Hgf beta-chain versions

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention relates to molecular biology and can be used in designing agent and methods of modulating body functions associated with HGF/c-met signalling pathway. The invention discloses HGF/c-met polypeptide-antagonists which are mutant forms of HGF which contain a mutation in the N-terminal part of the β-chain and/or in its dimerisation part. The disclosed polypeptides have lower biological activity compared to wild type HGG and can be used in modulating activity of c-met, cell proliferation, cell migration and angiogenic cell activity.

EFFECT: invention describes a method of obtaining HGF muteins using DNA recombinant technology and agents which are necessary for its existence.

22 cl, 8 dwg, 1 ex

 

Related applications

This application is nepredvidatelne application filed under 37 CFR 1.53 (b)(1), which claimed priority under 35 USC 119(e) on provisional application number 60/671 610, filed April 15, 2005, the contents of which are fully incorporated into the present description by reference.

The technical field to which the invention relates

The present invention relates, generally, to the field of molecular biology and regulation of growth factors. More specifically the present invention relates to modulators of signaling pathways HGF/c-met and to the use of these modulators.

Background of invention

Growth factor hepatocyte (HGF), also known as scatter factor or dissipation factor (SF), is a ligand for Met (Bottaro et al., 1991), a receptor tyrosine kinase that is encoded by protooncogenesc-met(Cooper et al., 1984a &b). Binding of HGF to Met induces phosphorylation of the intracellular domain of the kinase, leading to activation of complex intracellular pathways, and this, in turn, leads to growth, differentiation and migration of different cell types; in a number of surveys in recent years to give a detailed description of these processes (Birchmeier et al., 2003; Trusolino and Comoglio, 2002; Maulik et al., 2002). It has been shown that signaling pathway HGF/Met with fundamentally the value in embryonic development and tissue regeneration also involved in invasive tumor growth and metastasis and, as such, represents an interesting therapeutic target (Birchmeier et al., 2003; Trusolino and Comoglio, 2002; Danilkovich-Miagkova and Zbar, 2002; Ma et al., 2003).

HGF belongs to the family of the plasminogen-related growth factor and includes α-chain of size 69 kDa, containing N-terminal finger domain (N) and four Kringle domains (Kringle; K1-K4), and β-chain size of 34 kDa, which has great similarity with domains of the protease chymotrypsin-like serine protease family of Clan PA(S)/FamilyS1 (Nakamura et al., 1989; Donate et al., 1994; Rawlings et al., 2002). Like plasminogen and other zymogram serine protease HGF is secreted as single-stranded precursor (scHGF). Next scHGF associated with heparan sulfate proteoglycans, such as syndecan-1 (Derksen et al., 2002), on cell surfaces or in the extracellular matrix. Heparan-sulfate proteoglycans associated with the N-domain (Hartmann et al., 1998), which contributes to the high binding affinity of Met with amino acids located in K1 (Lokker et al., 1994). Although scHGF able to contact Met with high affinity, it may not activate the receptor (Lokker et al., 1992; Hartmann et al., 1992). The acquisition of HGF signaling activity occurs when the proteolytic cleavage (activation) scHGF in position Arg494-Val495 that leads to the formation of Mature HGF, Jerome is and α/β, linked by a disulfide bond (Lokker et al., 1992; Hartmann et al., 1992; Naldini et al., 1992). Proteinopathy domain of HGF (β-chain HGF) has no catalytic activity, because it does not contain the desired catalytic triad Asp[c102]-His[c57]-Ser[c195] (in the text of the description uses the standard numbering of chymotrypsinogen with the inclusion of parentheses), are detected in all of the serine proteases (Perona and Craik, 1995; Hedstrom, 2002), including Gln534[c57] and Tyr673[c195].

Due to the importance of the signaling activity of HGF this process should be tightly controlled HGF-converting enzymes and their respective physiological inhibitors. Activation of scHGF mediatedin vitrothe chymotrypsin-like serine-protease, comprising the activator growth factor hepatocyte (HGFA) (Miyazawa et al., 1993), matriptase/MT-SP1 (Takuchi et al., 1999; Lin et al., 1999), plasminogen activator urokinase type (Naldini et al., 1992), factor XIIa (Shimomura et al., 1995), factor XIa (Peek et al., 2002) and kallickrein plasma (Peek et al., 2002). As scHGF, these proteases are produced as inactive precursors; their enzymatic activity is also tightly regulated by other activating a protease and inhibitors of both types, as Martens-type (Kunitz)and serbinova type.

Serine protease and the process of their activation are described in the literature (Donate et al., 1994). Upon activation of serine proteases is the splitting of Imogene, with the latter the overall conformational rearrangement of the so-called "activating domain", which leads to the formation of suitably formed active site and the interaction region of the substrate/inhibitor. Activating domain includes three surface loops, denoted as [c140]-, [c180]- and [c220]-loop, and contains the insertion of the newly formed N-Terminus in a hydrophobic pocket (Huber and Bode, 1978). In a homologous pair of ligand/receptor for protein, stimulating macrophages (MSP)/Ron), β-chain MSP, similar to the serine protease, provides the basic energy for the receptor binding (Wang et al., 1997; Miller and Leonard, 1998). This process is a reversal system HGF/Met, where the high-affinity binding site of the receptor with the remnants of the Met is in the α-chain of HGF (Lokker et al., 1994; Okigaki et al., 1992).

The importance of signaling axis HGF/Met in the processes of cellular functioning in terms of standards and in the etiology of clinical disorders indicates its possible importance in the development of highly effective therapeutic agents based on the modulation of this axis. However, the complexity of the specified path, partly due to the poorly understood mechanism of interaction of HGF-HGF and HGF/Met, slows down progress in this direction and leads to the need to develop approaches based on a better understanding of the mechanism of interaction of HGF-HGF and HGF/Met. The description below meets this need and provides other benefits.

Description of the invention

Growth factor hepatocyte (HGF), a plasminogen-related growth factor, binds to the receptor tyrosine kinase Met (also known as C-Met, C-Met or C-met), which is involved in development, tissue regeneration and invasive tumor growth. Herself β-chain of HGF-type serine protease associated with the Met. In addition, what happens binding to Met, it is not clear what area and what specific residues in the β-chain of HGF necessary to implement the corresponding signal function on the way HGF/Met. The authors believe that certain areas/positions in the β-chain make an important contribution to the appropriate functional activity of HGF, whereas the contributions may involve or may not involve the binding of the β-chain of HGF with its corresponding receptor. In the present description, the results confirm the fact that mutation of the N-terminal site and/or site of dimerization of β-chain of HGF can disrupt biological function of HGF/Met, in the presence or in the absence of a material breach of binding HGF (in particular the β-chain of HGF C-Met. Mostly, but not necessarily, these mutations do not involve those provisions, which are believed to contain "activating domain or region of the active site of HGF on the who type.

Mutational analysis given in this description provides the basis for the development of a multitude of HGF mutants that are able to inhibit the interaction of HGF/HGF and HGF/C-met wild type on a wide range of functions. Examples of such mutants are given in the present description. These mutants are able to compete with wild-type HGF for binding to c-met, demonstrating a reduced ability to influence c-met-associated biological functions. This is particularly important in the case where it is not desired full or substantial inhibition of the axis of the HGF/c-met; this capability is very significant, since HGF and c-met is widely expressed in normal cells and tissues. Such mutants can also be used as a useful therapeutic agents for the treatment of pathological conditions where it is desirable reduction, but not complete absence of biological activity of HGF/c-met. The methods and compositions according to the present invention is based, at least in part, on those facts that were discovered by the authors and which are described below in more detail.

In one aspect the present invention relates to the molecule-antagonist of HGF/C-Met comprising a mutant HGF, which contains a mutation in the N-terminal site of the β-chain of HGF and/or at the site of dimerization of β-chain of HGF.

The mutation in the N-terminal site of the β-chain of HGF may be a l the buoy mutation, which violates the insertion of the N-terminal β-chain of HGF-binding pocket. In one embodiment, the resulting mutant β-chain of HGF binds to C-Met with reduced binding affinity compared to the β-chain of HGF wild type. In one embodiment, the resulting mutant β-chain of HGF binds to C-Met with essentially equivalent affinity, as in the case of the β-chain of HGF wild type. In one embodiment, the received full-HGF containing the mutated β-chain of HGF, binds to C-Met with reduced binding affinity compared to the full-size wild type HGF. In one embodiment, the received full-HGF containing the mutated β-chain of HGF, binds to C-Met with essentially equivalent affinity as the full-size wild type HGF. In one embodiment, the mutation is located in the P1' position (i.e. the position 495[c16]) or in the adjacent position, where this mutation leads to the production of fissionable HGF mutant, and where N is the end of the β-chain of HGF is not integrated into the active site or binding pocket. Examples of the inability to fit into the active site or binding pocket include, without limitation, the configuration in which the mutant is defective or one or both of (i) hydrophobic interactions and (ii) by formation of a salt bridge connecting the N-end with Asp672[c]194, that is, where N is the end contains a mutation, for example, includes the positive saragani is replaced or the native amino acid residue. In one embodiment, the alarm function via this mutant is impaired. In one embodiment, the mutation is in one or several positions P1', P2', P3' and P4' or beside them.

A mutation in the dimerization domain of the β-chain of HGF can be any mutation, which is expected to break the contact between the two β-chain of HGF, so that dimerization of two chains (and thus, two HGF molecules) is violated. The possibility of such mutations is determined by amino acid structure of HGF complexes, for example, as described in the work Kirchhofer et al., J. Biol. hem. (2004), 279(38):39915-24. Relevant amino acid positions include, without limitation, in the present description of the position. In one embodiment, the obtained mutant HGF has a reduced ability to timeresults with other β-chain of HGF. In one embodiment, the mutation in the region of the dimerization of β-chain of HGF significantly does not violate the binding obtained HGF mutant C-Met.

Domain dimerization refers to the plot of the β-chain of HGF, which interacts with other β-chain of HGF with the formation of a dimer (for example, in the complex activation of HGF/Met). The splitting of proHGF β-chain of HGF undergoes a conformational change. The N-terminal residue 495 in the β-chain of HGF forms a salt bridge with residue Asp 672. In some embodiments, the site of dimerization of β-chain of HGF includes amino acid residues, essentially consists of amino acid residues and which consists of at least one amino acid residue (until all amino acid residues), the corresponding residues of the β-chain of HGF in the area from about 495 to about 502 amino acids [loop c140], including Y619, T620, G621, [c180] loop including plot 662-665, or mixtures thereof. In one embodiment, the dimerization domain includes provisions localized close/near one or more of the provisions listed above, and would thus be expected to influence these one or more provisions. For example, in one embodiment, the dimerization domain may also include provisions 622 and 626.

In one aspect, the molecule antagonist of HGF/Met according to the present invention includes a mutation in the N-terminal site of the β-chain of HGF, where this mutation is in position V495, G498, R502 plus T503 and/or D672. The mutation may be provided in any form that will change the primary, secondary and/or tertiary structure of the N-terminal site of the β-chain of HGF. For example, in one embodiment, the mutation of the N-terminal site of the β-chain of HGF is a substitution, insertion and/or deletions, such as V495G, V495A, G498I, G498P, G498V, R502del plus T503del or D672N. In another embodiment, the mutation in the N-terminal site of the β-chain of HGF represents a deletion V495. A mutation that changes the primary, secondary and/or tertiary structure of the N-terminal site of the β-chain of HGF may also be in the position of any amino acid, which itself is not in the N-terminal site of the β-chain of HGF. For example, mutation D672 to what I eliminates formation of a salt bridge (for example, D672N) N-end of the β-chain of HGF is expected to change the primary, secondary and/or tertiary structure of the N-terminal site of the β-chain of HGF. Thus, mutations in the N-terminal site of the β-chain of HGF and plot the dimerization of β-chain of HGF does not have to be mutually exclusive. For example, as will be described in this text and as shown in figure 1, the mutation of certain provisions expected to affect the N-terminal domain and dimerization domain in the β-chain of HGF.

In one aspect, the molecule antagonist of HGF/Met according to the present invention includes a mutation in the dimerization domain of the β-chain of HGF, where this mutation is in position N497, G498, P500 or near T501 and R502 or R502. The mutation may be of any shape, which changes the primary, secondary and/or tertiary structure of the site of dimerization of β-chain of HGF. Examples of mutations that change the structure of the site of dimerization of β-chain of HGF include mutations that introduce a charged residue or a residue with a large side chain (e.g., volume), in the sequence of the wild type, where the charged residue may cause interactions by type of repulsion, and a large side chain may affect the occurrence of unfavorable steric interactions. In addition, cysteine mutations (for example, L622C, I664C, P500C and N497C) can also be entered so that they become available for modification with ecificatio thiol-alkylating reagents, such as agents, containing maleimide and halogenoacetyl group. In one embodiment, the mutation at the site of dimerization of β-chain of HGF is a substitution, insertions and/or deletions, such as N497R or K; G498A or S; P500W, H or E; the insertion between T501 and R502 (for example, the insertion of R and/or S); or R502del. In one embodiment, the mutation in position N497 cannot be N497F, A or E. In one embodiment, the mutation is in one or several positions on the plot 495-503, where this mutation may change the dimerization of β-chain of HGF and/or binding to the receptor. In another embodiment, mutations that affect the dimerization domain can be combined with a mutation in one or more positions outside the domain dimerization, for example, it may be a mutation in the cleavage site 494-495 or near it. For example, in the case of the mutant, which is expected to be necessasary (for example, the double mutant R494E:V495G) and which also contains a mutation in the dimerization domain, such a mutant will nevertheless demonstrate impaired biological function, even if he would be subjected to cleavage in vivo.

In some embodiments, the molecule antagonist of HGF/Met according to the present invention will include amino acids of the wild type at position 534, 578, 619, 673, 692, 693, 694, 695, 696, 699 and/or 702. In some embodiments, the molecule antagonist of HGF/Met according to the present invention includes mutati is in position L622 (for example, L622C or K); I623 (for example, I623C); D626 (for example, D626K); L622 plus D626 (for example, L622K plus D626K); K663 (for example, K663C); I664 (for example, I664C); R502 (for example, 502C); P500 (for example, P500C); N497 (for example, N497C); R494 plus I623 (for example, R494E plus I623C); N497 plus G498 (for example, N497R plus G498A or N497K plus G498A); N497 plus P500 (for example, N497R plus P500H or N497K plus P500H); G498 plus P500 (for example, G498A plus P500H); N497 plus G498 plus P500 (for example, N497R plus G498A plus RN or N497K plus G498A plus P500H); N497 plus L622 (for example, N497R plus L622K or N497K plus L622K); N497 plus D626 (for example, N497R plus D626K or N497K plus D626K); N497 plus L622 plus D626 (for example, N497R plus L622K plus D626K or N497K plus L622K plus D626K).

In one embodiment, the molecule antagonist of HGF/Met according to the present invention includes a mutation in the active site of HGF, alone or in combination with one or more of the mutations described in the present description. Mutations in the active site include a mutation at position 667 and/or 704. Suitable mutations include substitution of one or both of these provisions on or W.

