Methods and compositions for activation modulation of hepatocyte growth factor

FIELD: medicine.

SUBSTANCE: there is offered a method for identification of an inhibiting candidate substance which enables inhibits hepsin-activated HGF. The method is based on comparing the degree of activation of pro-HGF substratum in a sample containing hepsin, pro-HGF and the analysed candidate substance, with the degree of activation of pro-HGF substratum in the reference sample who not containing the candidate substance. Also there is offered an agent for inhibiting hepsin and hepatocyte growth factor reaction containing a sequence of Kunitz domain which represents a KD-1 sequence of Kunitz domain HAI-1 or HAI-1B, or one or both Kunitz domains HAI-2.

EFFECT: invention allows identifying physiological hepsin modulators.

6 cl, 14 dwg, 1 tbl, 1 ex

 

RELATED APPLICATIONS

This application is a non-provisional application filed under rule 1.53(b)(1) of article 37 of the CFR, which, in accordance with rule 119(e) of article 35 USC, claims the priority of provisional application number 60/591339 filed July 26, 2004, the contents of which are incorporated herein by reference.

The technical FIELD

The present invention mainly relates to the fields of molecular biology and regulation of growth factors. More specifically, the invention relates to modulators of the transmission signal HGF/c-met and applications of these modulators.

PRIOR

Hepsin (also known as TMRPRSS1) is expressed on the cell surface serine protease such as chymotrypsin, and is a representative of a family of transmembrane serine protease type II (TTSP), which also includes matriptase (also known as MT-SP1) and enteropeptidase [1]. Human gene Hasina, located on chromosome 19 in q11-13,2 [2], encodes the polypeptide of 417 amino acids [3], containing a short N-terminal cytoplasmic end of transmembrane region and an extracellular domain (Arg45 - Leu417), consisting of a cysteine-rich domain phagocytic receptor (SRCR domain) and proteasome domain. Proferment hepsin autocatalytically is activated through cleavage by Arg162 - Ile163 [4], forming a heterodimeric enzyme with protease domain, by a disulfide bond (Cys153-Cys277) associated with the SRCR domain. In addition to the covalent bond Cys-Cys, recently a certain crystal structure of hepsin revealed that the SRCR domain and proteiny domain share a broad contact area, about 1200Å2each domain [5]. As the contact area is located near the proximal relative to the membrane residues of the SRCR domain, proteiny domain and the active center of hepsin can be located near the cell surface (5). This is fundamentally different from the other located on the cell surface serine proteases, such as coagulation factors VIIa (FVIIa)1, IXa and Xa, active centers which are much higher (60-80 Å) membrane surface [6-8].

The physiological function of hepsin unclear. With the exception of coagulation factor VII, macromolecular substrates is not known and physiologically relevant inhibitors have not been identified. The role of hepsin in blood coagulation was proposed Kazama et al. (1995) [9], which showed that transfetsirovannyh Hasina cells can activate coagulation factor VII. However, mice deficient hepsin were viable and they did not reveal any blood coagulation disorder [10, 11], which raises doubts about the role of hepsin in normal hemostasis. However, it is possible that hepsin can contribute to the formation of fibrin in pathological situations, such as renal cell carcinoma [12], where the primary initiator of blood coagulation, tissue factor [13, 14], is missing. In addition, other studies suggest a functional link between Hasina and cell growth. It was reported that depending on the line of tumor cells and used experimental conditions, hepsin had stimulating growth [15] or suppressive growth [16] action. Further information on hepsin can be found, in particular, in PCT publication No. WO2004/009803; U.S. patent No. 6482630; U.S. patent No. 6423543; U.S. patent No. 5981830; publication of patent application U.S. No. 2004/0009911 A1; publication of patent application U.S. No. 2004/0001801 A1; publication of patent application U.S. No. 2003/0223973 A1; publication of patent application U.S. No. 2003/0175736 A1; publication of patent application U.S. No. 2003/0013097 A1 (also WO02/059373); publication of patent application U.S. No. 2003/0049645 (also WO02/064839) and the publication of the patent application U.S. No. 2004/0132156.

Recent experiments on gene expression have been identified hepsin as one of the most highly activated genes in prostate cancer [17-22]. Staining ofin siturevealed the expression of hepsin on the epithelial cells of the prostate glands [19]. Expression of hepsin correlated with neoplastic transformation [19], being higher in the tumors of patients with severe form of the disease and the lowest p and benign hyperplasia [18, 22]. In contrast, one study found that low expression of the protein Hasina correlates with high rates of Gleason and large tumors [20]. It's obvious contradiction with the used methods, i.e. immunohistochemistry [20], in contrast to counting RNA [18, 22], is unclear. In addition, hepsin also strongly activated in ovarian cancer [23] and in renal cell carcinoma, where it is mainly associated with the epithelial cells of the type [12].

On the surface of epithelial cells hepsin ideally situated to interact with components of the extracellular matrix and other associated with membrane proteins. It is known that the chymotrypsin-like serine proteases, including matriptase TTSP (synonym MT-SP1) [24, 25], which is structurally related to gasino, activate fibrinolytic enzymes, matrix metalloproteinases and latent forms of growth factors, such as a growth factor for hepatocytes (HGF). HGF stimulates cell proliferation, migration, angiogenesis, survival, and morphogenesis by activating the receptor tyrosine kinase Met (reviewed in [26, 27]). In addition to its importance in normal physiology, the way HGF/Met involved in invasive tumor growth and tumor metastasis [26]. HGF has a high similarity with serine protease with plasminogen and consists of α-chains containing N-is Onaway domain and four domain "kringle" and β-chains with homology to chymotrypsin-like-protease. It is secreted in the extracellular matrix in the form of inactive single-chain precursor (Pro-HGF) and requires activating cleavage by Arg494-Val495 for the formation of biologically competent, linked by a disulfide bond of heterodimeric α/β [28-31]. This stage is mediated conversion of Pro-HGF serine-proteases, such as activator growth factor hepatocyte (HGFA) [32], matriptase [33, 34], plasminogen activator urokinase type (u-PA) [35], factor XIIa [36], factor XIa and plasma kallickrein [34]. HGFA and matriptase inhibited expressed on the cell surface inhibitors type Konitza, such as two variants of splicing activator inhibitor growth factor hepatocyte HAI-1 [37, 38] and HAI-1B [34], and by HAI-2 [39]. HAI-2 (also known as placental bikunin) [40] is also strongly inhibits factor XIa and plasma kallickrein [41], whereas HAI-1B has weak inhibitory activity, or does it not have [34]. Thus, the biological availability of a pool of Pro-HGF in the extracellular matrix is regulated by the activity of the Mac Pro-HGF and their inhibitors.

The expression profile of hepsin in the tissues of malignant tumors, as described above, associated with its possible role in the action as a regulator of other growth factors, the dysregulation of which may underlie carcinogenesis, allows p is ecologici, the modulation of the interaction of hepsin with its substrate may be an effective therapeutic approach. In this respect there is a clear need to identify the physiological substrate of hepsin and/or physiological modulator(s). The invention meets this need and provides other benefits.

All cited in this document of references, including patent applications and publications, are incorporated herein by reference in full.

Description of the INVENTION

As described in this document, the physiological substrate for hepsin, protein cell surface strongly sverhagressivnomu in many malignant tumors, is a growth factor for hepatocytes, in respect of which it is known that it plays an important role in many aspects of the development of malignant tumors. In this paper it is shown that pepsin cleaves Pro-HGF activity, comparable with powerful physiological convertases Pro-HGF, HGFA (activator growth factor hepatocyte). Double-stranded (activated) HGF formed by Hasina, shows normal biological activity, including the induction of phosphorylation of tyrosine Met, stimulation of cell proliferation and stimulation of cell migration. In addition, in this document, as the physiological is practical regulators of enzymatic activity Hasina, identified containing two domains Konitza inhibitor, HAI-1 and HAI-2. The invention relates to methods and compositions based at least in part, on these discoveries, which are described below in more detail. It is shown that pepsin and its interaction with HGF and/or its physiological inhibitors can be a unique and effective target for more accurate adjustment in the development of preventive and/or therapeutic approaches to counteract pathological condition associated with abnormal or unwanted signal transmission path HGF/Met (also referred to as "c-met"). Thus, the invention relates to methods, compositions, kits and finished products for the identification and use of substances capable of modulating the path of HGF/c-met through the modulation of physiological interacting molecules involved in the regulation of HGF activation.

Thus, in one aspect the invention relates to a method of screening (or identification) possible inhibitory substances (i.e. antagonist)that inhibits activation of the HGF Hasina, where the method includes: (a) bringing the matter candidate in contact with the first sample containing pepsin and substrate Pro-HGF, and (b) comparison of the degree of activation of the substrate Pro-HGF in a sample with the degree of activation of the substrate Pro-HGF in atalantamarina, containing equal to the first sample number of hepsin and substrate Pro-HGF, but not in contact with the specified substance candidate, in accordance with the decrease in the degree of activation of the substrate Pro-HGF in the first sample compared to the reference sample indicates that the substance-the candidate is able to inhibit activation by Hasina single-chain HGF (Pro-HGF). In one embodiment, pepsin in the sample is effective for activation of Pro-HGF. The substrate Pro-HGF, suitable for use in these methods, can be in several forms, provided that it reproduces the property of the site of cleavage by Hasina on Pro-HGF. Examples of the substrate Pro-HGF include as non-limiting examples a full-sized single-chain HGF containing a form of the peptide bond R494-V495 wild type, and any fragment of HGF containing this peptide bond. Such a fragment can be of any length, for example, a length of at least approximately) 5, 7, 10, 15, 20, 25 amino acids or long (approx) from 4 to 25, 5 to 20, from 7 to 15 amino acids. Typically and preferably, the substrate Pro-HGF contains a peptide bond R494-V495, capable of splitting under the action of hepsin wild type. In one embodiment, the substrate Pro-HGF contains the cleavage site of human HGF corresponding to the consensus phase the cleavage proteases (i.e. basically residue in position P1and two hydrophobic amino acid residue in positions P1' and P2': P1- R494, P1' - V495, P2' - V496).

In another aspect, the invention relates to a method of screening substrate, blocking the activation of Pro-HGF by Hasina, where the method includes screening for substrate binding (preferably, but not necessarily, specifically) hepsin or Pro-HGF and blocking specific interaction (e.g., binding) of hepsin and Pro-HGF. In some embodiments, the implementation of the substance competes with Hasina for binding to HGF. In some embodiments, the implementation of the substance competes with Pro-HGF for binding to Hasina. In one embodiment, the substance contains, consists of or essentially consists of the amino acid sequence of at least approximately 60%, 70%, 80%, 90%, 95%, 99% similarity or sequence identity with respect to Pro-HGF (e.g., human), for example, a fragment of human HGF containing amino acid residues 494 (Arg)related peptide bond with 495 (Val). In some embodiments, the implementation, where the substance contains, consists of or essentially consisting of such amino acid sequence, a fragment of the mutated or has been deprived of at least part of the beta chain of HGF, so the selection has reduced the asset is the dominant of c-met activity compared to wild type HGF.

As is obvious to a person skilled in the art, screening tests, corresponding to the above analyses, can also include a first stage screening measures based on the formation of complex hepsin-HGF to obtain a first group of possible modulating substances, with subsequent second stage of screening based on the ability of the first group of possible modulating substances to modulate the activation of HGF and/or HGF conversion into a form that can activate the path of HGF/c-met. Appropriate indicators can be anything that is obvious to the person skilled in the art based on the knowledge about the formation of the enzyme-substrate complex and/or biological activity associated with the path of signal transmission HGF/c-met. The formation of the enzyme-substrate complex can be measured, for example, conventional biochemical tests (e.g., gel electrophoresis, chromatography, NMR etc). The biological activity of HGF/c-met include as non-limiting examples of phosphorylation of C-met phosphorylation of cellular molecules that are substrates of the kinase C-met, the cell growth (proliferation, survival, etc.), angiogenesis, cell migration, morphogenesis, cells, etc.

In one aspect the invention relates to antagonists of HGF/c-met violating transmission signal HGF/c-met. For example, the invention relates to molecules which, inhibiting the cleavage of Pro-HGF by Hasina (for example, splitting in position R494-V495). The molecule may exercise their inhibitory effect in a number of ways, including, as non-limiting examples, the binding or Hasina or Pro-HGF, so that inhibited the cleavage of Pro-HGF by Hasina, linking with complex hepsin-Pro-HGF, so that inhibited the cleavage of Pro-HGF, and/or the binding of Pro-HGF or Hasina (one by one or in combination)that inhibited the effects of splitting the HGF Hasina (for example, inhibition of release of HGF after splitting Hasina). In one embodiment, the molecule antagonist according to the invention did not inhibit the binding of HGF to c-met. For example, in one embodiment, the implementation of a molecule antagonist according to the invention is not an antibody or its fragment having a similar inhibitory and/or binding ability as the antibody produced by hybridoma cell line deposited under inventory number American Type Culture Collection ATCC HB-11894 (hybridoma 1A3.3.13) or HB-11895 (hybridoma 5D5.11.6). In one of the embodiments molecule antagonist according to the invention inhibits the biological activity associated with activation of HGF/c-met.

In one aspect, the antagonist according to the invention on the basis described in this document opening that inhibit the market activator growth factor hepatocyte (HAI-1, HAI-1B, HAI-2) are powerful inhibitors of the activation of Pro-HGF by Hasina. In one of the embodiments the invention relates to antagonist activation of Pro-HGF by Hasina where specified antagonist includes at least a part of the human HAI-1, HAI-1 or HAI-2 (including a full-sized). In one embodiment, the implementation of this part contains a sequence of domain Konitza (KD), capable of inhibiting the activation of Pro-HGF under the action of hepsin. In one embodiment, the implementation of the specified sequence domain Konitza represents a domain of Konitza 1 (KD1) HAI-1 or HAI-1B. In one embodiment, the implementation of the antagonist according to the invention contains a variant sequence KD1, at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% the sequence identity with the wild-type KD1 human HAI-1, where the specified variant sequence has at least comparable with wild-type KD1 ability to inhibit the cleavage of the human Pro-HGF under the action of hepsin. In one embodiment, the implementation of the antagonist according to the invention contains an option KD1 sequence with a sequence identity with the wild-type KD1 human HAI-1 is approximately from 70% to 99%, from about 75% to 98%, from about 80% to 97%, from 85% to 95%, where the specified sequence has at least the least comparable with wild-type KD1 ability to inhibit cleavage by Hasina human Pro-HGF. In one embodiment, the implementation of the specified sequence domain Konitza represents one or both domains of Konitza HAI-2. In one embodiment, the implementation of the antagonist according to the invention contains a variant sequence domain Konitza HAI-2, at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% the sequence identity with the corresponding domain(s) Konitza wild-type human HAI-2, where this variant sequence has at least comparable with HAI-2 wild-type ability to inhibit cleavage by Hasina human Pro-HGF. In one embodiment, the implementation of the antagonist according to the invention contains a variant sequence domain Konitza HAI-2 with sequence identity to the corresponding domain(s) Konitza wild-type human HAI-2 is approximately from 70% to 99%, from about 75% to 98%, from about 80% to 97%, from 85% to 95%, where the specified sequence has at least comparable with HAI-2 wild-type ability to inhibit cleavage by Hasina human Pro-HGF.

In some embodiments, the implementation of the antagonist according to the invention represents or contains a low-molecular compound, peptide, antibody, antibody fragment, aptamer, or a combination thereof. As described in this document antagonists m which should be obtained in the usual way using known in the field of methods (including described in more detail below) based on open interaction Hasina, the activator inhibitors of growth factor hepatocyte and Pro-HGF, as described in this document. For example, in some embodiments, the implementation of the antagonist according to the invention competes with Hasina for binding to HGF, but has no ability to cleave Pro-HGF in the area splitting Hasina. In some embodiments, the implementation of the antagonist according to the invention competes with Pro-HGF for binding to Hasina. For example, in one of the embodiments specified antagonist contains, consists of or essentially consists of the amino acid sequence of at least approximately 60%, 70%, 80%, 90%, 95%, 98%, 99% similarity or sequence identity is relatively Pro-HGF (e.g., human) and will definitely link hepsin, but it has no plot splitting hepsin (for example, a plot of P1containing the peptide bond of human wild-type HGF R494-V495) and/or it lacks the ability to activate c-met (for example, where metirovan β-chain of HGF, no β-chain of HGF or its part, and so on). In one embodiment, the implementation of the antagonist according to the invention contains, consists of or essentially consists of a fragment of HGF, are able to bind hepsin, where the specified portion is devoid of at least part of the β-chain of HGF, so that the fragment has reduced the activating c-met action what about the comparison with the wild type HGF.

Thus, the invention relates to mutant HGF able largely to link hepsin, but with reduced compared to wild-type HGF modulating activity against HGF/c-met, for example, the antagonist activity of HGF/c-met or HGF variant, with a reduction, but not with the absence of biological activity of HGF (for example, stimulating cell growth effect). In one embodiment, the implementation of the antagonist according to the invention are able to inhibit the biological activity (in vivoHGF wild type (such biological activity includes, as non-limiting examples, the stimulation of cell proliferation, increased survival of cells, stimulation of angiogenesis, induction/stimulation of cell migration). In one embodiment, the implementation of the antagonist according to the invention provides reduced stimulating cell growth activity (including as non-limiting examples of stimulating cell proliferation activity, stimulating cell survival activity, angiogenin stimulating, stimulating cell migration activity).

In some embodiments, the implementation of the antagonist according to the invention is produced by way of screening or identification according to the invention, as described in this document.

In one aspect of the mol is Kula-antagonist according to the invention is associated with a toxin, such as a cytotoxic agent. These molecules/substances can make or type in combination with additional reinforcing means, such as radiation and/or chemotherapeutic agent.

In one aspect the invention relates to a molecule capable of enhancing splitting Hasina Pro-HGF, where this molecule is able to inhibit the interaction of HAI-1, HAI-1B and/or HAI-2 Hasina. In some embodiments, the implementation of the amplifying molecule according to the invention represents or contains a low-molecular compound, peptide, antibody, antibody fragment, aptamer, or a combination thereof. For example, the amplifying molecule according to the invention can contain, or consist essentially consist of a fragment or variants of HAI-1, HAI-1B and/or HAI-2, where the specified fragment capable of binding hepsin, but not significantly inhibits the cleavage of Pro-HGF by Hasina. In one embodiment, the implementation of this molecule capable of competitive inhibition of binding of HAI-1, HAI-1B and/or HAI-2 wild type Hasina. In one embodiment, the implementation of the amplifying molecule according to the invention is an antibody that prevents the formation of a complex containing pepsin and HAI-1, HAI-1B and/or HAI-2. In one embodiment, the implementation of the amplifying molecule according to the invention is hepsin is whether its option (including any of them, defined below), where hepsin or variant is capable of cleavage of Pro-HGF section R494-V495.

The invention also relates to methods and compositions suitable for modulating disease States associated with impaired regulation of the transmission signal HGF/c-met. Thus, in one aspect the invention relates to a method of modulating the activation of c-met in a subject, where the method includes introduction to the subject modulating HGF/c-met molecule according to the invention (for example, a molecule antagonist, as described herein, inhibiting cleavage of Pro-HGF by Hasina), whereby modulated activation of c-met. In one embodiment, the implementation of this molecule is an antagonist of HGF/c-met inhibitory activity of HGF/c-met. In one embodiment, the implementation of this molecule represents amplifying molecule that increases the activity of the HGF/c-met. In one aspect the invention relates to a method of treating pathological conditions associated with activation of c-met in a subject, where the method includes introduction to the subject an antagonist of c-met according to the invention (for example, any of the antagonists in the cleavage of Pro-HGF by Hasina, as described herein), whereby inhibited the activation of c-met.

The transmission signal HGF/c-met is involved in many the creation of biological and physiological functions, including, for example, stimulation of cell growth (e.g., cell proliferation, survival, cells, cell migration, cell morphogenesis and angiogenesis. Thus, in another aspect, the invention relates to a method of inhibiting the activated c-met cell growth (e.g., proliferation and/or survival), where the method includes bringing the cells or tissue in contact with the antagonist according to the invention, whereby inhibited cell proliferation associated with activation of c-met. In another aspect the invention relates to a method of inhibiting angiogenesis, where the method includes introducing into the cell, tissue and/or body subject to the condition associated with abnormal angiogenesis antagonist according to the invention, whereby angiogenesis is inhibited. In another aspect the invention relates to a method of enhancing angiogenesis, where the method includes introducing into the cell, tissue and/or body of the subject in a state that could be improved with increased angiogenesis and/or which is associated with suboptimal degree of angiogenesis, enhancing molecules according to the invention, whereby angiogenesis is enhanced.

In one aspect the invention relates to the use of the antagonist according to the invention to obtain a Lek is stannage tools for therapeutic and/or preventive treatment of disease, such as tumor, cancer, impaired cell proliferation, immune (such as autoimmune) disorder and/or associated with angiogenesis violation. The antagonist may be any described herein form, including the antibody, antibody fragment, small molecule compound (for example, an organic molecule), a polypeptide (e.g., an Oligopeptide), nucleic acid (e.g., oligonucleotide, such as an antisense oligonucleotide or interfering RNA, aptamer, or a combination thereof.

In one aspect the invention relates to the use of amplifying molecule according to the invention to obtain drugs for therapeutic and/or prophylactic treatment of diseases, such as wound healing (e.g., RAS associated with diabetes, trauma etc). Amplifying molecule can be any described herein form, including the antibody, antibody fragment, small molecule compound (e.g., organic molecules), polypeptide (e.g., an Oligopeptide), nucleic acid (e.g., oligonucleotide, such as an antisense oligonucleotide or interfering RNA, aptamer, or a combination thereof.

In one aspect the invention relates to the use of nucleic acids according to the invention to obtain the drug among the STV for therapeutic and/or preventive treatment of disease, such as tumor, cancer, impaired cell proliferation, immune (such as autoimmune) disorder and/or associated with angiogenesis violation (e.g., wound healing).

