypeptide can be directly connected to each other or may be connected by spacers. In General, the term "spacer" (or linker) means one or more molecules, for example nucleic acids, or amino acids, or residues ones, such as polyethylene glycol, which can be embedded between about who or several constituent domains. For example, spacer elements of the sequence can be used to provide the required interest site between components to facilitate processing. The spacer can also be introduced to enhance the expression of the fused polypeptide of the host-cell to reduce steric interference, so that the component can accept their optimal tertiary structure and/or appropriately interact with their molecule-target. The spacers and methods of identification of required spacers see, for example, George et al. (2003) Protein Engineering 15: 871-879, included in this description by reference. The spacer sequence may contain one or more amino acids that are related in nature to the receptor component, or can be an added sequence used to amplify the expression of the fused polypeptide, provide required special interest sites, providing opportunities for education components of the optimal domains tertiary structures and/or to enhance the interaction of the component with its molecule target. In one embodiment, the spacer contains one or more peptide sequences between one or more components that contain 1-100 amino acids, preferably 1-25.

In more specific embodiments, R1 p is ecstasy an amino acid 27-126 SEQ ID NO: 8 or 1-126 SEQ ID NO: 8 (including the signal sequence 1-26); or amino acids 27-129 SEQ ID NO: 10 or 1-129 SEQ ID NO: 10 (including the signal sequence in position 1-26). In more specific embodiments, R2 is an amino acid 127-228 SEQ ID NO: 8, or amino acids 130-231 SEQ ID NO: 10. In more specific embodiments, R3 is an amino acid 127-225 SEQ ID NO: 13 (without signal sequence). In the case when, for example, R2 is placed on the N end of the fused polypeptide may be desirable that the signal sequence was preceded by a receptor component. Receptor component(s)associated with multimerization component, in addition, may contain a spacer elements component, such as the GPG sequence of amino acids 229-231 SEQ ID NO: 7.

Components that are partners in the merger, and Multimeida components

Partner in the merger is any component that enhances the functions of the fused polypeptide. Thus, for example, a partner in the merger may increase the biological activity of the fused polypeptide, to help his production and/or extraction or exacerbate pharmacological property or pharmacokinetic profile of the fused polypeptide, for example, by increasing its half-life in serum, the permeability of the tissue, ensure there is no potency or stability. In preferred embodiments, the partner in SL is anii selected from the group consisting of multimeasure component, whey protein or molecule capable of binding a serum protein.

In the case when the partner in the merger is whey protein or its fragment selected from the group consisting of α-1-microglobulin, AGP-1, orosomucoid, α-1-acid glycoprotein, binds the vitamin D protein (DBP), hemopexin, serum albumin, human (hSA), transferrin, ferritin, afamin, haptoglobin, α-fetoprotein of thyroglobulin, α-2-HS-glycoprotein, β-2-glycoprotein, hyaluronan binding protein, syntaxin, C1R, chain C1q binding galectin 3-Mac2 protein, fibrinogen, polymeric Ig receptor (PIGR), α-2-macroglobulin, protein, transporting urea, haptoglobin, IGFBP, phagocytic receptors of macrophages, fibronectin, giuntina, Fc, α-1-antichymotrypsin, α-1-antitrypsin, anti-thrombin III, apolipoprotein A-1, apolipoprotein B, β-2-microglobulin, ceruloplasmin, component of complement C3 or C4), esterase inhibitor CI, C-reactive protein, cystatin C and protein C. In a more specific embodiment, the partner in the merger is selected from the group consisting of α-1-microglobulin, AGP-1, orosomucoid, α-1-acid glycoprotein, binds the vitamin D protein (DBP), hemopexin, serum albumin, human (hSA), afamin and haptoglobin. The inclusion of a component, which is a partner in the merger may when the he desire to prolong the half-life of the fused polypeptide according to the invention in the serum. See, for example, U.S. patent No. 6423512, 5876969, 6593295 and 6548653 specifically included in this description by reference in full, in relation to examples of the polypeptide, fused with serum albumin. hSA is widely distributed in the body, especially in the intestines and blood components, and plays an important role in maintaining osmolarity and plasma volume. He slowly excreted from the liver in humans typically has a half-life in vivo 14-20 days (Waldmann et al. (1977) Albumin, Structure, Function and Uses; Pergamon Press; pp. 255-275).

In that case, when a partner in a merger is a molecule capable of binding a serum protein, a molecule may be a synthetic small molecule, a lipid or liposome, a nucleic acid, including synthetic nucleic acid, such as aptamer, peptide or oligosaccharide. In addition, the molecule may be a protein, such as, for example, FcγR1, FcγR2, FcγR3, polymeric Ig receptor (PIGR), ScFv and other antibody fragments that are specific in relation to serum protein.

In that case, when a partner in a merger is multimediali component (MC), it represents any natural or synthetic sequence capable of interacting with another MC with the formation of the structure of a higher order, such as dimer, trimer, etc. Suitable MC may include lacinova lightning, including domains latinboy lightning, recip is installed from c-jun or c-fos; sequence derived from the constant regions of light chains Kappa or lambda; synthetic sequence, such as the motives of the helix-loop-helix (Muller et al. (1998) FEBS Lett. 432: 45-49), coil-coil motifs, etc. or other conventional multimeadia domains known in this field. In some embodiments, the merged component contains a domain derived from an immunoglobulin, for example, IgG, IgM or IgA person. In specific embodiments, derived from immunoglobulin domain may be selected from the group consisting of the Fc domain of IgG, the heavy chain of IgG and light chains of IgG. Fc domain of IgG may be selected from the isotypes IgG1, IgG2, IgG3 and IgG4, as well as any allotype in each group isotypes. In one example, the VEGF trap according to the invention multimerization component is a Fc-domain of IgG4 (SEQ ID NO: 29).

The development of shorter minilogue VEGF

In one embodiment, the trap according to the invention, the shortened minicourse VEGF containing two or more fused polypeptide according to the invention, create, exposing the original trap with MC containing the C-region, the impact of the conditions under which hatshepsuts one or more MC containing the C-region. The resulting shortened minilogue may be a product of full and partial removal of the source of the trap.