Basically, the molecule antagonist of HGF/Met according to the present invention includes the HGF molecule containing a mutation in the β-chain of HGF, which reduces one or more biological characteristics normally associated with wild type HGF. For example, in one embodiment, the molecule has a reduced C-Met signaling ability (for example, phosphorylation of Met) compared with wild-type HGF. In d the natives embodiment, the molecule has a reduced ability to stimulate migration of cells compared to wild type HGF. In another embodiment, the molecule has a reduced ability to stimulate cell proliferation compared to wild type HGF. In another embodiment, the molecule has a reduced ability to stimulate angiogenesis compared to wild type HGF. Molecule antagonist of HGF/Met according to the present invention basically comprises at least a part of the chain of HGF, which is involved in binding to Met, attached to the mutated β-chain of HGF, according to the present description.

As shown by the results of the mutational analysis described in the present description, some parts of the β-chain of HGF and specific provisions of amino acids, play an important role in the modulation of the biological functions of HGF. Accordingly in one aspect the present invention relates to modulators of HGF/Met, which are specifically aimed at these areas. Such modulators include nucleic acids such as aptamers, polypeptides, such as binding peptides and antibodies.

In the context of the present description, the letter facing numeric indicator means corresponding amino acid of the wild type, the existing position of the amino acids, denoted by the given number, in the HGF polypeptide of wild type, and one or more letters (if present) after the numeric indicator to indicate the type of mutation/amino acid (for example, substitution of AMI is ocelote, a deletion (del) or insertion (ins)).

In one aspect the present invention relates to a mutant HGF, which has a modulatory activity against HGF/c-met, for example, the antagonist activity of HGF/c-met or HGF variant, demonstrating a reduction, but not the lack of biological activity of HGF (e.g., activity, stimulating cell growth). In one embodiment, the antagonist according to the present invention is able to inhibit the biological activity of HGF wild-typein vivoorin vitrosuch biological activity includes, without limitation, the phosphorylation of the receptor, the stimulation of cell proliferation, increased survival of cells, acceleration of angiogenesis, induction/enhancement of cell migration). In one embodiment, the mutant HGF provides reduced activity of promoting cell growth (e.g., cell proliferation, cell survival, angiogenesis, cell migration).

In one embodiment, the molecule antagonist according to the present invention competes with wild-type HGF for binding to Met. In some embodiments, this molecule inhibits multimerization receptor c-met (e.g., dimerization). In some embodiments, the specified molecule includes a variant (mutant) β-chain, with a reduced ability to interact (for example, to multimerization/dimerization) with β-chain another mod is ecoli. In some embodiments, this molecule inhibits multimerization β-chain of HGF (e.g., dimerization). In some embodiments, this molecule binds to c-met, but shows a reduced ability to influence the activation of c-met (e.g., as detected by reduced phosphorylation of c-met phosphorylation of mitogen-activated protein kinase (MAPK) and/or decrease dependent on HGF/c-met cell migration, cell proliferation, survival rates of cells, cell morphogenesis, angiogenesis, and the like).

In any molecule according to the present invention, where one or more provisions were metirovan relative to the corresponding sequence of the wild type, this mutation may be provided in any form that changes the functional effect of the corresponding residue of the wild type. The mutation can be obtained by any method known in this field (and/or may be determined empirically, for example by substitution, insertions, additions and/or deletions. In some embodiments, this mutation includes non-conservative substitution. Suitable substitutions include, without limitation, the substitution given in this description (in particular, in the following examples), for example, amino acids such as alanine or serine.

In one aspect, the molecule/substance (for example, fashion is atory HGF/c-met, in the present description) is associated with a toxin, such as a cytotoxic agent. These molecules/substances can be obtained in the composition or may be combined with additional reinforcing agent such as radiation and/or chemotherapeutic agent.

The present invention also relates to methods and compositions used to modulate pathological conditions associated with dysregulation of signaling axis HGF/c-met. Thus, the present invention in one aspect relates to a method of modulating the activation of c-met in a subject comprising administration to the subject molecule antagonist of HGF/c-met according to the present invention, thereby achieving modulation of the activation of c-met. In one embodiment, this molecule is an antagonist of HGF/c-met, which inhibits the activity of HGF/c-met. In one embodiment, the specified antagonist inhibits the specific binding of the β-chain of HGF wild type c-met. In one aspect the present invention relates to a method of treating pathological conditions associated with activation of c-met in a subject comprising administration to the subject an antagonist of c-met according to the present invention, resulting in inhibited activation of c-met.

Signaling pathway HGF/c-met is involved in multiple biological and physiological functions, including the possibility that for example, stimulation of cell growth (e.g., cell proliferation, cell survival, cell migration, cell morphogenesis and angiogenesis. Thus, in another aspect, the present invention relates to a method of inhibiting cell growth, activated C-met (for example, proliferation and/or survival), where the method includes contacting the cell or tissue with an antagonist according to the present invention, which is achieved by the inhibition of cell proliferation is associated with activation of c-met. In another aspect the present invention relates to a method of inhibiting angiogenesis, comprising introducing into the cell, tissue and/or in the body of a subject with a pathological condition associated with abnormal angiogenesis antagonist of HGF/c-met according to the present invention, which results in the inhibition of angiogenesis.

In one aspect the present invention relates to the use of the antagonist according to the present invention in the manufacture of a medicinal product for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor, cell proliferative violation, immune disorder (such as an autoimmune disease and/or disorder associated with angiogenesis. Specified antagonist can be any prevedeno is in this form, including the form of the antibody, antibody fragment, polypeptide (e.g., oligopeptides, mutant/variant polypeptide HGF), nucleic acids (aptamers) or combinations thereof.

In one aspect the present invention relates to the use of nucleic acid according to the present invention in the manufacture of drugs for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor, cell proliferative violation, immune disorder (such as an autoimmune disease and/or disorder associated with angiogenesis.

In one aspect the present invention relates to the use of the expression vector according to the present invention in the manufacture of drugs for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor, cell proliferative violation, immune disorder (such as an autoimmune disease and/or disorder associated with angiogenesis.

In one aspect the present invention relates to the use of host cell according to the present invention in the manufacture of drugs for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor, cell proliferative violation, immune disorder (such as an autoimmune disorder and/or disorders associated with what angiogenesis.

In one aspect the present invention relates to the use of the product according to the present invention in the manufacture of drugs for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor, cell proliferative violation, immune disorder (such as an autoimmune disease and/or disorder associated with angiogenesis.

In one aspect the present invention relates to the use of the kit according to the present invention in the manufacture of drugs for therapeutic and/or prophylactic treatment of diseases, such as cancer, tumor, cell proliferative violation, immune disorder (such as an autoimmune disease and/or disorder associated with angiogenesis.

In one aspect the present invention relates to a method of inhibiting cell proliferation, activated c-met, comprising contacting the cell or tissue with an effective amount of an antagonist according to the present invention, thereby achieving the inhibition of cell proliferation is associated with activation of c-met.

In one aspect the present invention relates to a method of treating pathological conditions associated with impaired regulation of c-met activation in a subject, where the method includes the introduction mentioned the subject an effective amount of an antagonist according to the present invention, as a result the treatment of the specified condition.

In one aspect the present invention relates to a method of inhibiting growth of a cell that expresses c-met or a growth factor for hepatocytes, or both of them, where the method includes contacting the specified cell with an antagonist of c-met according to the present invention, which leads to inhibition of growth of the specified cells. In one embodiment, the specified cell is brought into contact with HGF expressed by another cell (for example, through a paracrine effect).

In one aspect the present invention relates to a method of therapeutic treatment of a mammal having a cancerous tumor, which comprises a cell that expresses c-met or a growth factor for hepatocytes, or both of them, where the method includes the introduction of the specified mammal an effective amount of an antagonist according to the present invention, which leads to effective treatment specified mammal. In one embodiment, the specified cell is brought into contact with HGF expressed by another cell (for example, through a paracrine effect).

In one aspect the present invention relates to a method of treatment or prevention of cell proliferative disorders associated with increased expression or activity of c-met or growth factor hepatocyte is in, or both, where the method includes an introduction to the subject, if there is a need for such treatment, an effective amount of an antagonist according to the present invention, which leads to effective treatment or prevention of a specified cell-proliferative disorders. In one embodiment, the specified proliferative violation is a cancer.

In one aspect the present invention relates to a method of inhibiting cell growth, where the growth in this cell, at least partially, dependent upon the growth potentiating effect of c-met or growth factor hepatocyte, or both, where the method includes contacting the specified cell with an effective amount of an antagonist according to the present invention, which results in inhibition of growth of the specified cells. In one embodiment, the cell is brought into contact with HGF expressed by another cell (for example, through a paracrine effect).

In one aspect the present invention relates to a method of therapeutic treatment on the tumor of a mammal, where the growth in this tumor, at least partially, dependent upon the growth potentiating effect of c-met or growth factor hepatocyte, or both, where the method includes contacting the specified cell with an effective amount of an antagonist according to this the mu to the invention, to achieve effective treatment of this tumor. In one embodiment, the cell is brought into contact with HGF expressed by another cell (for example, through a paracrine effect).

The methods according to the present invention can be used to influence any suitable pathological condition, for example, cells and/or tissues associated with impaired regulation of HGF/c-met signaling pathway. In one embodiment, the cell that is targeted in the method according to the present invention, is a cancer cell. For example, the cancer cell can be a cell selected from the group consisting of: cancer cells are breast cancer cells, colorectal cancer cells, lung cancer cells, papillary carcinoma (e.g., cells of the thyroid gland)cells, colon cancer cells, pancreatic cancer cells, ovarian cancer, cervical cancer cells, cancer cells of the Central nervous system, cells of osteogenic sarcoma, carcinoma cells kidney cells, hepatocellular carcinoma cells, bladder cancer cells, gastric cancer cells, squamous cell carcinoma of the head and neck, melanoma cells, cells of multiple melanoma and leukemia cells. In one embodiment, the cell that is targeted in the method according to the present invention, is the way the th hyperproliferative and/or hyperplasias cell. In one embodiment, the cell that is targeted in the method according to the present invention, is depletions cell. In yet another embodiment, the cell that is the target of the method according to the present invention, is a metastatic cell.

The methods according to the present invention may also include additional stages of treatment. For example, in one embodiment, the method also includes a step in which the target cell and/or the target tissue (e.g., a cancer cell) is subjected to irradiation or exposure to a chemotherapeutic agent. In one embodiment, the molecule antagonist of HGF/Met according to the present invention is administered to a subject with one or more other therapeutic agents, for example with erlotinib (TARCEVA®), pemetrexed (ALIMTA®), bevacizumab (AVASTIN®), gefitinib (IRESSA®), trastuzumab (HERCEPTIN®) and rituximab (RITUXAN®). The introduction of therapeutic agents to the combination therapy can be carried out simultaneously or in sequential mode.

As described in the present description, c-met activation is an important biological process, the dysregulation of which leads to the development of many pathological conditions. Accordingly, in one embodiment, methods according to the present invention the cell, which is Misha is updated (for example, cancer cell), is a cell in which the activation of c-met increased compared with normal cell tissue of the same origin. In one embodiment, the method according to the present invention causes the death of target cells. For example, contact with the antagonist according to the present invention can lead to the inability of the cell to transmit a signal at c-met, which leads to cell death.

Dysregulation of c-met activation (and thus the signal function) may occur due to a variety of cellular changes, including, for example, the overexpression of HGF (cognatic c-met-ligand) and/or c-met. Accordingly, in some embodiments, the method according to the present invention includes effects on the cell, in those conditions, when c-met, or a growth factor for hepatocytes, or they both expressed in enhanced quantities of this cell (e.g. a cancer cell) as compared to normal cells in the tissue of the same origin. Cell expressing c-met, may be subject to regulation under the action of HGF, originating from many sources, such as autocrine or paracrine manner. For example, in one embodiment, methods according to the present invention the target cell is brought into contact associated with the growth factor for hepatocytes, expressed in another cell (e.g. the, through a paracrine effect). This is another cell may be derived from the same tissue or from tissue of different origin. In one embodiment, the target cell is brought into contact/connect with HGF, expressionism with the cell-target (e.g., via autocrine effect/loops).

In some embodiments, the antagonists of HGF/Met in accordance with the present invention include mutants of HGF, which contain modifications that enhance their inhibitory and/or therapeutic effect (including, for example, increased affinity, improved pharmacokinetic properties (such as half-life, stability, rate of clearance) reduced toxicity to the subject). Appropriate modification of this kind include, for example, glycosylation, paglierani, substitution unnatural, but functionally equivalent amino acid linking groups and other Such modifications are known in this field and can be determined empirically, if necessary.

In one aspect the present invention relates to compositions comprising one or more antagonists of HGF/c-met according to the present invention and a carrier. In one embodiment, the carrier is pharmaceutically acceptable.

In one aspect the invention relates to nucleic acids encoding the antagonist of HGF/c-met according to the present from which retenu. In one embodiment, the nucleic acid according to the present invention encodes an antagonist of HGF/c-met, which is the polypeptide or the polypeptide (for example, a mutant/variant HGF). In one embodiment, the nucleic acid according to the present invention encodes an antagonist of HGF/c-met, which is an antibody or its fragment or includes the antibody or fragment.

In one aspect the present invention relates to vectors comprising the nucleic acid according to the present invention.

In one aspect the present invention relates to cells of a host comprising the nucleic acid or vector according to the present invention. The vector may be of any type, for example, it may be a recombinant vector such as an expression vector. Can be used by any representative of the many well-known host cells. In one embodiment, a host cell is a prokaryotic cell, such as E. coli cell. In one embodiment, a host cell is a eukaryotic cell, such as mammal cells, such as cell Chinese hamster ovary (CHO).

In one aspect the present invention relates to methods of producing an antagonist of HGF/c-met according to the present invention. For example, the present invention relates to a method for the antagonist, which made the focus of an antibody (or fragment) or the antibody (or fragment), where the method includes the expression in a suitable cell host recombinant vector according to the present invention, encoding the indicated antibody (or fragment), and the allocation of the indicated antibodies. In another example, the present invention relates to a method for producing an antagonist of HGF/c-met, which is a polypeptide or the polypeptide (such as a mutant/variant HGF), where the method includes the expression in the appropriate cell host recombinant vector according to the present invention, encoding the specified polypeptide, and the allocation of the specified polypeptide.

In one aspect the present invention relates to a product comprising a container and a composition contained in the container where the specified composition includes one or more antagonists of HGF/c-met according to the present invention. In one embodiment, the composition includes a nucleic acid according to the present invention. In one embodiment, the composition comprising the antagonist of HGF/c-met, also includes the media, which in some embodiments is pharmaceutically acceptable. In one embodiment, the product according to the present invention includes instructions for introducing the composition to the subject.

In one aspect the present invention relates to a kit comprising a first container provided is a composition which includes one or more antagonists of HGF/c-met according to the present invention; and a second container containing a buffer. In one embodiment, the buffer is pharmaceutically acceptable. In one embodiment, the composition comprising the antagonist of HGF/c-met, also includes the media, which in some embodiments is pharmaceutically acceptable. In one embodiment, the kit also contains instructions for the introduction of the composition to the subject.

