Homodimeric protein having the activity of an inhibitor of angiogenesis (options), the dna molecule encoding homodimeric protein, a vector for expression homodimeric fused protein, the method transfection of mammalian cells and method for producing homodimeric fused protein

 

The invention relates to the field of biotechnology and can be used to obtain a fused protein inhibitors of angiogenesis. Protein is a glycosilated, in which the Fc region of immunoglobulin gamma fused with a target protein, comprising one or more molecules with activity inhibitor of angiogenesis that occurs from angiostatin or endostatin. Option homodimeric fused protein also includes the second target protein containing one or more molecules having the activity of an inhibitor of angiogenesis selected from angiostatin, endostatin, a fragment of plasminogen, possessing activity of angiostatin, a fragment of collagen XVIII, has the activity of endostatin. Molecules in the composition homodimeric fused protein associated with the Fc region of immunoglobulin gamma and each other directly or via a polypeptide of the bridge. Homodimeric protein produced by transfection of mammalian cells with a vector containing a DNA molecule encoding a target protein, culturing the mammalian cells for the production of fused protein and the allocation of fused protein. The invention provides efficient production and secretion of angiogenesis inhibitors in a variety of households who reak-before:always;">

The technical FIELD

The present invention relates mostly to methods and structures for obtaining and using the fused proteins containing an inhibitor of angiogenesis (development of blood vessels). More specifically, the invention relates to methods and structures for obtaining and using the fused proteins called “immunologiae, which contain the Fc region of an immunoglobulin and an inhibitor of angiogenesis.

The LEVEL of TECHNOLOGY

We have opened two active inhibitor of angiogenesis: angiostatin (O'reilly, etc. // Cell. 1994. So 79. S. 315), endostatin (O'reilly, etc. // Cell. 1997. So 88. S. 277). It was found that they naturally are produced by the primary tumor. Both proteins specifically inhibit the proliferation of endothelial cells and inhibit tumor growth by blocking angiogenesis - the formation of new blood vessels that nourish the tumor. Studies have shown that these inhibitors of angiogenesis are non-toxic even in very high doses and can inhibit the growth of metastases and primary tumors may regress to the size, indistinguishable under the microscope. Both inhibitors were identified as derived by proteolysis fragments of much larger intact molecules. It was found that angio the attention of oncologists, as it has been shown that they suppress in mice the growth of many different types of tumors, with no visible side effects or drug resistance. Traditional chemotherapy usually leads to acquired drug resistance, which is mainly caused by genetic instability of cancer cells. Therapy with angiogenesis inhibitors is not so much on the cancer cells, but on normal endothelial cells that support tumor growth. Because endothelial cells are genetically stable, methods of therapy using inhibitors of angiogenesis can lead to less drug resistance. Studies show that drug resistance did not develop in mice subjected to prolonged antiangiogenic therapy with endostatin. Moreover, the repeated cycles of treatment of mice with endostatin resulted in long “dormant” tumor, and after cessation of therapy, the tumor recurrence was not observed (Boehm and others // Nature. 1997. So 390. S. 404).

Despite the promising results of studies in mice, soluble active angiostatin, endostatin with qualifications suitable for clinical use, could not be obtained with Li were insoluble protein aggregates uncertain composition, unsuitable for injection people. For other kinds of products, including expression system based on the baculovirus and mammalian cells, was characterized by a very low level production of recombinant proteins (O'reilly, etc. // Cell. 1997. So 88. S. 277).

Low output used in this expression systems can be explained by the fact that angiostatin and endostatin are internal protein fragments of a much larger size. Truncated proteins can not be closed as needed in the absence of amino acid residues, cut off from precursor molecules. For example, angiostatin contains 26 cysteine residues which form a multiple disulfide bonds. The expression actually angiostatin may not provide optimal conditions for these multiple disulfide bonds were formed as needed at the stage of secretion. As for the recombinant protein of endostatin, when producing cells E. Li he precipitated during dialysis is probably due to the hydrophobicity of endostatin (O'reilly, etc. // Cell. 1997. So 88. S. 277).

The main obstacle in the use of angiostatin and endostatin in their current form is that to achieve the desired clinical effect is not the many months. For example, in the conventional schemes of mice to obtain optimum efficiency required dosage of endostatin 20 mg/kg daily (Boehm and others // Nature. 1997. So 390. S. 404). Because it requires urgent test the clinical efficacy of endostatin and angiostatin, it is important to develop products that give large amounts of clinical material qualification.

One of the systems of expression, is used to provide a high level of expression of the fused proteins in mammalian cells, is a structure of DNA which encodes a signal sequence, plot Fc (constant part) of an antibody and the target protein. Merged product of this design has the common name “immunotoxin”. As immunizing were successfully expressed several target proteins, including IL2 (interleukin 2), CD26, Tat, Rev, OSF-2, (-IG-H3, IgE receptor, PSMA and gp120. These are expressed by the constructions disclosed in U.S. patent No. 5541087 and No. 5726044, descriptions of which are incorporated here by reference.

The main purpose of the expression of the recombinant fused protein in mammalian cells was trying to put hybrid molecules new or useful properties such as the desired collapse, increased is suciu complement, protein binding And increased the half-life in blood flow and an increased ability to penetrate through the blood-brain barrier. Examples of recombinant fused proteins produced in mammalian cells, include immunoconjugate cytokines (Gillies, etc. // Proc. Natl. Acad. Sci. USA. 1992. So 89. S. 1428; Gillies, etc. // Bioconjugate Chemistry. 1993. So 4. S. 230), immunoadhesin (Capon, etc. // Nature. 1989. I. 337. S. 525), immunotoxins (Director, etc. // Nature. 1989. So 339. S. 394), and conjugate growth factor nerve (Friden, etc. // Science. 1993. I. 259. S. 373).

The purpose of the present invention is to obtain new DNA, ensuring efficient production and secretion of angiogenesis inhibitors in a variety of hosts mammalian cells. Another objective of the present invention is to provide methods of treatment of animals nucleic acids that encode, or amino acid sequences that determine protein inhibitors of angiogenesis, including negativnye, biosynthetic or otherwise obtained artificial proteins, as proteins created by rational design.

The INVENTION

The present invention provides methods and patterns, suitable for receipt and use of fused proteins containing beh protein inhibitors of angiogenesis. Protein inhibitors of angiogenesis can then be derived from the fused protein and combined with a pharmaceutically suitable carrier prior to administration to a mammal, such as man. Alternatively, the nucleotide sequence encoding, or amino acid sequences that define containing the angiogenesis inhibitor fused proteins can be combined with a pharmaceutically suitable carrier and administered to the mammal.

In one aspect the invention provides nucleic acid molecules, for example DNA or RNA encoding the protein according to this invention. The nucleic acid molecule encodes a signal sequence Fc region (constant part) immunoglobulin and at least one target protein, denoted here as protein-angiogenesis inhibitor selected from the group consisting of angiostatin, endostatin, a fragment of plasminogen activity of angiostatin, a fragment of collagen XVIII activity of endostatin and combinations thereof. In the preferred implementation of the nucleic acid molecule encodes sequentially in the direction from 5’-end to 3’-end, the signal sequence Fc region of immunoglobulin and posledovatelnostei, in the direction from 5’-end to 3’-end, the signal sequence, the sequence of the target protein and the Fc region of immunoglobulin.

In another preferred implementation of the Fc region of immunoglobulin contains the hinge region of immunoglobulin and preferably contains at least one constant part of the heavy chain of the immunoglobulin, for example, domain 2 constant part of the heavy chain (CH2), domain 3 constant part of the heavy chain (CH3and, depending on the type of immunoglobulin used for the formation of the Fc region, but not necessarily, domain 4 constant part of the heavy chain (CH4). In a more preferred implementation of the Fc region of immunoglobulin contains the hinge region, domain, CH2and the domain of CH3. Under certain circumstances, the Fc region of an immunoglobulin preferably devoid, domain CH1. Although the Fc region of immunoglobulin can be the Foundation of any class of immunoglobulins, such as IgA, IgD, IgE, IgG, and IgM are preferred Fc region of immunoglobulin-based IgG (gamma globulin).

In another implementation of the nucleic acid of the present invention may be included in functional communication capable of replication expressyou the enjoyment of the present invention provides cell-owners, perceiving such nucleic acid sequence, corresponding to the present invention.

In another aspect, the present invention relates to a fused protein containing the Fc region of immunoglobulin connected (either directly to a peptide or polypeptide through the bridge) with a target protein selected from the group consisting of angiostatin, endostatin, having the activity of angiostatin fragment of plasminogen having an activity of endostatin fragment of collagen XVIII, and combinations thereof. The target protein may be linked to its C-end N-end Fc region of immunoglobulin. However, in the preferred embodiment, the target protein is linked to its N-end C-end Fc region of immunoglobulin.