In one aspect the invention relates to the use of the expressing vector according to the invention to obtain drugs for therapeutic and/or prophylactic treatment of diseases such as tumor, cancer, impaired cell proliferation, immune (such as autoimmune) disorder and/or associated with angiogenesis violation (e.g., wound healing).

In one aspect the invention relates to the use of host cell according to the invention to obtain drugs for therapeutic and/or prophylactic treatment of diseases such as tumor, cancer, impaired cell proliferation, immune (such as autoimmune) disorder and/or associated with angiogenesis violation (e.g., wound healing).

In one aspect the invention relates to the use of the finished product according to the invention to obtain drugs for therapeutic and/or prophylactic treatment of diseases such as tumor, cancer, impaired cell proliferation, immune (such as autoimmune) bre is of and/or associated with angiogenesis violation (for example, wound healing).

In one aspect the invention relates to the use of the kit according to the invention to obtain drugs for therapeutic and/or prophylactic treatment of diseases such as tumor, cancer, impaired cell proliferation, immune (such as autoimmune) disorder and/or associated with angiogenesis violation (e.g., wound healing).

In one aspect the invention relates to a method of inhibiting the activated c-met cell proliferation, where the method includes bringing the cells or tissue in contact with an effective amount of an antagonist according to the invention, whereby inhibited cell proliferation associated with activation of c-met.

In one aspect the invention relates to a method of treating a pathological condition associated with dysregulation of the activation of c-met in a subject, where the method includes introduction to the subject an effective amount of an antagonist according to the invention, whereby the specified condition being treated.

In one aspect the invention relates to a method of inhibiting growth of cells expressing c-met or a growth factor for hepatocytes, or both of these factors, where the method includes bringing the specified cells in the context of the act with the antagonist according to the invention, thus causing inhibition of the growth of these cells. In one of the embodiments, the cell is in contact with HGF expressed by another cell (for example, through a paracrine effect).

In one aspect the invention relates to a method of therapeutic treatment of a mammal with a malignant tumor containing cells expressing c-met or a growth factor for hepatocytes, or both of these factors, where the method includes the introduction of the specified mammal an effective amount of an antagonist according to the invention, thereby effectively carrying out the treatment specified mammal. In one of the embodiments, the cell is in contact with HGF, downregulation of another cell (for example, through a paracrine effect).

In one aspect the invention relates to a method for the treatment or prevention of disorders of cell proliferation associated with increased expression or activity of hepsin, c-met and/or growth factor hepatocyte, where the method includes the introduction in need of such treatment to a subject an effective amount of an antagonist according to the invention, thereby exposing the breach cell proliferation effective treatment or prevention. In one embodiment, the implementation of the breach proliferation of pre what is a malignant tumor.

In one aspect the invention relates to a method of inhibiting cell growth, where the growth in this cell, at least partially, depends on the potential growth of the actions of hepsin, c-met and/or growth factor hepatocyte, where the method includes bringing the specified cell into contact with an effective amount of an antagonist according to the invention, thereby inhibiting the growth of specified cells. In one of the embodiments, the cell is in contact with HGF expressed by another cell (for example, through a paracrine effect).

In one aspect the invention relates to a method of therapeutic treatment of a tumor in a mammal, where the growth in this tumor, at least partially, depends on the potential growth effect of hepsin, c-met and/or growth factor hepatocyte, where the method includes bringing the specified cell into contact with an effective amount of an antagonist according to the invention, thus exposing the specified effective tumor treatment. In one of the embodiments, the cell is in contact with HGF expressed by another cell (for example, through a paracrine effect).

The methods according to the invention can be applied to influence any suitable pathological condition, for example, cells and/or tissue associated with the disturbance d is ulali of hepsin and/or the transmission path of the signal HGF/c-met. In one embodiment, the implementation of the cell, which is the target of the method according to the invention is a cell of a malignant tumor. For example, the cell is a malignant tumor can be a cell selected from the group consisting of breast cancer cells, colorectal cancer cells, lung cancer cells, cells of papillary carcinoma (e.g., thyroid cancer), cancer cells, colon cancer cells, pancreatic cancer, ovarian cancer cells, cancer cells of the cervix, malignant tumors of the Central nervous system, cancer cells prostate cancer cells, osteogenic sarcoma cells pochernkletocny carcinoma cells, hepatic cell carcinoma, malignant tumors of the bladder, carcinoma cells of the stomach, squamous cell carcinoma of the head and neck cells , melanoma cells and leukemia. In one embodiment, the implementation of the cell, which is the target of the method according to the invention, is a hyperproliferative and/or hyperplastic cell. In one embodiment, the implementation of the cell, which is the target of the method according to the invention, is a dysplastic cell. In yet another embodiment, the cell which is the target of the method according to the invention, is a metastatic cell.

FPIC is least according to the invention can further include additional processing stage. For example, in one embodiments, the method additionally includes the stage at which the cell or tissue which is the target (e.g., a malignant cell) is subjected to radiation or exposure to a chemotherapeutic agent.

As described herein, activation of c-met is an important biological process, the dysregulation of which leads to numerous pathological conditions. Thus, in one of the embodiments of the methods according to the invention, the cell-target (e.g., a malignant cell) is a cell in which compared to a normal cell of the same tissue origin increased activation of c-met. In one embodiment, the implementation of the method according to the invention causes the death of target cells. For example, contact with an antagonist according to the invention may result in the inability of cells to transmit a signal through c-met, which leads to cell death.

Dysregulation of activation of c-met (and thus signal transmission) may occur due to a number of cellular changes, including, for example, the overexpression of HGF (recognizable c-met ligand) and/or c-met. Thus, in some embodiments, the implementation of the method according to the invention includes the defeat of the cells where c-met or growth factor hepatocyte is in, or both of these factors specified by the cell (e.g., malignant cells) expressed more strongly compared to a normal cell of the same tissue origin. Expressing c-met cell may be regulated HGF from a variety of sources, i.e. autocrine or paracrine manner. For example, in one of the embodiments of the methods according to the invention, the cell-target contacts/associated with growth factor hepatocyte expressed in another cell (for example, through a paracrine effect). Stated another cell may be the same or different tissue origin. In one embodiment, the implementation of cell-target contacts/associated with HGF expressing in the cell a target (for example, due to autocrine effect/loops).

In one aspect the invention relates to a method comprising introducing to a subject amplifying molecule according to the invention. Suitable for treatment in this way include any pathological condition associated with abnormal/undesirable low physiological levels of angiogenesis-related activity of HGF/c-met in a subject. Examples of such conditions include as non-limiting examples of wound healing, cardiac hypertrophy, myocardial infarction, limb ischemia, a disease of perifericheskikh arteries, etc.

In one aspect the invention relates to compositions containing one or more antagonists or amplifiers according to the invention and a carrier. In one of the embodiments the carrier is pharmaceutically acceptable.

In one aspect the invention relates to nucleic acids encoding the antagonist or amplifying molecule according to the invention. In one embodiment, the implementation of the nucleic acid according to the invention encodes an antagonist or amplifying molecule representing or containing the polypeptide (for example, an Oligopeptide). In one embodiment, the implementation of the nucleic acid according to the invention encodes an antagonist or amplifying molecule representing or containing the antibody or fragment.

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

In one aspect the invention relates to a cell host containing a nucleic acid or vector according to the invention. The vector may be a vector of any type, for example a recombinant vector, such as expressing vector. You can apply any of a variety of master cells. In one of the embodiments a host cell is a prokaryotic cell, for example,E. coli. In one of the variants of the s implementation of a host cell is a eukaryotic cell, for example, mammal cells, such as cell Chinese hamster (CHO).

In one aspect the invention relates to methods for antagonist or intensifying molecules according to the invention. For example, the invention relates to a method for antagonist representing or containing the antibody (or fragment), where the method includes the expression in a suitable cell host recombinant vector according to the invention, encoding the indicated antibody (or fragment), and the allocation of the indicated antibodies. In another example, the invention relates to a method for antagonist or intensifying molecules representing or containing the polypeptide (such as an Oligopeptide), where the method includes the expression in a suitable cell host recombinant vector according to the invention, encoding the specified polypeptide (such as an Oligopeptide), and the allocation of the specified polypeptide (such as an Oligopeptide).

In one aspect the invention relates to a finished product with a container and a composition in the container, where the composition contains one or more antagonists or intensifying molecules according to the invention. In one of the embodiments the composition contains a nucleic acid according to the invention. In one variant the implementation of the composition, containing antagonist or amplifying molecule further comprises a carrier, which in some embodiments is pharmaceutically acceptable. In one embodiment, the implementation of the finished product according to the invention further comprises instructions for administration of the composition (e.g., antagonist or intensifying molecules) to the subject.

In one aspect the invention relates to a kit containing a first container containing a composition comprising one or more antagonists or intensifying molecules according to the invention and a second container containing a buffer. In one of the embodiments the buffer is pharmaceutically acceptable. In one of the embodiments the composition comprising the antagonist or amplifying molecule additionally contains a carrier, which in some embodiments is pharmaceutically acceptable. In one of the embodiments set further comprises instructions for administration of the composition (e.g., antagonist or intensifying molecules) to the subject.

BRIEF DESCRIPTION of FIGURES

Figure 1. The degree of purity of hepsin and its activity against factor VII. (A) Expressed by CHO cells soluble hepsin contains the entire extracellular domain (Arg45-Leu417) and the C-terminal His label8. On stained gels (restore the valid conditions) purified Hasina shows that hepsin spontaneously transformed in its double-stranded form by splitting on Arg163-Ile163 (confirmed by N-terminal sequencing). (B) Activation of proferment factor VII Hasina (40 nm) in the presence of PCPS vesicles and CaCl2at 37°C for a period of 2 hours. The provisions of proferment FVII and light chain (LC.) and proteasome domain (TS.) FVIIa marked. The molecular weight markers are shown as Mr× 10-3.

Figure 2. Specific activation of Pro-HGF by Hasina. Labeled125I Pro-HGF (0.05 mg/ml) were incubated in 20 mm Hepes, pH 7.5, 150 mm NaCl (Hepes buffer) with decreasing concentrations of hepsin and HGFA: stage 3-fold dilution of the enzymes from 40 nm on track 2 to 0.16 nm on the track 7. (A) Pepsin and (B) HGFA. After 4 hours at 37°C, the samples were analyzed by SDS-PAGE in reducing conditions, followed by exposure to x-ray film. The position of Pro-HGF α-chain of HGF β-chain of HGF (doublet) are marked. Track 1 is an aliquot directly selected at the beginning of the reaction. (C) Activation of plasminogen by t-PA (tissue plasminogen activator) and hepsin. The plasminogen (0,12 mg/ml) were incubated with t-PA (40 nm), Hasina (40 nm) or buffer (control) in Hepes buffer. At different points in time were selected aliquots and analyzed on SDS-PAGE (reducing conditions) with subsequent staining dye Simply Blue Safe Stain. D. the cone 1, buffer after 0.5 hours; track 2, t-PA after 0.5 hours; lane 3, hepsin after 0.5 hours; lane 4, the buffer after 5 hours; lane 5, t-PA after 5 hours; lane 6, hepsin after 5 hours. The provisions of plasminogen (Plg) and the heavy chain of plasmin (TC.) indicated.

Figure 3. Phosphorylation Met through Pro-HGF-activated Hasina and HGFA. (A) Unlabeled Pro-HGF (0.3 mg/ml) were digested with 40 nm of hepsin or 40 nm HGFA, receiving >95% conversion. Formed due to cleavage by Hasina (HGFhepsinand due to the splitting of HGFA (HGFHGFA) HGF were analyzed on SDS-PAGE (reducing conditions) and stained with the dye Simply Blue Safe Stain. (B) Phosphorylation of the Met receptor was measured in the analysis KIRA through effects on A549 cells to increasing concentrations of HGFhepsin(circle), HGFHGFA(squares) or scHGF (diamonds), unsplit single-chain form of HGF. The activity was expressed as a percentage of the maximum signal obtained with the control drug HGF. Values represent the arithmetic mean ± SD of three experiments.

Figure 4. Cell proliferation and migration with Pro-HGF, activated Hasina (HGFhepsinand HGFA (HGFHGFA). (A) Cell proliferation of BxPC3 cells in the presence of increasing concentrations of HGFhepsin(shaded columns) and HGFHGFA(empty columns). Values represent the average arithm the political two experiments. (B) Quantitative analysis of cell migration stimulated by increasing concentrations of HGFhepsin(shaded columns) and HGFHGFA(empty columns), added to the lower chamber of the system transwell cell migration. Values represent the arithmetic mean ± SD of three experiments. Activity in assays of cell proliferation and migration was expressed as percentage of control cells exposed to 100 ng/ml control drug HGF, which was included in each experiment.

Figure 5. Inhibition of the enzymatic activity of hepsin in amiloliticheskoi analysis. Pepsin (0.4 nm) were incubated with inhibitors for 30 min at room temperature and kinetic microplate reader were determined by enzymatic activity in relation to S2366 (0.2 mm ≈ Km). (■), sHAI-1B, (▼), sHAI-1B (R260A), (●), sHAI-1B (K401A), (●), sHAI-2.

6. Inhibition of the activation of Pro-HGF mutant forms of sHAI-1B and by sHAI-2. Labeled125I Pro-HGF (0.05 mg/ml) were incubated with Hasina (15 nm) and inhibitors for 4 hours at 37°C. Aliquots of the reaction mixture were analyzed as described in figure 1. Hepsin 15 nm were present in each sample. The level of inhibitors was 1 μm. Lane 1, sample at t = 0; 2, without inhibitor; 3, sHAI-1B; 4, sHAI-1B (R260A); 5, sHAI-1B (K401A); 6, sHAI-2. The position of Pro-HGF α-chain of HGF β-chain of HGF (doublet) indicated.

7. Od the n of the options for the implementation of the amino acid sequence of natural human hepsin.

Fig. Another variant implementation of the amino acid sequence of natural human hepsin.

WAYS of carrying out the INVENTION

The invention relates to methods, compositions, kits and finished products containing modulators transmission signal HGF/c-met, including methods of using such modulators.

This document provides a detailed description of these methods, compositions, kits and finished products.

The main methods

If not stated otherwise, in the practical implementation of the present invention apply generally accepted methods of molecular biology (including recombinant methods), Microbiology, cell biology, biochemistry and immunology, well-known experts in this field. Such methods are fully described in the literature, such 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, and periodic updates); "PCR: The Polymerase Chain Reaction", (Mullis et al., ed., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001).

Definitions

In this document the term "pepsin" refers to polypeptides with the natural sequence, polypeptide variants and fragments of the polypeptide with natural serial what inetu and polypeptide variants (which are defined in this document are optional), capable of cleavage of Pro-HGF way similar to Hasina wild type. Described in this document polypeptide of hepsin can be a polypeptide of hepsin, which is selected from a variety of sources, such as from human tissue samples or from another source, or obtained recombinant or synthetic methods. The terms "pepsin", "polypeptide of Hasina", "enzyme Hasina" and "protein Hasina also include variants of the polypeptide of hepsin, as described in this document.

"Polypeptide of hepsin with the natural sequence" includes a polypeptide with the same amino acid sequence as the polypeptide of hepsin obtained from natural conditions. In one of the embodiments the polypeptide of hepsin with the natural sequence contains the amino acid sequence of SEQ ID No. 1 (see Fig.7). In one of the embodiments the polypeptide of hepsin with the natural sequence contains the amino acid sequence of SEQ ID No. 2 (see Fig). Such a polypeptide Hasina with the natural sequence can be isolated from natural sources or to obtain recombinant or synthetic methods. The term "polypeptide Hasina with the natural sequence" specifically refers to naturally occurring truncated or secretively forms spiral the nogo polypeptide of hepsin (for example, the sequence of the extracellular domain), a naturally occurring forms of the variants (e.g., alternative splicing) and naturally occurring allelic variants of the polypeptide.

In this document "variant of the polypeptide of hepsin" or its variants means a polypeptide Hasina, as a rule, the active polypeptide Hasina, at least about 80% amino acid sequence identity with any of the sequences of polypeptides of hepsin with the natural sequence, as described herein. Such variants of the polypeptide of hepsin include, for example, polypeptides of hepsin, where the N - or C-Terminus of the natural amino acid sequence added or removed one or more amino acid residues. Typically, the variant polypeptide of Hasina has at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the sequence of the polypeptide of hepsin with the natural sequence, as described herein. Typically, the length of the polypeptide variants of hepsin is at least about 10 amino acids, alternatively, the length is, less than the least approximately 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 amino acids or more. Not necessarily in the polypeptide variants of hepsin is no more than one conservative amino acid substitutions compared to the natural sequence of the polypeptide of hepsin, alternatively, not more than 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions compared to the natural sequence of the polypeptide of hepsin.

"Percent (%) amino acid sequence identity" in relation to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in the sequence of the candidate 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 sequence identity, and without considering any conservative substitutions as part of the identity sequence. Alignment with the purposes of determining percent identity of amino acid sequences can be accomplished in various ways known to the experts in this field, for example, using a public computer or the cluster software such as software BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Specialists in this field can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment throughout the compared sequences. However, for the purposes of the present description value % identity of amino acid sequences were obtained using the computer program comparison sequence ALIGN-2, as described in U.S. patent No. 6828146.

In this document, the term "vector" is intended to refer to the nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a closed circular double-stranded DNA, which can be ligitamate additional segments of DNA. Another type of vector is a phage vector. Another type of vector is a viral vector, where the viral genome can be ligitamate additional segments of DNA. Certain vectors are capable of Autonomous replication in a cell-master in which they were introduced (e.g., bacterial vectors having a bacterial replication origin and epilimnia vectors mammals). Other vectors (e.g., napisanie vectors mammals) in the introduction to edu host can integrate into the genome of the host cell and, thus, replicated together with the genome of the host. Moreover, certain vectors are capable of directing the expression of genes with which they are functionally linked. Such vectors are denoted herein as "recombinant expressing vectors" (or simply, "recombinant vectors"). Basically, expressing vectors, suitable methods of recombinant DNA, are often in the form of plasmids. In the present description, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector.

"Polynucleotide" or "nucleic acid", used interchangeably herein, 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 the polymer by DNA or RNA polymerase or by synthesis reaction. Polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogues. If present, the modified nucleotide can be performed before or after Assembly of the polymer. The nucleotide sequence may be interrupted dinucleotide components. P is dinucleotid can be further modified after synthesis, for example, by conjugation with a label. Other types of modifications include, for example, "kierowanie, the replacement of one or more naturally occurring nucleotides a similar, mezhnukleotidnyh modifications such as, for example, mezhnukleotidnyh modification with uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoamide, carbamates, etc) and with charged linkages (e.g., phosphorothioate, phosphorodithioate etc), mezhnukleotidnyh modification containing side groups, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine and so on), mezhnukleotidnyh modification intercalators (e.g., acridine, psoralen, etc.), mezhnukleotidnyh modification containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc), mezhnukleotidnyh modification containing alkylating funds mezhnukleotidnyh modification with modified linkages (e.g., alpha anomeric nucleic acids, etc), as well as unmodified forms of polynucleotide(s). In addition, any of the hydroxyl groups typically present on sugars, it is possible to replace, for example, phosphonate groups, phosphate groups, protected by standard protective groups or activated for a education additional links with additional n is cleotide, or you can konjugierte with solid or semi-solid substrates. 5'- and 3'-terminal OH you can fosforilirovanii or replace amines or organic molecules kiperousa groups with the number of carbon atoms from 1 to 20. Other hydroxyl can also be derivateservlet standard protective groups. Polynucleotide can also contain forms analogues of sugars ribose or deoxyribose that, generally known in this field, including, 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 nucleoside analogues without reason, such as methylribose. One or more fosfolipidnyh relations can be replaced by alternative linking groups. These alternative linking groups include as non-limiting examples, embodiments of where the phosphate is replaced by P(O)S ("tiat"), P(S)S ("ditial"), "(O)NR2(amidate"), P(O)R, P(O)OR', CO or CH2(formatall"), in which each R or R' independently represents H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. Not all of the links in polynucleotide dollars which have to be identical. The above description applies to all mentioned in this document polynucleotides, including RNA and DNA.

Herein, "oligonucleotide"typically refers to short, usually single-stranded, as a rule, synthetic polynucleotides that, typically, but not necessarily, be less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. Above for polynucleotides description equally and fully applicable to oligonucleotides.

In this document, the term "growth factor hepatocyte" or "HGF", unless specifically stated or the context clearly otherwise, refers to any natural HGF polypeptide or variant (or naturally occurring or synthetic)capable of activating the transmission signal HGF/c-met in conditions that ensure the passage of the process. As a rule, the term "HGF wild-type" refers to a polypeptide containing the amino acid sequence of naturally occurring HGF protein. As a rule, the term "HGF sequence wild-type" refers to amino acid sequences found in naturally occurring HGF.

The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal is nitela (for example, full or whole monoclonal antibodies), polyclonal antibodies, multivalent antibodies, polyspecific antibodies (for example, bespecifically antibodies, provided that they exhibit the desired biological activity) and may also include certain antibody fragments (as described in more detail herein). The antibody can be human, humanized and/or affinity Mature.

"Antibody fragments" contain only part of a whole antibody, where this part preferably retains at least one, preferably most or all of the functions normally associated with this part when it is in a whole antibody. In one of the embodiments the antibody fragment contains antigennegative plot of whole antibodies and, thus, retains the ability to bind antigen. In another embodiment, the antibody fragment, for example, an antibody fragment containing the Fc region, retains at least one biological functions normally associated with the Fc-region, when it is in a whole antibody, such as FcRn binding, change the half-life of antibody function, ADCC and binding of complement. In one embodiment, the implementation of a fragment of the antibody is a monovalent antibody, which has a half-life ofi vivo essentially the same as the whole antibody. For example, such an antibody fragment may contain antigennegative shoulder associated with the Fc sequence, can give the fragment stabilityin vivo.