Brief description of drawings

Figure 1 (A) illustrates the characteristics of different mutants of HGF. Examples of mutants with N-terminal insertions in the β-chain of HGF and mutants on the site of dimerization of β-chain of HGF. Data "cell migration" refers to the migration of cells MDA-MB435 in the presence of full-HGF containing the indicated mutation(mutation), where the data are expressed as percentage relative to migration in the presence of wild type HGF. Data for the binding of HGF β/MetIgG" refers to the binding of the β-chain of HGF (containing the indicated mutation(mutation) with MetIgG, where the data are expressed as ratios of the IC50(mutant) to the IC50(for molecules wild-type), where the data obtained in the competitive test for binding. In the data "wild type" refers to the mutant 604S; mutants of HGF β also contain this mutation. It should be noted that in the above materials mutations decree is given in bold, if they expected to break a potential interaction type β-chain β-chain, and mutation are highlighted in italics and underlined (bold or plain font), if they expected to interfere with the insertion of the N-end. These assumptions are based on the dominant effect observed or expected for the corresponding mutations, that is determined by the impact or interaction type β-chain β-chain, or on the ability of the N-terminal β-chain embedded in the active site or binding pocket. However, any practitioner can easily determine, does a specific mutation of one or both of these effects, regardless of whether you specify it as such or not, figa. For example, in some cases, the mutation may affect the interaction type β-chain β-chain, and the insertion of the N-end, or in some cases, the mutation shown in figa, which is expected to have an effect on the interaction type β-chain β-chain, will also, as it can be empirically demonstrated to affect the insertion of the N-end. Similarly, the magnitude is clearly "broken" link Met are indicated in italics, and values explicitly "normal" binding are shown in bold, although the degree of "violations" and "normality" are relative concepts.

(B) Binding of full-HGF, sod is rasego specified mutation (mutation) with Met, the result of the evaluation test on competitive binding. Data are expressed as the ratio of the IC50(mutant) to the IC50(for molecules wild type).

(C) Inhibition of cell migration and proliferation full HGF containing the indicated mutation (mutation). The level of cell migration and proliferative activity, respectively, in the presence of mutant HGF and HGF wild-type (1 nm for estimation of migration in the presence of HGF wild type; 0,25 nm for assessment of cell proliferation in the presence of wild-type HGF) is expressed as the percentage relative to the activity observed in the presence of one wild-type HGF.

Figure 2 (A) Shows the inhibition of HGF-dependent Met phosphorylation in cells A HGF mutants; designation R494A:R494E relates to single-chain HGF. The level of phosphorylation of Met is listed as RLU (relative light units). (B) Shows the inhibition of HGF-dependent phosphorylation of Met in lung carcinoma cells A mutants of HGF. The level of phosphorylation Met specified as a percentage relative to control (which represents the number observed in the presence of 0.5 nm HGF wild type).

Figure 3 (a) and (B) Met Phosphorylation in cells A in the presence of HGF wild-type and mutant HGF. The level of phosphorylation of Met are shown as percentage relative to the maximum phosphorylation observed in the presence of HGF the IR type for each corresponding concentrations of HGF wild-type.

Figure 4 Angiogenic activity in the presence of mutant HGF. The level of angiogenesis is shown as the number of seedlings/granules in the presence of HGF mutants.

Ways of implementation of the present invention

The present invention relates to methods, compositions, kits and products used to modulate signaling pathways HGF/c-met.

Below is a detailed description relating to these methods, compositions, kits and articles.

The basic methodology

In practice, the implementation of the present invention can be used, unless specifically stated otherwise, standard methods of molecular biology (including recombinant techniques), Microbiology, cell biology, biochemistry and immunology, which are known to experts in this field. Such techniques are well described in the literature, in particular, in such works as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis (M.J. Gait, ed., 1984); "Animal Cell Culture (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F.M.Ausubel et al., eds., 1987, including periodic updates); "PCR: The Polymerase Chain Reaction", (Mullis et al., ed., 1994); "A Practical Guide to Molecular loning" (Perbal Bernard V., 1988).

Definitions

References to the names of the amino acids described in the present description, are adopted in this area designations, in the form of one or more forms, which is used interchangeably in this text: (I) full name (for example, tryptophan, serine, glycine and the like), (ii) the three-letter abbreviations (e.g., Trp, Ser, Gly, etc.), and (iii) a single-letter designation (for example, W, S, G and so on).

The term "percent (%) identity in amino acid sequence with respect to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in the intended sequence that are identical with amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing, if necessary, gaps, to achieve the maximum percent identity for each sequence, any conservative substitutions are not considered as part of the identity sequence. Alignment to determine percent identity in amino acid sequence can be performed by various methods known to experts in this field, for example, using widely available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Specialists in this area to determine the appropriate parameters needed to determine the level of identity with the alignment, including any relevant algorithms needed to achieve maximal alignment over the entire length comparison is imaamah sequences. For the purposes of the present invention % identity on amino acid sequence was determined using a computer program to compare sequences ALIGN-2, described in U.S. patent No. 6828146.

In the context of the present description, the terms "peptide" and "polypeptide" are used interchangeably, except that the term "peptide" typically refers to a polypeptide comprising less than 200 continuous amino acids. The term "peptide" typically refers to a continuous and relatively short sequence of amino acids connected by peptide bonds. In the typical case, but not necessarily, the peptide has a length of about 2-50 amino acids, 4-40 amino acids or 10-30 amino acids.

The term "vector" in the context of the present description is used to refer to a nucleic acid molecule that can carry another nucleic acid to which they are attached. One type of vector is a "plasmid", where this term refers to a circular double-stranded loop DNA, inside of which can be legirovanyh additional segments of DNA. Another type of vector is a phage vector. Another type of vector is represented by a viral vector, where additional DNA segments can be legirovanyh part of the viral genome. Certain vectors are capable of Autonomous replication in to atke-master, into which they are introduced (e.g., bacterial vectors containing bacterial Replicator, and episomal vectors mammals). Other vectors (e.g., episomal vectors mammals) can be integrated into the genome of a host cell upon introduction into the cell of the master, so when this is achieved the joint genome replication master. Moreover, certain vectors are capable of directing the expression of genes with which they are functionally linked. Such vectors are considered in the present description as "recombinant expression vectors" (or simply "recombinant vectors"). Most often, the expression vectors used in the methods of recombinant DNA, are in the form of plasmids. In this description, the terms "plasmid" and "vector" may be used interchangeably as the plasmid is a most commonly used form of vector.

The terms "polynucleotide" or "nucleic acid", used herein interchangeably, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or their analogs, or any substrate that can be incorporated into a polymer of DNA or RNA polymerase or during the synthetic reaction. Polynucleotide may include the step of modified nucleotides, such as methylated nucleotides and their analogues. In the case of this modification in the structure of nucleotides can be introduced before or after Assembly of the polymer. The nucleotide sequence can be interrupted components not nucleotide nature. Polynucleotide can also be modified after synthesis, for example, by conjugation with a label. Other types of modifications include, for example, "kierowanie", substitution of one or more natural nucleotide analog, mezhnukleotidnyh modifications, such as modifications under the influence of the input uncharged linkages (e.g., using methylphosphonates, phosphotriesters, phosphamidon, carbamates and the like), charged linkages (e.g., using phosphorothioate, phosphorodithioate and the like), and agents containing the attached fragments, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine and the like), intercalators (for example, acridine, psoralen, and the like), chelating agents (e.g., metals, radioactive metals, boron, oxidative metals, etc), alkylating agents, in particular containing modified linkages (e.g., alpha anomeric nucleic acids, etc), as well as using unmodified forms one or more polynucleotides. Besides, any guy who rossilini groups, originally present in the sugars may be replaced, for example, phosphonate groups, phosphate groups, protected by standard protective groups, or activated for more links with additional nucleotides, or they can be conjugated to a solid or semi-solid substrates. 5'- and 3'-Terminal OH can be phosphorylated or substituted amines or organic capping groups containing from 1 to 20 carbon atoms. Other hydroxyl can also be derivatization using standard protective groups. Polynucleotide can also contain ribose analogues or desoxyribose, mostly known in the field and include, for example, 2'-O-methyl, 2'-O-allyl-, 2'-fluoro - or 2'-isidoros-, carbocyclic analogues of sugars, alpha-anomeric sugars, epimeria sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugar, sedoheptulose, acyclic analogs and unfounded nucleoside analogues, such as methylribose. One or more fosfolipidnyh ties can be replaced by alternative linking groups. These alternative linking groups include, without limitation, variants, in which the phosphate is replaced by P(O)S("diatom"), P(S)S ("dayata"), "(O)NR2(amidate"), P(O)R, P(O)OR', CO or CH2("the form is malem"), where each of R or R' independently represent H or substituted or unsubstituted alkyl (1-20C), optionally containing ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. It is not necessary that all communication polynucleotide were identical. The above description applies to all polynucleotide described in the present description, including RNA and DNA.

The term "oligonucleotide" in the context of the present description generally refers to short, mostly single-stranded, the main synthetic polynucleotides, which generally, but not necessarily, have a length of less than about 200 nucleotides. The term "oligonucleotide" and "polynucleotide" are not mutually exclusive. The description given above in relation to polynucleotides, equally and fully applicable to oligonucleotides.

The term "growth factor hepatocyte" or "HGF" in the context of the present description, unless specifically indicated otherwise, refers to any native or variant (native or synthetic) HGF polypeptide, which is able to activate signaling pathway HGF/c-met in a condition that implemented such a process. The term "wild-type HGF" typically refers to a polypeptide that includes the amino acid sequence of the natural protein HGF. The term "sequence of wild-type HGF" mainly from OSISA to the amino acid sequence, found in natural HGF.

The phrase "substantially does not violate", "does not reduce to a significant extent", "substantially similar" or "essentially equivalent" and their variations in the context of the present description denotes a sufficiently high level of similarity between two numeric values, so that the person skilled in the art would consider the difference between the two values as having little biological significance or no biological significance, in relation to the biological characteristic measured by these indicators. The difference between the two values are preferably less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, and preferably less than about 10%. Examples of "two numeric values" include, in particular, is associated with the wild-type protein, and is associated with a mutant form of this protein.

The terms "antibody" and "immunoglobulin" are used in this description interchangeably in the broadest sense and include monoclonal antibodies (e.g., full-length antibodies or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, polyspecific antibodies (for example, bespecifically antibodies,provided they exhibit the desired biological activity) and may also include certain antibody fragments (as will be described below in more detail). The antibody can be human, humanized and/or ripened on the affinity.

"Fragments of antibodies include only a portion of an intact antibody, where a specified part preferably retains at least one and preferably most of the functions or all functions normally associated with this part when it is present in an intact antibody. In one embodiment, the fragment of the antibody includes antigen-binding site of the intact antibody and thus retains the ability to bind to the antigen. In another embodiment, the antibody fragment is a fragment that includes one Fc-region and retains at least one biological functions normally associated with the Fc-region, when this fragment is present in an intact antibody, such as FcRn-binding, modulation of the half-life of antibodies, ADCC function and binding of complement. In one embodiment, the fragment of the antibody is a monovalent antibody, which has a half-life ofin vivoessentially the same as for the intact antibody. For example, such an antibody fragment may include the antigen-binding shoulder, connected yennoe to the sequence Fc, which is able to impart stabilityin vivothe specified fragment.

The term "monoclonal antibody"used in the present invention refers to an antibody obtained from a population essentially homogeneous antibodies, that is, the individual antibodies comprising the population, which are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigen. In addition, unlike drugs polyclonal antibodies, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

In the present description monoclonal antibodies specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibody, whereas the remaining part of the one or more circuits are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass of the anti-Christ. ate, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (U.S. patent No. 4816567; and Morrisonet al., Proc. Natl. Acad. Sci. USA81:6851-6855 (1984)).

"Humanized" forms of antibodies, non-human (e.g. murine) antibodies that are chimeric antibodies that contain minimal sequence derived from the immunoglobulin other than human. For the most part, humanized antibodies are human immunoglobulins (recipient antibody)in which residues from a hypervariable area of the recipient replaced by residues from a hypervariable portion of the molecule other than human (donor antibody), such as a molecule of mouse, rat, rabbit, or Primate, non-human, having the desired specificity, affinity and capacity. In some embodiments, the remains of the skeleton of the plot (FR) of a human immunoglobulin are replaced by corresponding residues of the molecule other than human. Furthermore, humanized antibodies may include residues that are not present neither in the recipient antibody or in the donor antibody. Such modifications are created with the purpose of improving the characteristics of the antibodies. Basically, humanitariannet antibody includes essentially all of the at least one and typically devarapalli domains in which all or substantially all of the hypervariable loops correspond to the hypervariable loops in immunoglobulin molecules, non-human, and all or substantially all of the FR represent the FR sequence of the human immunoglobulin. Humanitariannet antibody optionally also includes at least a portion of constant region of immunoglobulin (Fc), typically a human immunoglobulin. Additional details are described in Joneset al., Nature321:522-525 (1986); Riechmannet al., Nature332:323-329 (1988); and Presta,Curr. Op. Struct. Biol. 2:593-596 (1992). Cm. the following review articles and references in these: Vaswani and Hamilton,Ann. Allegry, Asthma &Immunol. 1:105-115 (1998); Harris,Biochem. Soc. Transactions23:1035-1038 (1995); Hurle and Gross,Curr. Op. Biotech. 5:428-433 (1994).

"Human antibody" represents such an antibody that has an amino acid sequence corresponding to that of the antibody produced in the human body and/or obtained using any of the methods, create human antibodies described in the present description. This definition of human antibodies specifically excludes humanitariannet antibody comprising the antigen-binding residues of the molecule other than human.

Antibody ripe for the affinity" represents such an antibody, which contains one is whether more changes in one or more areas CDR, which lead to an increase in the affinity of the antibody for the antigen in comparison with the original antibody that does not contain such a change (changes). Preferred ripened on the affinity antibodies have affiniscape in the range of nanomolar or even picomolar concentrations in relation to the target antigen.

Mature affinity antibodies get by procedures known in the field. So, in Markset al. Bio/Technology10:779-783 (1992) describes the process of maturation on the affinity by moving the VH and VL domains. Can also be used random mutagenesis of CDR and/or residues of the frame area, as described in Barbaset al.,Proc. Nat. Acad. Sci, USA91:3809-3813 (1994); Schieret al., Gene169:147-155 (1995); Yeltonet al., J. Immunol.155:1994-2004 (1995); Jacksonet al., J. Immunol.154(7):3310-9 (1995) and Hawkinset al., J. Mol. Biol. 226:889-896 (1992).

"Blocking" antibody or antibody"antagonist" represents such an antibody that inhibits or reduces biological activity of the antigen with which it is associated. Preferred blocking antibodies or antibody antagonist essentially or completely inhibit the biological activity of the antigen.

"Antibody agonist" in the context of the present description refers to an antibody that mimics at least one of the functional activities of a polypeptide of interest.