In another embodiment, a protein may contain a second target protein is selected from the group consisting of angiostatin, endostatin, having the activity of angiostatin fragment of plasminogen and having the activity of endostatin fragment of collagen XVIII. In constructions of this type, the first and second target proteins can be the same protein or different proteins. For example, in a preferred implementation protein contains the first target protein - Angie is connected together, either directly either through a polypeptide of the bridge. Alternatively, both the target protein can be linked either directly or using a polypeptide of the bridge with the Fc region of immunoglobulin. In the latter case, the first target protein attached to the N-end of the Fc region of immunoglobulin, and the second target protein attached to the C-end of the Fc region of immunoglobulin.

In another embodiment, two fused protein may be linked or covalently, for example, disulfide, or a peptide bond or ecovalence, obtaining a multimeric protein. In the preferred embodiment, two fused protein covalently linked through one or more disulfide bonds between cysteine residues, preferably located inside the hinge regions of immunoglobulin inside the Fc regions of immunoglobulin both chains.

In a preferred embodiment, the target protein includes a fragment of plasminogen having a molecular weight of about 40 kDa, and may contain amino acid sequence as set forth in sequences SEQ ID NO: 3. In another preferred embodiment, the target protein includes a fragment of collagen XVIII, having the amino acid p is geostation or endostatin full length or their biologically active fragments. The source of the target protein to create certain fused proteins depends on the intended use of the target protein. For example, if the target protein must be entered people, the target protein should be preferably of human origin.

In another aspect the present invention provides methods of obtaining the fused protein containing the Fc region of an antibody and the target protein is selected from the group consisting of angiostatin, endostatin, having the activity of angiostatin fragment of plasminogen and having the activity of endostatin fragment of collagen XVIII. The method includes the steps: (a) obtaining mammalian cells containing a DNA molecule encoding such a protein with a signal sequence, with or without, and (b) culturing cells of a mammal to obtain a fused protein. Received target protein can then be collected, sorted, if necessary, otherwise and cleaned using conventional cleaning methods are well known and used in the field. If we assume that the protein contains the site of proteolytic cleavage, located between the Fc region of immunoglobulin and protein target, then the target protein can be visiplan from the merged BEM aspect the present invention provides methods of treating mammals, for example, people in need of therapy-based inhibitors of angiogenesis. For example, it is assumed that inhibitors of angiogenesis in accordance with the present invention it is possible to introduce people with the tumor. Treatment with an inhibitor of angiogenesis may slow or stop tumor growth and, under certain conditions, can lead to regression of the tumor. Treatment can include administration to a mammal an inhibitor of angiogenesis in a quantity sufficient to slow or stop tumor growth. Angiogenesis inhibitor can be in the form of a slit protein or nucleic acid, preferably functionally connected with expressing vector, in combination with a pharmaceutically suitable carrier.

The above and other objectives, features and advantages of the present invention will be clearer from the following detailed description, graphics and formulas.

A BRIEF DESCRIPTION of GRAPHIC MATERIALS

Fig.1A-1F are a schematic illustration of a typical fused protein with an inhibitor of angiogenesis, constructed in accordance with the present invention (see examples 10-15). They depict the following structure.

Fig.1A: Fc Is: Fc-endostatin-bridge Gly-Ser (glycine-serine)-the inner part of the 1st domain of angiostatin Kringle;

Fig.1D: Fc-endostatin-bridge Gly-Ser-1-th domain of angiostatin Kringle;

Fig.1E: Fc-endostatin-bridge Gly-Ser-angiostatin;

Fig.1F: angiostatin (PC-endostatin

Vertical lines indicate possible disulfide linkages between cysteine residues (C) inside the hinge region of the Fc molecules.

DETAILED description of the INVENTION

The present invention provides fused proteins, called here immunologiae, which is suitable for production of commercial quantities of inhibitors of angiogenesis with qualifications suitable for clinical application. Inhibitors of angiogenesis before use can be derived from protein structures immunizing. However, it is assumed that immunopotency or nucleic acid encoding immunopotency, you can directly enter a mammal in need of treatment an inhibitor of angiogenesis.

Therefore, the present invention provides a fused protein containing the Fc region of immunoglobulin and at least one target protein, referred to here as an inhibitor of angiogenesis. Angiogenesis inhibitor is preferably selected from the group consisting of angiostatin, endostatin, having the activity of angiostatin fragment plasminogen activity inhibitor of angiogenesis, which are currently known or will be opened later, can be expressed as a fusion protein described herein type.

Fig.1A-1F illustrate six typical embodiments of protein structures embodying the present invention. Because preferred are dimeric structures, they are all depicted as dimers, custom made a pair of disulfide bonds between cysteine located on adjacent subunits. In the drawings shown disulfide bridges that link the two parts of the Fc regions of immunoglobulin in the hinge region of immunoglobulin and, thus, are characteristic of the native forms of these molecules. Although the structure containing the hinge region of the Fc, the preferred and promising as therapeutic agents, the invention assumes that optionally may be selected staple in other places. In addition, in some circumstances, dimers or multimer, useful in the practice of the invention, can be obtained by non-covalent binding, for example by means of hydrophobic interactions.

As homodimers design are important variants of the invention, Fig.1 illustrates such a design, the am in this field, can often be difficult cleaning. However, can be designed suitable for the inhibition of angiogenesis in different species of mammals viable design, representing a mixture of homodimers and heterodimers. For example, one chain heterodimeric structure may contain endostatin, and the other may contain angiostatin.

In Fig.1A presents dimeric structure obtained in accordance with the procedure described in example 10. Each monomer of the dimer contains a Fc region of immunoglobulin (1), including the hinge region, the domain of CH2and the domain of CH3. Directly to the C-end of the field Fc (1) attached to the first section of angiostatin Kringle (2), including both internal and external rings. In Fig.1B presents the second variant of the invention (see example 11). It has the same Fc region, as in Fig.1A, but this time to the end of the Fc region (1) is attached only to the inner ring of the first region of angiostatin (3). Fig. 1C through 1E show various embodiments of protein structures in accordance with the present invention, which includes as a target protein, several inhibitors of angiogenesis, arranged in tandem and the United Masterpage plot of angiostatin Kringle (3). Fig.1D shows a protein containing the same area Fc, as shown in Fig.1A, and the target protein containing endostatin full length (4), polypeptide bridge (5) and the entire area of the 1st section of angiostatin Kringle (both internal and external rings) (2). Fig.1E is different from the structure shown in Fig.1D, the fact that a large part of the domain C-terminal part of the protein is a copy of angiostatin full length (7).

Although Fig.1A-1E represent design type Fc-X, where X is the target protein, it is assumed that the design of the type X-Fc can also be useful in the practice of the invention. In addition, it is assumed that the useful proteins in accordance with the present invention can also be designated by the formula X-Fc-X, where X may denote the same or different target proteins. Fig.1F shows such a structure that contains in the direction from N-Terminus to the C-end of angiostatin human full length (7), the Fc region of human immunoglobulin (6), including the hinge region, and the domain of endostatin human full length (4).

The term “angiogenesis inhibitor”, as used here, refers to any polypeptide chain, which reduces or suppresses the mammal formation of new blood senich vessels or tumor, preferably in or on a solid tumor. It is assumed that by using various well-known methods of analysis can be identified and used in this area are useful inhibitors of angiogenesis. Such assay methods include, for example, the test on the proliferation of endothelial cells of capillaries of cattle, the analysis using chorioallantoic membrane chickens (HAM) or analysis on the cornea of the eye mice. However, the preferred analysis on HAM (see, for example, O'reilly, etc. // Cell. 1994. So 79. S. 315-328 and O'reilly, etc. // Cell. 1997. So 88. S. 277-285, a summary of which is included here by reference). Briefly, embryos with intact yolks were removed from three white eggs fertilized and placed in Petri dishes. After incubation for 3 days at 37°C in an atmosphere of 3% CO2to the chorioallantoic membrane of an individual embryo was put methylcellulose disk coated with a putative inhibitor of angiogenesis. After incubation for approximately 48 hours chorioallantoic membranes examined under a microscope for the presence of zones of inhibition.

Preferred angiogenesis inhibitors that are suitable in the practice of this invention are, for example, angiostatin (O Reil No. 5854205). As established earlier, angiostatin, endostatin are specific inhibitors of proliferation of endothelial cells and may inhibit tumor growth by blocking angiogenesis, i.e. the formation of new blood vessels that feed tumors.