In this document the term "monoclonal antibody" refers to an antibody obtained from a group essentially homogeneous antibodies, i.e., the individual in the group antibodies are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed to a single antibody. In addition, unlike drugs polyclonal antibodies, which typically include different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a determinant on the antigen.

The monoclonal antibodies herein 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 specific class or subclass of antibody, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from other who Idov or belonging to another class or subclass of antibody, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (U.S. patent No. 4816567 and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

"Humanized" forms of non-human (e.g. murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-owned human immunoglobulin. Mainly, humanized antibodies are human immunoglobulins (recipient antibody)in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody)such as mouse, rat, rabbit or non-human Primate having the desired specificity, affinity and capacity. In some cases, remnants of the framework region (FR) of a human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues which are not found in the recipient antibody or in the donor antibody. These modifications are carried out to further improve the characteristics of the antibodies. Basically humanitariannet antibody contains essentially all, of at least one, and typically two, variable domains, where all or substantially all of the Hyper is variabelnah loops correspond to the hypervariable loops non-human immunoglobulin, and all or substantially all of the FR is FR sequence of the human immunoglobulin. Humanitariannet antibody optionally also contain at least a portion of constant region of immunoglobulin (Fc), typically that of a constant region of human immunoglobulin. For more detail, see 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). Cm. the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

"Human antibody" is an antibody with the amino acid sequence corresponding to the amino acid sequence of the antibody produced by a human and/or made using any of the methods for obtaining human antibodies, as described herein. This definition of human antibodies, in particular, excludes humanitariannet antibody containing non-human antigennegative remains.

"Affinity matured" antibody is an antibody with one or more changes in one or more CDR, which leads to increased affinity of antibodies against the antigen compared to the original antibody without this change(s). Preferred affinity Mature antibodies have nanomo is popular or even picomolar appendectomy in relation to the target antigen. Affine Mature antibodies get known in this field means. In Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by permutation domains VH and VL. Random mutagenesis of CDR and/or residues frame region described by: Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995) and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

"Blocking" antibody or an "antagonist" antibody is an antibody inhibiting or reducing the biological activity of the antigen with which it is associated. Preferred blocking antibodies or antagonist antibodies largely or completely inhibit the biological activity of the antigen.

Herein "antibody agonist" is an antibody that mimics at least one type of functional activity of interest polypeptide.

"Violation" is any condition that can be helped through treatment substance/molecule or method according to the invention. It includes chronic and acute disorders or diseases including those pathological conditions that create susceptibility of a mammal to the specified violation. Non-limiting examples of disorders to treatment according to this document, including the up in himself malignant and benign tumors; melakoski and lymphoid malignancy; neuronal, glial, astrocytic, hypothalamic violations and violations of other gland, macrophagal, epithelial, stromal and blastocele disorders and inflammatory, immunological and other associated angiogenesis disorders.

The terms "impaired cell proliferation and impaired proliferation" refers to violations associated with some degree of abnormal cell proliferation. In one embodiment, the implementation of a violation of cell proliferation is a malignant tumor.

Herein, "tumor" refers in General to the growth and proliferation of neoplastic cells, malignant or benign, and all pre-malignant and malignant cells and tissues. In this document, the terms "cancer", "cancerous", "violation of cell proliferation, impaired proliferation" and "tumor" are not mutually exclusive.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of malignant tumors include, as non-limiting examples, carcinoma, lymphoma, blastoma, sarcoma and leiko is. More specific examples of such malignant tumors include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, malignant tumor of the abdominal cavity, liver cell cancer, malignant tumor of the gastrointestinal tract, cancer of the pancreas, glioblastoma, cervical cancer, ovarian cancer, liver cancer, malignant bladder cancer, hepatoma, breast cancer, cancer of the colon, colorectal cancer, endometrial or uterine carcinoma of the salivary glands, malignant tumor, a malignant tumor of the liver, cancer of the prostate cancer, a malignant tumor of the external female genital organs malignant tumor of thyroid cancer, hepatic carcinoma and various types of malignant tumors of the head and neck.

Herein, "treatment" refers to clinical intervention aimed at changing the natural course of the disease under treatment of an individual or cell, and it can be done or for the prevention or in the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, reducing any direct is 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 implementation of the antibodies according to the invention are used to delay development of a disease or impairment.

"Effective amount" refers to an amount effective at 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 invention, the agonist or antagonist can vary, depending on such factors as stage of disease, age, sex and weight of the individual and the ability of a substance/molecule, agonist or antagonist to cause the individual's desired response. Therapeutically effective amount also represents the amount at which any toxic or detrimental effects of the substance/molecule, agonist or antagonist were outweighed by therapeutically favorable effects. "Prophylactically effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve a prophylactic result. Typically, but not necessarily as a prophylactic dose is used in the subject of the offering prior to or in the early stages of the disease, prophylactically effective amount is less than therapeutically effective amount.

In this document the term "cytotoxic agent" refers to a substance inhibitory or prevent the functioning of cells and/or calling the destruction of cells. The term is intended to include radioactive isotopes (e.g., At211I131I125, Y90That Re186That Re188Sm153Bi212, P32and radioactive isotopes of Lu), chemotherapeutic agents, for example, methotrexate, adriamycin, Vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, hlorambuzila, daunorubicin or other intercalating agents, enzymes and fragments, such as nucleotidase enzymes, antibodies and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants, and the various antitumor or anticancer means described below. The following are other cytotoxic funds. Destroying the tumor means cause the destruction of tumor cells.

"Chemotherapeutic agent" is a chemical compound suitable for the treatment of malignant tumors. Examples of himioterpevticeski funds include alkylating means, such as thiotepa and CYTOXAN® (CYTOXAN®) cyclophosphamide; alkyl sulphonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodepa, carboquone, matureup and uredepa; ethylenimines and methylmelamine, including altretamin, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and triethylenemelamine; acetogenins (especially bullatacin, bullatacin); Delta-9-tetrahydrocannabinol (dronabinol, MARINOL (MARINOL®)); beta-lapachone; lapachol; colchicine; betulin acid; camptothecin (including the synthetic analogue topotecan (COSMEGEN® (HYCAMTIN®)), CPT-11 (irinotecan, CAMPTOSAR® (CAMPTOSAR®)), acetylcystein, scopoletin and 9-aminocamptothecin); bryostatin; callistemon; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllin acid; teniposide; cryptophycins (especially 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, uramustine; nitrosoanatabine, such as carmustine, chlorozotocin, fotemustine, lomustin, nimustine and runnin the Steen; antibiotics such as andinavia antibiotics (for example, calicheamicin, especially calicheamicin gamma and calicheamicin omega (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin a; spiramycin; and the chromophore neocarzinostatin and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, autralian, azaserine, bleomycin, actinomycin, carubicin, karminomitsin, calcination, chromomycin, dactinomycin, daunorubicin, demoralizing, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN® (ADRIAMYCIN®), morphosyntactical, cyanomethaemoglobin, 2-pyrrolidinecarbonyl, liposomal injectable drug doxorubicin HCl (DOXIL® (DOXIL®)and desoxidation), epirubicin, zorubicin, idarubitsin, marsellaise, mitomycin, such as mitomycin C, mycofenolate acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, colomycin, radiobeacon, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate, gemcitabine (GEMZAR® (GEMZAR®)), tegafur (AFTERAL® (UFTORAL®)), capecitabine (XELODA® (XELODA®)), epothilone and 5-fluorouracil (5-FU); analogs of folic acid, such as deeperin, methotrexate, peripherin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, timipre, tioguanin; the taxes pyrimidine, such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenergicheskoe drugs, such as aminoglutetimid, mitotane, trilostane; the joints of folic acid, such as prolinnova acid; Eagleton; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; astroball; bisantrene; edatrexate; defaming; demicoli; diazinon; alternity; slipline acetate; etoposide; gallium nitrate; hydroxyurea; lentinan; londini; maytansinoid such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitrean; pentostatin; penomet; pirarubicin; losoxantrone; 2-acylhydrazides; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trihlortrietilamin; trichothecenes (especially toxin T-2, verrucarin a, roridin A and unguided); urethane; vindesine (ELDISINE® (ELDISINE®), FILESIN® (FILDESIN®)); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; Galitsin; arabinoside ("Ara-C"); thiotepa; taxoid, for example, paclitaxel (TAXOL® (TAXOL®)), constructed on the basis of albumin composition of nanoparticles of paclitaxel (ABRAXANE® (ABRAXANE™)) and d is setaccel (TAXOTERE® (TAXOTERE®)); chlorambucil; 6-tioguanin; mercaptopurine; methotrexate; platinum analogues, such as cisplatin and carboplatin; vinblastine (VELBAN® (VELBAN®)); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN® (ONCOVIN®); oxaliplatin; leucovorin; vinorelbine (NAVELBINE® (NAVELBINE®)); Novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; filtermaterial (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™ (ELOXATIN™)), combined with 5-FU and leucovorin.

Also in this definition included protivokomarinye means acting to regulate, reduce, block, or inhibit the effects of hormones that can stimulate the growth of malignant tumors and is often used in the form of systemic or General treatment. They can imagine themselves hormones. Examples include antiestrogens and selective estrogen receptor modulators (SERM), which includes, for example, tamoxifen (including tamoxifen NOLVADEX® (NOLVADEX®), raloxifene (EVISTA IS® (EVISTA®)), droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (FARESTON® (FARESTON®)); antiprogesterone means; negative regulators of estrogen receptors (ERD); antagonists of estrogen receptors, such as fulvestrant (FASLODEX® (FASLODEX®)); means operating to suppression or off the ovaries, for example, agonists releasing factor, luteinizing hormone (LHRH), such as leuprolide acetate (LUPRON® (LUPRON®), ELIGARD® (ELIGARD®)), goserelin acetate, buserelina acetate and triptorelin; other anti-androgens such as flutamide, nilutamide and bikalutamid and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutetimid, megestrol acetate (MEGASE® (MEGASE®), exemestane (AROMASIN® (AROMASIN®)), formestane, fadrozole, vorozole (RIVISOR® (RIVISOR®)), letrozole (FEMARA® (FEMARA®), and anastrozole (ARIMIDEX® (ARIMIDEX®)). In addition, such definition of chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS® (BONEFOS®) or OSTAC (OSTAC®), etidronate (DIDROCAL® (DIDROCAL®)), NE-58095, zoledronicaa acid/zoledronate (ZOMETA® (ZOMETA®), alendronate (FOSAMAX® (FOSAMAX®), pamidronate (AREDIA® (AREDIA®)), tiludronate (SKELID® (SKELID®), or risedronate (ACTONEL® (ACTONEL®); and also troxacitabine (1,3-dioxolane EN the log nucleoside cytosine); the antisense oligonucleotides, particularly those which inhibit expression of genes in the signal transduction pathways involved in impaired cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras and the receptor for epidermal growth factor (EGF-R); vaccines such as vaccines CERATOP® (THERATOPE®) vaccines and for gene therapy, for example, the vaccine ALLOVECTIN® (ALLOVECTIN®)vaccine LEUVECTIN (LEUVECTIN®) and vaccine VAXID® (VAXID®); a topoisomerase inhibitor 1 (for example, LURTOTECAN® (LURTOTECAN®)); rmRH (e.g., ABARELIX® (ABARELIX®)); lapatinib ditosylate (small molecule dual inhibitor tyrosine kinase ErbB-2 and EGFR, also known as GW572016); COX-2 inhibitors such as celecoxib (CELEBREX® (CELEBREX®); 4-(5-(4-were)-3-(trifluoromethyl)-1H-pyrazole-1-yl)benzosulfimide and pharmaceutically acceptable salts, acids or derivatives of any of the above.

In this document "growth inhibitory agent" refers to a compound or compositions that inhibit cell growth, the growth of which depends on the activation of HGF/c-metin vitroorin vivo. Thus, the growth inhibitory agent can be a tool that significantly reduces the percentage containing dependent HGF/c-met cells in S-phase. Examples of inhibiting the growth of funds include funds that are blocking the passage of the cell cycle (at a point other than the S-FA is s), such as tools, which induce arrest in the G1 phase and arrest in M-phase. Classic blockers M-phase include drugs periwinkle (vincristine and vinblastine), taxanes and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those funds, which carry out the arrest in the G1 phase, also carry out the arrest in S-phase, for example, alkylating DNA tools, such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-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" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially pp. 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs, both derived from the yew tree. Docetaxel (TAXOTERE® (TAXOTERE®), Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL® (TAXOL®), Bristol-Myers Squibb). Paclitaxel and docetaxel stimulate the Assembly of microtubules from tubulin dimers and stabilizes microtubules by preventing depolymerization, resulting in the inhibition of mitosis in cells.

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

Compositions and methods according to the invention

A. Antibodies

In one of the embodiments the present invention relates to antibodies that can be used according to the present description, as a therapeutic and/or diagnostic tools. Illustrative antibodies include polyclonal, monoclonal, humanized, bespecifically and heteroconjugate antibodies.

1. Polyclonal antibodies

Polyclonal antibodies are preferably induce in animals by multiple subcutaneous (sc) or intraperitoneally (ip) injections of the relevant antigen and an adjuvant. Can be effective to konjugierte the corresponding antigen (especially when using synthetic peptides) with a protein which is immunogenic in the species that are subjected to immunization. For example, the antigen can be konjugierte with hemocyanin marine saucers (KLH), serum albumin, bovine thyroglobulin, or soybean inhibitor of trypsin, using a bifunctional or derivatizing means, for example maleimidophenylmethacrylates ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2or R1N=C=NR, where R and R1represent skylinee group.

Animals subjected to immunization against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes complete adjuvant's adjuvant, and intradermal injection of a solution in a few places. A month later the animals are subjected supporting immunization from 1/5 to 1/10 the original amount of peptide or conjugate in complete Freund's adjuvant by subcutaneous injection at multiple locations. After passing from 7 to 14 days in animals take blood and serum analyzed for antibody titer. Animals are supportive of immunization before the release of the title on the plateau. The conjugates can also be obtained in the culture of recombinant cells in the form of a fused protein. To enhance the immune response it is convenient to use that aggregation tools such as alum.

2. Monoclonal antibodies

Monoclonal antibodies can be obtained using hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be obtained by using methods of recombinant DNA (U.S. patent No. 4816567).

In the hybridoma method, a mouse or other suitable animal host, such as a hamster, subjected to immunization, as described above, to obtain lymphocytes that produce or are capable of producing antibodies that specifically attributed audacia with protein, used for immunization. Alternatively, the lymphocytes can givein vitro. After immunization, lymphocytes secrete, and then drain line with myeloma cells using a suitable means, causing cell fusion, such as polyethylene glycol with the formation of a hybridoma cell (Goding, Monoclonal antibodies: Principles and Practice, p.59-103 (Academic Press, 1986)).

Thus obtained hybridoma cells were seeded and grown in a suitable medium for culturing, which preferably contains one or more substances that inhibit the growth or survival unused, original myeloma cells (also referred to as partner in the merger). For example, if the original myeloma cells lacking the enzyme hypoxanthineguanine (HGPRT or HPRT), selective medium for the cultivation for the hybridomas typically will contain gipoksantin, aminopterin and thymidine (Wednesday HAT), which prevent the growth of cells with deficiency of HGPRT.

Preferred myeloma cells-partners merger are cells that effectively merge, support stable high production of antibodies in selected cells that produce antibodies, and are sensitive to the selective medium, performing a selection from nalivshihsya the original cells. The preferred lines myeloma cells are what I murine myeloma lines, such as lines derived from murine tumors MOPC-21 and MPC-11, available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 and derivatives e.g., cells X63-Ag8-653, available from the American Type Culture Collection, Manassas, Virginia, USA. To obtain human monoclonal antibodies described cell lines of human myeloma and hypermile human-mouse (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Medium for cultivation, which grow hybridoma cells, assessed on the production of monoclonal antibodies directed to the antigen. Preferably, the binding specificity of monoclonal antibodies derived from hybridoma cells thus determined by or through analysis of the binding ofin vitrosuch as radioimmunological assay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of monoclonal antibodies, for example, can be determined by analysis of Scatchard, described by Munson et al., Anal. Biochem., 107:220 (1980).

Once identified hybridoma cells producing antibodies of the desired specificity, affinity and/or activity, the clones can be subclinical through serial dilutions and grown by standard methods (Goding, Monoclonal antibodies: Principles and Practice, p.59-103 (Academic Press, 1986)). Suitable for these purposes is her medium for cultivation include, for example, medium (D-MEM or RPMI-1640. In addition, the hybridoma cells can be grownin vivoas ascitic tumors in animals, for example, using the I.P. Pavlova. injection of cells of mice.

Monoclonal antibodies secreted by the subclones, the appropriate way to allocate environment for cultivation, ascitic fluid, or serum by using common methods purification of antibodies, such as, for example, affinity chromatography (e.g., using sepharose with protein A or protein G) or ion-exchange chromatography, chromatography on hydroxyapatite, the electrophoresis gel, dialysis, etc.

DNA encoding the monoclonal antibodies can easily be extracted and sequenced using conventional methods (e.g., using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a preferred source of such DNA. After DNA extraction can be placed in expressing vectors, which are then transferout in cell host, such as cellsE. colicells , COS monkey cells of the Chinese hamster (CHO) or myeloma cells that would otherwise not produce protein antibodies, to ensure the synthesis of monoclonal antibodies in the recombinant cell host. Review articles on recombinant expression in bacteria of DNA encoding ant the body, include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs. 130:151-188 (1992).

In additional embodiments, the implementation of the monoclonal antibodies or antibody fragments can be isolated from phage libraries of antibodies obtained using the methods described in McCafferty et al., Nature, 348:552-554 (1990). In Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) described the selection of mouse and human antibodies, respectively, using phage libraries. In subsequent publications described obtaining human antibodies with high affinity (nm range) rearranging circuits (Marks et al., Bio/Technology, 10:779-783 (1992)), as well as co-infection and recombinationin vivoas a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). Thus, these methods represent a competitive alternative to traditional methods of production of monoclonal antibodies by hybridomas for selection of monoclonal antibodies.

DNA encoding the antibody can be modified to obtain chimeric or fused polypeptide antibodies, for example, by replacing the human sequence constant domains of the heavy and light chains (CHand CL) homologous murine sequences (U.S. patent No. 4816567; and Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by SL the status coding sequence of the immunoglobulin with the whole or part of the coding sequence for a non-immunoglobulin polypeptide (heterologous polypeptide). Sequence non-immunoglobulin polypeptide can replace the constant domains of the antibody or they replace the variable domains of one antigennegative plot antibodies to obtain a chimeric bivalent antibody containing one antigennegative area, which is specific for one antigen and other antigennegative plot with specificity to a different antibody.

3. Human and humanized antibodies

Antibodies according to the invention can further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g. murine) antibodies are chimeric immunoglobulins, chains of immunoglobulins or fragments thereof (such as Fv, Fab, Fab', F(ab')2or other antigennegative subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of non-human species (donor antibody)such as mouse, rat or rabbit with the desired specificity, affinity and capacity. In some cases, Arcania remains Fv human immunoglobulin replaced by corresponding non-human residues. Humanized antibodies may also contain residues which are not found in the recipient antibody nor in the imported CDR or frame sequences. Basically humanitariannet antibody contains essentially all of at least one, and typically two, variable domain, in which all, or substantially all of the CDR regions correspond to the regions from non-human immunoglobulin and all or essentially all, of the areas FR constitute the region of the consensus sequence of human immunoglobulin. Optimally humanitariannet antibody will also contain at least a portion of constant region of immunoglobulin (Fc), typically that of a constant region 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 humanitarian non-human antibodies are well known in this field. As a rule, humanitariannet antibody has one or more amino acid residues introduced into it from a source that is not a person. These non-human amino acid residues are commonly referred to as "imported" residues, which are typically taken from an "import" variable domain. Humanitarian essentially can be performed according to the method of Winter and colleagues (Jones et al., Naure, 321:522-525 (1986); Riechmann et al., Nature. 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], replacing the CDRs or CDR sequences of rodents corresponding sequences of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (U.S. patent No. 4816567), where the corresponding sequence from a non-human species substituted substantially less than an intact human variable domain. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues substituted by residues from analogous sites of antibodies rodents.

The choice of human variable domains of light and heavy, for use in obtaining humanized antibodies is very important to reduce antigenicity and answer HAMA (human antibody to antibody mouse), when the antibody is intended for therapeutic use in humans. In accordance with the so-called method of "best match" sequence of the variable domain of the antibody rodent sceneroot against the complete library of known sequences of human variable domains. Identify the sequence of the human domain V which is closest to domain V of the rodent, and for gumanitarnogo antibody b is Ruth the human frame region (FR) (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). In another method uses a particular framework region derived from a consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same frame area can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).

In addition, it is important that antibodies were humanitarian with retention of high affinity binding in respect of the antigen and other favorable biological properties. To achieve this goal, in accordance with the preferred method, humanized antibodies are obtained from the application of the method of analysis of the source sequences and various speculative humanized products using three-dimensional models of the source and humanized sequences. Three-dimensional models of immunoglobulins are publicly available and well known to specialists in this field. Available computer programs which illustrate and display probable three-dimensional conformational structures of selected sequences of immunoglobulins candidates. The study of these images allows for analysis of the likely role of the residues in the functioning sequence of the immunoglobulin candidate, so what. the analysis of residues that influence the ability of the immunoglobulin candidate to bind antigen. Thus, it is possible to select and combine the remains of FR from the recipient and import sequences so as to achieve the desired characteristics of the antibodies, such as increased affinity for the antigen(s)target(s). Basically in effect on the binding of antigen directly and most substantially involved remains of the hypervariable region.

Consider various forms gumanitarnogo antibodies. For example, humanitariannet antibody may be an antibody fragment such as Fab, which is optional anywhereman with one or more cytotoxic means with the aim of obtaining immunoconjugate. Alternatively, humanitariannet antibody can be an intact antibody, such as an intact IgG1 antibody.