"Violation" or "patol the environmental condition" is any condition, which may benefit from treatment with the use of the substance/molecule or method according to the present invention. This state includes chronic and acute disorders or diseases including those pathological conditions in which the body of a mammal is predisposed to the disorder. Non-limiting examples of disorders to be treated according to the present invention, includes malignant and benign tumors or cancers; melakoski and lymphoid malignancies; neuronal, glial, astatically, hypothalamus and other glandular, macrophagal, epithelial, stromal and blastocele disorders; and inflammatory, immunological, neurodegenerative disorders, disorders associated with angiogenesis, and disorders associated with defects of metabolism in the mitochondria or metabolic pathways.

The term "cell-proliferative violation" and "proliferative violation" refers to disorders that are associated with abnormal to some extent the proliferation of cells. In one embodiment, the cell-proliferative violation is a cancer.

The term "tumor" in the context of the present description refers to the growth and proliferation of all cells neoplasm, regardless of whether she has a malignant sludge is benign, and also includes all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancer", "cell-proliferative violation", "proliferative violation" and "tumor" are not mutually exclude each other in the present description.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated growth/proliferation of cells. Examples of cancer include, without limitation, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include multiple myeloma, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, cancer of the colon, colorectal cancer, carcinoma of the endometrium or uterine carcinoma of the salivary gland, kidney cancer (e.g., renal carcinoma cells), liver cancer, prostate cancer, cancer vulval, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.

In the context of the present description, the term "treatment" refers to the procedure of clinical effects, undertaken with the purpose to alter the natural course of the process of an individual or in cells be treated, and may be performed either prophylactically or in the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, decrease the speed of disease progression, relief or weakening of the morbid state, and remission or improved prognosis. In some embodiments, the antibodies according to the present invention are used to delay development of a disease or impairment.

The term "effective amount" refers to an amount which is effective in those dosages and for periods of time necessary to achieve the desired therapeutic or prophylactic result.

"Therapeutically effective amount" of a substance/molecule according to the present invention, agonist or antagonist may vary, depending on a number of factors such as the nature of the disease condition, age, sex and weight of the individual, and the ability of a substance/molecule agonist or antagonist to call in the body of the individual desired response. Therapeutically effective amount is also an amount, if any toxic is or other harmful effects of the substance/molecule agonist or antagonist were outweighed by therapeutically favorable effects. "Prophylactically effective amount" refers to that amount which is effective, in the appropriate dosage and within a reasonable period of time to achieve the desired prophylactic result. In the typical case, but not necessarily, in the case of using prophylactic doses subjects before the onset of the disease or in its early stages, the prophylactically effective amount will be less than therapeutic amount.

The term "cytotoxic agent" in the context of the present description refers to a substance that inhibits or prevents a particular function of cells and/or causes destruction of cells. This term shall include radioactive isotopes (e.g., At211I131I125, Y90That Re186That Re188Sm153Bi212, P32and radioactive isotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin, Vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleotidase enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants, and RA is personal antitumor and anticancer agents as described later in this text. Other cytotoxic agents are also described in this text. Agent that destroys tumor cells, causing the destruction of tumor cells.

"Chemotherapeutic agent" is a chemical substance used in the treatment of cancer. Examples of chemotherapeutic agents 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, triethylenethiophosphoramide and triethylenemelamine; acetogenin (in particular, bullatacin, bullatacin); camptothecin (including the synthetic analogue topotecan); bryostatin; callistemon; CC-1065 (including its synthetic analogues of adozelesin, carzelesin and bizelesin); cryptophycin (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, halophosphate, estramustine, ifosfamide, mechlorethamine, hydrochloride oxide mechlorethamine; melphalan, novemberin, finestein, prednimustine, trofosfamide, oralloy mustard; nitrosoanatabine, still is as carmustine, chlorozotocin, fotemustine, lomustin, nimustine and ranimustine; antibiotics such as enediyne antibiotics (for example, calicheamicin, especially calicheamicin gamma II and calicheamicin omega II (see, e.g., Agnew,Chem Intl. Ed. Engl.,33:183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; spiramycin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, autralian, azaserine, bleomycin, actinomycin, carubicin, karminomitsin, casinopolis, chromomycin, dactinomycin, daunorubicin, demoralizing, 6-diazo-5-oxo-L-norleucine, doxorubicin ADRIAMYCIN® (including morpholino doxorubicin, cyanomethane doxorubicin, 2-pyrroline doxorubicin and desoxidation), epirubicin, zorubicin, idarubitsin, marsellaise, mitomycin, such as mitomycin C, mycofenolate acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, clamycin, 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 analogs, such as ancitabine, azacytidine, 6-Aza who ridin, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, propionate dromostanolone, epitiostanol, mepitiostane, testolactone; antigranulocyte agents, such as aminoglutethimide, mitotane, trilostane; wspanialy folic acid, such as prolinnova acid; Eagleton; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; astroball; bisantrene; edatrexate; defaming; demecolcine; diazinon; alternity; the acetate slipline; epothilone; etoposide; gallium nitrate; hydroxyurea; lentinan; londini; maytansinoid, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitrean; pentostatin; penomet; pirarubicin; losoxantrone; podofillina acid; 2-acylhydrazides; procarbazine; polysaccharide complex PSK® (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tinoisamoa acid; triatholon; 2,2',2"-trihlortrietilamin; trichothecenes (in particular, T-2 toxin, verrucarin And, roridin and unguided); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; Galitsin; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoid, for example, paclitaxel TAXOL® (Bristol-Myers Squibb Oncology, Princeton, N.J.), derived from albumin composition based on nanoparticles of paclitaxel that does not contain cremophor ABRAXANETM (American Pharmaceutical Partners, Schaumberg, Illinois) and docetaxel TAXOTERE® (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine GEMZAR®; 6-tioguanin; mercaptopurine; methotrexate; platinum analogues, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine NAVELBINE®; Novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; deformational (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of the aforementioned substances.

In the definition above, the term "chemotherapeutic agent" are also included antihormonal agents, which act in the direction of the regulation or inhibition of hormone action on tumors such as antiestrogens and selective modulators of estrogen receptor (SERM), including, for example, tamoxifen (including NOLVADEX tamoxifen®), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene FARESTON; aromatase inhibitors that inhibit the enzyme aromatase, which regulates the production of estrogen in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, acetate megestrol MEGASE®exemestane AROMASIN®; formestane, fadrozole, vorozole RIVISOR®; letrozole FEMARA® and anastrozole ARIMIDEX®; and antiandrogen, so is e as flutamide, nilutamide, bikalutamid, leuprolide, and goserelin; as well as troxacitabine (1,3-dioxolane similar casinomaha of a nucleoside); antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways involved in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as the inhibitor of the expression of VEGF (e.g., ribozyme ANGIOZIME®) and inhibitors of HER2 expression; vaccines such as vaccines, used for gene therapy, for example a vaccine ALLOVECTIN®vaccine, LEUVECTIN® vaccine VAXID®; PROLEUKIN® rIL-2; topoisomerase inhibitor 1 LURTOTECAN®; ABARELIX® rmRH; and pharmaceutically acceptable salts, acids or derivatives of the above compounds.

Used herein, the term "agent that inhibits axonal growth" refers to the compound or compositions that inhibit the growth of cells, when cell growth is dependent on activation of HGF/c-met,in vitroorin vivo. Thus, agent, inhibiting the growth may be any agent which reduces materially the percentage of HGF/c-met-dependent cells in S-phase. Examples of agents inhibiting growth include agents that block progression of the cell cycle (point other than S phase), such as agents that induce a stop at the G1 - and a stop at M-phase. Classical M-blockers phase include the Vinca alkaloids (vincristine and vinblastine, taxanes and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Agents that block the G1-phase, also have a certain effect on block S-phase, and include, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and Aga-C. further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" from Murakami et al. (WB Saunders: Philadelphia, 1995, in particular on page 13). Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from the yew tree. Docetaxel TAXOTERE®, Phone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel accelerate the Assembly of microtubules from tubulinea dimers and stabilizes microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.

Doxorubicin is an anthracycline antibiotic. Full chemical name of doxorubicin is (8S-CIS)-10-[(3-amino-2,3,6-trideoxy-α-L-oxohexanoyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetic)-1-methoxy-5,12-naphthacenedione.

Antagonists of HGF/Met-peptides/polypeptides (including antibodies)/p>

One aspect of the present invention refers to the selected peptide/polypeptide and modulate antibody interaction β-chain β-chain of HGF and interaction of HGF-Met. In one embodiment, the modulators (such as the peptides/polypeptides and antibodies) can be isolated from cells or tissues, as their sources, using a suitable purification systems, including standard methods of protein purification. In another embodiment, modulators receive as part of the techniques of recombinant DNA. Alternatively, recombinant expression modulators can be synthesized chemically using standard techniques of peptide synthesis.

Molecule antagonists of HGF/Met in accordance with the present invention include molecules that are illustrated in figure 1. The present invention also relates to mutant or variant protein in which any of the residues can be changed to the corresponding residues of these peptides/polypeptides, where still supported character encoding peptide/polypeptide that retains its modulating activity. In one embodiment, the variant antagonist peptide/polypeptide has at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% identity in amino acid sequence with the sequence of the reference antagonist peptide/polypeptide. Basically, the specified vari the NT shows essentially the same or greater binding affinity, as a reference antagonist peptide/polypeptide binding, which is, for example, at least 0,H, 0,8X, 0,9X, 1,0X, 1,25X or 1,5X from the binding affinity of the reference binding peptide/polypeptide/ligand, when evaluating test results linking by standard quantitative method for the assessment in the system of units/metric system, while retaining the desired degree of antagonistic activity.

Basically, the variants according to the present invention include those variants in which residues at a particular position of the sequence is replaced by other amino acids, and also include the possibility of inserting an additional one or more residues between two residues of the original protein/peptide, or deletions of one or more residues from the initial sequence, or adding one or more residues to the original sequence. Any amino acid substitutions, insertions or deletions are included in the scope of the present invention. In favorable conditions, the substitution represent a conservative substitution described in this text.

"Isolated" or "purified" peptide, polypeptide, protein, or biologically active fragment is separated and/or recovered from/of the components of its natural environment. Contaminante components include m is materials, which in a typical case prevents diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones and other protein and non-protein materials. Drugs, containing preferably less than 30%, by dry weight, unwanted contaminating material (contaminants), preferably less than 20%, 10% and preferably less than 5% contaminants are treated as essentially isolated. Isolated, obtained by recombinant methods peptides/polypeptides or biologically active portion is preferably essentially free of culture medium, i.e., culture medium is preferably less than 20%, preferably less than about 10% and preferably less than about 5% of the volume of the preparation of peptide/polypeptide. Examples of contaminants include fragments of cells, remnants of the culture medium and substances used and produced duringin vitrosynthesis of peptide/polypeptide.

Conservative substitution peptides/polypeptides shown in table a in the column entitled "Preferred substitutions". If such substitutions result in a change in biological activity, then the products can be more substantial changes, indicated as "Representative substitutions" in table a, or is described hereinafter with reference to different the classes of amino acids, and further, these products are subjected to screening.

Table a
The original balanceRepresentative substitutionsPreferred substitution
Ala (A)val; leu; ileval
Arg (R)lys; gln; asnlys
Asn (N)gln; his; asp; lys; arggln
Asp (D)glu; asnglu
Cys (C)sr; alaser
Gln (Q)asn; gluasn
Glu (E)asp; glnasp
Gly (G)alaala
His (H)asn; gln; lys; argarg
Ile (I)leu; val; met; ala; phe; nor shall Azin leu
Leu (L)norleucine; ile; vl; met; ala; pheile
Lys (K)arg; gln; asnarg
Met (M)leu; phe; ileleu
Phe (F)leu; val; ile; ala; tyrtyr
Pro (P)alaala
Ser (S)thr; cyscys
Thr (T)serser
TRP (W)tyr; phetyr
Tyr (Y)trp; phe; thr; serphe
Val (V)ile; leu; met; phe; ala; norleucineleu

Pronounced modification of the biological properties of the peptide/polypeptide accompanied by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide skeleton in the area of the substitution, n is the sample, in the form of a plate or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (C) the volume of the side chain. Natural residues are divided into groups on the basis of the General properties of their side chain:

(1) hydrophobic residues: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic residues: cys, ser, thr;

(3) acid residues: asp, glu;

(4) basic residues: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic residues: trp, tyr, phe.

Non-conservative substitution refers to the replacement of the representative of one of these classes to the representative of another class.

Options modulators of antibodies that do not have a significant impact on the activity of antibodies, can also be created on the basis of known information. For example, variants of the antibodies may be characterized by the fact that at least one amino acid residue in the molecule such antibodies substituted by another residue. In the case of antibodies, the sites of greatest interest from the point of view of the substitutional mutagenesis include mainly hypervariable sites, but can also be considered changes in the framework region (FR).

In the case of one type of antibody variant substitution includes replacement of one or more residues of the hypervariable area in the original antibody (in the example, if gumanitarnogo or human antibody). Basically, get one or more options selected for further development, have improved biological properties relative to the original antibody from which they were obtained. A convenient way to create such variants substitution involves a way of "ripening" affinity using the technique of phage view. Briefly, the method consists in the fact that several sites in the hypervariable segment (for example, 6-7 sites) are matirovanie with all possible amino acid substitutions at each site. Thus obtained antibodies analyzed using particle filamentous phages representing the fusion product of the gene III of M13 packaged within each particle. Options with the manifestation of the phage further subjected to skanirovaniya to evaluate their biological activity (e.g. binding affinity), as will be described later in the description. To identify possible sites in hypervariable region suitable for modification can be carried out by mutagenesis according to the method of alanine scanning to identify residues of the hypervariable area having a significant effect on binding to the antigen. Alternative or additionally, it may be useful to analyze crystallize the kind of structure of the complex antigen-antibody to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues are candidates for substitution in accordance with the methods described in the present description. Upon receipt of such options panel data of variants is subjected to screening in accordance with the present description, and antibodies with improved properties, demonstrated in one or more relevant tests can be selected for further development.

The nucleic acid molecules encoding amino acid sequence variants of antibodies, obtained using a variety known in the field of techniques. Such techniques include, without limitation, the selection is from a natural source (in the case of a natural amino acid sequence variants) or the receipt by oligonucleotide mutagenesis by PCR mutagenesis, and cassette mutagenesis of the previously received version or variant version of the antibody.

It may be desirable to incorporate one or more amino acid modifications in the Fc-part of the immunoglobulin polypeptides according to the present invention to create a variant Fc site. Variant Fc region can include a sequence human Fc-plot (for example, area of the human IgG1, IgG2, IgG3, or IgG4)that includes the amino acid modification (e.g. a substitution) at one or more linakis is now positions including cysteine of the hinge region.