Angiostatin was identified as a proteolytic fragment of plasminogen (O'reilly, etc. // Cell. 1994. So 79. S. 315-328 and U.S. patent No. 5733876, 5837682 and 5885795, a summary of which is included here by reference). Specifically, the angiostatin is an internal fragment of plasminogen with a molecular mass of 38 kDa, containing at least three of the areas of Kringle of plasminogen. Endostatin was identified as a proteolytic fragment of collagen XVIII (O'reilly, etc. // Cell. 1997. So 88. S. 277-285 and U.S. patent No. 5854205, a summary of which is included here by reference). Specifically, endostatin is a C-terminal fragment of collagen XVIII with a molecular mass of 20 kDa. The term “angiostatin” and “endostatin” as used here, means not only proteins are full length, but also their variants, and biologically active fragments, as well as biologically active fragments, respectively plasminogen and collagen XVIII. The term “biologically active fragment” as applied to the angiostatin means any Bel is our least 70% and most preferably at least 90% of the activity of angiostatin full length, a specific test in HAM. The term “biologically active fragment” as applied to the endostatin refers to any protein fragment of collagen XVIII or endostatin, which has at least 30%, more preferably at least 70% and most preferably at least 90% of the activity of full length endostatin, a specific test in HAM.

The term “variants” includes special variants and allelic variants, as well as other existing in nature or occurring other than the natural way ways, for example obtained from a conventional genetic engineering methods, which are at least 70%, or 60% identical, more preferably at least 75% or 65% identical, and most preferably at least 80% or 70% identical to any of the existing natural sequences of endostatin or angiostatin disclosed here.

To determine if the polypeptide required degree of similarity or identity with the polypeptide of the comparison (reference polypeptide), first of all conduct a parallel comparison of the considered amino acid sequence and amino acid sequence comparisons, iSpazio with substitution matrix BLOSUM62, described in Fig.2 work Henikoff and Henikoff “Amino acid substitution matrices from protein blocks” // Proc. Natl. Acad. Sci. USA. 1992. So 89. C. of 10,915-10919. For the present invention suitable value criterion unreliability of the gap is equal to 12, and the appropriate value for criterion unreliability lengthening of the lumen is equal to - 4. A computer program that performs a parallel comparison of sequences using the algorithm of Smith-Waterman (Smith-Waterman) and the BLOSUM62 matrix, such as GCG software (Oxford Molecular Group, Oxford, England), are commercial products and are widely used by specialists in this field.

After the parallel compare and reference sequences, it is possible to calculate the percentage of similarity. Sequentially mapped to individual amino acids in each sequence, determining their similarity to each other. If the value of the BLOSUM62 matrix, corresponding to the two mapped to amino acids, is equal to zero or is a negative value, then the similarity within pairs is denoted as zero. Otherwise, the similarity within pairs is denoted as 1.0. The total similarity value is the sum of the similarity within pairs for matching computers is under consideration or in the reference sequence. Normalized total value and represents the percentage of similarity. Alternatively, to calculate percent identity are again alternately maps aligned in a row parallel to the amino acids of each sequence. If amino acids are not identical, the value of identity within a pair is equal to zero; otherwise, the value of identity within a pair is 1.0. The total value of identity is the sum of identical aligned in a row parallel to the amino acids. Then the total value of identity normalize by dividing by the number of amino acids in a less considered or in the reference sequence. Normalized total value and represents the percentage identity. To calculate the percent similarity and identity insertions and deletions neglected. Accordingly, these calculations do not use the values of the criterion unreliability fill the gap, although they are used during the initial alignment of sequences.

Declared here target protein expressed as a fusion protein having the Fc region of immunoglobulin. As is known, the constant part of each heavy chain immunoglobulin consists of 4 or 5 domains. Domains are indicated placentas heavy chains are cross-homology among the classes of immunoglobulins, for example, domain CH2IgG homologous domain CH2IgA and IgD, as well as the domain of CH3IgM and IgE.

It is clear that in the sense used here, the term “Fc region of immunoglobulin” means a portion of carboxyl end of the constant region of the chain immunoglobulin, preferably a constant region of the heavy chain of the immunoglobulin, or portion thereof. For example, the Fc region of immunoglobulin may contain (1) the domain of CH1the domain CH2and the domain of CH3; (2) the domain of CH1and the domain of CH2; (3) the domain of CH1and the domain of CH3; (4) the domain of CH2and the domain of CH3or (5) a combination of two or more domains and the hinge region of the immunoglobulin. In the preferred implementation of the Fc region, reflected in the design of DNA that includes at least the hinge region of the immunoglobulin domain of CH2and the domain of CH3and preferably devoid of at least a domain of CH1.

It is generally accepted that the preferred class of immunoglobulin, which is a constant region of the heavy chain, is IgG (Ig) (subclasses 1, 2, 3 or 4). Can be used with other classes of immunoglobulins: IgA (Ig), IgD (Ig3field FCor homologous domains of any of immunoglobulin IgA, IgD, IgE or IgM.

Depending on the application can be used genes for the constant region not only humans, but other species such as mouse or rat. Typically, the Fc region used as a partner to join in DNA constructs for immunizing, can be from any mammal species. In the case when you don't want getting in the cage-the owner or the animal's immune response to the Fc region, the Fc region may be from the same species, and a host cell or animal. For example, if the animal host or a host cell - man, you can use Fc man; similarly in the case when the animal host or a host cell m is x constant regions, where one or more amino acid residues of the domains of the constant region of substituted or deleterow. One example could be the introduction of amino acid substitutions in the upper region CH2to obtain the variant Fc with reduced affinity for Fc receptors (Cole and others // J. Immunol. 1997. I. 159. S. 3613). Having a common experience in this field can prepare such structures, using well known methods of molecular biology.

Using Fc1 person as a sequence Fc region has several advantages. For example, if a protein containing the angiogenesis inhibitor together with Fc, should be used as a biologically active drug, the domain Fc1 can give a fused protein activity in the sense of effector functions. Such effector functions include such types of biological activity, as the binding of complement sold through antibodies cell-mediated cytotoxicity, transport in the placenta and increased half-life in serum. The domain Fc makes possible the detection method enzyme-linked immunosorbent assay ELISA and purification by binding to protein A of Staphylococcus aureus (Protein A). In some applications, however, Mogi Fc or link complement.

If immunizing inhibitors of angiogenesis, one of the functions Fc immunoglobulin as a partner binding is to stimulate proper folding of the protein inhibitor of angiogenesis to obtain active protein inhibitor of angiogenesis and to impart the solubility of the active components, at least in the extracellular environment. Since Fc as a partner to merge hydrophilic, immunotoxin with the inhibitor of angiogenesis is highly soluble, in contrast to, for example, from recombinant endostatin produced in E. coli (O'reilly // Cell. 1997. So 88. S. 277). All the examples were obtained by high levels of production immunities. Immunopotency inhibitors of angiogenesis were allocated (securityroles) on Wednesday with a concentration typically in the range from 30 to 100 µg/ml and could be successfully purified to homogeneity by chromatography with protein A. in Addition, immunopotency inhibitors of angiogenesis can be split and additionally cleaned using standard cleaning methods, including, for example, purification on heparin-sepharose, lysine-sepharose or affinity purification.

In addition to high levels of expression of the fused proteins in accordance with the present invention find also longer in the and, associated with Fc man has a half-life in mouse serum 33 h, and angiostatin human 4-6 h (O'reilly, etc. // Nature Medicine. 1996. So 2. S. 689). It is believed that the angiostatin with a molecular weight of 40 kDa, endostatin with a molecular weight of 20 kDa, due to their small size, are easily excreted from the body by filtering through the kidneys. In contrast, dimeric form Fc-angiostatin and dimeric Fc-endostatin have a molecular weight respectively 145 and 100 kDa, as there are two Fc region of immunoglobulin associated either with two molecules of angiostatin, or with two molecules of endostatin. Such bivalent design can show higher activity in binding to receptors of angiostatin or endostatin. If the activity in the suppression of angiogenesis via receptors fused proteins containing the Fc, potentially more effective in suppressing tumors than monovalent angiostatin or monovalent, endostatin themselves. In addition, if angiostatin and/or endostatin belong to the class of dimeric protein ligands, the physical effects of Fc on the angiostatin or endostatin will turn dimerization in an intramolecular process, shifting thus the equilibrium in the direction of the images of the sulfide bonds in the monomer using standard recombinant DNA technology can be introduced in the appropriate places cysteine residues.

In the same sense as it is used here, the term “multivalent” refers to a recombinant molecule that comprises two or more biologically active segment. Protein fragments forming multivalent molecule can be linked via a polypeptide bridge that connects the parts without causing a shift of the reading frame, and allows each part to function independently.