Alternatively, humanitarian, it is possible to obtain human antibodies. For example, currently it is possible to obtain transgenic animals (e.g. mice)that after immunization capable of producing a full repertoire of human antibodies in the absence of endogenous production of immunoglobulins. For example, described that the homozygous deletion of the gene of the connecting region of the heavy chain (JH) antibodies in chimeric and germ-mutant mice leads to the floor of the WMD inhibition of the production of endogenous antibodies. The transfer of a set of human germline immunoglobulin gene such embryonic mutants leads to production after stimulation by antigen human antibodies. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA. 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Measurement. 7:33 (1993); U.S. patent No. 5545806, 5569825, 5591669 (all GenPharm); 5545807 and WO 97/17852.

Alternatively, to obtain human antibodies and fragments of antibodiesin vitrofrom the repertoire of genes of variable (V) domains of immunoglobulins from unimmunized donors can apply the technology of phage display (McCafferty et al., Nature 348:552-553 [1990]). In accordance with this method, genes V-domain antibody clone in frame reading in the primary gene or minor envelope protein of filamentous bacteriophage, such as M13 or fd, and are as functional fragments of the antibodies on the surface ragovoy particles. Because filamentous particle contains a copy of the single-stranded DNA phage genome, selection on the basis of the functional properties of antibodies also leads to the selection of the gene encoding the antibody exhibiting those properties. Thus, phages mimic some properties of B-cells. Phage display can be performed in a variety of formats, considered, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). For phage display technique, you can use various sources of segments V-Geno is. Clackson et al., Nature, 352:624-628 (1991) identified a diverse set of antibodies to oxazolone from a small random combinatorial library of V genes derived from the spleens of immunized mice. You can construct a repertoire of V genes from unimmunized human donors and antibodies to various antigens (including its own antigens) can be distinguished, essentially following the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991) or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S. patent No. 5565332 and 5573905.

As indicated above, human antibodies can also be obtained through activatedin vitroB-cells (see U.S. patent 5567610 and 5229275).

4. Antibody fragments

In certain circumstances it is advantageous to use antibody fragments rather than whole antibodies. The smaller size of the fragments provides quick removal and may lead to improved access to solid tumors.

To obtain fragments of antibodies developed different ways. Typically, these fragments get through proteolytic cleavage of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can currently be obtained with the use of recombinant host cells. All antibody fragments, Fab, Fv and ScFv can be Express inE. coliand sekretarev the diamonds from her, thus, providing a light receiving large amounts of these fragments. Antibody fragments can be isolated from phage libraries of antibodies discussed above. Alternatively, fragments, Fab'-SH can be directly recovered from theE. coliand chemically bind with the formation of fragments F(ab')2(Carter et al., Bio/Technology 10:163-167 (1992)). In accordance with another approach, the fragments F(ab')2you can select directly from a culture of the recombinant host cells. In U.S. patent No. 5869046 described fragments Fab and F(ab')2with increased half-lifein vivocontaining residues of the epitope binding receptor salvation. Practitioners in this field are obvious other way to obtain fragments of antibodies. In other embodiments, implementation of the antibody of choice is a single-chain Fv fragment (scFv). Cm. WO 93/16185; U.S. patent No. 5571894 and U.S. patent No. 5587458. Only Fv and sFv are molecules with intact binding site that are constant region; thus, they are suitable for reduced nonspecific binding during usein vivo. You can construct fused proteins of sFv with the formation of compound effector protein with N - or C-end of the sFv. Cm. Antibody Engineering, ed. Borrebaeck, above. The antibody fragment may also be a "linear antibody", e.g., as described in the patent is U.S. 5641870. These linear fragments of antibodies can be a monospecific or bespecifically.

5. Bespecifically antibodies

Bespecifically antibodies are antibodies that have binding specificity for at least two different epitopes. Typical bespecifically antibodies can bind to two different epitopes of hepsin, HGF and/or complex hepsin:HGF, as described in this document. Other such antibodies may combine the binding site of these structures with the binding site for a different polypeptide. Alternative shoulder antibodies can be combined with an arm that binds to a trigger molecule on a leukocyte such as a molecule T-cell receptor (e.g. CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16)so as to focus and localize cellular defense mechanisms for expressing and/or linking hepsin and/or HGF cell. Bespecifically antibodies can also be used to localize cytotoxic agents to cells expressing and/or linking hepsin, HGF and/or complex hepsin:HGF. These antibodies are binding polypeptide shoulder and a shoulder connecting the cytotoxic agent (e.g., saporin, antibody to interferon-α, Vinca alkaloid, a chain of ricin A, methotrexate or hapten with a radioactive isotope). Biospecifics is their antibodies can be obtained as full-length antibodies or fragments of antibodies (e.g., bespecifically antibody F(ab')2).

In WO 96/16673 described bespecifically antibody to ErbB2/FcγRIII, and in U.S. patent No. 5837234 described bespecifically antibody to ErbB2/FcγRI. Bespecifically antibody to ErbB2/Fcα shown in WO98/02463. In U.S. patent No. 5821337 specified bespecifically antibody to ErbB2/CD3.

Methods of obtaining bespecifically antibodies known in the field. The conventional receiving full bespecifically antibodies is based on the co-expression of two pairs of heavy chain-light chain immunoglobulins, where the two chains have different specificnosti (Millstein et al., Nature 305:537-539 (1983)). Due to the random sorting of heavy and light chains of immunoglobulins these hybridoma (quadroma) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bespecifically structure. Purification of the correct molecule, which is usually carried out by stages affinity chromatography, is rather time-consuming, and the product yield is low. Such methods are described in WO 93/08829 and in Traunecker et al., EMBO J. 10:3655-3659 (1991).

According to another approach, the variable domains of the antibodies with the desired binding specificity (binding site of the antigen with the antibody) combined with the sequences of the constant domains of immunoglobulins. Preferably, the Association is carried out with a constant Domino the heavy chain Ig, containing at least part of the hinge region, region CH2 and CH3. It is preferable to have located at least one of the fused structures of the first constant region of the heavy chain (CH1)containing the site necessary for binding to the light chain. DNA encoding fused heavy chain of immunoglobulins and, if desirable, a light chain immunoglobulins, are inserted into the individual expressing vectors and cotransfected in a suitable cell host. This provides greater flexibility in adjusting the overall proportions of the three polypeptide fragments in the variants of implementation, when the optimal solution is desired especifismo antibodies provide unequal ratios used in the construction of three polypeptide chains. However, one expressing the vector it is possible to insert the coding sequences for two or all three polypeptide chains, when the expression of at least two polypeptide chains in equal ratios results in high outputs or when the ratio has no significant influence on the yield of a desirable combination of chains.

In the preferred embodiment, this approach bespecifically antibodies comprise the heavy chain hybrid immunoglobulin with a first binding specificity in one arm and a pair of heavy chain-light chain hybrids the first immunoglobulin (providing a second binding specificity) in the other shoulder. Found that this asymmetric structure facilitates the separation of the desired bispecific connections from unwanted combinations of chains of immunoglobulins, as the presence of light chain immunoglobulin in only one half of bespecifically molecules provides a simple way of separation. This approach is described in WO 94/04690. Learn more about getting bespecifically antibodies see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).

According to another approach described in U.S. patent No. 5731168, the contact surface between a pair of antibody molecules can be constructed so as to maximize the share of heterodimeric recovered from cultures of recombinant cells. The preferred surface of the partition contains at least part of the domain CH3. In this way one or several amino acids with small side chains on the boundary surface molecules of the first antibody is replaced by amino acids with large side chains (e.g. tyrosine or tryptophan). On the surface section of the second molecule antibodies create compensatory "cavities" of identical or similar with large side chains size by replacing amino acids with large side chains on amino acids with smaller side chains (e.g., alanine or threonine). This provides a mechanism to increase the output of heterodimer on Rel is the solution to other unwanted end-products, such as homodimer.

Bespecifically antibodies include sewn or heteroconjugate" antibodies. For example, one of the antibodies in heteroconjugate may be associated with Avidya, and the other with Biotin. Such antibodies have been proposed, for example, for immune system cells to unwanted cells (U.S. patent No. 4676980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Heteroconjugate antibodies can be obtained using any convenient method of knitting. Suitable cross-linking means well known in the field and described in U.S. patent No. 4676980, together with a number of ways stitching.

Methods of obtaining bespecifically antibodies, fragments of antibodies are also described in the literature. For example, bespecifically antibodies can be obtained using chemical binding. In Brennan et al., Science 229:81 (1985) described a method where the intact antibody proteoliticeski were digested with obtaining fragments F(ab')2. These fragments were recovered in the presence of forming complexes with the dithiol reagent, sodium arsenite to stabilize the present dithioles and prevent the formation of intermolecular disulfide bonds. Then we obtain Fab fragments' is converted into derivatives of dinitrobenzoate (TNB). One of the derivatives of Fab'-TNB then again converted into Fab'-thiol by restoring mercaptoethyl the Amin and mixed with equimolar amounts of the other derived Fab'-TNB obtaining especifismo antibodies. Get bespecifically antibodies can be used as a means for the selective immobilization of enzymes.

Recent progress has facilitated direct recovery fromE. colifragments, Fab'-SH, which can chemically bind with the formation bespecifically antibodies. At Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describes the obtaining of molecules completely gumanitarnogo especifismo antibody F(ab')2. Each fragment Fab' separately secrete fromE. coliand subjected to direct chemical bindingin vitrowith the formation of especifismo antibodies. Thus formed bespecifically antibody capable of contacting cells, sverkhekspressiya the ErbB2 receptor and normal human T-cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against targets breast cancer person. Also describes the various methods of obtaining and allocating fragments bespecifically antibodies directly from a culture of recombinant cells. For example, bespecifically antibody was obtained with the application of latinovich lightning. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992). Peptides with latinoware lightning from proteins Fos and Jun bound with parts of the Fab' of two different antibodies by gene fusion. Homodimeric antibodies were restored in the hinge region with the formation of monomers and then re-oxidized with the formation of heterodimeric antibodies. This method can also be used to obtain homodimeric antibodies. Technology "datel", described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), provides an alternative mechanism for obtaining fragments bespecifically antibodies. The fragments contain VHconnected to VLthrough a linker that is too short to allow pairing of the two domains of the same chain. Thus, the domains of the VHand VLone fragment are forced to pair with complementary domains of the VLand VHanother fragment, thereby forming two antigenspecific plot. Also described another strategy to obtain fragments bespecifically antibodies through the use of dimers of single-chain Fv antibodies (sFv). Cm. Gruber et al., J. Immunol., 152:5368 (1994).

Consider antibodies with more than two valencies. For example, you can get thespecification antibodies. Tutt et al., J. Immunol. 147:60 (1991).

6. Heteroconjugate antibodies

Heteroconjugate antibodies are also included in the scope of the present invention. Heteroconjugate antibodies consist of two covalently linked antibodies. For example, such antibodies have been proposed to immune system cells to unwanted cells [U.S. patent No. 4676980] and for the treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. Suppose that antibodies can is about to get in vitrousing known synthetic chemistry of proteins methods, including methods using cross-linking reagents. For example, using substitution reactions of disulfides or by formation of a thioester connection it is possible to construct immunotoxins. Examples suitable for this purpose reagents include aminothiols and methyl-4-mercaptopyrimidine and reagents described, for example, in U.S. patent No. 4676980.

7. Multivalent antibodies

Polyvalent antibody cell expressing the antigen associated antibodies may internalservice (and/or metabolismrelated) faster than bivalent antibody. Antibodies according to the present invention may be a polyvalent antibodies (which are often different from the IgM class) with three or more antihistamine areas (for example, tetravalent antibodies), which can easily be obtained by recombinant expression of a nucleic acid that encodes a polypeptide chain of the antibody. Multivalent antibody may contain a dimerization domain, and three or more antigenspecific plot. The preferred dimerization domain contains a Fc region or a hinge region (or consists of). Under these conditions, the antibody will contain the Fc-region and three or more antigenspecific plot with N-to the NCA from the Fc-region. Preferred polyvalent antibody according to this document contains from three to about eight, but preferably four, antigenspecific plot (or consists of). Polyvalent antibody contains at least one polypeptide chain (and preferably two polypeptide chains), where the polypeptide chain(s) contains two or more variable domains. For example, the polypeptide chain(s) may contain VD1-(X1)n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n represents 0 or 1. For example, the polypeptide chain(s) may contain: chain VH-CH1-flexible linker-VH-CH1-Fc region or chain VH-CH1-VH-CH1-Fc region. Multivalent antibody of this paper, preferably additionally contains at least two (and preferably four) of the polypeptide of the variable domains of the light chains. Polyvalent antibody in this document may, for example, contain from about two to about eight polypeptides, variable domains of the light chains. Covered in this document polypeptides, variable domains of the light chains contain a variable domain light chain and, optionally, addition of sod is rat CL domain.

8. Organization effector functions

It may be desirable to modify the antibody according to the invention in respect to effector function, e.g. so as to reinforce caused by antigen-mediated cell cytotoxicity (ADCC) and/or due to complement cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more replacement amino acids in the Fc region of antibodies. Alternative or additionally, in the Fc-region, you can enter the cysteine residue(s), thereby providing the possibility of the formation in this area disulfide bonds between the chains. Thus obtained homodimeric antibody may have an improved ability to internalize and/or increased due to complement the destruction of cells and is caused by antibodies cellular cytotoxicity (ADCC). Cm. Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes. B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be obtained by using heterobifunctional cross-linking means, as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, it is possible to construct an antibody that has two Fc regions and, thus, has increased abilities against complement lysis and ADCC. Cm. Stevenson et al., Anti-Cancer Drugs Design 3:219-230 (1989). To increase time Pology is no antibody in the serum, the antibody can be entered epitope binding receptor salvation (especially an antibody fragment), for example as described in U.S. patent 5739277. As used herein, the term "epitope-binding receptor salvation" refers to an epitope of the Fc region of the IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4)that is responsible for increasing the half-life of the IgG molecule in the serum ofin vivo.

9. Immunoconjugate

The invention also relates to immunoconjugates or conjugates of the antibody-drug (ADC)containing antibody conjugated with a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments), or a radioactive isotope (i.e radiocona).

The use of conjugates of the antibody-drug for local delivery of cytotoxic or cytostatic funds, i.e. medicines destruction or inhibition of tumor cells in the treatment of malignant tumors (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26:151-172; U.S. patent 4975278) theoretically provides targeted delivery of molecules of the drug in tumors, and intracellular accumulation of them in there, g is e systemic administration of these unconjugated drug in addition to tumor cells, seeking to remove, can lead to unacceptable levels of toxicity to normal cells (Baldwin et al., (1986) Lancet pp. (Mar. 15, 1986):603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (ed.s), p. 475-506). Thus, to achieve maximum efficiency and minimal toxicity. It was reported that these methods are suitable and polyclonal antibodies, and monoclonal antibodies (Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87). Used in these ways medicines include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986), above). Used for conjugates of the antibody-toxin toxins include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins, such as geldanamycin (Mandler et al. (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoid (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623) and calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). Toxins can exert their cytotoxic and cytostatic effect through mechanisms including tubulin binding, DNA binding or inhibition of topoisomerases. Some cytotoxic drugs during conjugation with large antibody Il the protein ligands of the receptors tend to become inactive or less active.

However, ZEVALIN® (ZEVALIN®) (ibritumomab tiuxetan, Biogen/Idec) is a conjugate of the antibody-radioactive isotope, consisting of murine monoclonal antibodies IgG1 Kappa directed to CD20 antigen found on the surface of normal and malignant B-lymphocytes, and radioactive isotope111In or90Y related by thiocarbamides linker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al (2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin. Oncol. 20(15):3262-69). Although the ZEVALIN has activity against B-cell non-Hodgkin lymphoma (NHL), the introduction leads to a severe and prolonged cytopenias in most patients. MYLOTARG (MYLOTARG™) (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), conjugate antibody-drug consisting of gumanitarnogo antibodies to CD33-related calicheamicin in 2000 approved for the treatment of acute myeloid leukemia by injection (Drugs of the Future(2000) 25(7):686; U.S. patents№ 4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001). Cantuzumab mertansine (Immunogen, Inc.), conjugate antibody-drug consisting of huC242 antibody via a disulfide linker SPP associated with the molecule maytansinoids medicinal substance, DM1, is undergoing phase II trials for the treatment of malignant tumors that Express CanAg, such as colon cancer, cancer under lodochnoy gland, stomach cancer and others. In the process of development for the potential treatment of prostate tumors is MLN-2704 (Millennium Pharm., BZL Biologis, Immunogen Inc.), conjugate antibody-drug consisting of a monoclonal antibody specific for the prostate membrane antigen (PSMA), associated with the molecule maytansinoids medicinal substance, DM1. Peptides auristatin, auristatin E (AE) and monomethylmercury (MMAE), synthetic analogs of dolastatin, conjugatively with chimeric monoclonal antibodies cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specific CD30 on malignant neoplasms hematologic origin) (Doronina et al (2003) Nature Biotechnology 21(7):778-784), and they are in therapeutic development.

Chemotherapeutic agents suitable for such immunoconjugates described above. Enzymatically active toxins and fragments thereof that can be used include A-chain of diphtheria toxin, not binding active fragments of diphtheria toxin A-chain, exotoxin a (fromPseudomonas aeruginosa)A-chain of ricin A-chain abrina, A-chain medecine, alpha sarcin, proteinsAleurites fordii, diantimony proteins, proteinsPhytolaca americana(PAPI, PAPII, and PAP-S), inhibitor ofMomordica charantiaCurtin, krotin, the inhibitor ofSapaonaria officinalis, gelonin, mitogillin, restrictocin,vanomycin, inomycin and tricothecene. To obtain radioconjugates antibodies have a wide range of radionuclides. Examples include212Bi131I131In90Y and186Re. Conjugates of the antibody and cytotoxic receive funds using a variety of bifunctional binding proteins funds, 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-azido connection (such asbis-(p-azidobenzoyl)hexanediamine), derivativesbis-page (such asbis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and connections with two atoms of active fluoride (such as 1,5-debtor-2,4-dinitrobenzene). For example, rezinovy immunotoxin can be obtained as described in Vitetta et al., Science, 238: 1098 (1987). Illustrative chelating agent for conjugation of radionucleotide with the antibody is labeled with carbon 14 1-isothiocyanatobenzene-3-metallienjalostuksessa acid (MX-DTPA). Cm. WO94/11026.

This document provides conjugates of the antibody and one or more low molecular weight toxins such as calicheamicin, maytansinoid, trichothecin and CC1065, and the derivatives of these t is xinou, having toxic activity.

Mitanin and maytansinoid

In one of the embodiments the antibody (full-length or fragments) according to the invention conjugated to one or more maytansinoid molecules.

Maytansinoid are inhibitors of mitosis, acting through inhibition of tubulin polymerization. Maytansine was first isolated from the East African shrub,Maytenus serrata(U.S. patent No. 3896111). Subsequently it was discovered that some microorganisms also produce maytansinoid, such as maytansine and esters with C-3 maytansine (U.S. patent No. 4151042). Synthetic maytansines and its derivatives and analogs are described, for example, in U.S. patents№ 4137230; 4248870; 4256746; 4260608; 4265814; 4294757; 4307016; 4308268; 4308269; 4309428; 4313946; 4315929; 4317821; 4322348; 4331598; 4361650; 4364866; 4424219; 4450254; 4362663 and 4371533, descriptions of which are explicitly incorporated herein by reference.

Conjugates maytansinoid-antibody

In order to increase their therapeutic index mitanin and maytansinoid conjugatively with antibodies that specifically bind with the antigens of tumor cells. Immunoconjugate containing maytansinoid and their therapeutic application, as described, for example, in U.S. patent No. 5208020, 5416064 and European patent EP 0 425 235 B1, descriptions of which are explicitly incorporated into the present document is as a reference. In Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugate containing maytansinoid designated DM1 associated with the monoclonal antibody C242 directed to human colorectal cancer. Found that the conjugate is vysokorychlostnym in respect of cultured cells of malignant tumors of the colon and demonstrates antitumor activity in the analysis of tumor growthin vivo. In Chari et al., Cancer Research 52:127-131 (1992) described immunoconjugate in which maytansinoid via a disulfide linker anywhereman with the murine antibody A7 binding to an antigen on the cell lines of malignant tumors of the large intestine of the person, or to another murine monoclonal antibody TA.1 that binds to the oncogene HER-2/neu. The cytotoxicity of the conjugate TA.1-maytansinoid testedin vitroin cell lines breast cancer human SK-BR-3, which expresses 3 × 105molecules of the surface antigen HER-2 on the cell. Conjugates with drug reached the degree of cytotoxicity similar to free maytansinoid drug, which can be increased by increasing the number of maytansinoid molecules per molecule of antibody. Conjugate A7-maytansinoid mice showed low systemic cytotoxicity.

Conjugates of the antibody-m is stanzino (immunoconjugate)

Conjugates of the antibody-maytansinoid get through chemical binding of an antibody to a molecule maytansinoid without a significant reduction in the biological activity of the antibodies or molecules maytansinoid. The average efficiency increased cytotoxicity to target cells without negative impacts on the feature or solubility antibodies showed 3-4 maytansinoid molecules conjugated per molecule of antibody, although one would expect that even one molecule of toxin/antibody will be increased cytotoxicity compared using unconjugated antibodies. Maytansinoid well known in this field and they can be synthesized by known methods or to isolate from natural sources. Suitable maytansinoid described, for example, in U.S. patent No. 5208020 and other patent and non-patent publications to which reference was made earlier in this document. Preferred maytansinoid are maytansines and analogues maytansine modified aromatic ring or other positions in the molecule maytansine, such as various esters maytansine.