In one embodiment, the variant Fc region can exhibit altered binding affinity with neonatal Fc receptor (FcRn). Such options Fc-sections may include modification of amino acids for any one or more amino acids in positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 436, 439 or 447 of the Fc-region, where the numbering of residues of the Fc-plot corresponds to the index EU, as described in Kabat. Options Fc-phase with reduced binding ability with FcRn may include modification of amino acids for any one or more amino acids in positions 252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 435, 436, 439 or 447 of the Fc-region, where the numbering of residues of the Fc-plot corresponds to the EU index as in Kabat. The above options Fc-phase can alternatively show increased binding to FcRN and include modification of amino acids for any one or more amino acids in positions 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 of the Fc-region, where the numbering of residues of the Fc-plot corresponds to the EU index as in Kabat.

Options Fc-phase with reduced ability to bind to FcγR may include modification of amino acids for any one or more amino acids at positions 28, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438 or 439 of the Fc-region, where the numbering of residues of the Fc-plot corresponds to the EU index as in Kabat.

For example, the variant Fc region can exhibit reduced binding to FcγRI and include modification of amino acids for any one or more amino acid positions 238, 265, 269, 270, 327 or 329 of the Fc-region, where the numbering of residues of the Fc-plot corresponds to the EU index as in Kabat.

Variant Fc region can exhibit reduced binding to FcγRII and include modification of amino acids for any one or more amino acids in positions 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 of the Fc-region, where the numbering of residues of the Fc-plot corresponds to the EU index as in Kabat.

Variant Fc region can exhibit reduced binding to FcγRIII and include modification of amino acids for any one or more amino acids in positions 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 293, 294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373, 376, 382, 388, 389, 416, 434, 435 or 437 Fc-region, where the numbering of residues of the Fc-plot corresponds to the Eu index as described in Kabat.

Options Fc-plot with a modified (e.g., increased or decreased) level of binding to C1q and/or complement-dependent cytotoxic the awn (CDC) are described in WO99/51642. Such options may include substitution of amino acids for any one or more of the amino acids in positions 270, 322, 326, 327, 329, 331, 333 or 334 of the Fc section. (See also Duncan & WinterNature322:738-40 (1988); U.S. patent No. 5648260; U.S. patent No. 5624821; and WO94/29351, where these works relate to options Fc-plot).

Design vector

In the present description of the polynucleotide sequence encoding the present invention the peptides/polypeptides may be obtained using standard recombinant techniques. The desired polynucleotide sequences can be selected from the appropriate cell sources and further sequenced. Cell sources for obtaining antibodies include antibody-producing cells, such as hybridoma cells. Alternatively, polynucleotide can be synthesized using nucleotide synthesizer or PCR methods. Sequences encoding immunoglobulins, after receiving them, is inserted into the recombinant vector capable of replication and expression of heterologous polynucleotides in the cell host. Many vectors are available and known in this field can be used for the purposes of the present invention. The choice of an appropriate vector is determined mainly by the size of the nucleic acid is you, embedded in the vector, and specific cell host transformed data vector. Each vector contains various components, defined by its function (amplification or expression of heterologous polynucleotide or both together) and its compatibility with a particular cell of the host in which it will be. Vector components generally include, without limitation, the Replicator (especially in the case when the vector is embedded in a prokaryotic cell), breeding gene-marker, a promoter, the binding site with the ribosome (RBS), signal sequence, the insertion of a heterologous nucleic acid and the sequence termination of transcription.

Basically, plasmid vectors containing replicon and control sequences derived from species compatible with the host-cell, used in combination with such cells masters. The vector typically contains a replication site, as well as marking sequences which are capable of providing phenotypic selection of the transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived fromE. coli. pBR322 contains genes encoding resistance to ampicillin (Amp) and tetracycline (et), and, thus, provides an easy way of identifying transformed cells. BR322, its derivatives, or other microbial plasmids or bacteriophage may also contain appropriate promoters, or can be modified in order to include promoters that can be used by the microbial organism for expression of endogenous proteins.

Additionally, phage vectors containing replicon and control sequences that are compatible with the microorganism host, can be used as transformation vectors, in combination with the specified cells of the host. For example, bacteriophage such as αGEM.TM.-11, can be used to create a recombinant vector, which then can be used to transform susceptible host cells such asE. coliLE392.

In accordance with the present invention can be either constitutive or inducible promoters, depending on the particular situation and their need may identify any person with an average level of knowledge in this area. In this area there are plenty of promoters recognized by a variety of potential cells of the host. The selected promoter can be further functionally connected with restroom DNA that encodes a polypeptide according to the present description, by removing the promoter from the source DNA by cleavage with restriction enzymes and subsequent UGT what hivaniem selected promoter sequence in the selected vector. As the sequence of the native promoter, and a sequence of many heterologous promoters may be used to perform the amplification and/or expression of the target genes. However, heterologous promoters are preferred, as they basically allow you to achieve a higher level of transcription and higher output downregulation of the target gene compared to the native promoter of the target polypeptide.

Promoters suitable for use in prokaryotic cells-hosts include the PhoA promoter, the promoter system β-galactosi and lactose promoter system tryptophan (trp) and hybrid promoters such astacortrcthe promoter. However, acceptable and other promoters that function in bacteria (such as other known bacterial and phage promoters). Their nucleotide sequences are known and published that allows the person skilled in the art to carry out their operational ligation with cisternae coding target light and heavy chains (Siebenlist et al., (1980) Cell 20:269)), using appropriate linkers or adapters to create any required restriction sites.

In some embodiments, each cistron comprising the recombinant vector comprises the sequence of the secretion signal, where this component sends tra is spartanovka expressed polypeptide through the membrane. Basically, the signal sequence may be a component of the vector, or can be a part of the DNA of the target polypeptide, which is embedded vector. The signal sequence selected, for the purposes of the present invention must be such a sequence that is recognized and processed (that is cleaved by a signal peptidase) the host-cell. In the case of a prokaryotic host cells that do not recognize and do not ProcessInput signal sequences native to the heterologous polypeptides, such a signal sequence is substituted prokaryotic signal sequence selected, for example, from the group consisting of the leader sequence of alkaline phosphatase, penitsillinazy, Ipp or thermostable enterotoxin II (STII), LamB, PhoE, PelB, OmpA and MBP.

Prokaryotic cells-hosts suitable for the expression of polypeptides include Archaebacteria and Eubacteria, such as gram-negative or gram-positive organisms. Examples of bacteria include Escherichia (e.g. E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia mercescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. Preferably use the gram-negative cells. Preferably a host cell must secrete minimum number is the number of proteolytic enzymes, preferably, the additional protease inhibitors in cell culture.

Production of peptide/polypeptide

Cell host is subjected to transformation or transfection by the above expression vectors and cultured in standard nutrient media modified as appropriate for induction of promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

The term "transfection" refers to the process of receiving the expression vector into the cell-the owner, regardless of whether in fact the expression of the coding sequences. Specialists known various methods of transfection, such as deposition method using a CaPO4method and electroporation. It is believed that the transfection was successful, if the cell is the owner signs of this vector.

The term "transformation" means embedding the DNA in prokaryotic organism-host, so that said DNA is made replicable, either as an extra-chromosomal element, or integrated with the chromosome condition. Depending on the type of host cell transformation is performed with the use of standard techniques that are suitable for such cells. Method calcium handling, which uses chlorine is the ID of calcium, mainly applicable for bacterial cells that contain difficult to overcome barriers in the form of cell walls. Another transformation method involves the use of polyethylene glycol/DMSO. Another method is electroporation.

Prokaryotic cells used to obtain peptides/polypeptides, according to the present invention are grown in environments known to specialists in this area, which are suitable for culturing a selected host cells. Examples of suitable media include broth, Luria ((LB) plus the necessary nutritional supplements. In preferred embodiments, the medium also contains breeding agent, selected based on the design of the expression vector, which allows to achieve selective growth of prokaryotic cells containing the expression vector. For example, add ampicillin to the medium for growth of cells expressing the ampicillin-resistant gene.

You can also include any necessary additives, in addition to the sources of carbon, nitrogen and inorganic phosphate, in appropriate concentrations, which are either singly, or in mixture with other components or the environment, such as a complex nitrogen source. Optional culture medium may contain one or more reducing agents selected from the group consisting of glutathione, qi is Thein, the applied, thioglycollate, dithioerythritol and dithiothreitol.

Prokaryotic cells are the owners of cultivated under suitable temperatures. For growth of E. coli, for example, the preferred temperature ranges are from about 20°C. to about 39°C., more preferably from about 25°to about 37°C., and more preferably at about 30°C. the Medium may be any pH ranging from about 5 to about 9, mainly depending of the host body. In the case ofE. coli, the pH is preferably from about 8 to about 7.4 and more preferably is approximately 7,0.

If the expression vector is used inducible promoter, expression of the protein induce under conditions suitable for activation of the promoter. For example, if control transcription using the PhoA promoter, the transformed cells may be cultured in an environment with limited phosphate, for induction. Can be used in many other inductors defined by the used design vector, which is known in this field.

In the present description, the peptides/polypeptide expressed in the microorganism can secretariats and recover from periplasm host cells. The process of recovery of protein in the typical case involves the destruction of ICRI is the body with the use, basically, techniques such as osmotic shock, scoring or lysis. After the resolution of the cells, remove cellular debris or whole cells by centrifugation or filtration. Proteins can be further purified, for example, using affinity chromatography resin. Alternatively, proteins can be transferred into the culture medium and separated from her. Cells can be removed from the culture and then the culture supernatant was filtered and concentrated for further purification the proteins. Expressed polypeptides can be isolated and identified using well known methods such as fractionation on columns for immunoaffinity chromatography or ion-exchange columns; using the method of precipitation by ethanol; HPLC method with phase reversal, chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; electrophoresis SDC-PAGE; precipitation ammonium sulfate; gel filtration using, for example, Sephadex G-75; affinity chromatography on resins based on hydrophobic interaction; chromatography based on affinity to the ligand, an antigen, immobilized on matrix and Western blot analysis.

In addition to prokaryotic host cells, eukaryotic system cells-hose is in can also be set methods known in this area. Suitable organisms-hosts include mammalian cell lines such as CHO and insect cells such as cells, are described below.

Purification of the peptide/polypeptide

The resulting peptides/polypeptides can be further purified to obtain preparations that are essentially homogeneous for the purposes of further testing and use. Standard methods of protein purification are known in this field and can be used accordingly. The procedures below are only examples of suitable cleaning procedures: fractionation on immunoaffinity columns or ion-exchange columns, the method of precipitation with ethanol, HPLC method with phase reversal, chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; electrophoresis SDC-PAGE; precipitation with ammonium sulfate and gel filtration using, for example, Sephadex G-75.

The methods of the present invention

The present invention relates to various methods, based on the fact that the mutation in some parts of the β-chain of HGF leads to a modification of the biological activities of the molecules, so that these mutant molecules demonstrate antagonistic effects on modulation path HGF/Met.

Can be used a variety of substances or molecules including peptides, polypeptide and the like) as therapeutic agents in accordance with the methods of the present invention. These substances can be made by known methods of producing pharmaceutically useful compositions, where such a product to the corresponding mixture with a pharmaceutically acceptable carrier. Therapeutic compositions suitable for storage, prepared by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington''s Pharmaceutical Science16thedition, Osol, A. Ed. (1980)), with the receipt of the products in the form of lyophilised compositions or aqueous solutions. Use an acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations, and include buffers such as phosphate, citrate, and buffers on the basis of other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides (containing less than 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; soleares the matter of counterions, such as sodium; and/or nonionic surfactants such as TWEENTM, PLURONICSTMor PEG.

The composition used for the introduction ofin vivomust be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes, prior to lyophilization and restore or after them.

In the present description, therapeutic composition is placed in a container having a sterile access port, for example in a bag or bottle of intravenous solution, having a tube, peelable hypodermal needle for injection.

The introduction can be carried out in accordance with known methods, for example, by injection or infusion mode intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial routes of administration or introduction into the damage, using local injection or by use of a slow release.

Dosages and desired drug concentration in pharmaceutical compositions according to the present invention can vary, depending on the specific method of administration. Determination of the appropriate dosage or method of administration in the state to carry out any practitioner. The results of the experiments on animal is x provide a reliable basis for the determination of effective doses for treatment of humans. Interspecies analysis of effective doses can be performed using the principles established by Mordenti and Chappelle (Mordenti, J. and Chappel, W. "The use of interspecies scaling in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, New York 1989, pp.42-96).

In that case, when practicing the introduction of a substance or molecule according to the present inventionin vivonormal amount in the dosage can vary from about 10 ng/kg to 100 mg/kg body weight of the mammal or more per day, preferably about 1 μg/kg/day to 10 mg/kg/day, depending on the method of introduction. In the literature there is guidance on specific dosages and methods of delivery. See, for example, U.S. patent No. 4657760; 5206344 or 5225212. It is expected that different compositions will be effective for different compounds used for treatment of different disorders, because the introduction is directed to one organ or tissue, for example, may necessitate a delivery method other than the delivery directed to another organ or tissue.

When it is desirable long-term administration of substances or molecules in the composition with extended release where the release characteristics suitable for the treatment of specific diseases or disorders requiring the introduction of a substance or molecule, the possibility of microencapsulation this ve is esta or molecules. Microencapsulation of recombinant proteins for achieving sustained release has been successfully performed with human growth hormone (rhGH), interferon - (rhIFN-), interleukin-2, and MN rgp 120 (Johnson et al.,Nat. Med., 2:795-799; Yasuda,Biomed. Ther.27:1221-1223 (1993); Hora et al.,Bio/Technology,8:755-758 (1990); Clelan, "Design and Production of Single Immunization Vaccines Using Polylactid Polyglycolid microsphere was Systems", inVaccines Design: The Subunit and Adjuvant Approach,Powell and Newman, eds. (Plenum Press: New York, 1995), pp.439-463; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. patent No. 5654010).

The composition of these proteins with slow release were developed using polymer polylactic-coglycolide acid (PLGA), due to its bioavailability and wide range of biodegradable properties. Products of biodegradation of PLGA, lactic and glycolic acid, can be quickly removed from the body. In addition, the ability to degradation of this polymer can be adjusted to achieve compounding periods from several months to several years, depending on its molecular weight and composition (Lewis, "Controlled release of bioactive agents from lactide/glycolid polymer" in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems(Marcel Dekker: New York, 1990), pp.1-41).

Identification of sites within the β-chain of HGF, which are crucial for the function of HGF in the signal path HGF/Met, indicates those sites in the β-chain of HGF, which may be directed action is their antagonists. Examples of potential antagonists include an oligonucleotide (which may be an aptamer)that binds to N-terminal section and/or section of the dimerization of β-chain of HGF and, in particular, antibodies including, without limitation, poly - and monoclonal antibodies and antibody fragments, single-chain antibodies, antiidiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and fragments of such antibodies. Aptamers are nucleic acid molecules that are able to communicate with the target molecule. Obtaining and therapeutic use of aptamers are well known in this field (See, for example, U.S. patent No. 5475096, which also describes therapeutic efficacy of Macugen® (Eyetech, New York) in the treatment of age-related macular degeneration).