In the same sense as it is used here, the term “bivalent” refers to a recombinant molecule containing the slit structure of the present invention two of the target protein, for example a molecule Fc-X, where X is selected independently from angiostatin, endostatin or their variants. Since there are two sites X, connected to the Fc region of immunoglobulin (which itself is usually a dimer fragments of heavy chains comprising at least part of the hinge region domain and CH3and possibly, but not necessarily the domain of CH2), the bivalent molecule (see, for example, Fig.1A). If the slit design in accordance with the present invention has the form Fc-X-X derived molecule with an Fc dimer tetravalency. Two proteins that form the molecule Fc-X-X, can be linked via a peptide of the er, if one part of endostatin in the construction of Fc-endostatin can bind to the receptor on the cell with a certain amount of affinity, the second section of endostatin of the same construction Fc-endostatin can be used for much more contact with the second receptor on the same cell (apparent affinity). This is a consequence of the physical proximity of the second section of the endostatin receptor after the first section of endostatin already contacted. In the case of binding of the antibody to the antigen apparent affinity is increased by about 104time.

In the sense as used here, the terms “multimer” and “multimeric” refers to a stable Association of two or more polypeptide chains, either covalent manner, for example, by covalent interactions, for example, the formation of disulphide bonds or non-covalent manner, for example, through hydrophobic interactions. Have in mind that the term “multimer” includes both homomultimers, where the polypeptides are the same, and heteropolymer, where the polypeptides are different.

In the same sense as it is used here, the term “dimer” refers to a specific multimeric molecule, where the two protein polypeptide chain stable is immunoglobulin and the Fc fragment is usually a dimer fragments of the heavy chain, including at least part of the hinge region domain and CH3and possibly, but not necessarily, the domain of CH2. It is known that many protein ligands bind to their receptors in the form of dimers. If a protein-ligand X in a natural way diminished, the areas of X in the molecule Fc-X will timeresults much stronger, because the process of dimerization depends on the concentration. Physical sblizhenie two sites X, connected attached to the Fc region of immunoglobulin, turns dimerization in an intramolecular process, greatly shifting the equilibrium towards the formation of dimers and increasing their binding to receptors.

It is clear that the present invention uses conventional methods of recombinant DNA to obtain containing Fc-fused proteins, suitable for practical application of the invention. Fused design with Fc preferably created at the level of DNA, and the resulting DNA integrates in expressing vectors and Express to obtain immunities. It should be understood that the term “vector” in the sense it is used here, refers to any nucleic acid containing the nucleotide sequence can be introduced into the cell host and recombine vectors include linear nucleic acid, plasmids, phagemid, Comedy, RNA vectors, viral vectors, etc., Examples of viral vectors, non-limiting all the possibilities include retrovirus, adenovirus and adeno-associated virus. It is clear that in the sense as it is used here, the term “gene expression” or “expression” of the target protein indicates the transcription of the DNA sequence, the translation of mRNA transcripts and secretion of the product - fused protein containing the Fc.

Suitable expressing vector is pdCs (Lo etc. // Protein Engineering. 1988. So 11. S. 495, the details are included here by reference), in which transcription of the gene Fc-X used the enhancer/promoter of the human cytomegalovirus and the SV40 polyadenylation signal. Used the sequence of the enhancer and promoter of human cytomegalovirus was derived from nucleotides from -601 to +7 sequence proposed by Boshart, etc. // Cell. 1985. So 41. S. 521, a summary of which is included here by reference. The vector also contains a mutant gene dihydrofolate-reductase as selectivity marker (Simonsen and Levinson // Proc. Natl. Acad. Sci. USA. 1983. So 80. S. 2495, details are included here by reference).

Suitable a host cell can be transformed or transfected with a DNA sequence of infusion of the TCI-owners for use in the present invention include immortal hybrid cells, cell myeloma NS/0 cells, 293 cells Chinese hamster ovary cells, HeLa and COS cells.

Slit proteins in accordance with the present invention preferably are conventional methods of recombinant DNA. Slit proteins are preferably produced at expressive in the cell-the owner of the DNA molecule that encodes a signal sequence region of the immunoglobulin Fc and the target protein (referred to here is also an inhibitor of angiogenesis). The preferred design can be encoded in the direction 5’3’ signal sequence Fc region of an antibody and the target protein. Alternatively, design can be encoded in the direction 5’3’ signal sequence of the target protein and the Fc region of immunoglobulin.

In the same sense as it is used here, the term “signal sequence” means a peptide segment that provides secretion of the protein immunizing inhibitor of angiogenesis, and then ottsepleny after the broadcast in the cell host. The signal sequence in accordance with the present invention is polynucleotide that encodes the amino acid sequence, the INIC is that usable in the present invention, include the signal sequence of the light chain of the antibody, such as antibody 14.18 (Gillies, etc. // J. Immunil. Meth. 1989. So 125. S. 191-202), signal sequence of the heavy chain of the antibody, such as signal sequences of heavy chain antibodies MARS (Sakano, etc. // Nature. 1980. I. 286. C. 5774) and any other known in the field of signal sequence (for example, see Watson // Nucleic Acids Research. 1984. So 12. C. 5145). All cited works are included here by reference.

Signal sequences are well studied in this area. It is known that they usually contain from 16 to 30 amino acid residues and may contain more or fewer amino acid residues. A typical signal peptide consists of three sections: basic N-terminal segment, a Central hydrophobic segment and a more polar C-terminal site. The Central hydrophobic region contains from 4 to 12 hydrophobic residues that reinforce the signal peptide in the lipid bilayer membrane during transport of the synthesized polypeptide. After initiation of the signal peptide is typically cleaved in the lumen of the endoplasmic reticulum cellular enzymes known as signal peptidases. Potential sites of cleavage signalnoise neutral amino acid residues at positions -1 and -3 and does not contain in this area Proline residues. Signal peptidase splits this signal peptide is between amino acids -1 and +1. Thus, the signal sequence as part of the sequences encoded by the DNA can be derived in the process of secretion from the amino end of the protein immunizing. This leads to secretion of the protein immunovative, consisting of the Fc region and the target protein. A detailed discussion of the sequence of the signal peptide is given von Heijne // Nucleic Acids Res. 1986. So 14. C. 4683. The contents of this work are included here by reference.

As it is obvious to experts in this area, the suitability of a particular signal sequence for use in the present invention may require some conventional experimental verification. This experimental verification includes determining the ability of the signal sequence to control the secretion of immunizing and also the determination of the optimal configuration of the encoding genomic DNA or cDNA sequence, which can be used to achieve efficient secretion immunities. Additionally, professionals in this field can create a synthetic signal peptide, following the rules of von Heijne (see link above), and to test the effectiveness of this is the existence of a “signal peptide”, “leader sequence” or “leader peptide”.

The connection of the signal sequence and the Fc region of immunoglobulin sometimes referred to as the secretory cassette. Approximate secretory cassette suitable in the practice of the present invention is polynucleotide encoding in the direction of 5’3’ signal sequence of a gene light chain immunoglobulin and Fc region1 gene1 human immunoglobulin. Region Fc1 immunoglobulin gene1 preferably contains at least a portion of a hinge domain, and at least part of the domain of CH3or alternatively, at least part of the hinge domain, a domain of CH2and domain of CH3. DNA encoding the secretory cassette may be in the configuration of the genomic DNA or in the configuration of the cDNA.

In another implementation, the DNA sequence encodes the site of proteolytic cleavage, which is placed between the secretory cassette and a protein inhibitor of angiogenesis. The site of cleavage, providing proteolytic cleavage of the coding is about, the term “site of proteolytic cleavage,” as it is used here, means an amino acid sequence that is primarily split proteolytic enzyme or other agents proteolytic action. Appropriate sites of proteolytic cleavage include amino acid sequences that are recognized by proteolytic enzymes such as trypsin, plasmin or enterokinase K. Known many couples site splitting/splitting agent. For example, see U.S. patent No. 5726044, the disclosure of which is incorporated here by reference. If the sequence of the target protein is a molecule predecessor angiostatin, endostatin or active variants, necessary protein product can be obtained by splitting by using proteolytic enzyme in the endogenous actions, such as elastin or plasmin or urokinase.

The present invention also includes fused proteins containing various combinations of recombinant angiostatin and endostatin or their fragments, which can be produced in large quantities. Despite the demonstrated efficacy in suppressing tumor growth, the blocking mechanism of angiogenesis, angiostatic structural motifs, each of which can be solely responsible for linking or promoting the binding of proteins to endothelial cells and expression of antiangiogenic effect. Therefore, this invention includes the target proteins that are biologically active fragments of angiostatin, such as 1-th domain of the Kringle 2 domain of Kringle, 3rd Kringle domain, and combinations thereof, and endostatin, which are physiologically similar to the existing in nature angiostatin and endostatin full length.