In this area there are many linker groups to obtain conjugates antibody-maytansinoid, for example, include groups described in U.S. patent No. 5208020 or E. the European patent EP 0 425 235 B1 and Chari et al., Cancer Research 52:127-131 (1992). Linker groups include disulfide groups, thioester group unstable to acids group, photolabile group, resistant to peptidases group or unstable to esterases group, as described in the above patents, which are preferred disulfide and thioester groups.

Conjugates of the antibody and maytansinoid can be obtained using a variety of bifunctional binding proteins funds, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylacetamide HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde),bis-azido connection (such asbis-(p-azidobenzoyl)hexanediamine), derivativesbis-page (such asbis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and compounds with two atoms of active fluoride (such as 1,5-debtor-2,4-dinitrobenzene). Especially preferred connecting means include N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 [1978]) and N-Succinimidyl-4-(2-pyridylthio)pentanoate (SPP) for the formation of disulfide bonds.

The linker can be attached to the molecule maitan inode in different positions, depending on the type of communication. For example, the ester bond can be formed by reaction with a hydroxyl group, using the common methods of binding. The reaction may occur at position C-3 hydroxyl group, C-14, modified with hydroxymethyl, C-15, modified hydroxyl group, and the position of the C-20 hydroxyl group. In a preferred embodiment, the communication form in position C-3 maytansine or similar maytansine.

Calicheamicin

Another interest immunoconjugate contains antibody conjugated with one or more molecules calicheamicin. Family of antibiotics calicheamicin capable of forming double-stranded DNA breaks in subpicomolar concentrations. To obtain conjugates with family calicheamicin, see U.S. patent 5712374, 5714586, 5739116, 5767285, 5770701, 5770710, 5773001, 5877296 (all issued by American Cyanamid Company). Structural analogues calicheamicin that can be used include as non-limiting examples, γ1Iα2Iα3IN-acetyl-γ1I, PSAG and θI1(Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al, Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patents issued to American Cyanamid). Another anticancer drug, which can conjugio is the substance of the antibody, is QFA, which is antifolate. The point of application of actions in calicheamicin and QFA are inside cells, but it is hard for them to cross the plasma membrane. Therefore, the capture cells of these funds in the mediated antibody internalization greatly enhances their cytotoxic action.

Other cytotoxic funds

Other antitumor agents that can be konjugierte with antibodies according to the invention include BCNU, streptozocin, vincristine and 5-fluorouracil, the family of the tools, collectively known as the set of LL-E33288 described in U.S. patent 5053394, 5770710 and espiramicina (U.S. patent 5877296).

Enzymatically active toxins and fragments thereof that can be used include A-chain of diphtheria toxin, nesviazana active fragments of diphtheria toxin A-chain, exotoxin a (fromPseudomonas aeruginosa)A-chain of ricin A-chain abrina, A-chain medecine, alpha sarcin, proteinsAleurites fordii, diantimony proteins, proteinsPhytolaca americana(PAPI, PAPII, and PAP-S), inhibitor ofMomordica charantiaCurtin, krotin, the inhibitor ofSapaonaria officinalis, gelonin, mitogillin, restrictocin, vanomycin, inomycin and tricothecene. See, for example, WO 93/21232 published October 28, 1993.

The present invention additionally relates to immunoconjugate formed between the antibody and the the group with nucleotidase activity (for example, a ribonuclease or a DNA endonuclease such as desoksiribonukleaza; Ncasa).

For the selective destruction of tumor antibody may contain highly radioactive atom. To obtain radioconjugates antibodies there are many radioactive isotopes. Examples include At211I131I125, Y90That Re186That Re188Sm153Bi212, P32, Pb212and radioactive isotopes of Lu. When the conjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, for example, tc99mor I123or a spin label for a method of obtaining images of nuclear magnetic resonance (NMR) (also known as the method of obtaining images using magnetic resonance imaging, mri), such as again, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Radioactive or other labels can be entered in the conjugate by known methods. For example, the peptide can be biosynthetically or to synthesize by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 instead of hydrogen. Labels such as tc99mor I123That Re186That Re188and In111you can attach via a cysteine residue in the peptide. Yttrium-90 can is about to attach via a lysine residue. For the introduction of iodine-123, you can apply the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57. In "Monoclonal antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) described other ways.

Conjugates of the antibody and cytotoxic tools can be accessed using a variety of bifunctional binding proteins funds, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylacetamide HCl), active esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde),bis-azido connection (such asbis-(p-azidobenzoyl)hexanediamine), derivativesbis-page (such asbis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and connections with two atoms of active fluoride (such as 1,5-debtor-2,4-dinitrobenzene). For example, rezinovy immunotoxin can be obtained as described in Vitetta et al., Science, 238: 1098 (1987). Illustrative chelating agent for conjugation of radionucleotide with the antibody is labeled with carbon 14 1-isothiocyanatobenzene-3-metallienjalostuksessa acid (MX-DTPA). Cm. WO94/11026. The linker may be a "biodegradable linker"facilitating release of the cytotoxic drug in the cell. For example, the can is to use unstable to acid linker, sensitive peptidase linker, photolabile linker, dimethyl linker or containing a disulfide linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. patent No. 5208020).

Compounds according to the invention explicitly include as non-limiting examples of the ADC obtained using cross-linking reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, fairs are forthcoming-Siab, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-fairs are forthcoming-Siab, sulfo-SMCC, sulfo-SMPB, and SVSB (Succinimidyl-(4-vinylsulfonic)benzoate)which are commercially available (e.g., Pierce Biotechnology, Inc., Rockford, IL., U.S.A). Cm. pages 467-498, 2003-2004 Applications Handbook and Catalog.

OBTAINING CONJUGATES ANTIBODY-DRUG

In the conjugates of the antibody-drug (ADC) according to the invention, the antibody (Ab) via a linker (L) conjugated with one or more molecules of the drug (D), for example, from about 1 to about 20 molecules of the drug to the antibody. The ADC of formula I can be obtained in various ways, using the reaction conditions and reagents of organic chemistry known to specialists in this area, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent with the formation through the formation of covalent bonds Ab-L with subsequent reaction with a molecule drugs D; and (2) react the Yu nucleophilic group of the molecule drugs with a bivalent linker reagent with the formation through the formation of covalent bonds D-L followed by reaction with the nucleophilic group of an antibody.

Ab-(L-D)pI

Nucleophilic groups on the antibodies include as non-limiting examples: (i) N-terminal amino group, (ii) amine groups of the side chains, for example, lysine, (iii) thiol groups of the side chains, for example, cysteine, and (iv) hydroxyl sugars or amino groups, when the antibody is glycosylated. Amine, thiol and hydroxyl groups are nucleophilic and capable to react with the formation of covalent bonds with electrophilic groups on linker molecules and linker reagents including: (i) active esters such as NHS esters, esters, HOBt, halogenfree and halides; (ii) alkyl and benzylchloride, such as halogenated; (iii) aldehyde, ketone, carboxyl and maleimide group. Some antibodies are recoverable megamachine disulfides, i.e. cysteine bridges. Antibodies can be made reactive for conjugation with linker reagents through processing regenerating agent such as DTT (dithiothreitol). Thus, each cysteine bridge will theoretically generate two reactive thiol of the nucleophile. Additional nucleophilic group is s can be fed into the antibody by reaction of lysine residues with 2-aminothiophenol (reagent trout), which leads to the conversion of the amine to the thiol.

Conjugates of the antibody-drug can also be obtained by modifying antibodies with the introduction of electrophilic groups, which can react with nucleophilic substituents on the linker reagent or drug. You can oxidize sugar glycosylated antibodies, for example, with the use of oxidizing reagents periodate, with the formation of aldehyde or ketone groups that can react with the amino group of the linker reagents or molecules of the drug. Received minovia group of Schiff bases can form a stable connection or they can be recovered, for example by borohydride reagents with the formation of a stable amine linkages. In one embodiment, the implementation of the reaction of the carbohydrate portion of a glycosylated antibody with galactosaminidase or metaperiodate sodium can lead to the formation of protein carbonyl (aldehyde or ketone) groups that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal residues of serine or threonine, can react with metaperiodate sodium, which leads to the formation of aldehyde instead of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146;U.S. patent 5362852). This aldehyde can react with a molecule of a drug or linker by a nucleophile.

Similarly nucleophilic groups on the molecule drugs include as non-limiting examples: amino, Tilney, hydroxyl, hydrazide, oximo, hydrazine powered, thiosemicarbazones, hydrazinecarboxamide and arylhydrazines groups capable of reaction with the formation of covalent bonds with electrophilic groups on linker molecules and linker reagents including: (i) active esters such as NHS esters, esters, HOBt, halogenfree and halides; (ii) alkyl and benzylchloride, such as halogenated; (iii) aldehyde, ketone, carboxyl and maleimide group.

Alternatively, a fused protein containing the antibody and cytotoxic agent can be obtained, for example, by recombinant methods or peptide synthesis. The DNA chain may contain the corresponding region encoding the two parts of the conjugate, located next to each other or separated by a region that encodes a linker peptide which does not violate the desired properties of the conjugate.

In yet another embodiment, the antibody can be konjugierte with the "receptor" (such as streptavidin) for utilization in the pre-treatment of tumors, where p is they introduce a conjugate of the antibody-receptor with subsequent removal from the bloodstream unbound conjugate using a cleanser, and then enter a "ligand" (e.g. avidin)which anywhereman with a cytotoxic agent (e.g., radionucleotide).

10. Immunoliposome

Described in this document antibodies can also be in the form of immunoliposome. "Liposome" is a small carrier, consisting of various types of lipids, phospholipids and/or surfactant, which is suitable for delivering the drug to a mammal. Components of liposomes, usually located in the two-structure, similar to the arrangement of lipids in biological membranes. Containing antibody liposomes get known in this field by the methods as in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77:4030 (1980); U.S. patent No. 4485045 and 4544545 and WO97/38731, published on 23 October 1997. In U.S. patent No. 5013556 described liposomes with increased residence time in the bloodstream.

Particularly useful liposomes can be obtained by the evaporation method with reversed phase with a lipid composition comprising phosphatidylcholine, cholesterol and derivationally PEG phosphatidylethanolamine (PEG-PE). Liposomes ekstragiruyut through filters with defined pore size to yield liposomes of the desired diameter. Fragments Fab' antibody according to the present invention can be konjugierte with liposomes as described in Martin et al.,J. Biol. Chem. 257:286-288 (1982), the exchange reaction of disulfides. In the liposome contains optional chemotherapeutic agent. Cm. Gabizon et al., J. National Cancer Inst. 81 (19): 1484 (1989).

B. Binding oligopeptides

Binding oligopeptides according to the invention are oligopeptides that bind, preferably specifically with Hasina, HGF and/or complex hepsin:HGF, as described in this document. Binding oligopeptides can be synthesized chemically using known techniques for the synthesis of oligopeptides or you can get and clear with the use of recombinant technology. The length of the binding oligopeptides, as a rule, is at least about 5 amino acids, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 amino acids or more, wherein such oligopeptides are able to bind, preferably specifically, to a polypeptide as described herein. Binding oligopeptides may be identified without undue experimentation using well known methods. It should be noted that the methods of screening libraries of oligopeptides is and oligopeptides, can specifically bind to a polypeptide target are well known in the art (see, for example, U.S. patents№ 5556762, 5750373, 4708871, 4833092, 5223409, 5403484, 5571689, 5663143; PCT publication no WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. USA, 82:178-182 (1985); Geysen et al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA. 87:6378; Lowman, H.B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A.S. et al. (1991) Proc. Natl. Acad. Sci. USA. 88:8363 and Smith, G. P. (1991) Current Opin. Biotechnol., 2:668).

In this respect, a well-known technique for ensuring that the screening of large libraries of oligopeptides to identify representative(s) of these libraries can specifically bind to a polypeptide target is bacteriotherapy (phage) display. Phage display is a method by which the variants of the polypeptides in the form of a fused protein membrane proteins presented on the surface bacteriophobic particles (Scott, J.K. and Smith, G. P. (1990) Science, 249: 386). The utility of phage display technique lies in the fact that a large library of selectively randomized variants of proteins (or accidentally cloned cDNA) can be quickly and effectively to check on those molecules that are associated with the molecule-target with high affinity. View Pat the ne (Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA. 87:6378) or protein (Lowman, H.B. et al. (1991) Biochemistry. 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A.S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on phage used for screening millions of polypeptides or oligopeptides on polypeptides or oligopeptides with specific binding properties (Smith, G. P. (1991) Current Opin. Biotechnol., 2:668). Check phage libraries of random mutants requires a strategy for the design and reproduction of a large number of cases, the procedure for affinity purification using directional receptor and means for assessing the results strengthen the binding. U.S. patent No. 5223409, 5403484, 5571689 and 5663143.

Although most methods of phage display using filamentous phage, also known system of phage display-based phage lambda (WO 95/34683; U.S. patent 5627024), system of phage display-based phage T4 (Ren et al., Gene, 215: 439 (1998); Zhu et al., Cancer Research, 58(15): 3209-3214 (1998); Jiang et al., Infection &Immunity, 65(11): 4770-4777 (1997); Ren et al., Gene, 195(2):303-311 (1997); Ren, Protein Sci., 5:1833 (1996); Efimov et al., Virus Genes, 10: 173 (1995)) and the system of phage display-based phage T7 (Smith and Scott, Methods in Enzymology, 217: 228-257 (1993); U.S. patent 5766905).

Nowadays, there are many other improvements and variations on the basic concept of phage display. These enhancements improve the ability of systems to display skanirovat peptide library is the place to associate with the selected molecules to target and present functional proteins with the possibility of screening these proteins on desirable properties. For reactions phage display technique developed devices for combinatorial reactions (WO 98/14277), and the libraries of phage display was used for analysis and control of bimolecular interactions (WO 98/20169; WO 98/20159) and properties of strained helical peptides (WO 98/20036). In WO 97/35196 described the method of allocation of affine ligand, in which a library of phage display is in contact with one solution, in which the ligand binds with a molecule-target, and the second solution, in which the affinity ligand is not associated with the molecule target for the selective extraction bind ligands. In WO 97/46251 described by way of biopanning library random phage display affinity purified antibody, and then highlight the phages bound peroxidase followed by the process of microplanning with the use of the wells of a microplate for selecting phage that bind with high affinity. It was also reported the use of protein AStaphylococcus aureusas affinity labels (Li et al. (1998) Mol Biotech., 9:187). In WO 97/47314 described the use substracted wichitaclony libraries to distinguish between enzyme specificity using combinatorial libraries, which can be a library of phage display. In WO 97/09446 described method of selection of enzymes suitable for use in detergents using phage display. Additional selection methods specifically attributed audacia proteins described in U.S. patent No. 5498538, 5432018 and WO 98/15833.

Methods of obtaining peptide libraries and screening these libraries are described in U.S. patents№ 5723286, 5432018, 5580717, 5427908, 5498530, 5770434, 5734018, 5698426, 5763192 and 5723323.

C. Binding of low molecular weight compounds

Binding of low-molecular compounds preferably are organic molecules other than oligopeptides or antibodies as defined herein that bind, preferably specifically, to Hasina, HGF and/or complex hepsin:HGF, as described in this document. Communicating the organic low-molecular compounds can be identified and chemically synthesized using known methodology (see, for example, PCT publication no WO 00/00823 and WO 00/39585). The amount of binding of the organic low molecular weight compounds, as a rule, is less than about 2000 daltons, alternatively, less than about 1500, 750, 500, 250 or 200 daltons, wherein such organic low-molecular-weight compounds that are capable of binding, preferably specific to the polypeptide, as described herein, without undue experimentation, to identify with the application of known methods. It should be noted that the methods of screening libraries of organic low molecular weight compounds for molecules capable of SV is experienced in continuation with the target polypeptide, well known in the art (see, for example, PCT publication no WO 00/00823 and WO 00/39585). Communicating the organic low-molecular compounds may represent, for example, aldehydes, ketones, oximes, hydrazones, semicarbazones, carbazide, primary amines, secondary amines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols, thioethers, disulfides, carboxylic acids, esters, amides, urea, carbamates, carbonates, ketals, thioketal, acetals, thioacetals, aryl halides, arylsulfonate, alkylhalogenide, alkyl sulphonates, aromatic compounds, heterocyclic compounds, anilines, alkenes, alkynes, diols, aminoalcohols, oxazolidine, oxazoline, thiazolidine, thiazoline, enamines, sulfonamides, epoxides, aziridines, isocyanates, sulphonylchloride, diazo-compounds, anhydrides, etc.

D. Screening for antibody binding oligopeptides and binding of low molecular weight compounds with desirable properties.

The above described methods for producing antibodies, oligopeptides and low molecular weight compounds according to the invention. Optionally, you can further select the antibodies, oligopeptides or other low molecular weight compounds with specific biological characteristics.

Inhibiting the growth effects of the antibodies, oligopeptides or other nizkomolekularnah what about the compounds according to the invention can be assessed well-known in this field means, for example, using cells expressing hepsin and/or Pro-HGF endogenous or after transfection of the corresponding gene(s). For example, suitable lines of tumor cells and transfetsirovannyh Hasina and/or HGF polypeptide cells can be treated with monoclonal antibody, Oligopeptide or other low-molecular compound according to the invention at various concentrations for a few days (e.g., 2-7) and dye crystal violet or MTT or analyzed using any other colorimetric analysis. Another method of measuring proliferation represents the comparison of capture3H-thymidine by the cells treated in the presence or absence of antibodies, binding of oligopeptides or binding of low molecular weight compounds according to the invention. After treatment, cells are collected and acquired scintillation counter quantitatively determine the level to become involved in DNA radioactivity. Appropriate positive controls include processing the selected cell lines inhibiting the growth of the antibody for which it is known that it inhibits the growth of this cell line. Inhibition of growth of tumor cellsin vivoyou can define various well-known in this field means. The tumor cell may be a tumor CL is woven, sverkhekspressiya hepsin and/or polypeptide Pro-HGF. Antibody binding Oligopeptide or binding organic small molecule compound will inhibit cell proliferation expressing hepsin and/or HGF tumor cellsin vitroorin vivocompared to the untreated tumor cell by about 25-100%, more preferably about 30-100%, and even more preferably by about 50-100% or 70-100%, in one of the embodiments when the antibody concentration from about 0.5 to 30 μg/ml Inhibition of growth can be measured when the antibody concentration from about 0.5 to 30 μg/ml or about 0.5 nm to 200 nm in cell culture, where the growth inhibition determined in 1-10 days after exposure of the antibodies on tumor cells. The antibody inhibits the growth ofin vivoif the introduction of antibodies at a concentration from about 1 μg/kg body weight to about 100 mg/kg body weight results in reduction in tumor size or reduction of proliferation of tumor cells within approximately 5 days to 3 months from the first injection of the antibody, preferably within about 5 to 30 days.

For selection of antibodies, binding of oligopeptides or binding organic low-molecular compounds, inducere the observed cell death, you can analyze the loss of integrity of the membrane relative to the control, as indicated, for example, the capture of iodide of propecia (PI), Trypanosoma blue or 7AAD. Analysis of capture PI can be carried out in the absence of complement and immune effector cells. Expressing hepsin and/or polypeptide Pro-HGF tumor cells incubated with the same medium or medium containing the appropriate antibody (e.g, at about 10 μg/ml), binding Oligopeptide or binding organic small molecule compound. Cells are incubated for a period of time in 3 days. After each treatment, the cells washed and aliquots added to coated 35-mm sieve tubes 12 × 75 (1 ml per tube, 3 tubes in the treated group) to remove aggregates of cells. Then in a test tube add PI (10 μg/ml). Then the samples can be analyzed using the flow cytometer FACSCAN® and software FACSCONVERT® CellQuest (Becton Dickinson). Those antibodies, binding oligopeptides or binding organic low-molecular-weight compounds that induce statistically significant levels of cell death as determined by capturing PI, you can choose as inducing cell death antibody binding oligopeptides or binding organic low-molecular compounds.

For screening of the antibodies is a, oligopeptides or other organic low-molecular-weight compounds that bind to the epitope on the polypeptide associated interest antibody can be a standard analysis of cross-blocking, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). This analysis can be used to determine if a test antibody, Oligopeptide or other organic low-molecular compound bind to the same site or epitope as a known antibody. Alternative or additionally can be performed epitope mapping known in the field of ways. For example, to identify contact residues of the sequence of the antibody can be subjected to mutagenesis, such as alanine scanning. Mutant antibody source tested for binding to a polyclonal antibody to ensure proper installation. In another method, peptides corresponding to different regions of the polypeptide, can be used in competition assays with the test antibodies or with a test antibody and an antibody with a characterized or known epitope.

E. Dependent antibody-mediated enzyme proletarienne therapy (ADEPT)

Antibodies of the present invention can also be used in ADEPT by conjugation of the antibody with the trigger about the medicine enzyme converts a prodrug (e.g., peptidyl chemotherapeutic agent, see WO 81/01145) in the active antitumor drug. See, for example, WO 88/07378 and U.S. patent No. 4975278.

Enzymatic component immunoconjugate suitable for ADEPT, includes any enzyme capable of acting on a prodrug in such a way to convert it into its more active, cytotoxic form.

Enzymes suitable in the method according to this invention, include as non-limiting examples of alkaline phosphatase, suitable for conversion into free drugs containing phosphate prodrugs; arylsulfatase suitable for conversion into free drugs containing sulfates prodrugs; sitoindosides suitable for conversion in anticancer drug, 5-fluorouracil, non-toxic 5-fertilizin; proteases such as proteaseSerratiathermolysin, subtilisin, carboxypeptidase and cathepsin (such as cathepsin B and L), suitable for conversion into free drugs containing peptides prodrugs; D-alanismorissette suitable for converting prodrugs that contain D-amino acid substituents; splitting carbohydrates enzymes, such as β-galactosidase and neuraminidase, which are suitable for conversion to its adnie medicines glycosylated prodrugs; β-lactamase, suitable for conversion into free drugs derivatizing β-lactam drugs; and penicillinases, such as amidase penicillin V or amidase penicillin G, suitable for conversion into free medicines medicines derivateservlet their nitrogen in amines have had phenoxyacetyl or phenylacetylene groups, respectively. Alternatively, for the conversion of the prodrugs according to the invention into free active drugs can be used antibodies with enzymatic activity, also known in this area as "abzyme" (see, for example, Massey, Nature 328:457-458 (1987)). For delivery of abzyme in the accumulation of tumor cells can be obtained conjugates antibody-Abim, as described in this document.