MC containing the C-region may be any MC, capable of interaction is to Modestovich with another MC with the formation of the structure of a higher order, for example, dimer or trimer. C-region may be created in MC in any desired position. In light of the instructions presented in the examples below, a person skilled in the art will be able to choose a website to create a C-region on the basis of the desired properties of the resulting shortened traps, such as molecular weight, Monomeric or dimeric structures, etc.

In a specific embodiment, the C-region is the site of cleavage by thrombin (LVPRGS) (SEQ ID NO: 6), built-in domain FcΔC1 after the N-terminal sequence CPPC (SEQ ID NO: 1). In this embodiment, the design of the full-sized original trap VEGF is expressed in the cell in the form of Fc-tagged protein, thus enabling the capture and purification, for example, using a column with protein A. After the formation of the dimer and covalent binding of one or both cysteine residues CPPC sequence (SEQ ID NO: 1) dimer is exposed to thrombin in the conditions under which hatshepsuts one or both domains FcΔC1, so shortened form dimeric minilovely having a molecular weight of about 50-90 kDa and has affinity to VEGF, comparable to the affinity of the original traps. The specialist in this area can regulate the conditions of cleavage, so that it is preferable to form the partial product of the cleavage or the full product is th splitting, this version of the terms of splitting is chosen based on the need for a specific product with specific properties, such as molecular weight.

In a specific embodiment, the C-region is the site of cleavage by thrombin (LVPRGS) (SEQ ID NO: 6), built-in domain FcΔC1 on the N-end with respect to the CPPC sequence (SEQ ID NO: 1). After the formation of the dimer and covalent binding of one or both cysteine residues CPPC sequence (SEQ ID NO: 1) dimer is exposed to thrombin under conditions which arise one or both domains FcΔC1 and formed shorter Monomeric minilovely. Monomeric shortened minilogue formed thus contains the receptor component, a small fragment of the Fc and has a size of approximately 25 kDa and exhibits reduced affinity to VEGF compared with the truncated dimeric trap and a full-sized original trap. It is shown that such Monomeric trap received in the form of recombinant protein has KDapproximately 1 nm.

Creating minilogue VEGF

In one embodiment, the invention relates to minilogues VEGF, having one or more domains of the receptor components (R1R2)Xand/or (R1R3)Ywhere X≥1, Y≥1 and R1, R2 and R3 have the meanings defined above, and optionally a partner in the merger, which preferably is Domino the MC, which is an amino acid sequence ranging in length from 1 to about 200 amino acids, containing at least one cysteine residue, where at least one cysteine residue capable of forming a disulfide bond with a cysteine residue present in another MS fused polypeptide (cMC). cMC can be at the N-end or C-end of the fused polypeptide or between two domains of the receptor components. In one specific embodiment, the cysteine added to the C-end component of the VEGF receptor, for example R1R2Cthat allows fused to the polypeptide to form a covalent dimers through the formation of covalent disulfide bonds between the cysteine residue at the C-end of a fused polypeptide and a cysteine residue at the C-end of another fused polypeptide. In this illustrative example, minilogue is a dimer fused polypeptide shown in SEQ ID NO: 2, where each fused polypeptide (R1R2-cMC or R1R2C) has a molecular mass of approximately 23,0 kDa.

In another embodiment, the cMC is a sequence of 4 amino acids (XXXX) (SEQ ID NO: 11), where X means any amino acid and the sequence contains at least one cysteine residue. In a specific embodiment, the cMC is added to the C-end domain of the receptor component. In a more specific embodiment, the sequence of 4 amino acids, not only is em a ACGC (SEQ ID NO: 4) and the cMC form two disulfide bonds with cysteine residues, present in the second fused to the polypeptide. As shown below (table 2), both given as an example of minilovely show affinity to VEGF, comparable to the affinity of the original trap.

Therapeutic use

Minilovely VEGF according to the invention therapeutically applicable for the treatment of any disease or condition which is improved, reduced, inhibited or prevented by removal, inhibition or reduction of VEGF. A non-limiting list of specific conditions, improve the inhibition or reduction of VEGF, includes a clinical condition characterized by excessive proliferation of endothelial cells of blood vessels, vascular permeability, edema or inflammation, such as brain edema associated with injury, stroke or tumor; edema associated with inflammatory diseases such as psoriasis or arthritis, including rheumatoid arthritis; asthma; generalized edema associated with burns; ascites and pleural effusion associated with tumors, inflammation or trauma; chronic inflammation of the Airways; the syndrome capillary leak; sepsis; kidney disease associated with increased leakage of protein; and eye diseases such as age related macular degeneration and diabetic d is ineptia.

Compositions according to the invention therapeutically applicable for the treatment of a wide variety of diseases associated with elevated levels of VEGF. For example, inflammation with abnormal increase in Th2 and remodeling of the Airways characteristic of the pathogenesis of asthma (see, e.g., Elias et al. (1999) J. Clin. Invest. 104: 1001-6). Elevated levels of VEGF detected in tissues and biological samples of patients with asthma, which directly correlate with disease activity (Lee et al. (2001) J. Allergy Clin. Immunol. 107: 1106-1108) and inversely correlated with the diameter of the respiratory tract and sensitivity of the respiratory tract. In addition, it was assumed that VEGF contributes to the swelling of the tissue in asthma.

Another disease associated with elevated levels of VEGF, is adenocarcinoma of the ducts of the pancreas (PDAC). Specified malignant tumor often has a hearth enhanced proliferation of endothelial cells and often sverkhekspressiya VEGF (Ferrara (1999) J. Mol. Med. 77: 527-543). PDAC is the cause of more than 20% of deaths due to malignant tumors of the gastrointestinal tract, which makes this disease is the fourth most common cause associated with malignant tumor mortality in the United States and other industrialized countries. The experimental data indicate the important role of VEGF in the development of malignant tumors of page the bridges : cancer thus, the VEGF inhibitor is promising as a therapeutic agent to reduce the growth of the tumor within the pancreas and regional and distal metastasis.

Smaller deglycosylation minilogue, expressed in E. coli (example 4), the glycosylated minilogue expressed in CHO cells (example 5), or based on the Monomeric receptor trap (example 6) is optimised for local/intravitreal delivery, i.e. a shorter half-life in serum for faster clearance and minimizing undesirable systemic effects. In addition, due to its smaller minilogue has the ability to penetrate through the internal limiting membrane (ILM) in the eye and diffuse through the vitreous body to the retina/pigment epithelial layer of the retina (RPE), which will help to cure the disease of the retina. In addition, minicourse can be used for local injection in the treatment of such eye diseases as neovascularization of the choroid of the eye, diabetic macular edema, proliferative diabetic retinopathy, neovascularization of the cornea/graft rejection. In addition, minicourse can be applied in any situation where you need a temporary (short-term) blocking VEF, for example, to avoid chronic exposure to VEGF blockade, for example, in the treatment of psoriasis.