Another implementation of the present invention provides bifunctional hybrid design of inhibitors of angiogenesis. In the sense used here bifunctional hybrid molecule or structure means a protein produced by the Union of two protein subunits and two subunits can occur from different proteins. Each protein subunit has its own independent function, so that the hybrid molecule functions of two subunits can be additive or synergistic. Such hybrid proteins with functions will allow us to study the synergistic effect of angiostatin and endostatin in animal models. Preferred bifunctional hybrid may contain at least two Rostik. For example, in the preferred implementation of the respective target protein sequence encodes at least part of angiostatin, connected in one frame is read at least part of endostatin, and both domain - angiostatin and endostatin - exhibit antiangiogenic activity or inhibition of angiogenesis. Two subunits can be linked polypeptide bridge.

It should be understood that in the sense used here, the term “polypeptide bridge” means a peptide sequence that can bind together two proteins or a protein with the Fc region. Polypeptide bridge preferably contains mostly amino acids such as glycine and/or serine. Preferably the polypeptide bridge contains a set of peptides of glycine and serine length of approximately 10-15 units. For example, see U.S. patent No. 5258698, the disclosure of which is incorporated here by reference. However, it is assumed that the optimal length of the bridge and the amino acid composition can be determined by conventional experimental research.

It was found that if the different parts of angiostatin expressed as a fusion molecule with Fc, achieved high levels of expression presumably through CSOs, the Fc region can be glycosylated and at physiological pH values have a high charge. Therefore, the Fc region may contribute to the dissolution of hydrophobic proteins.

The present invention also provides methods of production of angiostatin and endostatin other than humans, species, in the form of a fused protein with the Fc. Such fused proteins angiogenesis inhibitors other than human species useful for preclinical studies of angiogenesis inhibitors, as research activity and toxicity of protein drugs before trials in humans must be conducted in systems with animal models. The human protein may not have activity in mice in model experiments, since the protein can stimulate the immune response and/or exhibit different pharmacokinetics, distorting the test results. Therefore, the equivalent protein of the mouse is the best substitute for human protein for model tests on mice.

For comparison soluble fused FC proteins(hu)-angiostatin(hu), Fc(hu)-endostatin(hu), FC(mu)-angiostatin(mu), FC(mu)-endostatin(mu) (hu means human, a mu - mouse) with insoluble proteins produced by the expression system of E. coli, was used a standard model carcinoma leeast tumors in a model of lung carcinoma Lewis, than the corresponding proteins obtained in E. Li. In addition, laboratory mice are linear and their tumors indutsirovannye, and not spontaneous. Therefore, the efficiency in the mouse model may not match the possible efficacy against human tumors. Preclinical studies on dogs give more accurate information about the effectiveness of these angiogenesis inhibitors on spontaneous tumors, as in dogs there are a large number of naturally occurring, spontaneous tumors. Methods of obtaining murine (mu) fused proteins Fc(mu)-angiostatin(mu) and FC(mu)-endostatin(mu) and canine (CA) fused proteins Fc(ca)-angiostatin(sa) and FC(sa)-endostatin(sa) of the present invention will facilitate pre-clinical study of angiogenesis inhibitors in murine and canine systems.

The present invention provides methods of treating disease conditions mediated by angiogenesis, by introducing a DNA, RNA or proteins of the present invention. Mediated angiogenesis painful condition include, for example, solid tumors, generated by the circulatory system tumors, metastatic tumors, benign tumors, including hemangiomas, acoustic neuroma, neurofibro americasa retinal pathology - retinopathy, retinopathy of premature maturation, degeneration corneal staining, rejection of transplanted corneal associated with tumor vascular glaucoma); return delayed fibroplasia, rubella syndrome of Osler-Webber; myocardial angiogenesis; plaque neovascularization; telangiectasia; associated with hemophilia angiofibroma and granulation of wounds; excessive or abnormal stimulation of endothelial cells, bowel adhesions, atherosclerosis, sclerodermalike and hypertrophic scars, i.e., keloids.

Design DNA disclosed herein may be useful for gene therapy procedures, in which the gene of angiostatin or endostatin is introduced into the cell is one of many ways, for example, in the form of native DNA, which is connected with the promoter or in the form of DNA in a viral vector. Once in the cell, the gene of angiostatin and/or endostatin expressed, and in vivo protein is produced with normal biological function. The DNA construct of the present invention gives high levels of expression of the fused protein. Fused proteins of the present invention may also be useful for treating medical conditions mediated by angiogenesis, and may have a higher clinical efficacy, che is hibitory angiogenesis according to the present invention have a longer half-life in serum than other recombinant inhibitors of angiogenesis or natural inhibitors of angiogenesis. Bivalent and dimeric forms proposed in the present invention, should have a higher affinity binding due to bivalent and dimeric structure. Bifunctional hybrid molecules of the present invention can have a higher clinical efficacy due to possible synergistic effects of two different inhibitors of angiogenesis associated linked Fc region or a flexible polypeptide bridge.

The compositions of the present invention can be introduced to the animal by any suitable ways: directly (for example, by injection, implantation or applique on the surface of the tissue) or systemically (for example, parenterally or orally). If the composition must be introduced parenterally, for example intravenously, subcutaneously, ophthalmologist, intraperitoneally, intramuscularly, into the oral cavity, rectal, vaginal, ocular depression, vnutriarterialno, intracranial way, in the spinal cord, inside the heart, inside the shell, inside the cavity, intracapsular, intranasal or aerosol, comp is dasti) or solution. Thus, the carrier or the carrier physiologically acceptable so that when it is added during the introduction of the necessary part of the patient does not reflect adversely on the balance of electrolytes or volume of the patient. Therefore, the liquid medium for the agent can be a normal physiological salt solution (e.g. aqueous solution 9,85% NaCI, 0.15 M, pH 7-7,4).

The preferred dose immunizing for insertion in the range of 50 ng/m2to 1 g/m2, more preferably from 5 g/m2up to 200 mg/m2and most preferably from 0.1 mg/m2up to 50 mg/m2. The preferred dose of a single introduction of nucleic acids encoding immunopotency are in the range from 1 mg/m2up to 100 mg/m2, more preferably from 20 g/m2up to 10 mg/m2and most preferably from 400 g/m2up to 4 mg/m2. It is emphasized, however, that the optimal route of administration and dosage can be determined by conventional experimental study that corresponds to the qualification in this field.

Further, the invention is illustrated by the following not limiting examples.

Example 1. The expression of the fused protein FC(hu)-endostatin(hu).

Angosta is under instructions Lo and other // Protein Engineering. 1998. So 11. S. 495. Fc denotes a fragment of the Fc gamma immunoglobulin (DNA sequence set forth as sequence of SEQ ID NO: 1; amino acid sequence is shown below as the sequence of SEQ ID NO: 2). To fit cDNA (complementary DNA) for the expression of endostatin with getting fused protein Fc-Endo used polymerase chain reaction (PCR) (the cDNA sequence shown as sequence of SEQ ID NO: 3; corresponding amino acid sequence disclosed in the sequence SEQ ID NO: 4). Direct primer served as either 5’-CC CCG GGT AAA CAC AGC CAC CGC GAC TTC (the sequence SEQ ID NO: 5; encoded amino acids are disclosed in the sequence SEQ ID NO: 6) or 5’-AAG CTT CAC AGC CAC CGC GAC TTC (the sequence SEQ ID NO: 7; encoded amino acids are disclosed in the sequence SEQ ID NO: 8), where a website or XmaI-HindIII site followed by sequence encoding a N-terminal endostatin. Primer with XmaI site makes it possible ligation (joining a ligase) endostatin cDNA to the XmaI site at the end of the sequence that encodes a domain of CH3the Fc region of immunoglobulin G (IgG). Primer with a HindIII site makes it possible ligation of cDNA of endostatin to the HindIII site of the vector pdCs-F connection fused protein. Reverse primer served 5’-CTC GAG HUNDRED CTT GGA GGC AGT CAT G (sequence SEQ ID NO: 9), which was designed to introduce a stop codon broadcast (anticodon, a HUNDRED) immediately after the end of endostatin, and that was followed by an XhoI site. The PCR products were cloned and sequenced, and the fragment XmaI-XhoI was Legerova with the received vector pdCs-Fc, split restrictase XmaI and XhoI. Similarly, fragment HindIII-XhoI encoding endostatin was Legerova respectively in the split vector pdCs-huFc(D4K). Stable clones expressing Fc-Endo or FC(D4K)-endostatin were obtained by electroporation of the cells, NS/0, followed by selection in growth medium containing 100 nm methotrexate. The level of expression of protein was determined by the method of ELISA for Fc man (example 3) and were confirmed by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (SDS), which showed the presence of a protein product with a molecular weight of approximately 52 kDa. The clones with the best products were subcloned by the method of exhaustive dilution.