The enzymes according to this invention can covalently bind to antibodies are well known in this field means, such as application discussed above heterobifunctional cross-linking reagents. The alternative with the use of well-known in the field of methods of recombinant DNA can be designed fused proteins containing at least antigennegative region of the antibodies according to the invention, associated, at least functionally active part of the enzyme according to the invention (see, for example Neuberger et al., Nature 312:604-608 (1984).

F. Options antibodies

Except as described in this document antibodies, it is assumed that it is possible to obtain the variants of the antibodies. Variants of antibodies can be obtained by introducing appropriate nucleotide substitutions in the coding DNA and/or by synthesis of the desired antibodies. Specialists in this field it is clear that replacement of amino acids can alter post-translational processing of the antibody, such as changing the number or position of glycosylation sites or changes in the characteristics of attachment to the membrane.

Changes in antibodies described herein can be performed, for example, using any of the techniques and guidelines for conservative and non-conservative mutations, see, for example, in U.S. patent No. 5364934. Change can be a substitution, deletion or insertion of one or more codons encoding the antibody, which leads to a change in amino acid sequence compared to the natural sequence of the antibody or polypeptide. Optional change carried out by replacing at least one amino acid with any other amino acid in one or more domains of the antibody. A guide to determining which amino acid residue may be inserted, substitute or remove without negative impact on chelation is optimum activity can be found when comparing the sequences of the antibodies with homologous sequence is known protein molecules and minimizing the number of changes of amino acid sequence, made in regions of high homology. Replacement of amino acids can be the result of replacing one amino acid with another amino acid with similar structural and/or chemical properties, such as the substitution of leucine for serine, i.e. conservative replacement amino acids. Inserting or deleting optional can be in the range from approximately 1 to 5 amino acids. Valid change can be determined by the system creating insertions, deletions or substitutions of amino acids in the sequence and testing the obtained variants on the activity, show the source sequence.

This document provides antibody fragments and polypeptides. Such fragments may be truncated at the N-end or C-end or they may lack internal residues, for example, when compared with a full-sized natural antibody or protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity of the antibody or polypeptide.

Antibody fragments and polypeptides can be obtained by any of several conventional ways. The desired peptide fragments can be synthesized chemically. An alternative approach involves obtaining fragments of the antibodies or polypeptides by enzymatic split the I, for example, by processing of the protein by the enzyme for which it is known that he cleaves proteins at sites defined by particular amino acid residues, or by cleavage of the DNA with suitable restriction enzymes and highlight the desired fragment. Another suitable method involves the selection and amplification of a DNA fragment encoding the desired antibody fragment, or polypeptide, by polymerase chain reaction (PCR). In PCR for 5' and 3' primers used oligonucleotides that define the desired ends of the DNA fragment. Preferably, antibody fragments and polypeptides possess at least one biological and/or immunological activity as described herein native antibody or polypeptide.

In specific embodiments, the implementation of interest conservative substitutions are presented in the table below under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then spend a more substantial changes, indicated in this table as illustrative replacement or as further described below with respect to classes of amino acids, and the products are subjected to screening.

The original balancePreferred replacement
Ala (A)Val; Leu; IleVal
Arg(R)Lys; Gln; AsnLys
Asn (N)Gln; His; Asp, Lys; ArgGln
Asp (D)Glu; AsnGlu
Cys (C)Ser; 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; norleucineLeu
Leu (L)norleucine; Ile; Val; Met; Ala; PheIle
Lys (K)Arg; Gln; Asn Arg
Met (M)Leu; Phe; IleLeu
Phe (F)Trp; Leu; Val; Ile; Ala; TyrTyr
Pro (P)AlaAla
Ser(S)ThrThr
Thr (T)Val; SerSer
Trp(W)Tyr; PheTyr
Tyr(Y)Trp; Phe; Thr; SerPhe
Val (V)Ile; Leu; Met; Phe; Ala; norleucineLeu

Substantial modifications in function or immunological identity of the antibody or polypeptide is conducted by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide of the frame in the area of replacement, such as the conformation of the sheet or helix, (b) the charge or hydrophobicity of the molecule at the site of the target or (c) the size of the side chain. Amino acids can be grouped according to similarity of the properties of their side chains (in A. L. Lehninge, in Biochemistry, second edition, p. 73-75, Worth Publishers, New York (1975)):

(1) non-polar: Ala (A)Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M)

(2) uncharged polar: Gly (G), Ser (S)Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q)

(3) acidic: Asp (D)Glu (E)

(4) basic: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues may be divided into groups based on common properties of the side chains:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions involve replacement of the representative of one of these classes for another class. Such substituted residues can also be entered in the plots conservative substitutions or, more preferably, in the remaining (non-conservative) areas.

Changes can be performed using known in the field of methods, such as mediated by oligonucleotides (site-specific) mutagenesis, alanine scanning, and mutagenesis by PCR. To obtain the variant DNA of the antibody or polypeptide on the cloned DNA can be site-specific mutagenesis [Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], mutagenesis on the basis of restriction selection [Wells et al., Philos. Trans. R. Soc. London SerA, 317:41 (1986)] or other known methods.

To identify one or more amino acids in a continuous sequence, you can also apply the analysis scanning amino acid. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Alanine is typically a preferred scanning amino acid of this group, because it does not have a side chain after the beta carbon atom and less likely that he will change the conformation of the main chain variant [Cunningham and Wells, Science, 244:1081-1085 (1989)]. Alanine is typically also prefer, as it is the most common amino acid. Furthermore, it is often found, and in-depth, and surface positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If replacement by alanine does not give satisfactory amounts of options, you can use isothermic amino acid.

You can also substitute any cysteine residue not involved in maintaining the proper conformation of the antibody or polypeptide, as a rule, serine, to increase the stability of the molecule oxidation and prevent improper stitching. On the contrary, the antibody or polypeptide to increase its stability cysteine bond(s) can be added (especially to the Yes antibody is an antibody fragment, such as an Fv fragment).

Particularly preferred variant type substitution includes replacement of one or more residues of the hypervariable region of the initial antibody (for example, humanitariannet or human antibody). Typically, the received option(s)selected for further improvements, will have improved biological properties relative to the source of the antibody from which it is obtained. A convenient way of obtaining such substituted variants include affinity maturation using phage display. In summary, some parts of the hypervariable region (e.g., 6-7 sites) are matirovanie with obtaining all possible amino acid substitutions at each site. Thus obtained variants of monovalent antibodies in the form of exhibit on the particles of filamentous phage in the form of a fused protein product of the gene III of M13 packaged within each particle. Exposed on the phage variants then sceneroot on their biological activity (e.g. binding affinity of), as described in this document. To identify areas candidate hypervariable region for the modification can be performed mutagenesis to alanine scanning to identify residues of the hypervariable region, making a significant contribution to the binding to the antigen. Al is ternative or additionally may be beneficial to analyze a crystal structure of the complex antigen-antibody to identify contact points between the antibody and the polypeptide antigen. Such contact areas and areas located nearby, are candidates for replacement in accordance with the developed hereunder ways. Upon receipt of such options panel of variants is subjected to screening as described herein, and antibodies with the best in one or more appropriate tests properties can be selected for further refinement.

The nucleic acid molecules encoding amino acid sequence variants of antibodies, produced by many known in the field of methods. These methods include as non-limiting examples, the selection is from a natural source (in the case of naturally occurring amino acid sequence) or getting through mediated by oligonucleotides (or site-specific) mutagenesis, mutagenesis by PCR and cassette mutagenesis previously received options or betweenthey version antibodies.

G. Modification of the antibodies and polypeptides

In the scope of the present invention is enabled covalent modification of the antibody and the polypeptide. One type of covalent modification includes a reaction outlined amino acid residues of the antibody or polypeptide with an organic derivatizing agent capable of reacting with selected side chains or the N - or Schonewille residues of the antibody or polypeptide. Deriving from the use of bifunctional suitable means, for example, to bind antibody or polypeptide with a water-insoluble matrix or surface of the substrate for use in the methods of purification of antibodies and Vice versa. Commonly used cross-linking means include, for example, 1,1-bis(diazoacetic)-2-Penilaian, glutaraldehyde, esters of N-hydroxysuccinimide, for example, esters with 4-azidoaniline acid, homobifunctional imidiately comprising esters of disuccinimidyl, such as 3,3'-dithiobis(succinimidylester), bifunctional maleimides, such asbis-N-maleimido-1,8-octane and tools such as methyl-3-[(p-azidophenyl)dithio]propionamide.

Other modifications include desametasone glutaminergic and asparaginase residues to the corresponding glutaminase and aspartame residues, respectively, hydroxylation of Proline and lysine, phosphorylation of hydroxyl groups of serine or treoninove residues, methylation of the α-amino groups of the side chains of lysine, arginine and histidine [T.E. Creighton, Proteins: Structure and Molecular Properties. W.H. Freeman & Co., San Francisco, p. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the antibody or polypeptide, are included in this volume is about invention, involves changing the original profile of the glycosylation of the antibody or polypeptide. "Modifying the original glycosylation profile" is used herein to identify deletions of one or more carbohydrate molecules on the initial sequence of the antibody or polypeptide (or by removing the subject of a glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the original sequence of the antibody or polypeptide. In addition, the phrase includes quantitative changes in glycosylation source of proteins, which includes the change in the nature and proportions of different carbohydrate molecules present.

Glycosylation of antibodies and other polypeptides, as a rule, is either N-linked or O-linked. N-linked refers to the attachment of a molecule of carbohydrate to the side chain of an asparagine residue. Recognized sequences for enzymatic attachment molecule of carbohydrate to the side chain of asparagine are Tripeptide sequence asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except Proline. Thus, the presence or any and which of these Tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-atsetilgalaktozamin, galactose, or xylose to hydroxynicotinate, often serine or threonine, although you can also use 5-hydroxyproline or 5-hydroxylysine.

Adding to the antibody or antibody glycosylation sites convenient to carry out, changing the amino acid sequence so that it contains one or more of the above Tripeptide sequences (for N-linked glycosylation sites). Change can also be carried out by adding to the sequence of the original antibody or polypeptide of one or more residues of serine or threonine or replace them (for O-linked glycosylation sites). Amino acid sequence of the antibody or polypeptide optionally can be modified through changes at the DNA level, particularly by mutations to DNA that encodes the antibody or the polypeptide at preselected bases such that formed the codons that are translated into the desired amino acids.

Other ways of increasing the number of carbohydrate molecules to the antibody or the polypeptide are chemical or enzymatic joining of glycosides to the polypeptide. Such methods are described in this area, for example in WO 87/05330 published the Anna 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., p. 259-306 (1981).

Removal of carbohydrate molecules, located on the antibody or the polypeptide can be chemical or enzymatic means or by mutational substitution of codons encoding amino acid residues that serve as targets for glycosylation. Methods of chemical deglycosylation known in this field and are described, for example, Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate molecules from polypeptide can be accomplished by the use of a variety of endo - and ectoparasites, as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of the antibody or polypeptide comprises linking the antibody or polypeptide to one of a variety of non-protein polymers, for example polyethylene glycol (PEG), polypropyleneglycol or polyoxyalkylene, in the manner specified in U.S. patents№ 4640835; 4496689; 4301144; 4670417; 4791192 or 4179337. The antibody or polypeptide can also be enclosed in microcapsules obtained, for example, by means koatservatsii or interfacial polymerization (for example, hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacrylate) microcapsules, respectively), in colloidal systems drug delivery (for example, liposomes, albumin microspheres, microemulsions, NAS the particles and nanocapsules) or in microemulsion. Such methods are described in Remington''s Pharmaceutical Sciences. 16th edition, Oslo, A., Ed., (1980).

The antibody or polypeptide of the present invention also can be modified by the method of formation of chimeric molecules containing antibody or polypeptide that is fused with other the heterologous polypeptide or amino acid sequence.

In one embodiment, the implementation of such a chimeric molecule contains protein antibody or polypeptide with a polypeptide tag, providing the epitope, which can be selectively contact the antibody to the label. The epitope-tag, usually placed at the N - or C-end of the antibody or polypeptide. The presence of such labeled epitope forms of the antibody or polypeptide can be detected using antibodies to the polypeptide tag. Provision of the epitope-tagged makes it easy to identify the antibody or polypeptide by affinity chromatography using antibodies to label, or another type of affinity matrix that binds to the epitope-tag. In this area there are various polypeptide tags and their corresponding antibodies. Examples include tags polyhistidine (poly-his) or polyhistidine (poly-his-gly); polypeptide tag flu HA and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the label c-myc and antibodies 8F9, 3C7, 6E10, G4, B7 and 9E10 thereto [Evan et al., Molecular and Cellular Biology. 5:3610-3616 (1985)]; and the label on the basis of which glycoprotein D (gD) of herpes simplex virus and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other polypeptide tags include Flag peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the peptide epitope KT3 [Martin et al., Science, 255:192-194 (1992)]; peptide epitope α-tubulin [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)] and the tag from the peptide of the protein of the gene 10 T7 [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].

In an alternative embodiment, the chimeric molecule may contain protein antibody or polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as "immunoadhesin"), such a protein can be created with the Fc-region of IgG molecules. Hybrids with Ig preferably include the substitution of a soluble (transmembrane domain deleted or activated) forms of the antibody or polypeptide instead of the at least one variable region of the Ig molecule. In a particularly preferred embodiment, the hybrid immunoglobulin includes the area of the hinge, CH2 and CH3 or hinge, CH1, CH2 and CH3 IgG1 molecule. To obtain hybrids with immunoglobulins, see also U.S. patent No. 5428130, published on June 27, 1995.

H. antibodies and polypeptides

Further description mainly relates to the production of antibodies and polypeptides by culturing cells transformed or transfacial is the R vector, containing encoding the antibody or the polypeptide nucleic acid. Of course I believe that in order to obtain antibodies and polypeptides can be used alternative methods, which are well known in this field. For example, a suitable amino acid sequence or part thereof can be obtained by direct peptide synthesis using solid-phase synthesis methods [see, for example, Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)]. The synthesis of proteinsin vitrocan be performed using manual methods or automatically. Automated synthesis can be performed, for example, with the use of the synthesizer, Applied Biosystems Peptide Synthesizer (Foster City, CA) according to the manufacturer's instructions. Different parts of the antibody or polypeptide can be synthesized separately chemically and combine using chemical or enzymatic methods to produce the desired antibody or polypeptide.

1. The allocation of encoding the antibody or polypeptide DNA

Encoding the antibody or polypeptide DNA can be obtained from a cDNA library derived from tissue, for which there is confidence that it contains mRNA antibody or polypeptide and expresses it on detektiruya level. Thus, the DNA of a human antibody or polypeptide can be conveniently obtained from a cDNA library derived from tissue is a ne. Encoding the antibody or polypeptide gene can also be obtained from a genomic library or by known methods of synthesis (e.g., automated synthesis of nucleic acids).

Libraries can be skanirovat using probes such as oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or to encode them with protein. Screening of the cDNA library or genomic library with the selected probe can be performed using standard methods as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative method of selection of the gene encoding the antibody or the polypeptide consists of the application of the PCR method [Sambrook et al., above; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

Methods for screening cDNA libraries are well known in this field. Selected as probes oligonucleotide sequence must be of sufficient length and sufficiently unambiguous to minimize false positive reactions. The oligonucleotide probe is preferably labeled so that it can be detected by hybridization with DNA in scrinium the library. Methods of labeling are well known in this area and include the use of radioactive labels, like what's labeled 32P ATP, biotinylation or enzyme labels. Conditions of hybridization, which includes a moderately strict and highly stringent described in Sambrook et al., above.

Identified in such methods of screening libraries of sequences can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other closed, databases of sequences. The identity of the sequence or at the level of amino acids or nucleotides) within defined regions of the molecule or across the length of the sequence can be determined using known in the field of methods and as described in this document.

Nucleic acid protein coding sequence can be obtained by screening selected cDNA libraries or genomic libraries using for the first time described in this document are derived amino acid sequence and, if necessary, conventional ways of extension primers, as described in Sambrook et al., above, for the detection of precursors and processing intermediates of mRNA that may not be subjected to reverse transcription into cDNA.

2. The selection and transformation of host cells

Cell owners transferout or transform is written in this document expressing or cloning vectors for obtaining antibodies or polypeptide and cultured in conventional nutrient media appropriately modified for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Culturing conditions, such as environment, temperature, pH, etc., specialists in this field can be selected without undue experimentation. In General, principles, protocols, and practical guidance for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., above.

Methods of transfection of eukaryotic cells and transformation of prokaryotic cells generally known to experts in this field, for example, CaCl2CaPO4by liposomes and electroporation. Depending on the host cell, the transformation is carried out using standard methods appropriate to such cells. For prokaryotes, as a rule, use the treatment with calcium using calcium chloride as described in Sambrook et al., above, or electroporation. For transformation of certain plant cells use the infection with the use ofAgrobacterium tumefaciensas described by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published 29 June 1989. For mammalian cells without such cell walls, you can apply the method of precipitation with calcium phosphate by Graham and van der Eb, Virology, 52:456-457 (1978). The main aspects is respecti system cells of the host mammal is described in U.S. patent No. 4399216. Transformation of yeast, usually carried out by the method of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA). 76:3829 (1979). However, for the introduction of DNA into cells can also be used in other ways, such as by microinjection into the nucleus, electroporation, fusion of bacterial protoplasts with intact cells or by polycation, for example, polybrene, poliorcetes. About the various methods for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).

Appropriate cell hosts for cloning and expression of the DNA in the vectors of the present document include prokaryotes, yeast, and cells of higher eukaryotes. Suitable prokaryotes include as non-limiting examples of eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobacteriaceae such asE. coli. The public are different strains ofE. colisuch as a strain ofE. coliK12 MM294 (ATCC 31446);E. coliX1776 (ATCC 31537); strainE. coliW3110 (ATCC 27325) and K5 772 (ATCC 53635). Other suitable prokaryotic cell hosts include Enterobacteriaceae, such asEscherichiafor example,E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonellafor example,Salmonella typhimurium, Serratiafor example,Serratia marcescansandShigellaandBacillisuch asB. subtilisandB. licheniformis(for example,B. licheniformis41P described in DD 266710, is published 12 April 1989), Pseudomonassuch asP. aeruginosaandStreptomyces. These examples are illustrating and not limiting. Strain W3110 is especially preferred owner or the original owner, as it is a common strain host for fermentation products of recombinant DNA. Preferably, a host cell secretes a minimum number of proteolytic enzymes. For example, strain W3110 may be modified by the introduction of genetic mutations in the genes coding for endogenous host proteins, with examples of such hosts include strains ofE. coliW3110 1A2, which has the complete genotypetonA; strainE. coliW3110 9E4, which has the complete genotypetonA ptr3; strainE. coliW3110 27C7 (ATCC 55244), which has the complete genotypetonA ptr3 phoA E15 (argF-lac)169 degP ompT kanr; strainE. coliW3110 37D6, which has the complete genotypetonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kanr; strainE. coliW3110 40B4, which is strain 37D6 with a deletion mutationdegPare not resistant to kanamycin, and the strain ofE. coliwith mutant periplasmatic the protease described in U.S. patent No. 4946783, published on 7 August 1990. Alternatively, suitable methods of cloningin vitrofor example, PCR or other polymerase reactions of nucleic acids.

Full-length antibodies, antibody fragments and fused with the antibody proteins to monopropionate in bacteria, specifically, when glycosylation and effector function of the Fc is not needed, for example, when therapeutic antibody kongugiruut with a cytotoxic agent (e.g., a toxin) and immunoconjugate alone is effective in the destruction of tumor cells. Full-size antibodies have a longer half-life in the bloodstream. Getting inE. coliis faster and more economical. About the expression of fragments of the antibodies and polypeptides in bacteria, see, for example, U.S. patent No. 5648237 (Carter et. al.), U.S. patent No. 5789199 (Joly et al.) and U.S. patent No. 5840523 (Simmons et al.), which describes the area of translation initiation (TIR) and signal sequences for optimizing expression and secretion, these patents are incorporated herein by reference. After the expression of the antibody isolated from the cell massE. coliin a soluble fraction and can be cleaned, for example by column with protein A or G, depending on the isotype. Final cleaning can be performed similarly to the method of purification of antibodies expressed, for example, in CHO cells.