A serious problem, leading to adverse outcome after surgery for glaucoma is previously inflammation and angiogenesis, as well as too rapid wound healing. Accordingly, VEGF trap according to the invention can be effectively used as an adjuvant during operation of glaucoma to prevent early heme - and lymphangiogenesis and recruitment of macrophages to the filtration cushion after surgery for glaucoma and improve the outcome of the operation.

Combination therapy

In many embodiments, the VEGF trap may be introduced in combination with one or more additional compounds or therapies, including a second molecule of VEGF trap, a chemotherapeutic agent, surgery, catheter devices and irradiation. Combination therapy involves the introduction of a single pharmaceutical dosage of the drug, which contains a VEGF trap, and one or more additional funds; and the introduction of VEGF trap and one or more tools in their own separate pharmaceutical dosage preparations. For example, VEGF trap, and a cytotoxic agent, chemotherapeutic agent or inhibiting the growth medium is in can be administered to the patient together in a single dosage drug such as a combined preparation or each tool can be introduced in the form of a single dosage of the drug. In that case, when used individually dosed medications, VEGF-specific fused polypeptide according to the invention and one or more additional means can be introduced simultaneously or in separate periods of time-shift, for example, sequentially.

The term "cytotoxic agent" used in this sense refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. It is implied that the term shall include radioactive isotopes (e.g., I131I125, Y90and Re186), chemotherapeutic agents and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal or their fragments.

"Chemotherapeutic agent" is a chemical compound that is applicable for the treatment of malignant tumors. Examples of chemotherapeutic tools include alkylating agents, such as thiotepa and cyclophosphamide (Cytoxan®); alkyl sulphonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodepa, carboquone, matureup and uredepa; ethylenimines and methylmelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylene is isformed and triethylenemelamine; nitrogen mustards such as chlorambucil, chlornaphazine, chlorpropamide, estramustine, ifosfamide, mechlorethamine, hydrochloride oxide mechlorethamine, melphalan, novemberin, finestein, prednimustine, trofosfamide, oralloy mustard; nitrosoanatabine, such as carmustine, chlorozotocin, fotemustine, lomustin, nimustine, ranimustine; antibiotics such as aclacinomycin, actinomycin, autralian, azaserine, bleomycin, actinomycin, calicheamicin, carubicin, karminomitsin, casinopolis, chromomycin, dactinomycin, daunorubicin, demoralizing, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, zorubicin, idarubitsin, marsellaise, mitomycin, mycofenolate acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, colomycin, radiobeacon, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); analogs of folic acid, such as deeperin, methotrexate, peripherin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, timipre, tioguanin; pyrimidine analogues such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, propionate dromostanolone, epitiostanol, mepitiostane, testolactone; antiadrenergic, the e as aminoglutethimide, mitotane, trilostane; DOPOLNITEL folic acid, such as folinovaya acid; Eagleton; glycoside aldophosphamide; aminolevulinic acid; amsacrine; astroball; bisantrene; edatrexate; defaming; demecolcine; diazinon; alternity; the acetate slipline; etoposide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitrogen; pentostatin; penomet; pirarubicin; podofillina acid; 2-acylhydrazides; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trihlortrietilamin; urethane; vindesine; the dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; Galitsin; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (Taxol®, Bristol-Myers Squibb Oncology, Princeton, N. J.) and docetaxel (Taxotere®; Aventis, Antony, France); chlorambucil; gemcitabine; 6-tioguanin; mercaptopurine; methotrexate; platinum analogues, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; Novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; deformational (DMFO); retinoic acid; espiramicina; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above funds. Also in this definition include the ENES antihormone means, that act, regulating or inhibiting hormone action on tumors such as antiestrogens, such as tamoxifen, raloxifene, inhibiting aromatase 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bikalutamid, leuprolide, goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above funds.

"Growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of cells, especially malignant tumors, either in vitro or in vivo. Examples of inhibiting the growth of funds include funds that are blocking the passage of the cell cycle (in a different phase other than S phase), such as tools, which induce a delay in G1 and delay in M-phase. Classic blockers phase M include the Vinca alkaloids (vincristine and vinblastine), Taxol® and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. These funds, which inhibit G1, also apply to the delay in S-phase, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.

The routes of administration

The invention relative is seeking to methods for treating, includes introduction to the subject an effective amount of VEGF trap according to the invention. In a preferred aspect of the trap is substantially purified (e.g., essentially does not contain substances that limit its effect or produce undesired side effects). The subject is preferably a mammal and most preferably a human.

There are various delivery systems that can be used to administer the funds according to the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of Express connection-mediated receptor endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction can be enteral or parenteral and include, without limitation intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intraocular and oral ways. The compounds may be introduced in any convenient way, for example by infusion or bolus injection, by absorption through epithelial or skin-mucous lining (e.g., mucosa of the oral cavity, the mucous membrane of the rectum and intestines and so on), and can be introduction the s together with other biologically active agents. The administration can be systemic or local. The introduction may be urgent or chronic (e.g., daily, weekly, monthly etc) or in combination with other tools. Can also be applied pulmonary introduction, for example, using the inhaler or nebulizer and the drug agent to the spray.

In another embodiment, the active agent can be delivered in a vesicle, in particular liposomes, in a controlled release system or pump. In another embodiment, when the active agent according to the invention is a nucleic acid encoding a protein, nucleic acid can be introduced in vivo to support the expression of the encoded it protein, by constructing it as part of the corresponding vector for the expression of nucleic acid and introducing it in such a way that it becomes intracellular, for example, using a retroviral vector (see, for example, U.S. patent No. 4980286), direct injection or by use of microparticle bombardment, or capping lipids, or through receptors on the cell surface or transfairusa agents, or by entering it in connection with the peptide, such homeobox, which is known to enter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88: 1864-1868), etc. Alternative nucleic acid may be introduced within the b cells and incorporated into the DNA of the host cell for expression by homologous recombination.