Example 2. Cell culture and transfection.

For the time of transfection the plasmids were introduced into cells 293 human kidney by the method of co-precipitation of plasmid DNA with calcium phosphate (Sambrook, etc. // Molecular Cloning - A and with the instructions of the provider.

To obtain stably transfected clones plasmid DNA was introduced by electroporation into cells, NS/0 myeloma mouse. Cells, NS/0 were grown in medium Needle in the modification of Daleko with the addition of 10% calf serum. Approximately 5×106cells were washed once with phosphate-buffered saline (PBS) and resuspendable in 0.5 ml PBS. Then, 10 μg of linearized plasmid DNA was incubated with the cells in the cell unit Gene Pulser (the space between the electrodes of 0.4 cm, the firm BioRad, Hercules, CA, USA) with ice cooling for 10 minutes In installing a Gene Pulser (BioRad, Hercules, CA) was performed electroporation parameters of 0.25 V and 500 μf. Cells kept for 10 min in ice, after which they resuspendable in the growth medium and were sown on tablets with 96 cells. The selection of stably transfected clones was performed by growth in the presence of 100 nm methotrexate (ITC), which was added 2 days after transfection. The reagent was added 3 times every 3 days, resistant to MTC clones appeared after 2-3 weeks. To identify highly active producers supernatant of clones analyzed using ELISA method for Fc. Highly productive clones were isolated and maintained in growth medium containing 100 nm MTC.

Example 3. Methods the implementation of the TC clones used 3 different modifications of ELISA method. To measure the amount of proteins containing the Fc man (huFc), ELISA was used for huFc. To measure the amount of proteins containing mouse Fc (muFc) and Fc dog (caFc) used a modified method ELISA with antibodies respectively to muFc and to the caFc. Methodology ELISA for huFc described in detail below.

A. Preparation (coating) tablets for the reaction.

On tablets ELISA with 96 cells inflicted goat antibody affinity purity to human IgG - AffiniPure Goat anti-Human IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at a concentration of 5 μg/ml in PBS in a volume of 100 μl into each of the 96 wells of the tablet (Nunc-Measurement plate MaxiSorp, Nalge Nunc International, Rochester, NY, USA). Antibody coated tablets were washed 4 times with 0.05% tween-20 in PBS and blocked with 1% solution of bovine serum albumin (BSA) and 1% goat serum in PBS (200 μl per cell). After incubation with blocking buffer for 2 h at 37°C. the tablets were washed 4 times with 0.05% tween in PBS and dried off with paper towels.

C. Incubation with test samples and secondary antibodies.

The investigated samples were diluted to the desired concentration in the test buffer containing PBS, 1% BSA, 1% goat serum and 0.05% tween. With the chimeric antibody of known concentration (with anti-huFc activity) Polo,9 ng/ml were prepared by serial dilution in the test buffer. Diluted samples and standards were introduced into the cells of the tablet, 100 µl in each well, and the tablet then incubated for 2 h at 37°C. After incubation, the tablet was washed 8 times in 0.05% solution of tween in PBS. In each cell was then added 100 μl of secondary antibody conjugated with horseradish peroxidase (HRP) goat antibodies to human IgG (horse radish buffer (HPR)-conjugated goat anti-human IgG, Jackson ImmunoResearch Laboratories, West Grove, PA, USA), diluted approximately 1:120000 in the test buffer. For each batch, conjugated with HRP antibodies to human IgG was necessary to determine the exact dilution of the secondary antibody. After incubation for 2 h at 37°C. the tablet was washed 8 times in 0.05% solution of tween in PBS.

C. Manifestation.

The substrate solution was prepared by dissolving 30 mg (1 tablet) of o-phenylenediamine dihydrochloride (OPD) in 15 ml solution: 0.025 M citric acid/0.05 M Na2HPO4buffer, pH 5, containing 0.03% immediately before use was added N2About2. The substrate solution was added to the plate in a volume of 100 μl of the cell. For the manifestation of the painting was kept for 30 min in the dark at room temperature. The developing time can be changed depending on differences in quantities of dye, secondary antibodies, and so on, the Reaction was stopped by the named device (“plate reader”) with the installation of wavelengths as 490, and 650 nm, is programmed to subtract the background optical density at 650 nm from the measured optical density at 490 nm.

Procedure when ELISA for muFc was the same, except that the tablet ELISA coated with goat antibody to mouse immunoglobulin G (AffiniPure Goat anti-murine IgG (H+L), Jackson ImmunoResearch, West Grove, PA, USA) at a concentration of 5 μg/ml in PBS, 100 μl of the cell, and the secondary antibodies were conjugated with horseradish peroxidase goat antibody Fc fragmentmouse IgG (horse radish buffer-conjugated goat anti-muIgG, FC, Jackson ImmunoResearch, West Grove, PA, USA) used at a dilution of 1:5000. Similarly in ELISA for caFc tablets ELISA was covered with rabbit antibodies specific for the Fc fragment of IgG dogs (AffiniPure Rabbit anti-dot IgG, Fc Fragment specific, Jackson ImmunoResearch, West Grove, PA, USA), at a concentration of 5 μg/ml in PBS, 100 μl of the cell, and the secondary antibodies were conjugated with horseradish peroxidase rabbit antibodies to the Fc fragment of canine IgG (horse radish buffer-conjugated AffiniPure rabbit anti-dog IgG, Fc fragment specific, Jackson ImmunoResearch, West Grove, PA, USA), used at a dilution of 1:5000.

Example 4. The expression of the fused protein Fc human - human angiostatin.

Angiostatin human DNA sequence below as th is was expressively as a fusion protein Fc man the human angiostatin (huFc-huAngio), no essentially as described in example 1. To accommodate angiostatin cDNA (sequence SEQ ID NO: 3) for expression in vectors pdCs-huFc or pdCs-huFc(D4K), PCR was used. Related direct primers were 5’-CC CCG GGT AAG AAA GTG TAT CTC TCA GAG (sequence SEQ ID NO: 12; encoded amino acids are shown in the sequence SEQ ID NO: 13) and 5’-CCC AAG CTT AAA GTG TAT CTC TCA GAG (sequence SEQ ID NO: 14; encoded amino acids are disclosed in the sequence SEQ ID NO: 15), where the Xmal site or a HindIII site followed by the sequence encoding the N-end of angiostatin. The reverse primer was 5’-CCC CTC GAG HUNDRED CGC TTC TGT TCC TGA GCA (sequence SEQ ID NO: 16), which was designed to put a stop codon broadcast (anticodon, a HUNDRED) immediately after the end of angiostatin, followed by the Xhol site. The PCR products were cloned and sequenced, and the obtained fragment XmaI-XhoI and the fragment HindIII-XhoI encoding angiostatin were legirovanyh with vectors, respectively pdCs-huFc and pdCs-huFc(D4K). Stable clones NS/0, expressing the huFc-huAngio and huFc(D4K)-huAngio, selectively and analyzed as described in examples 2 and 3.

Example 5. The expression of the fused protein mouse Fc - endostatin mouse.

Indostat alnost SEQ ID NO: 18) and Fc mouse DNA sequence presented below as a sequence of SEQ ID NO: 19, the encoded amino acids are represented in the sequence SEQ ID NO: 20) were expressed as a fusion protein Fc mouse - endostatin mouse (muFc-muEndo), as substantial parts described in example 1. To accommodate endostatin cDNA (sequence SEQ ID NO: 4) for expression in vector pdCs-muFc(D4K) used PCR. Direct primer was 5’-CCC AAG CTT CAT ACT CAT CAG GAC TTT (sequence of SEQ ID NO: 21; encoded amino acids are disclosed in the sequence SEQ ID NO: 22), where after the HindIII site is a sequence encoding a N-terminal endostatin. The reverse primer was 5’-CCC CTC GAG HUNDRED TTT GGA GAA AGA GGT (sequence of SEQ ID NO: 23), which was designed to put a stop codon broadcast (anticodon, a HUNDRED) immediately after the end of endostatin, followed by an XhoI site. The PCR product was cloned and sequenced, and the obtained fragment HindIII-XhoI encoding endostatin, ligated into the vector pdCs-muFc(D4K). Stable clones NS/0 expressing muFc(D4K)-muEndo, selectively and analyzed using ELISA method for muFc, as described in examples 2 and 3.

Example 6. The expression of the fused protein mouse Fc - angiostatin mouse.