In addition to prokaryotes, suitable hosts for cloning and expression encoding the antibodies or polypeptides of the vectors are eukaryotic microorganisms such as filamentous fungi or yeast. A commonly used lower eukaryotic organism is the master of the m is Saccharomyces cerevisiae. Others include theSchizosaccharomyces pombe(Beach and Nurse, Nature, 290: 140 [1981]; EP 139383, published 2 may 1985); hostsKluyveromyces(U.S. patent No. 4943529; Fleer et al., Bio/Technology. 9:968-975 (1991))such as, for example,K. lactis(MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 [1983]),K. fragilis(ATCC 12424),K. bulgaricus(ATCC 16045),K. wickeramii(ATCC 24178),K. waltii(ATCC 56500),K. drosophilarum(ATCC 36906; Van den Berg et al., Bio/Technology, 8:135 (1990)),K. thermotoleransandK. marxianus; yarrowia(EP 402226);Pichia pastoris(EP 183070; Sreekrishna et al., J. Basic Environ., 28:265-278 [1988]);Candida;Trichoderma reesia(EP 244234);Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);Schwanniomycessuch asSchwanniomyces occidentalis(EP 394538 published 31 October 1990) and filamentous fungi such as, for example,Neurospora, Penicillium, Tolypocladium(WO 91/00357 published 10 January 1991) and the hostsAspergillussuch asA. nidulans(Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 [1984]) andA. niger(Kelly and Hynes, EMBO J., 4:475-479 [1985]). In this document are suitable methylotrophic yeast, and they include as non-limiting examples of yeast able to grow on methanol selected from the genera, includingHansenula, Candida, Kloeckera, Pichia, Saccharomyces, TorulopsisandRhodotorula. A list of specific species that represent examples of this class of yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

Suitably the cell hosts for the expression of glycosylated antibodies or polypeptide come from multicellular organisms. Examples of invertebrate cells include insect cells such asDrosophilaS2 andSpodopteraSf9, as well as plant cells, such as cell cultures of cotton, corn, potato, soybean, Petunia, tomato, and tobacco. Identified many baculovirus strains and variants and corresponding permissive cells are the owners of insects, such owners, asSpodoptera frugiperda(caterpillar),Aedes aegypti(the mosquito),Aedes albopictus(the mosquito),Drosophila melanogaster(fruit fly) andBombyx mori. Public is the variety of viral strains for transfection, such as option L-1 NPVAutographa californicaand strain Bm-5 NPVBombyx morisuch viruses can be used in the present document as viruses according to the present invention, particularly for transfection of cellsSpodoptera frugiperda.

However, the greatest interest is represented vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a standard procedure. Examples of suitable cell lines of mammalian hosts represent a line of monkey kidney CV1 transformed by SV40 (COS-7, ATCC CRL 1651); the line of embryonic human kidney (293 cells or 293 cells, subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); kidney cells baby hamster (BHK, ATCC CCL 10); the cells of the Chinese hamster ovary/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); the cells and Sertoli mouse (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); kidney cells of monkeys (CV1 ATCC CCL 70); kidney cells of the African green monkey (VERO-76, ATCC CRL-1587); carcinoma cells human cervical (HELA, ATCC CCL 2); cells, dog kidney (MDCK, ATCC CCL 34); liver cells buff rats (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); the cells of the human liver (Hep G2, HB 8065); tumor of the mammary gland of the mouse (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); the cells, MRC 5; FS4 cells and line human hepatoma (Hep G2).

Cell owners transform described above expressing or cloning vectors for the production of antibodies or polypeptide and cultured in conventional nutrient media, appropriately modified for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

3. The selection and use can replicate vector

Nucleic acid (e.g., cDNA or genomic DNA)encoding the antibody or polypeptide can be inserted into can replicate the vector for cloning (amplification of the DNA) or for expression. The public are the set of vectors. For example, the vector may be in the form of plasmids, Comedy, viral particles or phage. A suitable nucleic acid sequence can be inserted into a vector in a variety of ways. Basically, DNA is inserted into suitable participants is K(s) recognition restriction endonuclease using known in the field of methods. Components of vectors typically include as non-limiting examples of one or more signal sequences, the plot of the beginning of replication, one or more marker genes, an enhancer element, a promoter and termination sequence transcription. For the construction of suitable vectors containing one or more of these components, use the standard methods of ligation, well-known experts in this field.

The polypeptide can be obtained by recombinant means, not only directly, but also as a fused polypeptide with a heterologous peptide, which may be a signal sequence or other polypeptide with a specific cleavage site at the N-end of the Mature protein or polypeptide. Basically, the signal sequence may be a component of the vector, or it may be part encoding the antibody or polypeptide DNA, which is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected from the group leader peptides alkaline phosphatase, penitsillinazy, lpp or thermostable enterotoxin II. For yeast secretion the signal sequence may represent, for example, leader peptide, dragiev the th invertase, leader peptide alpha-factor (including the leader peptides of α-factorSaccharomycesandKluyveromyceswhere the latter is described in U.S. patent No. 5010182) or leader peptide of acid phosphatase, a leader peptide of glucoamylaseC. albicans(EP 362179, published 4 April 1990), or the signal peptide described in WO 90/13646 published 15 November 1990. When expression in mammalian cells for direct secretion of the protein, you can use the signal sequence mammals, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leader peptide.

And expressing, and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected cells of the host. Such sequences are well known for a variety of bacteria, yeast and viruses. Plot start replication from the plasmid pBR322 is suitable for most gram-negative bacteria, the site of the beginning of replication plasmid 2μ suitable for yeast, and various viral plots beginning of replication (SV40, polyoma, adenovirus, VSV or BPV) are suitable for cloning vectors in mammalian cells.

Expressing and cloning vectors usually contain selective gene, also called selective brand is. Typical selective genes encode proteins that (a) cause resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficit or (c) provide critical nutrients not available from complex media, e.g., the gene encoding D-alaninate forBacilli.

Examples of suitable selective markers for mammalian cells are markers that enable the identification of cells capable of capturing encoding the antibody or the polypeptide nucleic acid, such as DHFR or thymidine kinase. A suitable cell host when using wild-type DHFR is a line of CHO cells with deficiency of the activity of DHFR obtained and grown as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). Suitable selective gene for use in yeast is a gene oftrp1present in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. Genetrp1provides marker selection for mutant strain of yeast with the lack of ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].

Expressing and cloning vectors usually contain a promoter functionally linked to the coding antibody or polypeptide sequence of the nucleic KIS is the notes, to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the promoter system β-lactamase and lactose [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature. 281:544 (1979)], the promoter system, the alkaline phosphatase, the tryptophan (trp) [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also contain a Shine-dalgarno sequence (S.D.) functionally linked to DNA that encodes the antibody or polypeptide.

Examples of suitable promoter sequences for use in yeast hosts include the promoters for 3-phosphoglycerate [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or other enzymes of glycolysis [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphatedehydrogenase, glucokinase, piruvatcarboksilazy, phosphofructokinase, glucose-6-fortismere, 3-phosphoglyceromutase, piruwatkinaza, triazolopyrimidine, phosphoglucomutase and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage of controlled growth conditions transcriptions represent p motornya region for alcohol dehydrogenase 2, sociogram C, acid phosphatase, catabolic enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the utilization of maltose and galactose. Suitable for use for expression in yeast vectors and promoters are additionally described in EP 73657.

Transcription of the antibody or polypeptide with vectors in the cells of the host mammal is controlled, for example, by promoters obtained from the genomes of viruses such as virus polyoma, avian pox (UK 2211504, published 5 July 1989), adenovirus (such as adenovirus 2), human papilloma virus of cattle, sarcoma virus of birds, cytomegalovirus, a retrovirus, hepatitis B virus and the monkey virus 40 (SV40), heterologous mammalian promoters, e.g. the actin promoter or an immunoglobulin promoter, and promoters of heat shock proteins, provided such promoters are compatible with systems of the host cell.

Transcription of DNA encoding the antibody or polypeptide, in higher eukaryotes can be increased by inserting into a vector enhancer sequence. The enhancers are active in the CIS-position elements of DNA, usually a length of from about 10 to 300 BP that act on a promoter, increasing transcription. There are many enhancer on the of sledovatelnot from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, as a rule, apply the enhancer of virus eukaryotic cells. Examples include the SV40 enhancer on the late side of the plot early replication (BP 100-270), the enhancer early promoter of cytomegalovirus enhancer of virus polyoma during the later part of the plot, the beginning of replication and adenovirus enhancers. The enhancer can be inserted into the vector at a position 5' or 3' to the coding antibody or polypeptide sequence, but preferably it is from 5' from the promoter.

Expressing the vectors used in eukaryotic cells-the masters (yeast, fungi, insects, plants, animals, human or nucleated cells from other multicellular organisms)will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are usually located at the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as fragments of polyadenylation in the untranslated portion of the mRNA that encodes the antibody or polypeptide.

Other methods, vectors and cells of the host, suitable for adaptation to the synthesis of antibodies or polypeptides in recombinant vertebrate cell cultures described in Gething et al., Naure, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117060 and EP 117058.

4. Culturing host cells

Cell owners used to obtain the antibody or polypeptide of this invention can be grown in many environments. For culturing host cells suitable commercially available medium such as ham's F10 (Sigma), minimal maintenance medium ((MEM), (Sigma), RPMI-1640 (Sigma) and modified Dulbecco Wednesday Needle ((DMEM), Sigma). In addition, for culturing host cells as a medium for the cultivation you can use any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. patent No. 4767704; 4657866; 4927762; 4560655 or 5122469; WO 90/03430; WO 87/00195 or reference to U.S. patent 30985. Any of these environments, as necessary, can be supplemented with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, salts of calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as a drug GENTAMICIN™ (GENTAMYCIN™)), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. You can also add any other necessary the additives in suitable concentrations, which well-known experts in this field. Culturing conditions, such as temperature, pH, etc. represent the conditions previously used for the host cell selected for expression, and they usually obvious to specialists in this field.

5. Detection of amplification/gene expression

Amplification and/or gene expression can be measured in a sample directly, for example, a common southern-blotting, Northern-blotting to quantify the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot-blotting (DNA analysis), or by hybridization of thein situ, using appropriately labeled probe, based on information provided in this document sequences. Alternatively, it is possible to use antibodies that recognize specific duplexes, including DNA duplexes, RNA duplexes, and hybrid duplexes, DNA-RNA or DNA duplexes protein. In turn, the antibodies can be marked, and the analysis can be conducted so that the duplex bound to the substrate so that the formation on the surface of the duplex can be used to detect the presence of antibodies bound to the duplex.

Alternatively, for the direct quantitative determination of expression of a gene product the expression of genes can be measured by immunological methods, such as immunohistochemical occasianally or tissue slices and analysis of cell culture or body fluids. Antibodies suitable for immunohistochemical staining and/or analysis of sample fluids may be either monoclonal or polyclonal and may be obtained from any mammal. Antibodies can conveniently get to the native sequence polypeptide or to a synthetic peptide based on information provided in this document DNA sequence or to exogenous sequence fused to DNA polypeptide and encoding a specific epitope antibodies.

6. Purification of antibodies and polypeptide

Form of the antibody and the polypeptide can be isolated from the environment for cultivation or from lysates of cells-owners. If they are membrane-bound, they can be separated from the membranes by use of suitable solutions of detergents (such as Triton-X 100) or by enzymatic cleavage. Used for expression of the antibody or polypeptide cells can destroy various physical or chemical means, such as cyclic freeze-thaw, ultrasonic radiation, mechanical damage or lyse the cell means.

It may be desirable to purify the antibody and the polypeptide from proteins or polypeptides from recombinant cells. Examples of suitable purification methods are the following methods: by separation on ion-exchange column; osuzhdeni the ethanol; HPLC reverse phase; chromatography on silica or cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; precipitation ammonium sulfate; gel filtration using, for example, Sephadex G-75; protein sepharose columns to remove contaminants such as IgG; and metalhalide column to bind labeled epitopes forms antibodies and polypeptide. You can apply different methods to purify proteins and such methods are known in this field and are described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). Selected stage(s) of cleaning will depend, for example, from the nature of the use of the method of obtaining and get specific antibody or polypeptide.

When using recombinant methods, the antibody can be obtained in intracellular periplasmatic space and right to secrete into the environment. If the antibody receive intracellularly, as a first stage to remove debris from particulate or host cells, or fragments of lysis, for example, by centrifugation or ultracentrifugation. In Carter et al., Bio/Technology 10:163-167 (1992) describe the method of selection of antibodies that are secreted into periplasmatic spaceE. coli. Briefly, the cell mass is thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl the IDA (PMSF) over about 30 minutes. Cell debris can be removed by centrifugation. When the antibody is secreted into the medium, supernatant of each expression systems are generally first concentrated using a commercially available concentrate the protein filter, for example a device for ultrafiltration Amicon or Millipore Pellicon. Any of the above stages for the inhibition of proteolysis, you can add a protease inhibitor such as PMSF, and to prevent the growth of additional pollutants can add antibiotics.

The composition of the antibody obtained from the cell can be cleaned, for example, using chromatography on hydroxyapatite, electrophoresis gel, dialysis, and affinity chromatography, where the affinity chromatography is the preferred method of cleaning. The need for protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domains, which is present in the antibody. Protein A can be used for purification of antibodies, which are based on the heavy chain γ1, γ2, or γ4 human (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all isotypes of mice and γ3 person (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which is attached an affine ligand, most often represents the agarose, but also available to other matrices. Mechanically stable matrices such as glass with a controlled size is m then, or poly(Stradivari)benzene, can achieve faster flow rates and shorter processing times than can be achieved with the use of agarose. When the antibody contains a domain CH3, suitable for cleaning resin Bakerbond ABX™ (J. T. Baker, Phillipsburg, NJ). Depending on the antibody to highlight also other available methods for protein purification, such as separation on ion-exchange column, ethanol precipitation, HPLC reverse phase; chromatography on silica; chromatography on heparinase (heparin SEPHAROSE™), chromatography on anyone - or cation-exchange resin (such as a column with poliasparaginovaya acid), chromatofocusing, SDS-PAGE and precipitation with ammonium sulfate.

After any preparatory stage(s) containing cleaning of interest antibody and pollutants mixture can be subjected to a hydrophobic interaction chromatography with a low pH using an eluting buffer at a pH within about 2.5 to 4.5, preferably carried out at low salt concentrations (e.g., from about 0-0,25M salt).

I. Pharmaceutical compositions

Therapeutic formulations of the antibodies, binding oligopeptides that bind organic or inorganic low molecular weight compounds and/or polypeptides used according to the present invention, receive for storage by mixing the antibody,polypeptide, the oligopeptides or organic/inorganic molecular compounds with the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations, and include buffers such as acetate, Tris, phosphate, citrate, and buffers on the basis of other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as chloride of octadecyltrimethoxysilane; chloride hexadecane; benzalkonium chloride; chloride benzathine; phenol, butyl or benzyl alcohol; alkylarene, such as methyl or propyl paraben; catechin; resorcinol; cyclohexanol; 3-pentanol and m-cresol); polypeptides of low molecular weight (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelators, such as EDTA; create Tonino the tee, such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactant such as Polysorbate; salt forming counterions such as sodium; complex compounds with metals (for example, complexes of Zn-protein) and/or non-ionic surface-active agents such as TWEEN®, PLURONICS® or polyethylene glycol (PEG). The composition may contain the antibody at a concentration of from 5 to 200 mg/ml, preferably from 10 to 100 mg/ml

The compositions according to the present document may also contain more than one active connection in accordance with the need for a particular indication being treated, preferably such active compounds with a custom action that do not affect each other adversely. For example, in addition to antibodies, binding of oligopeptides or binding organic or inorganic low-molecular compounds, it may be desirable to include one additional antibody, e.g., a second antibody that binds to a different epitope on the same polypeptide or an antibody to some other target such as a growth factor that affects the development of certain malignancies. Alternative or additionally, the composition may further comprise a chemotherapeutic agent, cytotoxic the mini-tool, cytokine, growth inhibitory agent, protivogelmintnoe tool and/or cardioprotector. Accordingly, these molecules are present in combination in amounts that are effective for the intended purpose.

The active ingredients can also be enclosed in microcapsules obtained, for example, ways koatservatsii or interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacrylate) microcapsules, respectively, in colloidal systems drug delivery (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in microemulsion. Such methods are described in Remington''s Pharmaceutical Sciences. 16th edition, Oslo, A., Ed., (1980).

You can get drugs with a slow release. Suitable examples of drugs with a slow release include a semi-permeable matrices of solid hydrophobic polymers containing the antibody, where the matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of matrices with a 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, degradiruem the ethylene vinyl acetate, degradiruete copolymers Molo is Noah acid-glycolic acid such as LUPRON-DEPOT (LUPRON DEPOT®) (injectable microspheres composed of a copolymer of lactic acid-glycolic acid and leuprolide acetate) and poly-D-(-)-3-hydroxybutiric acid.

Compositions for application to the introduction ofin vivomust be sterile. This is easily accomplished by filtration through sterile filtration membranes.

J. Treatment with antibodies, binding oligopeptides and communicating organic/inorganic small molecule compounds

To determine the expression of the polypeptide (hepsin and/or HGF) in malignant tumors there are many detectionin methods of analysis. In one embodiment, the implementation of the overexpression of the polypeptide can be analyzed immunohistochemically (IHC). Immersed in paraffin tissue sections from a tumor biopsy may be subjected to IHC analysis and agree with the criteria for intensity of staining of the polypeptide set forth below:

A 0 - staining is not observed or observed staining membranes with less than 10% of tumor cells.

Figure 1+ - faint/barely perceptible staining membranes reveal more than 10% of tumor cells. Cells stained only part of their membrane.

Indicator 2+ - from weak to moderate complete staining of membranes see more than 10% of tumor cells.

Indicator 3+ - from the front to the strong full staining of membranes see more than 10% of tumor cells.

Tumors with indices of 0 or 1+ for expression of the polypeptide can be described as neverexpire polypeptide, whereas tumors with 2+ or 3+ can be described as sverkhekspressiya polypeptide.

Alternative or additionally, fixed in formalin, immersed in paraffin tumor tissue to determine the extent (if any) overexpression of the polypeptide in the tumor, it is possible to conduct analyses of FISH, such as the INFORM® (sold Ventana, Arizona) or PATHVISION® (Vysis, Illinois).

The overexpression or increased amounts of the polypeptide can be assessed using detectiong analysisin vivofor example, through the introduction of a molecule (such as an antibody, Oligopeptide or organic low-molecular compound)which binds with a molecule for detection and marks its detectable label (e.g. a radioactive isotope or a fluorescent label), and external scanning the patient for localization of the label.

As described above, antibodies, oligopeptides and organic low molecular weight compounds according to the invention have various non-therapeutic applications. The antibodies, oligopeptides and organic/inorganic molecular compounds according to the present invention may be suitable for determining the stage of expressing the polypeptide of malignant op is Halsey (for example, when radiodiagnosis). The antibodies, oligopeptides and organic low molecular weight compounds are also suitable for cleaning or thus of the polypeptide from cells, for detection and quantification of the polypeptidein vitrofor example, in an ELISA or Western-blotting, for the destruction and removal of expressing the polypeptide of cells from a population of mixed cells as a stage of purification of other cells.

Currently, depending on the stage of a malignant tumor, the treatment of a malignant tumor involves one or a combination of the following therapies: surgery to remove cancerous tissue, radiation therapy and chemotherapy. Therapy with an antibody, Oligopeptide or organic low molecular compound may be particularly preferred for older patients who poorly tolerate the toxicity and side effects of chemotherapy, and metastatic disease, when radiation therapy has limited usefulness. Directed to the tumor antibodies, oligopeptides and organic/inorganic molecular compounds according to the invention is suitable to facilitate expressing the polypeptide of malignant tumors at initial diagnosis or at relapse. For therapeutic applications, the antibody, Oligopeptide sludge is organic/inorganic small molecule compound can be applied separately or in combined treatment, for example, hormones, anti-angiogenesis or radioactively-labeled compounds or with surgery, cryotherapy, and/or radiotherapy. Treatment with the antibody, Oligopeptide or organic/inorganic small molecule compound can be combined with other forms of traditional treatment consistently with the previous or subsequent traditional treatment. In the treatment of malignant tumors, especially in patients with particularly high risk, use of chemotherapeutic drugs such as TAXOTERE® (TAXOTERE® (docetaxel), TAXOL® (TAXOL®) (paclitaxel), estramustine and mitoxantrone. In the present method according to the invention for the treatment or relief of malignant tumor patients with malignant tumor, you can enter the antibody, Oligopeptide or organic/inorganic small molecule compound, in combination with the treatment of one or more of the above chemotherapeutic agents. In particular, the proposed combined treatment with paclitaxel and modified derivatives (see, for example, EP0600517). The antibody, Oligopeptide or organic/inorganic small molecule compound should be entered with a therapeutically effective dose of a chemotherapeutic drug. In another embodiment, the implement is placed antibody Oligopeptide or organic/inorganic small molecule compound is administered in combination with chemotherapy to enhance the activity or effectiveness of a chemotherapeutic drug, such as paclitaxel. In the Physicians' Desk Reference (PDR) lists the doses of these funds, which are used in the treatment of various malignant tumors. The dosage and dosage of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the specific subject of the treatment of malignant tumors, the extent of the disease and other factors well known to practitioners in this field, and can be determined by the doctor.

In one specific embodiment, the patient is given conjugated with a cytotoxic agent conjugate containing an antibody, Oligopeptide or organic/inorganic small molecule compound. Preferably immunoconjugate associated with the protein, internalized cell, which leads to increased efficiency immunoconjugate in the destruction of malignant tumor cells with which it is associated. In a preferred embodiment, the cytotoxic agent affects nucleic acid in the cells of a malignant tumor or counteracts it. Examples of such cytotoxic among the STV described above and include maytansinoid, calicheamicin, ribonuclease and endonuclease DNA.

The antibodies, oligopeptides, organic/inorganic low-molecular compounds or their conjugates with a toxin is administered to the person by known methods, such as intravenous administration, e.g., in the form of a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebroventricular, subcutaneous, intra-articular, vnutricinovialnoe, intrathecal, oral, topical, or inhalation methods. Intravenous or subcutaneous administration of the antibodies, oligopeptides or organic low-molecular compounds is preferred.

With the introduction of the antibodies, oligopeptides or organic/inorganic low-molecular compounds can be combined with other therapeutic regimes. The combined introduction includes joint introduction using separate formulations or a single pharmaceutical composition and sequential introduction in any order, where, preferably, there is a time period while both (or all) active funds simultaneously exert their biological activity. Preferably the combined treatment leads to a synergistic therapeutic effect.

Also, it may be desirable to combine the introduction of antibodies or antibodies,oligopeptides or organic/inorganic low molecular weight compounds with the introduction of antibodies, directed to another tumor antigen associated with a particular malignancy.