As shown in the present description, HGF/Met antagonist is a substance/molecule according to the present invention is a peptide or polypeptide (including antibody). Methods for such peptides and polypeptides are known in this area and include the screening of peptide and polypeptide libraries for binding to the relevant target antigen. In one embodiment, suitable antigens target will include the β-chain of HGF (or part of it, which includes the N-terminal site and/or participation is OK dimerization), described below in more detail. In one embodiment, suitable antigens of the target will be Met include, for example, the extracellular domain of Met. Libraries of peptides and polypeptides are well known in this field and can also be obtained well-known in this region methods (see, for example, Clark et al., U.S. patent No. 6121416; Garrard et al., 5750373, 5733743, 5837242, 5969108, 6172197, 5580717 and 5658727). Libraries of peptides and polypeptides attached to a heterologous protein component, such as a protein ragovoy shell known in this field and are described, for example, Clark et al. and Garrard et al., above. Variants of the first selected binding peptide or polypeptide can be obtained by screening mutants of the peptide or polypeptide with achievement of the interesting characteristics (for example, with increased affinity for binding to the target, with improved pharmacokinetics, reduced toxicity, improved therapeutic index, and so on). For example, the interesting feature may provide the ability to connect with the Met, but with reduced ability to activate HGF-related biological activity, such as cell proliferation, Met phosphorylation, cell migration and angiogenesis. Methods of mutagenesis are known in this field, and in the present description describes the areas within the β-chain of HGF suitable for mutations in a particular position, Assoc the new N-terminal by insertion in the β-chain of HGF and/or dimerization type β-chain - β-chain. In addition, methods of scanning mutagenesis (such as methods based on alanine scanning) can be particularly useful for evaluating structural and/or functional significance of individual amino acid residues in the composition of the peptide or polypeptide.

Determining the ability of the potential of the substance/molecule according to the present invention modulate the signaling activity of HGF/c-met and/or biological activity associated with the specified signal mechanism can be made when testing the modulating ability of the substance/molecule in testsin vitroorin vivothat is known in this field and are described, for example, in the following activities: Okigaki et al., above; Matsumoto et al., above; Date et al., FEBS let. (1997), 420:1-6; Lokkert et al., above; Hartman et al., above; Kirchhofer et al., J. Biol. Chem. (2004), 279:39915-24; Stamos et al. (2004) EMBO J. 23:2325-35; Kirhhofer et al., FEBS Lett. (2005) 579:1945-50; Nakatsu et al. (Microvasc. Res. 66:102, 2003).

Antibodies against β-chain of HGF

The present invention relates to methods involving the use of antibodies. Typical antibodies include polyclonal, monoclonal, humanized, bespecifically and heteroconjugate antibodies.

1. Polyclonal antibodies

Antibodies may include polyclonal antibodies. Methods of obtaining polyclonal antibodies known to specialists in this field. Polyclo the social antibody can be obtained from a mammal, for example, through one or more injection immunizing agent and, if desired, an adjuvant. In a typical case, the immunizing agent and/or adjuvant is administered to the mammal by multiple subcutaneous or intraperitoneal injections. Immunizing agent may include the β-chain of HGF (or part thereof) or protein. It can also be useful to carry out the conjugation immunizing agent to a protein, in respect of which it is known that it is immunogenic to the mammal to be immunized. Examples of such immunogenic proteins include, without limitation, hemocyanin snails, serum albumin, bovine thyroglobulin, and soybean inhibitor of trypsin. Examples of adjuvants that may be used include beta-blockers and adjuvant MPL-TDM (monophosphoryl-lipid A, synthetic dikarenakan trehalose). The Protocol of immunization can be selected by the specialists in this field without difficult experiments.

2.Monoclonal antibodies

Alternatively, the antibody can be a monoclonal antibody. Monoclonal antibodies can be obtained by methods involving the use of hybridomas described by Kohler and Milshtein (Kohler and Milstein,Nature,256:495 (1997)). In accordance with this hybridoma techniques are conducting typical is mmunicatio mouse hamster, or other appropriate body-the owner of the immunizing agent for the effect of the lymphocytes that produce or are capable of producing antibodies that, in turn, specifically associated with immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

Immunizing agent will typically include the β-chain of HGF (or part thereof) or protein on the basis of their merger. Basically, use either peripheral blood lymphocytes (PBL), if desired cells of human origin, or spleen cells or cells lymph node, if desired sources in mammals other than man. Next lymphocytes merge with immortalizing cell line using a suitable agent for the merger, such as polyethylene glycol, with the formation of hybridoma cell [Goding,Monoclonal Antibodies: Principles and PracticeAcademic Press, (1986) pp.59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and cells of human origin. Usually use the myeloma cell line rat or mouse. Hybridoma cells may be cultured in an appropriate culture medium that preferably contains one or more substances that inhibit the growth is whether survival Nikitich immortalized cells. For example, if the parental cells do not contain the enzyme hypoxanthineguanine (HGPRT or HPRT), the culture medium for the hybridomas typically includes gipoksantin, aminopterin and thymidine ("medium HAT"), where these substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are cell lines that are able to effectively merge, support stable high level expression of the antibodies selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are a myeloma line mouse, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California, and American Type Culture Collection, Manassas, Virginia. Human myeloma cell lines and heteromyinae cell line mouse-man is also described for the purpose of obtaining human monoclonal antibodies [Kozbor,J. Immunol.,133:3001 (1984); Brodeur et al.,onoclonal Antibodies Production Techniques and Application,Marcel Dekker, Inc., New York, (1987) pp.51-63].

Cultural environment in which the cultured hybridoma cells can be further analyzed for the presence of monoclonal antibodies directed against β-chain of HGF. Preferably, the binding specificity of monoclonal antibodies produced by g is Britanie cells, determined by thus or test linkin vitrosuch as radioimmunoassay (RIA) or ELISA assay (ELISA). Such techniques and assays known in the field. Binding affinity of monoclonal antibodies can be, for example, determined according to the method Scatchard (Scatchard) (Munson and Pollard,Anal. Biochem.,107:220 (1980)).

After identifying the desired hybridoma cells corresponding clones can be subjected to subclavian procedures limit cultivation, with their further cultivation by standard methods [Goding, above]. Suitable culture media for this purpose include, for example, the environment of a needle in a modification of Dulbecco and RPMI1640 medium. Alternatively, the hybridoma cells may be grownin vivoin the form of ascites in the body of a mammal.

Monoclonal antibodies secreted by the subclones may be isolated or further purified from culture medium or ascitic fluid using standard techniques for the purification of immunoglobulins, such as, for example, chromatography using protein a-Sepharose, chromatography on hydroxyapatite, gel electrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies can also be obtained using methods of recombinant DNA, such as methods, opisanie U.S. patent No. 4816567. DNA encoding the monoclonal antibodies according to the present invention, can be readily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells according to the present invention serve as a preferred source of such DNA. After DNA extraction can be put into expression vectors, which are then used for transfection of host cells, such as COS cells, monkey cells Chinese hamster ovary (CHO) or myeloma cells that are not otherwise produce immunoglobulin protein, to achieve the synthesis of monoclonal antibodies in the recombinant cell host. DNA can also be modified by replacing the coding sequence for the constant domains of the heavy and light chain of the human molecule homologous murine sequences [U.S. patent No. 4819567, Morrison et al., above] or by covalent joining the coding sequence of the immunoglobulin full or part of the coding sequence for a polypeptide other than an immunoglobulin. Such immunoglobulin polypeptide may be substituted for the constant domains of the antibodies according to the present image is meniu or may be substituted for the variable domains of antigen-United website antibody according to the present invention with the formation of a chimeric bivalent antibody.

Antibodies may represent a monovalent antibody. Methods of obtaining monovalent antibodies are known at the present time. For example, one such method involves recombinant expression of the light chain immunoglobulin and modified heavy chain of the immunoglobulin. The heavy chain can be mainly truncated at any point in the Fc section, so as to prevent the joining of the heavy chain. Alternatively, spend the substitution of the relevant cysteine residues with another amino acid residue or deleteroute that also prevents the stitching.

Can also be used in methods of thein vitroto obtain a monovalent antibody. Cleavage of antibodies with getting their fragments, particularly Fab fragments, can be accomplished using standard techniques known in this field.

Antibodies can also be obtained by screening of phage libraries for the presence of antibodies or fragments of antibodies that bind to the appropriate/desirable affinity with the β-chain of HGF (or their equivalent). Such techniques are known in this field and are described, for example, in U.S. patents№№ 5750373; 5780279; 5821047; 6040136; 5427908; 5580717 and contained in the references.

3. Human and humanized antibodies

Antibodies with β-chain of HGF according to the present invention may also include humanized antibodies richlovsky antibodies. Humanized forms of antibodies, non-human (e.g. murine)are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2or other antigen-binding sequences of antibodies)that contain minimal sequence derived from the immunoglobulin other than human. Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from the plot, complementarity determining (CDR) of the recipient are replaced by residues from a CDR of the body, which refers to species other than human (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases the remains of the frame Fv region of the human immunoglobulin are replaced by corresponding residues of the molecule, non-human molecule. Humanized antibodies may also include residues that are not found neither in the recipient antibody nor in the imported CDR or sequences of frame field. Basically, humanitariannet antibody includes essentially all or at least one, and typically two, variable domain, in which all or substantially all of the CDR parts correspond to those in immunoglobulin is e, non-human, and all or substantially all of the FR regions are to the consensus sequence of human immunoglobulin. Humanitariannet antibody optimally also includes at least a portion of constant region of immunoglobulin (Fc), in the typical case of a human immunoglobulin [Jones et al.,Nature,321:522-525 (1986); Riechmann et al.,Nature,332:323-329 (1988); and Presta,Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods of humanizing antibodies, non-human, known in this area. Basically humanitariannet antibody contains one or more amino acid residues built into it from a source that differs from the human. These amino acid residues of non-human molecule, also referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed according to the method of winter and co-authors (Winter and co-workers) [Jones et al.,Nature, 321,522-525 (1986); Riechmann et al.,Nature,332:323-327 (1988); Verhoeyen et al.,Science,239:1534-1536 (1988)] by replacing one or more CDR sequences of rodents corresponding sequences in the human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. patent No. 4816567), where essentially less than intact the th human variable domain has been substituted by the corresponding sequence from molecules other than human. Practically, humanized antibodies typically are human antibodies in which some CDR residues and possibly FR residues substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known in this field, including the use of phage libraries [Hoogenboom and Winter,J. Mol. Biol.,227:381 (1991); Marks et al.,J. Mol. Biol.,222:581 (1991)]. Methods Kula with co-workers and börner with co-authors (Cole et al., and Boerner et al.) also applicable for obtaining human monoclonal antibodies (Cole et al.,Monoclonal antibodies and Cancer Therapy,Alan R. Liss, p.77 (1985) and Boerner et al.,J. Immunol.,147(1):86-95 (1991). Similarly, human antibodies can be obtained by embedding loci of human immunoglobulin in transgenic animals, such as mice, where the endogenous immunoglobulin genes have been partially or completely inactivated. When the provocation is observed production of human antibodies, which is in all respects very closely reflects what exists in the human body, including gene rearrangement, Assembly or antibody-based test and repertoire. This approach is described, for example, in U.S. patents№№ 5545807; 5545806; 5569825; 5625126; 5633425; 5661016 and in the following scientific publications: Markset al.,Bio/Technology 10, 779-783 (1992); Lonberg .,Nature, 368, 856-859 (1994); Morrison,Nature,368, 812-13 (1994); Fishwild et al.,Nature Biotechnology14, 845-51 (1996); Neuberger,Nature Biotechnology14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol.13, 65-93 (1995).

Antibodies can be tucked "maturation" affinity using well-known methods of selection and/or mutagenesis, as described above. Preferred "ripened" by affinity antibodies are affinity, which is five times, more preferably 10 times, more preferably 20 or 30 times more than the activity of the original antibody (mainly mouse, gumanitarnogo or human antibodies), from which it was received "matured" antibody.

4. Bespecifically antibodies

Bespecifically antibodies are monoclonal, preferably human or humanized antibodies that have binding specificnosti at least two different antigens. In this case, one of the binding specificdate refers to the β-chain of HGF, and the other is for any other antigen, and preferably to a protein on the cell surface, or receptor or receptor subunit.

Methods of obtaining bespecifically antibodies known in the field. Standard recombinant method of obtaining bespecifically antibodies based on simultaneous expression of two pairs of heavy chain/light chain immuno is lobeline, where the two heavy chains have different specificity [Molstein and Cuello,Nature, 305:537-539 (1983)]. The result is a random rearrangement of the heavy and light chains of immunoglobulin these hybridoma (quadroma) produce a potential mixture of ten different antibody molecules, of which only one has the correct bespecifically structure. Purification of the correct molecule is usually done through several stages affinity chromatography. Similar procedures are described in WO93/08829, published 13 may 1993, and in Traunecker et al.,EMBO J.,10:3655-3659 (1991).

The variable domains of the antibodies with the desired binding specificnosti (joint sites of the antibody-antigen) can be attached to the sequence of the constant domain of immunoglobulin. The fusion preferably is performed with a constant domain of a heavy chain immunoglobulin comprising at least part of the hinge sections of CH2 and CH3. Preferably the constant part of the first heavy chain (CH1)containing the site necessary for binding to the light chain was present in at least one of the fused products. DNA encoding fused products on the basis of the heavy chains of immunoglobulin, and, if desirable, the light chain immunoglobulin is inserted into separate expression vector and subjected to joint transfection into an appropriate organism-the household is in. Additional details create bespecifically antibodies are described, for example, in the work of Suresh and co-authors (Suresh et al.,Methods in Enzymology,121:210 (1986)).

In accordance with another approach, described in WO 96/27011, the interface between a pair of antibody molecules can be arranged in such a way as to maximize the percentage of heterodimers recovered from cultures of recombinant cells. The preferred interface comprises at least part of the plot CH3 constant domain of the antibody. In accordance with this method, one or more small side chains of the amino acids from the interface of the first molecule or antibody-based test replace larger side chains (for example, the side chains of tyrosine or tryptophan). On the surface of the second molecule antibodies create compensatory "cavities" of identical or similar size to one or more large side chains, by the replacement of large side chains of amino acids smaller (e.g., alanine or threonine). This creates a mechanism to enhance the output of heterodimer relative to other unwanted end-products such as homodimers.

Bespecifically antibodies can be obtained as full-length antibodies or fragments of antibodies (e.g., F(ab')2bespecifically antibodies). Methods of creating bespecifically antibodies, fragments of antibodies description is found in the scientific literature. For example, bespecifically antibodies may be produced using chemical binding. Brennan and co-authors (Brenann et al.,Science229:81 (1985)describe a procedure in accordance with which the intact antibody is subjected to proteolytic cleavage with the formation of fragments F(ab')2. These fragments regenerate in the presence of sodium arsenite, as agent forming a complex with a dithiol, to stabilize nearby dithioles and prevent the formation of intermolecular disulfide bonds. The obtained Fab' fragments are then converted into derivatives of dinitrobenzoate (TNB). One of the Fab'-TNB derivatives is then convert to the Fab'-thiol by restoring using mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to education especifismo antibodies. Received bespecifically antibodies can be further used as agents for the selective immobilization of enzymes.