In a specific embodiment, it may be desirable introduction of pharmaceutical compositions according to the invention locally to the required treatment area; specified can be performed, for example, without limitation, by local infusion during surgery, by topical application, for example, by injection, catheter, or by means of an implant, the implant is porous, non-porous, or gelatinous material, including membranes, such as silicone membranes, fibers or substitutes skin.

Composition applicable in the practical implementation of the methods according to the invention may be a liquid containing the agent according to the invention in solution, in suspension or in both. The term "solution/suspension" refers to a liquid composition in which the first portion of the active agent present in the solution, and the second portion of the active agent is present in the form of particles in suspension in a liquid matrix. The liquid composition also includes a gel. The liquid composition may be water or in the form of ointments. In addition, the composition may take the form of solid particles, which can be introduced into the eye, for example between the eye and eyelid or in the conjunctival SAC, where the released VEGF trap. The release of such particles usually occurs to the cornea or through Lezno the liquid, either directly to the cornea, with which the solid particle is usually in direct contact. Solid particles that are suitable for implantation in the eye, usually mainly consist of biorazlagaemykh or nabirasulillah polymers. Aqueous solution and/or suspension may be in the form of eye drops. The required dosage of active agent can be measured by introducing a known quantity of drops in the eye. For example, in the case of droplet volume, 25 ál introduction 1-6 drops will deliver 25-150 μl of the composition.

Aqueous suspension or solution/suspension, applicable in the practical implementation of the methods according to the invention may contain one or more polymers as suspendida agents. Applicable polymers include water-soluble polymers such as cellulose polymers, and water-insoluble polymers, such as cross-crosslinked carboxyl-containing polymers. Aqueous suspension or solution/suspension according to the present invention are preferably viscous or mucoadhesive or more preferably both viscous and mucoadhesive.

In another embodiment, the composition is applicable in the practical implementation of the methods according to the invention, is gelatinizing in situ water composition. This composition contains gelatinizing agent in a concentration effective the La promotion of gelation upon contact with the eye or with the lacrimal fluid. Suitable gelatinizing agents include, but are not limited, thermotherapies polymers. The term "gelatinizing in situ" is used in this sense includes not only liquids of low viscosity, which form gels upon contact with the eye or lacrimal fluid, but also includes a more viscous fluid, such as politique and thixotropic gels that have significantly increased the viscosity or density of the gel when injected into the eye.

The methods of diagnosis and screening

The VEGF trap according to the invention can be used diagnostically and/or screening methods. For example, the trap may be used to monitor the levels of VEGF in clinical research to assess the effectiveness of treatment. In another embodiment, the methods and compositions according to the present invention is used for selection of individuals for introduction to clinical research, to identify people with, for example, too high or too low levels of VEGF. Traps can be used in methods known in the field related to the localization and activity of VEGF, for example, when visualizing, measuring its levels in appropriate physiological samples, in diagnostic methods, etc.

Trap according to the invention can be used in the screenshot is novom analysis in vivo and in vitro, to estimate the number present unbound VEGF, for example, in a screening method for identifying test agents that can decrease the expression of VEGF. More widely trap according to the invention can be used in any analysis or the way in which you want quantitative measurement and/or allocation of VEGF.

The pharmaceutical composition

The present invention also relates to pharmaceutical compositions containing minicourse VEGF according to the invention. Such compositions contain a therapeutically effective amount of one or more minilogue and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a regulatory Agency of the Federal government or the state government or listed in the Pharmacopoeia list US or other generally recognized Pharmacopoeia for use in animals and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient or filler, which is administered therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils from petroleum, oils of animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Lov the leading pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition optionally may also contain small amounts of moisturizers, or emulsifying agents, or agents for tebufelone pH. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, drugs extended release and the like. Examples of suitable pharmaceutical carriers are described in "Remington''s Pharmaceutical Sciences, E. W. Martin.

Minilogue VEGF according to the invention can be prepared in the form of a neutral or salt form. Pharmaceutically acceptable salts include salts formed with free amino groups, such as salts derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acid, etc. and salts formed with free carboxyl groups, such as the salts obtained with the hydroxides of sodium, potassium, ammonium, calcium, iron, Isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

In addition, water composition, applicable for the practical implementation of the methods according to the invention, are compatible with the eye of pH and osmotic pressure. One or more acceptable to the eyes of the AG is new for adjusting pH and/or buffering agents may be introduced into the composition according to the invention, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate and sodium lactate; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers enter in the quantity required to maintain the pH of the composition is acceptable to the eye. One or more acceptable to the eye salts can be included in the composition in an amount sufficient to bring the osmotic pressure of the composition to an acceptable eye limits. Such salts include salts having cations of sodium, potassium or ammonium and the anion chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfate.

The number of traps that will be effective if it is planned therapeutic applications, you can define a standard clinical methods based on this description. In addition, optional can be used in vitro, help to identify the optimal dose limits. In General, a suitable dose limits for intravenous typically be approximately 50-5000 mg of active compound per kilogram of body weight. The appropriate dose limits for intranasal usually approximately from 0.01 PG/the g body weight to 1 mg/kg of body weight. Effective doses may be extrapolated from dose dependence curves obtained in test systems in vitro or in animal models.

For systemic injections therapeutically effective dose can be first identified in vitro. For example, a dose can be formulated in animal models to achieve the limits of circulating concentrations that include the IC50determined in cell culture. Such information can be used for more accurate determination of the dose that is acceptable for a person. The initial dose can be estimated on the basis of the data in vivo, for example, in animal models, using methods that are well known in the field. The person skilled in the art can easily optimize administration to humans based on the data obtained on animals.

The dose amount and interval may be adjusted individually to provide levels of compounds in plasma, which are sufficient to maintain therapeutic effect. Local injection or selective absorption of the effective local concentration of the compounds may not be related to plasma concentration. The person skilled in the art will be able to optimize therapeutically effective local dosages without undue experimentation.

The number of input link is, of course, will vary depending on the subject being treated, the weight of the subject, severity of the disease, the route of administration and the decision of the attending physician. therapy can be repeated periodically until the symptoms are identified, or even when they are not registered. Therapy may be used alone or in combination with other medicines.