Angiostatin mouse DNA sequence presented below as posledovatelnosti mouse (muFc-muAngio), as substantial parts described in example 1. To accommodate angiostatin cDNA (sequence SEQ ID NO: 6) for expression in vector pdCs-Fc(D4K) used PCR. Direct primer was 5’-CCC AAG CTT GTG TAT CTG TCA GAA TGT AAG CCC TCC TGT CTC TGA GCA (sequence SEQ ID NO: 26; encoded amino acids are disclosed in the sequence SEQ ID NO: 27), where after the HindIII site is the sequence encoding the N-end of angiostatin. The reverse primer was 5’-CCC CTC GAG HUNDRED CCC TCC TGT CTC TGA GCA (sequence SEQ ID NO: 28), which was designed to put a stop codon broadcast (anticodon, a HUNDRED) immediately after the end of angiostatin, followed by an XhoI site (CTCGAG). The PCR product was cloned and sequenced, and the obtained fragment HindIII-XhoI encoding angiostatin, ligated into the vector pdCs-muFc(D4K). Stable clones NS/0 expressing muFc(D4K)-muAngio, selectively and analyzed using ELISA method for muFc, as described in examples 2 and 3.

Example 7. Expression of Fc dog (caFc).

For preparation of mRNA used monocyte cells in the peripheral blood of dogs (RVMS), isolated from the blood of dogs. After synthesis of the first chain cDNA with reverse transcriptase and oligo(dt) was performed PCR for amplificatorecassawireless SEQ ID NO: 29; the encoded amino acids are disclosed in the sequence SEQ ID NO: 30), which is immediately upstream from the sequence encoding the hinge region of the Fc dog entered the AflII site, and the reverse primer -5’-CTC GAG TCA TTT ACC CGG GGA ATG GGA GAG GGA TTT CTG (sequence SEQ ID NO: 31), in which, after the stop codon broadcast Fc dog (anticodon, TCA) introduced a XhoI site. The reverse primer also introduces a silent mutation to create XmaI restriction site, facilitating the design vector pdCs-caFc(D4K) using a linker-adaptor and prishivki to DNA constructs encoding endostatin or angiostatin dogs. Like the design of the pdCs-huFc, which was described in detail in the work of Lo and employees (Lo etc. // Protein Engineering. 1998. So 11. S. 495) expressing vector pdCs-caFc was constructed as follows. Fragment AflII-XhoI encoding Fc dog, was made by ligase with XbaI fragment-AflII coding for the signal peptide of light chain, and vector pdCs, split XbaI-XhoI. Received expressing vector pdCs-caFc then used for transfection of 293 cells. About 3 days after transfection the culture fluid was purified by chromatography with protein A. the Expression of Fc dog (the DNA sequence presented on how the sequence of SEQ ID NO: 32; aminosalicylates gel with SDS and subsequent analysis by Western-blotting using conjugated with peroxidase rabbit antibody to dog IgG, specific to the Fc fragment (peroixidase-cohjugated rabbit anti-dog IgG, Fc fragment specific, Jackson ImmunoResearch, West Grove, PA, USA).

Example 8. The expression of the fused protein Fc dog - endostatin dogs.

The coding sequence for endostatin dogs (the DNA sequence presented herein as a sequence of SEQ ID NO: 34; amino acid sequence presented herein as a sequence of SEQ ID NO: 35) was transformed into the fragment HindIII-XhoI for expression in the form of a fused protein with Fc, as essentially described in example 5. On the 3’-end, immediately after the codon encoding the C-terminal lysine residue, was introduced stop codon, for example, by using PCR, followed by restriction site NotI. At the 5’-end, however, there was a DraIII restriction site suitable for redevelopment. Was chemically synthesized oligonucleotide duplex containing sticky ends HindIII and DraIII; he was used to prishivki to restriction fragment DraIII-XhoI, which encode the rest of endostatin cDNA dogs. Used duplex shown below:

The first triplet CAC in duplex encodes N-terminal histidine residue of endostatin dogs. Fragment HindIII-XhoI encoding endostatin dogs full length, was therefore, to Express what is aEndo, selectively and analyzed using ELISA method for the caFc, as described in examples 2 and 3. The presence of the protein product was determined by electrophoresis in polyacrylamide gel with SDS and confirmed by analysis using Western blotting.

Example 9. The expression of the fused protein Fc dogs angiostatin dogs.

Encoding angiostatin dogs full length cDNA (DNA sequence presented herein as a sequence of SEQ ID NO: 39; the amino acid sequence presented herein as a sequence of SEQ ID NO: 40) was used for expression in the form of a fused protein with the caFc, as essentially described in the above examples. Briefly, the 3’-end, directly behind the codon encoding the C-terminal lysine residue, introduced a stop codon (e.g., by PCR), which instead of the XhoI site was followed by restriction site NotI, because angiostatin cDNA dogs were internal restriction site XhoI. At the 5’-end directly upward from the N-Terminus of angiostatin was introduced a HindIII site with the preservation of the reading frame. Then the fragment HindIII-NotI encoding angiostatin dogs full length, for expressive sewn in vector pdCs-caFc, split HindIII-NotI (NotI site was introduced into it near the Xhol site by ligating through Moana in examples 2 and 3. The presence of the protein product was determined by electrophoresis in polyacrylamide gel with SDS and confirmed by analysis using Western blotting.

Example 10. The expression of the fused protein muFc-domain K1 muAngio.

Angiostatin contains the first four of the five Kringle domains of plasminogen. To determine whether any one or more of the Kringle domains are responsible for the observed antiangiogenic activity of angiostatin, it is possible to test to produce either a single Kringle domains, or combinations thereof. To demonstrate the suitability of Fc as a partner in the fused protein expression in the first Kringle domain of mouse angiostatin (K1) was carried out as follows. The first Kringle domain in the murine angiostatin ends in the 87th position residue glutamic acid Glu-87 (the sequence SEQ ID NO: 25). In cDNA from this provision, there is a convenient NsiI restriction site, so that after cleavage by the restriction enzyme Nsil 3’-end redundancy one thread a length of 4 nucleotides were removed by T4 polymerase to create the end with the same cut DNA strands. Directly following the GAA triplet coding for Glu-87, was introduced in a downward direction of the stop codon translation proshivkoi the ligase to palindromes bridge TGA CTC GAG military angiostatin, i.e. only the first Kringle domain, was then sewn to the expression vector pdCs-muFc(D4K). Analysis using ELISA method for muFc and electrophoresis in polyacrylamide gel with SDS showed that there have been high levels of expression of both temporary and stable expression.

Example 11. The expression of the fused protein muFc-the inner part K1 muAngio.

Domain Kringle consists of multiple loops, including the outer loop and inner loop. In the first Kringle domain of mouse angiostatin inner loop is limited to cysteine residues Cys-55 and Cys-79, which form with each other a disulfide bond at the base of the loop. Cys-67 internal loop other forms disulfide bond with Cys residue of the outer loop, forming the structure of Kringle. To check whether the inner loop of any antiangiogenic activity, it was expressed in the form of a fused protein containing muFc and the inner loop of the first Kringle domain (K1), as described below. For mutation engine (mutational transformations) TGC (Cys-67) to AGC (Ser) by PCR was used as template DNA fragment encoding the first Kringle domain, together with the mutagenic primer having the sequence 5’ GGG CCT TGG AGC TAC ACT (ACA sequence of SEQ ID NO: 42; the encoded amino is if the inner loop of the first Kringle domain expressed without an external loop. For the introduction of a HindIII site in a frame is read directly from the 5’-side of the codon for Ser-43 (AGT) was used upstream primer having the sequence 5'GCGGATCCAAGCTT AGT ACA CAT CCC AAT GAG GG (sequence SEQ ID NO: 44; encoded amino acids are disclosed in the sequence SEQ ID NO: 45). Immediately after the HindIII site in the upward direction was also introduced BamHI site. The BamHI site is useful for prishivki the ligase to the BamHI site at the end of the sequence for flexible bridge Gly-Ser, shown below in example 12. In this way the DNA fragment HindIII-XhoI encoding section of the mouse angiostatin from Ser-43 to Glu-87, was sewn to the expression vector pdCs-muFc(D4K). Analysis using ELISA method for muFc and electrophoresis in polyacrylamide gel with SDS showed that there have been high levels of expression of fused protein muFc with internal loop K1 as temporary and stable expression.

Example 12. The expression of the fused protein muFc-muEndo-bridge GlySer-inner loop domain K1 muAnqio.

The hybrid molecule muFc-muEndo-inner loop domain K1 consists of the fused protein muFc-muEndo attached polypeptide bridge containing residues of glycine and serine, to the inner loop of the first Kringle domain of mouse endostatin. The structure of DNA was as follows.