Fab'fragments can be directly isolated from cellsE. coliand then chemically related to education bespecifically antibodies. Chalabi with co-authors (Shalaby et al.,J. Exp. Med. 175:217-225 (1992)describe the receipt of a fully humanized molecules especifismo antibody F(ab')2. Fab'-fragment separately secreted by cells in theE. coli, then gooderham directed chemical binding in vitrowith the formation of especifismo antibodies. The thus created bespecifically antibody capable of contacting cells carrying the overexpression of the ErbB2 receptor and normal human T-cells, as well as to run the lytic activity of human cytotoxic lymphocytes against tumor cells in the breast of man, as a target.

Also described various methods of acquisition and allocation of fragments bespecifically antibodies directly from a culture of recombinant cells. For example, bespecifically antibodies obtained using latinovich zippers (Kostelnyet al.,J. Immunol.148(5):1547-1553 (1992)). Lacinova tipperne peptides from proteins Fos and Jun connect with Fab'portions of two different antibodies by gene fusion. Homodimeric antibodies restore hinge section to form monomers and then re-oxidized with the formation or antibody-based test of heterodimers. This method can also be used to obtain homodimeric antibodies. The technique of "dential"described by Hollinger with co-authors (Hollinger et al.,The OEWG. Natl. Acad. Sci USA90:6444-6448 (1993)), offers an alternative mechanism for creating fragments bespecifically antibodies. These fragments include the variable domain of the heavy chain (VH)connected to the variable domains of the light chain (V L) using a linker, which is short enough to make pairing between the two domains on the same chain. Respectively (VH) and (VL) domains of one fragment are forced to pair with the complementary (VL) and (VH) domains of another fragment, which leads to the formation of two antigen-binding sites. Also described another strategy to create slices bespecifically antibodies through the use of dimers of single-chain Fv (sFv). (See Gruberet al.,J. Immunol. 152:5368 (1994)).

It also considers antibodies with more than two valencies. For example, can be obtained thespecification antibodies (Tutt et al.,J. Immunol. 147:60 (1991)).

Typical bespecifically antibodies can bind to two different epitopes on the β-chain of HGF or epitope on the β-chain of HGF and epitope on a different polypeptide (e.g., c-met or α-chain HGF).

5.Heteroconjugate antibodies

Heteroconjugate antibodies are also included in the scope of the present invention. Heteroconjugate antibodies consist of two covalently United antibodies. Such antibodies, for example, have been proposed in order to direct the immune system cells to unwanted cells [U.S. patent No. 4676980] and for the treatment of HIV infection [WO 91/00369; WO 92/200372; EP 03089]. Such antibodies can be obtainedin vitrousing the m known methods in synthetic protein chemistry, including methods using cross-linking agents. For example, immunotoxins can be made by reaction of disulfide exchange or by formation of a thioester linkages. Examples suitable for this purpose reagents include immunocyt and methyl-4-mercaptopyrimidine, as well as the agents described, for example, in U.S. patent No. 4676980.

6.Impact on effector function using techniques of genetic engineering

The antibody according to the present invention it may be desirable to modify its effector function, so as to improve, for example, the effectiveness of the antibodies in the treatment of cancer. For example, one or more cysteine residues may be introduced in the Fc site, that creates the possibility for the formation of messagewall disulfide bonds in a given area. Thus obtained homodimeric antibody may have an improved ability to internalize and/or increased ability to complement-dependent lysis of cells and antibody-induced cellular toxicity (ADCC) (See. Caronet al.,J. Exp. Med.,176:1191-1195 (1992) and Shopes,J. Immunol.,148:2918-2922 (1992)). Homodimeric antibodies with enhanced anti-tumor activity may also be obtained using heterobifunctional cross-linking agents as described, for example, in Wolffet al., Cancer Research 53:2560-2565 (1993).

Alternate is but the antibody can be obtained by genetic engineering methods, so it will contain dual Fc plots and, thus, may have enhanced abilities to complement-dependent lysis and ADCC (See. Stevensonet al.,Anti-Cancer Drug Design, 3:219-230 (1989)).

7.Immunoconjugate

The present invention also relates to immunoconjugates, including antibody conjugated with a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e radioconjugates).

Chemotherapeutic agents used in the creation of such immunoconjugates were described above. Enzymatically active toxins and fragments, which can be used include the a chain of diphtheria, nesviazana active fragments of diphtheria toxin, exotoxin a chain of (Pseudomonas aeruginosa), And a chain of ricin And the chain abrina And chain modeccin, alpha sarcin, proteinsAleurites fordiiproteins of diantin, proteinsPhytolaca americana(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, krotin, inhibitor sapaonaria officinalis, gelonin, mitogillin, restrictocin, vanomycin, inomycin and tricothecene. There are many radionuclides to obtain radioconjugates antibodies. Examples included the t 212Bi131I131In90Y and186Re. Conjugates of the antibody and cytotoxic agent are created with the use of a large number of bifunctional protein-coupling agents such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylacetamide HCL), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-etidocaine (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium compounds (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as talien-2,6-diisocyanate), and bis-active fluorine-containing compounds (such as 1,5-debtor-2,4-dinitrobenzene). For example, rezinovy immunotoxin can be obtained according to the method described in Villetta with co-authors (Vitettaet al.,Science,238:1098 (1987)). Labeled by carbon-14 1-isothiocyanatobenzene-3-metallitron-triaminotoluene acid (MX-DTPA) is a representative chelating agent used for conjugation of the radionuclide and antibodies. Cm. WO94/11026.

In another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for utilization for pre-targeting tumor, when the conjugate of the antibody-receptor is administered to the patient, followed by removal from the bloodstream nesw the related conjugate using the output agent, then enter a "ligand" (e.g. avidin)which was anywhereman with a cytotoxic agent (e.g. a radionuclide).

8. Immunoliposome

In the present description, the antibodies can also be made in the form of immunoliposome. Liposomes containing the antibody, get known in this field techniques, such as described in Epsteinet al.,Proc. Natl. Acad. Sci. USA82:3688 (1985); Hwanget al.,Proc. Natl. Acad. Sci. USA,77:4030 (1980); and U.S. patent No. 4485045 and 4544545. Liposomes with increased residence time in blood is described in U.S. patent No. 5013556.

Especially commonly used liposomes can be obtained by the method of evaporation with phase reversal when using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivationally the phosphatidylethanolamine (PEG-PE). Liposomes extruded through filters with defined pore size to obtain liposomes of the desired diameter. Fab'-fragments of the antibodies according to the present invention can be conjugated to the liposomes according to the method described Martinet al.,J. Biol. Chem.,257:286-288 (1982) on the procedure of the reaction disulfide exchange. Chemotherapeutic agent (such as doxorubicin) may not necessarily be contained in the composition of the liposomes. Cm. Gabizonet al.,J. National Cancer Inst.,81(19):1484 (1989).

9. The pharmaceutical compositions of the Academy of Sciences is Itel

Antibodies can be administered for the treatment of various disorders in the form of pharmaceutical compositions.

If using whole antibodies as inhibitors, preferred antibodies capable of internalization. However lipofectin or liposomes can also be used to deliver the antibody according to the present invention in cells, if this is desirable. In that case, when using fragments of antibodies, the preferred smallest inhibitory fragment. For example, on the basis of sequences of the variable segment antibodies can be generated peptide and polypeptide molecules that retain the ability to bind β-chain of HGF and/or inhibit interaction between the β-chain of HGF and c-met, to interfere with the insertion of the N-terminal β-chain of HGF and/or inhibit the interaction type β-chain β-chain of HGF. Such peptides and polypeptides can be synthesized chemically and/or can be obtained by the methods of recombinant DNA (see, e.g., Marascoet al.,Proc. Natl. Acad. Sci. USA90:7889-7893 (1993)). Described in this application, the composition may contain more than one active connection in accordance with the need for specific indications to be treated, preferably with the presence of additional activities that do not have each other adverse effects. Alternative or additionally, the composition which I can include the agent, which increases its function, such as, for example, cytotoxic agent, cytokine, chemotherapeutic agent, or an agent that inhibits axonal growth. Such molecules are introduced in combination, in amounts which are effective for the intended purpose.

The active ingredients may also be entered in the microcapsules obtained, for example, by koatservatsii or cross-thread method of polymerization, for example, in hydroxymethylcellulose or gelatin microcapsules or poly-(methylmethacrylate) microcapsules, respectively, in the composition of the colloidal drug-delivery systems (e.g. liposomes, albumen microspheres, microemulsions, nanoparticles and nanocapsules) or in the composition of microemulsion. Such techniques are described in the manual Remington (Remington'sPharmaceutical Scienceabove).

Compositions suitable for use to introduce thein vivomust be sterile. Sterility can be achieved by carrying out filtering through membrane for sterile filtration.

Can be obtained drugs slow release. Suitable examples of drugs with slow release include semi-permeable matrices of solid hydrophobic polymers containing the antibody, where this matrix is the product of a particular form, for example, has the form of films and the and microcapsules. Examples of matrices with slow release include polyesters, hydrogels (for example, poly(2-hydroxyethylmethacrylate) or poly(vinyl alcohol)), polylactide (U.S. patent No. 3773919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-biodegradable, the ethylene vinyl acetate, degradable copolymers of lactic acid-glycolic acid such as LUPRON DEPOTTM(injectable microspheres composed of a copolymer of lactic acid-glycolic acid and acetate leuprolide) and poly-D-(-)-3-hydroxybutiric acid. While polymers such as ethylene vinyl acetate and a copolymer of lactic acid-glycolic acid capable of releasing molecules within 100 days, certain hydrogels release proteins for shorter time periods. In that case, when encapsulated antibodies remain in the body for a long time, they can denaturing or to form aggregates as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. In this regard, should be developed rational stabilization strategy, defined by the respective operating mechanism. For example, if it is shown that the aggregation mechanism involves the formation of intermolecular S-S connection through theodoulidis currency, stabilization mo is for can be achieved by modifying sulfhydryl residues, lyophilization from acidic solutions, controlling moisture content, using appropriate additives, and by developing specific polymer matrix compositions.

Below are examples of the methods and compositions according to the present invention. It should be understood that in practice the implementation of the present invention may be suitable for other options, while the above text gives only a General description of the invention.

EXAMPLES

MATERIALS AND METHODS

Mutants of HGF get essentially according to the method described in Kirchhofer et al., J Biol. Chem. (2004), 279:39915-24; Stamos et al. (2004) EMBO J. 23:2325-35. Tests for binding to Met and the migration of cells MDA-MB435 performed using reagents and methods described in Kirchhofer et al., above and Stamos et al., above. The test Met phosphorylation is performed using A549 cells, essentially as described in Kirchhofer et al., above. Similarly, the reach of inhibiting the phosphorylation of Met, except that the HGF mutants to assess the inhibition of phosphorylation add in a dose-dependent mode using a 50 ng/ml HGF. Inhibition of cell proliferation is carried out in the test for BxPC3, essentially as described in Kirchhofer et al., FEBS Lett. (2005) 579:1945-50). Test for angiogenesisin vitrocarried out essentially as described by Nakatsu et al., Microvasc. Res. 66:102, 2003). Mutants of HGF added to the culture medium at the concentration is 10 μg/ml every other day for 6 days of the experiment. At the end of the experiment determine the number of seedlings on the granule and Express the result as the average ±SD of 4 independent experiments.

Results

The mutant β-chain of HGF (in the form of one β-chain and in full-length HGF), with the mutation, assess their ability to communicate with MetIgG compared to the β-chain of HGF wild-type (one β-chain and a full-length HGF), using the test-based competitive ELISA. To minimize any possible formation of a dimer linked by a disulfide bond, β-chain of HGF wild type and mutant β-chain of HGF analyze amid C604S; thus, HGF β wild-type is actually HGF β C604S. In addition, evaluate the selected full-size double-stranded mutant HGF in the test cell migration to determine the effects, if any, on biological function by mutation in the β-chain. The data obtained is shown in figa, B, C. the Mutant HGF G498I inhibits HGF-dependent phosphorylation of Met-dependent doses as shown in figure 2, shows the results for HGF mutant R424A:R494E (single-chain HGF). Mutants of HGF G498I and G498P activate Met to a much lesser extent than wild-type HGF, the test results Met phosphorylation, as shown in figure 3, shows the results for HGF mutant R424A:R494E. HGF-dependent Met phosphorylation is modulated dependent on the PS. Mutants of HGF G498I and G498P also inhibit the proliferation of BxPC3 cells, characterized by the level of activity of 2.5% and 56% relative to the activity of wild type HGF. In addition, selected full-sized mutants of HGF at a concentration of 10 μg/ml (D672N, V495G, G498I, R424A:R494E) inhibit angiogenesis in the test in vitro (figure 4), further confirming the importance of the β-chain of HGF (and selected mutations) for the implementation of the full biological function of HGF.

A PARTIAL LIST of WORKS CITED

1. Polypeptide antagonist of HGF/C-Met, representing the mutant HGF containing a mutation in the N-terminal site of the β-chain of HGF and/or at the site of dimerization of β-chain of HGF, where a mutation in the N-terminal site of the β-chain of HGF is a V495G, V495A, G498I, G498P, G498V, R502del + T503del or D672N and where the mutation at the site of dimerization (3-chain HGF is a N497R, N497K, G498A, G498S, P500W, RN, RE, insertions, for example, R and S, between T and R502, or R502del.

2. Polypeptide antagonist according to claim 1, characterized in that the mutation in the N-terminal site of the β-chain of HGF violates the insertion of the N-terminal β-chain of HGF in HGF-binding pocket and where the resulting mutant HGF has significantly reduced biological function compared to wild type HGF.

3. Polypeptide antagonist according to claim 2, otlichuy is the, what biological function is cell proliferation, migration, cells, phosphorylation of Met or angiogenesis.

4. Polypeptide antagonist according to any one of the preceding paragraphs, characterized in that the mutation in the N-terminal site of the β-chain of HGF violates the insertion of the N-terminal β-chain of HGF in HGF-binding pocket and where the resulting mutant HGF binds to C-Met with substantially reduced binding affinity compared to wild-type HGF.

5. Polypeptide antagonist according to any one of claims 1 to 3, characterized in that the mutation in the N-terminal site of the β-chain of HGF violates the insertion of the N-terminal β-chain of HGF in HGF-binding pocket and where the resulting mutant HGF binds to C-Met with essentially equivalent affinity as wild type HGF.

6. Polypeptide antagonist according to any one of claims 1 to 3, wherein the molecule comprises a mutation at the site of dimerization of β-chain of HGF, where the resulting mutant HGF has significantly reduced biological function compared to wild type HGF.

7. Polypeptide antagonist according to claim 6, wherein the biological function is a cellular proliferation, cell migration, phosphorylation of Met or angiogenesis.