Transfection of cells and gene therapy

The present invention relates to the use of nucleic acids encoding the fused polypeptide according to the invention for transfection of cells in vitro and in vivo. These nucleic acids can be incorporated into any of a number of well known vectors for the transfection of target cells and organisms. Nucleic acid transferout into cells ex vivo and in vivo through interaction of the vector and the target cell. The composition is administered (for example, by injection into a muscle) to a subject in amounts sufficient to cause a therapeutic response. Quantity adequate for implementation, is defined as "therapeutically effective dose or amount".

In another aspect, the invention relates to a method of reducing levels of VEGF in humans or other animal, comprising the transfection of cells a nucleic acid that encodes a fused polypeptide according to the invention, in this case, nucleic acid content is t inducible promoter, functionally linked to a nucleic acid that encodes a fused polypeptide or minicourse. Methods of gene therapy for the treatment or prevention of human disease, see, for example, in Van Brunt (1998) Biotechnology 6: 1149-1154.

Sets

The invention also relates to articles of manufacture containing packaging material and a pharmaceutical agent, located in the packing material, while the pharmaceutical preparation contains at least one VEGF trap, consisting of two or more fused polypeptides according to the invention, the packaging material contains a label or an insert in the packaging, which indicated that the VEGF-specific fused polypeptide can be used to treat mediated VEGF disease or condition.

Transgenic animals

The invention includes a transgenic animal other than human, expressing the trap according to the invention. The transgenic animal can be obtained by introducing the nucleic acid into the male pronucleus of a fertilized egg, such as microinjection, retroviral infection, and providing opportunities for the development of the ovum from pseudoelement foster females. Any regulatory or other sequences used in expressing vectors can form part of a transgenic sequence. Timespecific the Naya regulatory sequence(s) can be operatively linked to a transgene, to control transgene expression in specific cells. Transgenic animal other than human, expressing the fused polypeptide or minicourse according to the invention, applicable for a variety of applications, including use as a means of obtaining such a fused polypeptide. In addition, the transgene can be placed under the control of the inducible promoter so that expression of the fused polypeptide or minilovely can be adjusted, for example, the introduction of small molecules.

Specific options

In the following experiments we created a smaller trap VEGF and investigated their ability to bind VEGF. Such minilovely preferably used in specific applications. For example, some conditions or illnesses can preferably be treated with a local injection of VEGF trap in a specific organ, tissue or cell, rather than systemic administration. In one illustrative example of minilogue according to the invention was created by a smaller trap VEGF direct cleavage dimenisonal VEGF trap that has an area splitting (C-region), created in the domain Fc (example 2). Shortened trap showed comparable affinity to VEGF and the half-life as a full-sized original trap. In examples 3-5 describes the design of the fused polypeptide, are the component of the VEGF receptor and multimediali component, consisting of one or two cysteine residues. Measuring the affinity showed that non-fused polypeptide expressed in E. coli, or glycosylated polypeptide expressed in CHO cells, were comparable to the affinity of binding to VEGF, as a full-sized original trap. Example 6 also illustrates Monomeric VEGF trap, consisting of (R1R2)2which is able to bind and inhibit VEGF. In example 7 describes the design minilovely VEGF (SEQ ID NO: 26), having a high binding affinity of VEGF compared to full-sized trap (SEQ ID NO: 10).

Other features of the invention will be apparent in the further description of exemplary options that are given to illustrate the invention and not intended to be limiting thereof.

EXAMPLES

The following example is provided in order to provide specialists in this field a complete disclosure and description of how to obtain and apply the methods and compositions according to the invention, and is not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure the accuracy in terms of numerical values (e.g., amounts, temperature, etc.), but should take into account some experimental errors and TCI is onine. Unless otherwise stated, parts are parts by weight, molecular weight is an average molecular weight, temperature is given in degrees Celsius and the pressure is atmospheric or close to atmospheric.

Example 1. The design of Flt1D2.Flk1D3.FcΔC1(a)

The original design of VEGF trap Flt1D2.Flk1D3.FcΔC1(a) (SEQ ID NO: 7-8), VEGFR1R2.FcΔC1(a) (SEQ ID NO: 9-10) and Flt1D2.VEGFR3D3.FcΔC1(a) (SEQ ID NO: 12-13) is described in detail in PCT publication WO/0075319 specifically included in this description by reference in full. Also in WO/0075319 describes how to design and expression constructs of the nucleic acid encoding the VEGF trap, methods of recording and measuring binding of the trap VEGF with VEGF, how to determine the stoichiometry of binding of VEGF using BIAcore analysis and pharmacokinetic analyses.

Example 2. Split by thrombin a dimer minilogue VEGF

Design VEGFR1R2.FcΔC1(a) (SEQ ID NO: 9-10) modified by insertion site of cleavage by thrombin after CPPC (SEQ ID NO: 1) of the Fc domain. Purified VEGF trap (5 µg) were incubated with thrombin (Novagen) in 20 mm Tris-HCl, pH of 8.4, 50 mm NaCl, 2.5 mm CaCl2during 16 h at 37°C. Controls included protein to control splitting (CCP) and protein source of VEGF trap, incubated without thrombin. SDS-page analysis (Tris-glycine 4-20% gel; 5 µg of protein per lane) confirmed the correct splitting (results not p is cauldrons).

Determination of affinity. The Kd of binding of each VEGF trap with hVEGF165 was determined as described in WO/0075319, for the source of VEGF trap, unsplit trap VEGF that contains the site of cleavage by thrombin ("unsplit trap VEGF"), split minilovely VEGF and recombinant Monomeric R1R2-myc myc his. More specifically, the ability of traps to block VEGF165-dependent phosphorylation of the receptor was determined using primary endothelial cells (HUVEC). VEGF165incubated in the presence of various concentrations of tested traps and the mixture was added to HUVEC in order to stimulate the phosphorylation of VEGFR2 tyrosine. When substochiometric concentrations of VEGF trap unbound VEGF induced phosphorylation of the receptor. However, when the molar ratio 1:1 or more traps to VEGF ligand observed complete blockage of signal transmission receptor, having found that one molecule of dimer compounds are capable of blocking one molecule VEGF165man. Thus, the high affinity binding of VEGF trap in respect of VEGF leads to the formation of the complex, which prevents the interaction of VEGF with receptors on the cell surface. Equivalent results were obtained in the case of identical experiments on the inhibition of phosphorylation for the source of VEGF trap, unsplit trap VEGF and rassal is authorized to minilovely VEGF. The results are shown in table 1.