On the residues of glycine and serine, fragment HindIII-BspHI length 540 base pairs encoding endostatin sewed the ligase to the oligonucleotide duplex with overlap formed by the oligonucleotides indicated in the sequences SEQ ID NO: 46 and SEQ ID NO: 48. Amino acid bridge, encoded by the sequence SEQ ID NO: 46, shown in the sequence SEQ ID NO: 47.

Fragment HindIII-BamHI encoding the mouse endostatin and bridge Gly-Ser, was subcloned into a standard cloning vector. Then, the introduction of a fragment of BamHI-XhoI encoding internal loop domain of K1 in example 11, used the BamHI site. The resulting fragment HindIII-XhoI encoding a protein muEndo-bridge Gly-Ser-inner loop K1, was sewn to the expression vector pdCs-muFc(D4K). Analysis using ELISA method for muFc and electrophoresis in polyacrylamide gel with SDS showed that there have been high levels of expression of fused protein muFc-muEndo-bridge Gly-Ser-inner loop K1 as temporary and stable expression.

Example 13. The expression of the fused protein muFc-muEndo-bridge GlvSer domain K1 muAngio.

The hybrid molecule muFc-muEndo-KI consists of a fused protein rnuFc-muEndo attached polypeptide bridge containing residues of glycine and serine, the first Kringle domain of mouse endostatin. Design DNA was when the HindIII-XhoI, codereuse the first Kringle domain of murine angiostatin (example 10), through the following adapter:

The adaptor has a sticky end HindIII’, which when legirovanii does not generate a HindIII site. So the resulting fragment HindIII-XhoI encoding muEndo-bridge GlySer-K1, was sewn to the expression vector pdCs-muFc(D4K). Analysis using ELISA method for muFc and electrophoresis in polyacrylamide gel with SDS showed that there have been high levels of expression of fused protein muFc-muEndo-bridge Gly-Ser-K1 as temporary and stable expression.

Example 14. The expression of the fused protein muFc-muEndo-bridge GIySer-muAnqio.

The hybrid molecule muFc-muEndo-bridge GIySer-muAngio is a muFc-muEndo attached containing residues of glycine and serine polypeptide bridge to mouse angiostatin. Design DNA was essentially assembled as follows. The end of the BamHI fragment HindIII-BamHI encoding muEndo-bridge Gly-Ser (example 12) was sewn to the fragment HindIII-XhoI, codereuse murine angiostatin, through the adapter described in example 13. The resulting fragment HindIII-XhoI encoding muEndo-bridge GlySer-muAngio was sewn for expression in vector pdCs-muFc(D4K). Analysis using ELISA method for muFc and electrophoresis in polyacrylamide gel with SDS so far is with a steady expression.

Example 15. The expression of the fused protein huAngio-huFc-huEndo.

The hybrid molecule huAngio-huFc-huEndo is a human angiostatin attached peptide bond huFc-huEndo. Design DNA was collected as follows. Fragment HindIII-XhoI encoding angiostatin person without stop codon, was first obtained by PCR, so that the codon for the first amino acid residue of angiostatin directly followed the sequence CTCGAG Xhol site. HindIII at the 5’-end sewn to the XbaI fragment-AflII, codereuse signal light chain peptide (Lo etc. // Protein Engineering. 1998. So 11. S. 495), through the adapter AflII-HindIII’:

Sticky end HindIII’ adaptor when legirovanii not recreate the HindIII site. On the 3’-end of the XhoI site was sewn to the site AflII fragment AflII-XhoI encoding huFc-huEndo, through the adapter XhoI’-AflII:

Sticky end XhoI’ adaptor not recreate when legirovanii the XhoI site. The resulting fragment XbaI-XhoI encoding protein: signal peptide-human angiostatin-huF-endostatin human cloned for ekspressirovali in vector pdCs. Analysis by ELISA for huFc and electrophoresis in polyacrylamide gel with SDS showed that there have been high levels explica.

In one of the cycles pharmacokinetic study of linear mice with implanted tumors Lewis lung size 100-200 mm3introduced by injection into the tail vein of protein huFc-huAngio at a dose of 720 μg per mouse. The size of the tumors and the dosage used in these studies huFc-huAngio chose to model real-world methods of treatment described by O'reilly (O'reilly // Nature Mediicine. 1996. So 2. S. 689). Blood was collected by bleeding from the feedback loop through 1/2, 1, 2, 4, 8, 24 and 48 h after injection. Blood samples were analyzed using ELISA method for huFc with subsequent analysis of Western blot testing. It was found that the half-life of huFc-huAngio in the bloodstream of mice is about 32 h, and Western analysis showed that over 90% of the molecules huFc-huAngio remain in circulation in the blood stream intact.

Pharmacokinetic studies were also repeated on Swiss (Swiss) mice without tumors at a dose of 200 μg per mouse. In this case, it was found that huFc-huAngio has a half-life in the blood line about C.

Equivalents

The invention can be implemented in other specific forms without departing from its ideas or essential characteristics. Therefore, the above examples all aspects are considered delaetsa applied the formula, instead of the previous description, and meaning, so that it includes all able to receive changes that are within the meaning and scope of equivalence formulas.

Claims

1. Homodimeric protein having the activity of an inhibitor of angiogenesis, obtained by recombinant methods in which each Monomeric chain of glycosilated contains the Fc region of immunoglobulin gamma, which includes the hinge region, the domain of CH2and the domain of CH3and the target protein, comprising one or more molecules having the activity of an inhibitor of angiogenesis, which occurs from angiostatin or endostatin, are related to each other either directly or via a polypeptide of the bridge, with the specified target protein attached, either directly or via a polypeptide of the bridge to the N-end or From the end of the specified Fc region of immunoglobulin.

2. Homodimeric protein under item 1, characterized in that the specified target protein contains two molecules with activity inhibitor of angiogenesis associated with each other, either directly or through politcal selected from the group including angiostatin, endostatin, a fragment of plasminogen, possessing activity of angiostatin, a fragment of collagen XVIII, has the activity of endostatin.

4. Homodimeric protein according to any one of paragraphs.1-3, characterized in that the target protein attached to the C-end of the Fc region of immunoglobulin gamma.

5. Homodimeric protein having the activity of an inhibitor of angiogenesis, obtained by recombinant methods in which each Monomeric chain of glycosilated contains the Fc region of immunoglobulin gamma, which includes the hinge region, the domain of CH2and the domain of CH3and the first target protein, comprising one or more molecules having the activity of an inhibitor of angiogenesis, which occurs from angiostatin or endostatin, are related to each other either directly or via a polypeptide of the bridge, and the second target protein, comprising one or more molecules having the activity of the inhibitor of angiogenesis is selected from the group including angiostatin, endostatin, a fragment of plasminogen, possessing activity of angiostatin, a fragment of collagen XVIII, has the activity of endostatin, and related to each other either directly or via a polypeptide of the bridge, and Odie the target protein attached, either directly either through a polypeptide of the bridge to the end of the Fc region of immunoglobulin gamma.

6. Homodimeric protein under item 5, characterized in that the first and/or second target protein consists of two molecules with the activity of the inhibitor of angiogenesis is selected from the group including angiostatin, endostatin, a fragment of plasminogen, possessing activity of angiostatin, a fragment of collagen XVIII, and are related to each other either directly or via a polypeptide connection.

7. Homodimeric protein according to any one of paragraphs.1-6, characterized in that the molecule having the activity of angiostatin, contains the amino acid sequence presented in the sequence SEQ ID NO: 11, and the molecule having the activity of endostatin, contains the amino acid sequence presented in the sequence SEQ ID NO:4.

8. The DNA molecule encoding homodimeric protein as defined in any of paragraphs.1-7, including (a) the Fc region of immunoglobulin gamma, which includes the hinge region, the domain of CH2and the domain of CH3; (b) the target protein.

9. The DNA molecule under item 8, characterized in that the DNA sequence encoding the Fc region of an immunoglobulin that comprises a nucleotide sequence that represent the ü DNA encoding the target protein comprises the nucleotide sequence presented in the sequence SEQ ID NO: 10 and/or SEQ ID NO: 3.

11. The vector for the expression of homodimeric fused protein as defined in any of paragraphs.1-7, capable of replication in mammalian cells containing a DNA molecule as defined in any of paragraphs.8-10, and the DNA sequence encoding a signal peptide.

12. The way transfection of mammalian cells, comprising introducing into the specified cell expression vector, as defined in paragraph 11.

13. The method of obtaining homodimeric fused protein as defined in any of paragraphs.1-7, includes the following steps: (a) transfection of mammalian cells with a vector as defined in paragraph 11 (b) culturing mammalian cells for production of the specified fused protein; (c) the allocation specified fused protein.



 

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