8. Polypeptide antagonist according to any one of claims 1 to 3, characterized in that the polypeptide comprises a mutation at the site of dimerization of β-chain of HGF, where the resulting mutant HGF has a reduced ability to dimerization with d the natives β-chain of HGF.

9. Polypeptide antagonist according to any one of claims 1 to 3, characterized in that the mutation at the site of dimerization chain HGF, essentially, does not violate the binding obtained mutant HGF to C-Met.

10. Polypeptide antagonist according to any one of claims 1 to 3, characterized in that the mutation in position N497 is not a mutation N497F, or E.

11. Polypeptide antagonist according to any one of claims 1 to 3, characterized in that the molecule consists of amino acids of the wild type in terms 534, 578, 619, 673, 692, 693, 694, 695, 696, 699 and/or 702.

12. Polypeptide antagonist according to any one of claims 1 to 3, characterized in that the polypeptide has a reduced C-Met signaling ability compared with wild-type HGF.

13. Polypeptide antagonist according to any one of claims 1 to 3, characterized in that the polypeptide has a reduced ability to stimulate migration of cells compared to wild type HGF.

14. Polypeptide antagonist according to any one of claims 1 to 3, characterized in that the polypeptide has a reduced ability to stimulate cell proliferation compared to wild type HGF.

15. Polypeptide antagonist according to any one of claims 1 to 3, characterized in that the polypeptide has a reduced ability to stimulate angiogenesis compared to wild type HGF.

16. The method of modulation of c-met activation in vitro, including the introduction of a polypeptide antagonist of HGF/c-met according to any one of claims 1 to 15, whereby is achieved by modulation of the activation of c-met.

17. The modulation of cell proliferation in vitro, including the introduction of a polypeptide antagonist of HGF/c-met according to any one of claims 1 to 15, whereby is achieved by modulation of cell proliferation.

18. The modulation of cell migration in vitro, including the introduction of a polypeptide antagonist of HGF/c-met according to any one of claims 1 to 15, whereby is achieved by modulation of cell migration.

19. The modulation method angiogenic activity of cells in vitro, including the introduction of a polypeptide antagonist of HGF/c-met according to any one of claims 1 to 15, whereby is achieved by modulation of the angiogenic activity of cells.

20. Nucleic acid encoding a polypeptide antagonist of HGF/c-met according to any one of claims 1 to 15.

21. A host cell comprising the nucleic acid according to claim 20, for expression of the polypeptide antagonist of HGF/c-met according to any one of claims 1 to 15.

22. A method of obtaining a polypeptide antagonist of HGF/c-met according to any one of claims 1 to 15, including expression in the cell-host nucleic acid that encodes a polypeptide antagonist, and secretion of the polypeptide antagonist of the cells.



 

Same patents:

FIELD: medicine.

SUBSTANCE: in order to predict insufficiency of saturation with oxygen of peripheral blood of pregnant women who have herpes-virus infection, content of glucose-6-phosphat-dehydrogenase and TNFα in peripheral blood is determined. Discriminant equation D=-0.589·G-6-PDG+{+1.23·TNFα} is solved. If value of discriminant function is equal or greater than 89.95, predicted is development of oxygen insufficiency, accompanied with reduction in peripheral blood of pregnant women of pO2 with titre of antibodies to Herpes simplex virus 1:12800 to 27.63±0.7 mm Hg, and of HbO2 to 90.2±0.47% (control: pO2 - 39.22±0.5 mm Hg; HbO2 - 95.3±0.27%).

EFFECT: increased accuracy of determining possibility of development of oxygen insufficiency in pregnant woman with herpes-virus infection.

3 tbl

FIELD: medicine.

SUBSTANCE: in estimation of local inflammation activity in case of background diseases of neck of uterus sampling of analysed biomaterial is performed by bringing of device for native material removal to external neck of uterus fauces. Said device has elongated holder in form of rod with length, exceeding length of vagina. From distal side of rod, at angle to it, located is operation element in form of roller. Proximal end of rod serves as manipulator-handle. Sampled material is placed until complete submergence into tightly closable test tube, filled with 1.0 ml of 0.155 M solution of sodium chloride. Mixture is centrifuged for 10 minutes. Obtained materials are analysed by method of solid phase ELISA analysis on boards by means of set of reagents "Vector-Best". Concentration of cytokine of interleukin-6 in analysed samples is determined spectrophotometrically. If value is higher than 50 pg/ml to 92 pg/ml, conclusion about presence of chronic inflammation process is made. If value is higher than 92 pg/ml, conclusion about active course of inflammatory process and expressed immune response is made.

EFFECT: application of method allows to increase quality of analysed native material and accuracy of obtained result without complicating analysis process.

2 cl, 3 ex

FIELD: medicine.

SUBSTANCE: invention can be used for diagnostics of presence of destructive process in uterine appendages in case of localised peritonitis if gynecological genesis, and for selection of optimal treatment tactics. In order to realise the method content of C-reactive protein (CRP) in blood serum is determined by turbodimetric method, if values of C-reactive protein content are 130 mg/l and higher, purulent-destructive process in uterine appendages in case of localised peritonitis is diagnosed, if values of C-reactive protein content are lower than 130 mg/l diagnosed is purulent salpingitis in case of pelvioperitonitis.

EFFECT: application of method allows to detect presence of purulent-destructive process in uterine appendages in case of pelvioperitonitis in urgent way, which determines tactics of further treatment.

2 ex

FIELD: medicine.

SUBSTANCE: invention can be used for obtaining diagnostic and/or therapeutic agents, in particular antibodies, which specifically interact with proteins of cell surface of target cells. Claimed is method of screening phage display library and including it method of isolation of library element, which is specific partner of binding of target cell surface protein, and method of isolation of unknown cell surface protein, specifically interacting with library element. Method of screening phage display library according to invention includes contact of said library with cells of interest with further separation of cells, which bound with one or several elements of expression library, from non-bound elements by separation through organic phase, and differs by introduction of additional stage, which consists in carrying out before said separation of at least one stage of material washing, and ensures essential increase of method efficiency.

EFFECT: higher efficiency.

30 cl, 7 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to ophthalmology, can be used for prediction of progression of myopia acquired in schools in 10-14 and 15-17 year old children. Blood serum is analysed for cartilage glycoprotein-39 with the use of sandwich-type enzyme immunoassay. If its concentration is within 34.7-53.4 ng/ml in 10-14-year-old children and 22-56.3 ng/ml in 15-17-year-old children, a permanent course of myopia is predicted. The concentration 18.5-33.9 ng/ml in 10-14-year-old children and 53.7-69 ng/ml in 15-17-year-old children enables to predict a progressive course of myopia.

EFFECT: use of the invention improves accuracy of the prediction of clinical course.

4 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to diagnostic techniques and concerns a diagnostic techniques technique for respiratory function of peripheral blood erythrocytes in pregnant women in herpes virus infection episode in the third trimester. The technique consists in determination of glutathione reductase and herpes virus antibody titre concentration by the enzyme immunoassay in peripheral blood in pregnant women suffered from a herpes virus infection episode. If glutathione reductase concentration is 8.36±0.137 E/g Hb (ref. - 7.82±0.185 E/g Hb), herpes virus antibody titre is 1:6400. If herpes virus antibody titre is increased to 1:12800, erythrocyte glutathione reductase concentration is decreased 4.48±0.22 E/g Hb.

EFFECT: technique is characterised by high sensitivity and allows predicting the development of impaired respiratory activity of erythrocytes in the pregnant women with the herpes virus infection episode.

FIELD: medicine.

SUBSTANCE: simultaneously two parametres are determined: for men - in ejaculate, and for women - in discharge of cervical canal, namely, slgA and lactoferrin. If level of secretory immunoglobulin A is 3.01±0.50 mcg/ml and lower and simultaneous level of lactoferrin is 6876±89 ng/ml and higher, gonococcus infection with systemic manifestations is diagnosed, if said condition is absent, localised gonorrhea is diagnosed.

EFFECT: application of claimed method allows carrying out differential diagnostics of various forms of gonococcus infection in case of its oligosymptomatic course.

3 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: before and after treating chronic heart disease, modified LP(a) are determined as follows. Blood serum 0.6 ml is treated with 0.1% Triton X-100 0. 2 ml, incubated for 15 minutes at 20°C. Then 7% polyethylene glycol 6000 is added, incubated with the Sudan Black stain at 40°C for 1 h; it is followed with electrophoretic separation of LP in agarose gel in a cavity 4×20 mm. The decreasing level of modified LP(a) by 40% and more in comparison with the primitive level, treating chronic heart disease treatment is considered as effective.

EFFECT: higher accuracy of estimating clinical effectiveness of chronic heart disease.

1 tbl, 6 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: there is offered a method to assess of enzyme's ability to the level of phosphorylation of polypeptide that implies a reaction of the analysed enzyme and a substratum presented with a biotin-conjugated fragment of 516 to 777 residues of a human insulin 1 receptor substratum (hIRS-1-p30), binding of the reaction product and immobilised streptavidin and detection of the level of phosphorylation by antibodies specific to the phosphorylated polypeptide residues.

EFFECT: according to the invention, the method allows identifying tyrosine and serine proteinases and can be taken as a basis of a test system for new modulators of their activity.

9 cl, 8 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: activity of enzymes MAO A and MAO B is determined in tumor tissue in menopausal and reproductive patients with uterine body cancer in stage III. If the coefficient value is 4.48 - 6.90 maintaining remission for at least 12 months after treatment termination is forecasted in treated patients, and if the coefficient value is 18.70 - 50.27 recurrence of disease minimally in 7 months is predicted.

EFFECT: use of the method enables to determine efficacy of integrated treatment of patients with uterine body cancer in menopause and reproductive periods.

2 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: purified RNA synthesised in vitro by nonviral transfection is introduced into placental and cord cells. RNA comprises at least one sequence providing cell transition to a pluripotent state.

EFFECT: method allows increasing efficiency of preparing selected somatic pluripotent cells, reducing probability of oncogenesis and other negative consequences of cell reprogramming, and eliminating invasiveness at the stage of preparation of the cell material.

10 dwg, 3 ex

FIELD: agriculture.

SUBSTANCE: nucleotide sequences, which code polypeptides of resistance to glyphosate are used in structures of DNA or expression cassettes for transformation or expression in organisms, including microorganisms and plants.

EFFECT: transformed organisms become resistant to herbicide.

17 cl, 5 dwg, 11 tbl, 23 ex

FIELD: medicine.

SUBSTANCE: method facilitates linkage of sequences, coding immunoglobulin variable regions, T-cells receptors or B-cells receptors. Method is instrument of higher effectivity for making sequence data libraries. Capability of multiple RT-PCR with chain extension by interruption with employment of matrix, derived from single cell, provides highly effective creation of sister pairs libraries.

EFFECT: method is effective for linkage of two or few nucleotide sequences, coding domens or subunits of heteromeric protein as a result of single reaction performance.

51 cl, 25 dwg, 27 tbl, 14 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically obtaining hemopoietic cells from blood, and may be used in medicine. The homopoietic cell CD34+ is extracted from peripheral blood of cancer patients undergone a growth factor treatment course. The obtained cell is transduced by a ligand which induces apoptosis with participation of the tumour necrosis factor, and is used to treat tumours.

EFFECT: invention enables to obtain a hemopoietic cell CD34+ which has anti-tumuor activity.

6 cl, 3 dwg, 8 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to a method of treating ophthalmopathies. The method of treating an ophthalmopathy in a mammal by recovery from mammal's bone marrow of a population of haematopoietic stem cells of a negative lineage which includes precursor endotheliocytes, transfection of the prepared population with a gene which functionally codes a fragment of antiangiogenic protein T2 of human tryptophanyl-tRNA-synthetase, and the following introduction in a vitreous body of the population of transfected cells in amount sufficient for symptomatic relief. The method of transgene delivery in a retinal vasculature of the mammal by introducing in the mammal's vitreous body of the population of haematopoietic stem cells of the negative lineage including precursor endotheliocytes where the cells are transfected with the antiangiogenic fragment of protein T2 of human tryptophanyl-tRNA-synthetase.

EFFECT: method of treating the ophthalmopathy is novel and highly effective.

2 cl, 27 dwg, 2 tbl, 10 ex

FIELD: agriculture.

SUBSTANCE: invention includes compositions, methods and cell lines related to insect control. Substance of invention includes compositions that contains vegetable essences purposely acting at least at one receptor of insects selected from the following - tyramine receptor , olfactory receptor Or83b or olfactory receptor Or43a, as a result of which intracellular levels cAMP, Ca+2 or both vary in insects. Also invention includes method of insect control, line of insects cells including sequence of nucleic acid of above mentioned receptors and methods for selection of composition by activity in respect to insects with application of specified line of insect cells.

EFFECT: development of softer sparing compositions that are not toxic for mammals and other types.

20 cl, 34 dwg, 1 tbl, 33 ex

FIELD: food industry.

SUBSTANCE: strain Streptococcus thermophilus which produces lactic acid is described. Sequence of nucleic acids made of the strain producing polysaccharides are also described as well as food or pharmaceutical composition and milk product containing such strain.

EFFECT: strain has strong structural properties.

16 cl, 4 dwg, 6 tbl, 5 ex

FIELD: biotechnologies.

SUBSTANCE: this invention is related to the field of biotechnology and may be used in production of various protein products with the help of recombinant DNA technology. New sequences of DNA are defined and generated, which are related to matrix attachment region, which are characterised by ability to improve producing of protein in eukaryotic cells.

EFFECT: methods are suggested for transfection of eukaryotic master cells, including new method of multiple transfection, based on use of active sequenes of DNA MAR according to invention and providing for substantial increase of recombinant protein expression level compared to similar cells transfected by traditional methods.

11 cl, 21 dwg, 9 tbl, 17 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of genetic engineering and medicine. Described is animal, non-human, having sequence of nucleic acid encoding presenilin 1, carrying mutations, corresponding to M233T and L235P mutations in PS1 protein of mouse. Animal also contains sequence of nucleic acid, encoding whole gene or part of gene, encoding APP. APP protein represents APP751, originates from human and carries mutations Swedish and London. Animal is intended for application in fight against Alzheimer's disease. Also described are PS1 protein and encoding it nucleic acid.

EFFECT: invention can be used in medicine for discovering compounds intended for Alzheimer's disease treatment.

20 cl, 50 dwg, 1 tbl, 8 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.

EFFECT: invention allows for making gamma-carboxylated vitamin K dependent protein in production quantities.

29 cl, 5 dwg, 6 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention can be used in medical and biologic industry for preparing antineoplastic drugs. Plasmid DNA pFK2 providing synthesis of recombinant analogue of human kappa casein fragment, in Escherichia coli cells is designed; and a method for preparing a recombinant product with using it is described. The recombinant analogue of human kappa-casein fragment recovered from Escherichia coli cells transformed by recombinant plasmid DNA pFK2 has molecular weight of approximately 16 kDa; consists of residual methionine, human kappa-casein fragment with 24 on 134 amino acid residue and C-terminal histidine path and exhibits apoptotic activity in relation to malignant cells.

EFFECT: higher anticancer activity of the compounds.

3 cl, 6 dwg, 4 ex

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