Table 1
TrapKinetic dissociation rate (1/s)T1/2(h)
Source VEGF trapthe 5.51×10-5±0,94%3,5
Unsplit VEGF trapis 4.93×10-5±0,70%a 3.9
Split minilogue VEGF5,46×10-5±0,62%3,53
The monomer R1R2-myc myc his6,74×10-3±0,38%0,028

Example 3. Construction of plasmids encoding minilovely VEGF

Minilovely VEGF was designed from the precursor source of VEGF trap, VEGFR1R2.FcΔC1(a) (SEQ ID NO: 9-10), in which the three amino acids glycine-alanine-Proline was used as linker between Flk1D3 and FcΔC1(a). This plasmid pTE115 used to construct minilogue VEGF as linker DNA sequence contains a sequence of recognition by restriction enzyme Srf I, which facilitated the construction of VEGF trap. All other otnoshenijami VEGF, encoded pTE115, identical to the VEGF trap VEGFR1R2.FcΔC1(a) (SEQ ID NO: 9-10), is described in detail in PCT publication WO/0075319.

Designed two minilovely VEGF domains for multimerization consisting of either a single cysteine residue (R1R2C) (SEQ ID NO: 2)or amino acids ACGC (SEQ ID NO: 4) (R1R2ACGC) (SEQ ID NO: 5), added to the C-Termini of the receptor components Flt1D2.Flk1D3. Both of the resulting construction can form homodimers molecules, stable one (R1R2C) or two (R1R2ACGC) intermolecular disulfide bonds.

Plasmid pTE517 was obtained by deletion of the fragment length of 690 BP-induced DNA cleavage pTE115 with Srf I and Not I, and the embedding of a fragment of synthetic DNA formed by annealing oligonucleotides R1R2NC (SEQ ID NO: 14) and R1R2CC (SEQ ID NO: 15). The resulting plasmid encodes R1R2C, which consists of domains Flt1D2.Flk1D3, followed by a cysteine residue (SEQ ID NO: 23). In this way received plasmid pTE518 removing fragment length 690 BP-induced DNA cleavage pTE115 with Srf I and NotI, followed by legirovaniem fragment of synthetic DNA formed by annealing oligonucleotides R1R2NACGC (SEQ ID NO: 16) and R1R2CACGC (SEQ ID NO: 17). The resulting plasmid encodes R1R2ACGC, which consists of domains Flt1D2.Flk1D3, followed by amino acids ACGC (SEQ ID NO: 25).

Also designed plasmids to control the effect of the expression of these minilogue E. coli. The primers used were R1R2N-Nco1 (SEQ ID NO: 18) and R1R2CNot1 (SEQ ID NO: 19), to amplify a DNA fragment pTE115, which encodes amino acids G30 on K231 relative to the source of VEGF trap (SEQ ID NO: 10). Amplification of this sequence led to the merging of the initial methionine codon at the 5'-end and merging of cysteine codon, followed by a stop codon on the 3'-end (SEQ ID NO: 2). Then, the obtained DNA fragment was cloned in the sites Nco I and Not I expressing plasmids E. coli pRG663 getting pRG1102, so that the expression R1R2Cwas dependent on transcription from the promoter Φ1.1 phage T7. Induction of gene expression with pRG1102 leads to the accumulation R1R2cys in the cytoplasm of the strain of the host E. coli RFJ238. Similarly primers R1R2N-Nco1 (SEQ ID NO: 18) and R1R2ACGC-Not1 (SEQ ID NO: 20) were used to amplify a DNA fragment from pTE115, which encodes amino acids G30 on K231 (SEQ ID NO: 10), resulting in the merging of the initial methionine codon at the 5'-end and merge codons ACGC (SEQ ID NO: 4), followed by a stop codon on the 3'-end (SEQ ID NO: 5). Then the resulting fragment was cloned in the sites Nco I and Not I expressing plasmids E. coli pRG663 getting pRG1103, so that the expression R1R2ACGCdepended on transcription from the promoter Φ1.1. phage T7. Induction of gene expression as pRG1102 and pRG1103 led to the accumulation R1R2Cor R1R2ACGCaccordingly, in the cytoplasm of the strain of the host E. coli RFJ238.

Example 4. Clear the as and characterization of minilogue VEGF from E. coli

And R1R2Cand R1R2ACGCexpressed in the form of cytoplasmic proteins in E. coli and purified the same way. Induction of the promoter Φ1.1 phage T7 or pRG1102 or pRG1103 in E. coli K12 strain RFJ238 led to accumulation of the protein in the cytoplasm. After induction the cells were collected by centrifugation, resuspendable in 50 mm Tris-HCl, pH 7.5, 20 mm EDTA and literally passing through the homogenizer for cells Niro-Soavi. Taurus inclusion were collected from lysed cells by centrifugation, once washed with distilled H2O, and then was dissolved in 8M of guanidine-HCl, 50 mm Tris-HCl, pH 8.5, 100 mm sodium sulfite, 10 mm tetrathionate sodium and incubated at room temperature for 16 hours. Clarified adosados was fractionally on S300 column, equilibrated 6M guanidinium-HCl, 50 mm Tris-HCl, pH 7.5. The fractions containing R1R2Cthat United and were dialyzed against 6M urea, 50 mm Tris-HCl, pH 7.5. Cialisovernight protein was diluted to 2M urea, 50 mm Tris-HCl, pH 8.5, 2 mm cysteine, and then slowly stirred for 7 days at 4°C. Subjected to refolding protein were dialyzed against 50 mm Tris-HCl, pH 7.5, and then were applied to a column of SP-separate, equilibrated with 50 mm Tris-HCl, pH 7.5, and was suirable gradient of NaCl from 0 to 1 M in 50 mm Tris-HCl, pH 7.5. The fractions containing R1R2Cwere combined, concentrated and applied on a column of Superdex 200, equilibrated with 50 mm Tris-Cl, pH 7.5, 150 mm NaCl. The fractions containing the dimer of minilovely, collected and combined. Using SDS-page identified the molecular weight of purified minilovely of approximately 46 kDa.

Spent BIAcore analysis (as described in WO/0075319)to determine the affinity trap against VEGF, and the results showed that minilovely R1R2Cand R1R2ACGChad the affinity to VEGF, comparable to the affinity of full-length VEGF trap (table 2).

Table 2
TrapKinetic dissociation rate (1/s)T1/2(h)
The VEGF trapto 4.23×10-54,53
R1R2C3,39×10-5of 5.68
R1R2ACGCto 3.41×10-5the 5.65

Example 5. Expression of minilogue VEGF in CHO K1

Expression of minilogue VEGF encoded pTE517 and pTE518 depends on transcription from the CMV-MIE person and leads to the secretion of minilogue into the culture medium by expression in CHO cells. When the expression in the form of secreted proteins in CHO K1 both is innovaci found in air-conditioned environments and determination of their molecular weight in SDS-page showed as expected that proteins were glycosylated. The analysis in SDS-page also showed that minilovely capable of forming homodimers molecules, stabilized by intermolecular disulfide bond(s) between the C-terminal cysteine. In particular, minilogue R1R2Ceffectively formed covalent dimers when the expression in the form of a secreted protein in CHO cells.

Example 6. Construction and expression of single-stranded minilovely VEGF

Also designed minicourse VEGF that does not require the domain to multimerization (SEQ ID NO: 24). This minicourse designed direct merge one domain Flt1D2.Flk1D3 (R1R2) (amino acids 30-231 SEQ ID NO: 24) with the second domain Flt1D2.Flk1D3 (R1R2) (amino acids 234-435 SEQ ID NO: 24) with the linker Gly-Pro between receptor domains in tandem (amino acids 232-233 SEQ ID NO: 24).

To construct the gene encoding tandem domains Flt1D2.Flk1D3, synthesized DNA fragment (Blue Heron Biotechnology), which encodes a single domain Flt1D2.Flk1D3, which minimized the homology of the DNA with a DNA that encodes a domain Flt1D2.Flk1D3 found in pTE115. The obtained synthesized DNA fragment cloned in the form of a fragment of Srf I-Not I sites Srf I-Not I pTE115 to get pTE570, which expresses minicourse VEGF R1R2-R1R2 with the CMV-MIE. When this plasmid is respiciunt cells CHO K1, minilogue VEGF R1R2-R1R2 is accumulated in the culture medium.

Example 7. Construction and expression of minilovely VEGF

Minicourse VEGF designed, as described above, the direct fusion of a single domain Flt1D2.Flk1D3 (R1R2) (amino acids 30-231 SEQ ID NO: 26) with C-terminal sequence of nine amino acids, ending CPPC. When this plasmid transferout cells CHO K1, minilogue with VEGF SEQ ID NO: 26 is secreted into the culture medium. Subsequent purification by electrophoresis in non SDS-page, and a simple analysis of light scattering revealed molecule traps with a molecular weight of about 64 kDa. The specified molecular weight of the evidence that was formed covalent dimer between two fused polypeptides with SEQ ID NO: 26. Similar experiments were performed with plasmids encoding the fused polypeptides with SEQ ID NO: 27 and 28, and similarly showed that these molecules were formed homodimers traps. Determine the affinity for binding of VEGF-165 person with traps EGF, consisting of dimers with SEQ ID NO: 10 and SEQ ID NO: 26, shown in table 3.

Table 3
The VEGF trapka (1/Ms)kd (1/s)KD (M)
by 2.73×10+7to 1.79×10-56,55×10-13
SEQ ID NO: 262,00×10+76,56×10-6of 3.28×10-13
SEQ ID NO: 262,61×10+75,77×10-6of 2.21×10-13

1. An isolated nucleic acid molecule encoding a fused polypeptide capable of binding growth factor endothelial cells of blood vessels (VEGF), where the polypeptide consists of components (R1R2)Xand, optionally, multimeasure component (M is), which is an amino acid sequence ranging in length from 1 to 200 amino acids that has at least one cysteine residue, where X≥1, R1 means an immunoglobulin-like (Ig) domain 2 of the VEGF receptor Flt-1, representing amino acids 27-126 SEQ ID NO:8, 27-129 SEQ ID NO:10; and R2 means Ig domain 3 of the VEGF receptor Flk-1, representing amino acids 127-228 SEQ ID NO:8 or 130-231 SEQ ID NO:10 and where the fused polypeptide does not contain multimeasure component when X=2, and multimediali component is an amino acid sequence ranging in length from 1 to 15 amino acids in the case when X=1.

2. An isolated nucleic acid molecule according to claim 1, where multimediali component (MS) selected from the group consisting of HSHS, ACGC and SRRS.

3. An isolated nucleic acid molecule according to claim 1, in which X is 1 and multimerization component is the amino acid sequence of length 1-15 amino acids with 1-2 cysteine residues.

4. An isolated nucleic acid molecule according to claim 1, in which X is 2 and which does not contain multimeasure component.

5. Fused polypeptide capable of binding growth factor endothelial cells of blood vessels (VEGF), having the amino acid sequence defined by a sequence of nucleic acid according to claims 1-4.

6. Fused polypeptide according to claim 5, having the amino acid is of the selected SEQ ID NO:26, 27 or 28.

7. Replicable expression vector capable of expression in a transformed cell host containing a nucleic acid molecule according to claims 1-4.

8. The method of obtaining the fused polypeptide capable of binding growth factor endothelial cells of blood vessels (VEGF), which includes stages of introduction into a suitable expression system expressing the vector according to claim 7, implementation of the expression of the fused polypeptide VEGF and extracting the obtained fused polypeptide.

9. Trap growth factor endothelial cells of blood vessels (VEGF), containing multimer of two or more fused polypeptide according to claim 5.

10. The VEGF trap according to claim 9, which is a dimer.

11. Dimeric VEGF trap containing two fused polypeptide having the amino acid sequence of SEQ ID NO:26, 27 or 28.

12. Pharmaceutical composition for treating a VEGF-mediated disease or condition, containing an effective amount of VEGF trap according to claim 9 or 10 and a pharmaceutically acceptable carrier.

13. A method of treating a VEGF-mediated disease or condition involving the introduction of a pharmaceutical composition according to item 12 to the needy in this subject.

14. The method according to item 13, in which VEGF-mediated disease or condition is a disease or condition of the eye.

15. The method according to 14, in which the disease or condition of the eye is is provided with age-related macular degeneration.

16. A kit for treating a VEGF-mediated disease or condition, comprising:
(a) packaging material; and
(b) a pharmaceutical agent, in packaging material; however, the pharmaceutical preparation contains at least one VEGF trap according to any one of p-11, and where the packaging material contains a label or an insert in the packaging in which it is written that the VEGF-specific fused polypeptide can be used for the treatment of VEGF-mediated disease or condition.



 

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EFFECT: invention simplifies and increases efficiency of the technology of purifying target proteins, and also allows for obtaining biologically active hybrid proteins, suitable for making medicinal agents.

3 cl, 1 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: vitamin K dependent protein is made by separating a cultivated eukaryotic cell that contains an expressing vector that contains a nucleic acid molecule coding vitamin K dependent protein and associated sequences regulating expression. The associated sequences contain the first promoter and the nucleic acid molecule coding gamma-glutamylcarboxylase, and the second promoter. The first promoter represents a pre-early promoter of human cytomegalovirus (hCMV), and the second promoter is a pre-early promoter SV40. Herewith the expressing relation of vitamin K dependent protein and gamma-glutamylcarboxylase is 10:1 to 250:1.

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29 cl, 5 dwg, 6 tbl, 7 ex

FIELD: medicine; microbiology.

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3 cl, 3 dwg, 3 ex

FIELD: genetic engineering.

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9 cl, 6 ex

FIELD: medicine.

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FIELD: biology.

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25 cl, 8 dwg, 7 tbl, 6 ex

FIELD: medicine.

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EFFECT: novel compounds possess useful biological properties.

14 cl, 1 dwg, 12 tbl, 19 ex

FIELD: medicine.

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6 cl, 1 dwg, 4 tbl, 5 ex

FIELD: pharmacology; biotechnology.

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6 cl, 11 dwg, 9 ex

FIELD: biotechnology.

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4 cl, 8 dwg, 1 tbl, 2 ex

FIELD: medicine.

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37 cl, 6 ex, 1 dwg

FIELD: chemistry, organic, viruses.

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FIELD: biotechnology, in particular gene engineering.

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2 cl, 3 dwg, 6 ex

FIELD: gene engineering.

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EFFECT: higher efficiency.

FIELD: biotechnology, virology, medicine.

SUBSTANCE: invention relates to attenuated virus derived from modified Ankara vaccina virus. Said virus are not able for reproduction by replication in human cell lines. Also disclosed are application of virus or recombinant variants thereof as drug or vaccine, as well as method for inducing of immune response in patients with defected immunity, in patients having immunity to vaccine virus, or in patient during antiviral therapy.

EFFECT: variant of Ankara vaccina virus effective in medicine and veterinary.

86 cl, 15 dwg, 1 tbl, 2 ex

FIELD: biotechnology, medicine, in particular viral disease treatment.

SUBSTANCE: invention relates to recessive dividing retroviral vector useful in inhibition of wild-type retrovirus replication. Vector contains retroviral long terminal repeat sequences; retroviral packing signal; nucleotide sequence encoding (expressing) genetic antiviral agent; and optionally the second nucleotide sequence. Disclosed are method for production of said vector and reproduction thereof. Further isolated and purified nucleic acid (NA) molecule providing of selective advantage in regard to viral generation packing into virions is disclosed. Uses of retroviral vector in particular for specific antibody production are described.

EFFECT: new genetic antiviral agents generating prolonged and stable immunological responses in regard, for example, to AIDS and cancer viruses.

97 cl, 11 ex

FIELD: biotechnology, medicine, in particular viral disease treatment.

SUBSTANCE: invention relates to recessive dividing retroviral vector useful in inhibition of wild-type retrovirus replication. Vector contains retroviral long terminal repeat sequences; retroviral packing signal; nucleotide sequence encoding (expressing) genetic antiviral agent; and optionally the second nucleotide sequence. Disclosed are method for production of said vector and reproduction thereof. Further isolated and purified nucleic acid (NA) molecule providing of selective advantage in regard to viral generation packing into virions is disclosed. Uses of retroviral vector in particular for specific antibody production are described.

EFFECT: new genetic antiviral agents generating prolonged and stable immunological responses in regard, for example, to AIDS and cancer viruses.

97 cl, 11 ex

FIELD: genetic engineering, proteins, medicine, pharmacy.

SUBSTANCE: invention relates to a method for preparing a fused protein representing immunoglobulin Fc-fragment and interferon-alpha and can be used in treatment of hepatitis. Method involves construction of a fused protein comprising immunoglobulin Fc-fragment prepared from Ig G1 or Ig G3 in direction from N-end to C-end and the end protein comprising at least one interferon-alpha. Fc-fragment and the end protein are joined directly or by a polypeptide bridge. The fused protein is used for preparing a pharmaceutical composition used in treatment of liver diseases and in a method for targeting interferon-alpha into liver tissues. Invention provides preparing the fused protein eliciting with biological activity of interferon-alpha providing its concentrating in liver and showing enhanced solubility, prolonged half-time life in serum blood and enhanced binding with specific receptors.

EFFECT: improved targeting method, valuable biological properties of fused protein.

10 cl, 5 dwg, 9 ex

FIELD: genetic engineering, proteins, medicine, pharmacy.

SUBSTANCE: invention relates to a method for preparing a fused protein representing immunoglobulin Fc-fragment and interferon-alpha and can be used in treatment of hepatitis. Method involves construction of a fused protein comprising immunoglobulin Fc-fragment prepared from Ig G1 or Ig G3 in direction from N-end to C-end and the end protein comprising at least one interferon-alpha. Fc-fragment and the end protein are joined directly or by a polypeptide bridge. The fused protein is used for preparing a pharmaceutical composition used in treatment of liver diseases and in a method for targeting interferon-alpha into liver tissues. Invention provides preparing the fused protein eliciting with biological activity of interferon-alpha providing its concentrating in liver and showing enhanced solubility, prolonged half-time life in serum blood and enhanced binding with specific receptors.

EFFECT: improved targeting method, valuable biological properties of fused protein.

10 cl, 5 dwg, 9 ex

FIELD: gene engineering, in particular apoptosis inducing gene delivery vectors useful for cancer, hyperplasia, metaplasia and displasia diagnosis and treatment.

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FIELD: food industry.

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EFFECT: strain has strong structural properties.

16 cl, 4 dwg, 6 tbl, 5 ex

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