In another embodiment, therapeutic methods of treatment of the present invention include co-administration of the antibody (or antibodies, oligopeptides or organic/inorganic low molecular weight compounds and one or more chemotherapeutic agents or inhibiting the growth of funds, including joint introduction mixtures of different chemotherapeutic agents. Chemotherapeutic agents include estramustine phosphate, prednimustine, cisplatin, 5-fluorouracil, melphalan, cyclophosphamide, hydroxycarbamide and hydroxycarbamide (such as paclitaxel and docetaxel) and/or anthracycline antibiotics. To obtain such chemotherapeutic agents and use their dosing protocols by manufacturer's instructions or as determined practitioners empirically. Receiving and dosing protocols for such chemotherapy are also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992).

The antibody, Oligopeptide or organic/inorganic small molecule compound can be combined with protivovandalnymi connection, for example antiestrogens compound such as tamoxifen; antiprogesterone connection, yet is how onapristone (see EP 616812): or antiandrogens connection, such as flutamide, in dosages known for such molecules. When a malignant tumor to treatment is androgenization malignant tumor, the patient could previously be subjected antiandrogenna treatment, and after a malignant tumor will be androgenization the patient can enter the antibody, Oligopeptide or organic/inorganic small molecule compound (and optionally other means, as described in this document).

Sometimes the patient may also be useful in conjunction enter cardioprotectant (to prevent or reduce myocardial dysfunction associated with treatment) or one or more cytokines. In addition to the above therapeutic regimes for the treatment of a patient to treatment with an antibody, Oligopeptide or organic/inorganic small molecule compound simultaneously with or after it can be subjected to surgical removal of malignant cells and/or radiation therapy. Suitable dosages for any of the above therapeutic agents are currently used dosages and they can be reduced due to the combined action (synergy) tools and antibodies, oligopeptides or organic/inorganic molecular compounds.

For the prevention or treatment of disease, the dosage and method of administration selects the attending physician based on known criteria. A suitable dose of the antibodies, oligopeptides or organic/inorganic low molecular weight compounds will depend on the type of disease to treatment, as defined above, the severity and course of the disease, enter whether the antibody, Oligopeptide or organic/inorganic small molecule compound for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, Oligopeptide or organic/inorganic small molecule compound and choice of physician. Accordingly, the antibody, Oligopeptide or organic/inorganic small molecule compound is administered to the patient once or several injections. Preferably, the antibody, Oligopeptide or organic/inorganic small molecule compound is administered by intravenous infusion or by subcutaneous injection. Depending on the type and severity of the disease, the initial dose of a candidate for introduction to the patient through one or more separate injections or by continuous infusion may be from about 1 μg/kg body weight to about 50 mg/kg of body weight (for example, about 0.1-15 mg/kg/dose) of antibody. Re the im dosing may include the introduction of an initial loading dose of approximately 4 mg/kg, subsequent weekly support a dose of approximately 2 mg/kg of antibody. However, the fit may be other dosing regimens. The usual daily dose may be in the range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For re-introductions for several days or more, depending on the condition, treatment continues until, until the desired suppression of disease symptoms. The progress of this treatment can easily be controlled by traditional methods and analyses and on the basis of well-known doctors and other specialists in this field criteria.

In addition to the introduction of the patient protein antibodies, the present invention relates to the introduction of antibodies by gene therapy. This introduction encoding the antibody nucleic acid is in the expression "the introduction of a therapeutically effective amount of antibodies". See, for example, WO96/07321, published March 14, 1996, regarding the application of gene therapy to obtain intracellular antibodies.

There are two main approaches to introduction of the nucleic acid (optionally contained in a vector) into the cells of the patient:in vivoandex vivo. Deliveryin vivonucleic acid is injected directly into the patient, usually at the site where the antibody is necessary. For the treatment ofex vivothe patient takes the cells in these selected cells are introduced nucleic acid and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes which are implanted to the patient (see, for example, U.S. patent No. 4892538 and 5283187). There are many available methods for introducing nucleic acids into living cells. The methods differ depending on whether the transfer whether the nucleic acid in cultured cellsin vitroorin vivoin the cells of the intended host. Methods suitable for the transfer of nucleic acid into mammalian cellsin vitroinclude the use of liposomes, electroporation, microinjection, merge cells, DEAE-dextran, the method of deposition of calcium phosphate, etc. Traditionally used vector for gene deliveryex vivois a retroviral vector.

Preferred at the present time the methods of transfer of nucleic acidsin vivoinclude transfection with viral vectors (such as adenovirus, herpes simplex I or adeno-associated virus) and is based on the lipid system (suitable lipids for mediated lipid transfer genes represent, for example, DOTMA, DOPE and DC-Chol). For an overview of currently known protocols tagging of genes and gene therapy, see Anderson et al., Science 256:808-813 (192). See also WO 93/25673 and quoted it references.

Antibodies according to the invention can be in various forms, covered by the definition of "antibody" in this document. Thus, antibodies include full-length or intact antibody, antibody fragments, the natural sequence of the antibody or amino acid variants, humanized, chimeric or fused antibodies, immunoconjugates and their functional fragments. The merged antibody sequence of the antibody is fused with a heterologous polypeptide sequence. Antibodies can be modified in the Fc-region to provide the desired effector functions. As discussed in more detail in one of the sections of this document, with the appropriate Fc-regions, a simple antibody associated with the cell surface can induce cytotoxicity, for example, due to antibodies cellular cytotoxicity (ADCC) or through the involvement of the complement when driven by the complement of cytotoxicity or by any other mechanism. Alternatively, where it is desirable to eliminate or reduce effector function, in order to minimize side effects or therapeutic complications, you can use some other Fc-field.

In one embodiment, the implementation of the anti-Christ. ate competes for binding or firmly binds to the same epitope the antibodies according to the invention. Also discusses the antibody with the biological characteristic of the provided antibodies according to the invention, in particular, including orientation to the tumorin vivoand any inhibition of cell proliferation or cytotoxic characteristics.

Herein are described methods of obtaining the above antibodies.

Provided antibodies, oligopeptides and organic/inorganic low-molecular compound suitable for the treatment of expressing hepsin and/or HGF malignant tumors or to alleviate one or more symptoms of malignant tumors in mammals. The methods according to the invention include use in the treatment and/or alleviation of symptoms of metastatic tumors associated with these tumors antagonists. The antibody, Oligopeptide or organic/inorganic small molecule compound is an antagonist capable to contact at least a part of malignant cells expressing the polypeptide(s) (hepsin and/or HGF) in mammals. In one of the embodiments, the antibody, Oligopeptide or organic/inorganic small molecule compound is effective in the destruction and the destruction of expressing the polypeptide and/or tumor cells or in the will eurout the growth of such cells in vitroorin vivowhen binding to the polypeptide. Such an antibody includes a simple antibody (not conjugated to any agent). Simple antibodies, which have a cytotoxic or any abscopal cell growth properties, you can also connect with a cytotoxic agent to make them even more effective in the destruction of tumor cells. Cytotoxic properties can be given to the antibody, for example, by conjugation of the antibody with a cytotoxic agent, to form immunoconjugate, as described in this document. In some embodiments, the implementation of the cytotoxic agent or a growth inhibitory tool is a small molecule compound. In some embodiments, the implementation used toxins, such as calicheamicin or maytansinoid and their analogs or derivatives.

The invention relates to compositions containing the antibody, Oligopeptide or organic/inorganic small molecule compound according to the invention and a carrier. For the treatment of a malignant tumor in need of such treatment the patient can enter the composition, where the composition may contain one or more antibodies provided in the form of immunoconjugate or in the form of simple antibody. In additional embodiments, the implementation of the composition can containing the ü these antibodies, oligopeptides or organic/inorganic low molecular weight compounds in combination with other therapeutic means, such as a cytotoxic or inhibiting growth of the funds, including chemotherapeutic agents. The invention also relates to compositions containing the antibody, Oligopeptide or organic/inorganic small molecule compound according to the invention and a carrier. In one of the embodiments the composition is a therapeutic composition that contains a pharmaceutically acceptable carrier.

Another aspect of the invention is a nucleic acid encoding the antibody. The scope includes nucleic acids encoding the H chain and L, and especially the remains of the hypervariable regions, circuits, encoding natural sequence antibodies, and variants, modifications and humanized version of the antibody.

The invention also relates to methods suitable for the treatment of malignant tumors and / or alleviate one or more symptoms of a malignant tumor in a mammal, comprising the administration to a mammal a therapeutically effective amount of the antibodies, oligopeptides or organic/inorganic low molecular weight compounds. Therapeutic compositions of the antibodies, oligopeptides or organic/inorganic low the molecular connection, you can enter a short (sharply) or permanently or intermittently, in accordance with the decision of the attending physician. Also provided methods of inhibiting the growth and destruction of the polypeptide (hepsin and/or HGF)- expressing and/or the responding cells.

The invention also relates to kits and finished products containing at least one antibody, Oligopeptide or organic/inorganic small molecule compound. Contain the antibodies, oligopeptides or organic/inorganic low-molecular compound sets are used, for example, for analysis of cell killing, for cleaning or thus of the polypeptide from the cells. For example, for isolation and purification of the polypeptide, the kit can contain an antibody, Oligopeptide or organic/inorganic small molecule compound associated with granules (for example, granules sepharose). You can provide kits which contain the antibodies, oligopeptides or organic/inorganic low molecular weight compounds for the detection and quantification of the polypeptidein vitrofor example, in an ELISA or Western-blotting. Such antibody, Oligopeptide or organic/inorganic small molecule compound, suitable for detection, it is possible to provide with a label, such as fluorescent or radioactive label.

K. the Finished product and sets

Another variant implementation of the image is to be placed is a finished product, containing substances suitable for the treatment of expressing the polypeptide (hepsin and/or induction of a malignant tumor, a cancer of the prostate and ovary. The finished product includes a container and a label or an insert in the packaging for the container or attached to the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers can be formed from a variety of materials, such as glass or plastic. The container contains a composition effective for the treatment of malignant tumors, and may have a sterile inlet (for example, the container may be a bag with intravenous solution or a bottle with a stopper that can be puncturing needle for subcutaneous injection). At least one active agent in the composition is an antibody, Oligopeptide or organic/inorganic small molecule compound according to the invention. A label or an insert in the packaging indicates that the composition is used to treat malignant tumors. The label or the liner in the package additionally contain instructions for administration of the composition of the antibodies, oligopeptides or organic/inorganic low-molecular compound to a patient with a malignant tumour. In addition, the finished product may further comprise a second container containing farmacevtichesky acceptable buffer, such as bacteriostatic water for injection, phosphate-saline buffer, ringer's solution and dextrose. Optionally, you can include other materials desirable from a commercial point of view or standpoint, including other buffers, diluents, filters, needles and syringes.

Also provides kits that are useful for various purposes, for example, for analyses of the expression of the polypeptide or destruction of cells, for cleaning or thus of the polypeptide from the cells. For isolation and purification of the polypeptide, the kit can contain an antibody, Oligopeptide or organic/inorganic small molecule compound associated with granules (for example, sivaratnam granules). You can provide kits which contain the antibodies, oligopeptides or organic/inorganic low molecular weight compounds for the detection or quantitative determination of the polypeptidein vitrofor example, in an ELISA or Western-blotting. As for the finished product, the kit contains a label or an insert in the packaging for the container or attached to the container. The container contains a composition comprising at least one antibody, Oligopeptide or organic/inorganic small molecule compound according to the invention. Additionally, this may include containers, for example, containing thinners and puff the market, control antibodies. The label or the liner in the package can provide a description of the composition, and instructions intended use thein vitroor detection.

L. Polypeptides and encoding the polypeptides, nucleic acid - specific forms and applications

The nucleotide sequence (or a complementary thereto), encoding the polypeptides according to the invention have many applications in the field of molecular biology and applications in therapy, etc. Encoding polypeptides nucleic acid is also suitable for the production of the polypeptides described herein by recombinant methods, where such polypeptides can be used, for example, to generate antibodies, as described in this document.

Full size natural gene sequence of the polypeptide or part thereof can be used as hybridization probes for a cDNA library to highlight other cDNA (for example, cDNA encoding a naturally occurring variants of the polypeptide or polypeptide from another species), having a desired sequence identity and the natural sequence of the polypeptide described herein. Optional length of the tip is from about 20 to about 50 bases. Hybridization probes can be obtained, at least partially, the new areas full of natural nucleotide sequence, where such areas can be determined without undue experimentation or from genomic sequences including promoters, enhancer elements and introns of the natural sequence of the polypeptide. As an example, the screening method includes the allocation of the coding region polypeptide gene using the known DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes can be marked by various labels, including radionucleotides, such as32P or35S, or enzymatic labels such as alkaline phosphatase, associated with the probe binding systems avidin/Biotin. Labeled probes with a sequence complementary to the gene sequence of the polypeptide of the present invention, can be used for screening libraries of human cDNA, genomic DNA or mRNA to determine which representatives of such libraries hybridize with this probe. Methods of hybridization additionally detailed in the examples below. Any EST sequence described in the present application, in a similar way can be used as probes with the use described in this document ways.

Other suitable fragments encoding polypeptides nucleic acids include antisense or sense oligonucleotides containing single-stranded sequence (nucleic acid or RNA, or DNA)capable of contacting the sequence of the target mRNA of the polypeptide (sense) or DNA polypeptide (antisense). Antisense or sense oligonucleotides, according to the present invention contain a fragment of the coding region of the DNA that encodes hepsin, Pro-HGF or binding fragments as described herein. This fragment typically contains at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. The opportunity to get antisense or a sense oligonucleotide, based on the cDNA sequence that encodes the protein, for example, described in Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechniques 6:958, 1988).

Binding of antisense or sense oligonucleotides with sequences of target nucleic acids leads to the formation of duplexes that block transcription or translation to a target sequence in a number of ways, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. Such methods are included in the scope of the present invention. Thus, the antisense oligonucleotides can be used to block expression of the protein, where the protein may play a role in the induction of malignant tumors in mammals. Antis is USLOVIE or sense oligonucleotides also include oligonucleotides with modified sugar-fosfomifira frame (or other bonds of sugars, such as described in WO 91/06629)where such communication sugars are resistant to endogenous nucleases. Such oligonucleotides with sustainable connections sugars stablein vivo(i.e. able to withstand enzymatic destruction), but retain sequence specificity to be able to contact the nucleotide sequences of target.

Preferred intergenic sites for binding of antisense sequences include the region containing the initiation codon/start broadcast (5'-AUG/5'-ATG) or the termination codon or stop codon (5'-UAA, 5'-UAG and 5-UGA/5'-TAA, 5'-TAG and 5'-TGA) open reading frame (ORF) of the gene. These areas are part of the mRNA or gene, containing from about 25 to about 50 contiguous nucleotides in the forward direction (i.e. the 5' or 3') from the initiation codon or translation termination. Other preferred region for binding of antisense sequences include: introns; exons; junctions of introns and exons; open-reading frames (ORF) or "coding region," which is a region between codon of translation initiation and termination codon broadcast; 5'cap of mRNAs containing methylated at the N7 guanosine residue associated with the 5'-terminal residue of the mRNA via a 5'-5' trifosfatnogo communication and includes the very structure of the 5'-cap, and that is as the first 50 nucleotides, adjacent to the cap; 5'- noncoding region (5'UTR), the portion of the mRNA in the direction of 5'from codon of translation initiation and, thus, containing nucleotides between the 5'-cap and the initiation codon of the mRNA or corresponding nucleotides on the gene, and 3'- untranslated region (3'UTR), the portion of the mRNA in the direction of the 3'of codon translation termination and, thus, containing nucleotides between codon translation termination and 3'end of an mRNA or corresponding nucleotides on the gene.

Specific examples of preferred antisense compounds suitable for inhibiting expression of the polypeptide include oligonucleotides containing modified frames or unnatural magnolioideae communication. Oligonucleotides with modified frames include oligonucleotides containing a phosphorus atom in the cage, and oligonucleotides that do not contain a phosphorus atom in the skeleton. For the purposes of this description, and, as some people think in this area, modified nucleotides without phosphorus atom in their magnoliopsida the frame can also be viewed as oligonucleotide. Preferred modified frames oligonucleotides include, for example, phosphorothioate, the chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphonic methylated and other alkylated foston is you, including 3'-alkylenediamine, 5'-alkylenediamine and chiral phosphonates, phosphonate, phosphoramidate, including 3'-amino, phosphoramidate and aminoalkylphosphonic, conopophagidae, cyanoacetate, cyanoacetaldehyde, selenophosphate and borophosphate with the normal 3'-5' linkages, 2'-5' linked analogs and oligonucleotides with inverted polarity, where one or more mezhnukleotidnyh linkages is a 3' to 3', 5' to 5' or 2' to 2'. Preferred oligonucleotides with inverted polarity contain a single link 3' 3' 3'-terminal mezhnukleotidnyh connection, i.e. a single inverted nucleoside residue which may be missing base (the nucleic base is missing or instead is a hydroxyl group). Also included are various salts, mixed salts and free form acids. Representative United States patents that describe a receipt containing phosphorus linkages include as non-limiting examples of U.S. patents№: 3687808; 4469863; 4476301; 5023243; 5177196; 5188897; 5264423; 5276019; 5278302; 5286717; 5321131; 5399676; 5405939; 5453496; 5455233; 5466677; 5476925; 5519126; 5536821; 5541306; 5550111; 5563253; 5571799; 5587361; 5194599; 5565555; 5527899; 5721218; 5672697 and 5625050, each of which is incorporated herein by reference.

Preferred modified frames of oligonucleotides that do not contain the ATO is and phosphorus, have the frames formed by short chain alkyl or cycloalkyl magnolioideae links, mixed magnolioideae ties with the heteroatom and alkyl or cycloalkyl or one or more short-chain magnolioideae ties with the heteroatom or a heterocycle. They include oligonucleotides with morpholinium bonds (partially generated from the sugar portion of a nucleoside); siloxane skeletons; sulfide, sulfoxide and sulfonic frames; formatline and toformation frames; metainformation and-toformation frames; rebaseline frames; containing alkenes frames; sulphamate frames; methylaniline and metilimidazylio frames; sulphonate and sulfonamidnuyu frames; amide frames and the other containing parts of mixed components N, O, S, and CH2. Representative United States patents that describe the receipt of such oligonucleotides include as non-limiting examples of U.S. patents№: 5034506; 5166315; 5185444; 5214134; 5216141; 5235033; 5264562; 5264564; 5405938; 5434257; 5466677; 5470967; 5489677; 5541307; 5561225; 5596086; 5602240; 5610289; 5602240; 5608046; 5610289; 5618704; 5623070; 5663312; 5633360; 5677437; 5792608; 5646269 and 5677439, each of which is incorporated herein by reference.

In other preferred antisense-oligonucleotides and sugar, and magnoliopsida communication, i.e. frame n is cleotide units, replaced by new groups. Base save for hybridization with the corresponding connection of the nucleic acid target. One such oligomeric compounds, oligonucleotide mimetic, which shows that he has an excellent hybridization properties, is designated here as peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced by containing amide linkages frame, specifically on aminoantipyrine frame. Nucleic bases are stored and linked directly or indirectly to Aza nitrogen atoms of amide side of the frame. Representative United States patents that describe the receipt of PNA compounds include, as non-limiting examples, U.S. patent No.: 5539082; 5714331 and 5719262, each of which is incorporated herein by reference. Additional guidance on PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

Preferred antisense oligonucleotides comprise phosphorothioate frames and/or frames with heteroatoms, specifically CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- [known as a methylene(methylimino) or MMI frame], -CH2-O-N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2- I-O-N(CH3)-CH2-CH2- [where natural fosfodiesterzy frame represent the stations as-O-P-O-CH 2-], described in the aforementioned U.S. patent No. 5489677 and amide frames in the above U.S. patent No. 5602240. Preferred antisense oligonucleotides with morpholinium frame structures in the above mentioned U.S. patent No. 5034506.

Modified oligonucleotides may also contain one or more substituted sugar molecules. Preferred oligonucleotides contain in position 2'- one of the following: OH; F; O-alkyl, S-alkyl or N-alkyl; O-alkenyl, S-alkenyl or N-alkenyl; O-quinil, S-quinil or N-quinil or O-alkyl-O-alkyl, where alkyl, alkenyl and quinil may represent a substituted or unsubstituted alkyl from C1to C10or alkenyl and quinil from C2to C10. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2and O(CH2)nON[(CH2)nCH3)]2where n and m are from 1 to about 10. Other preferred antisense oligonucleotides contain in position 2'- one of the following: lower alkyl from C1to C10substituted lower alkyl, alkenyl, quinil, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2N3 , NH2heteroseksualci, heterocyclochain, aminoethylamino, polyallylamine, substituted silyl that cleave RNA group, reporter group, intercalator, a group for improving the pharmacokinetic properties of the oligonucleotide, a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents with similar properties. A preferred modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e. the group of alkoxyalkane. Additional preferred modification includes 2'-dimethylaminoethoxy, i.e. the group O(CH2)2ON(CH3)2, also known as 2'-DMAOE, as described in the examples below in this document, and 2'-dimethylaminoethoxide (also known in this area as 2'-O-dimethylaminoethoxide or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2).

Additional preferred modification includes a closed nucleic acid (LNA) in which the 2'-hydroxyl group of the sugar ring is linked to the 3' or 4' carbon atom, thereby forming a bicyclic sugar molecule. The connection preferably represents a linking 2' oxygen atom and the 4' carbon atom of methylene (-CH2-)ngroup, where n represents 1 or 2. LNA and receive OPI is ANO WO 98/39352 and WO 99/14226.

Other preferred modifications include 2'-methoxy (2'-O-CH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-allyl (2'-CH2-CH=CH2), 2'-O-allyl (2'-O-CH2-CH=CH2) and 2'-fluoro (2'-F). Modification of the 2' may be in arabino-position (top) or RIBO-position (bottom). The preferred modification of the 2'-arabino is a 2'-F. Similar modifications can also be produced in other provisions of the oligonucleotide, specifically in the 3'-position of sugar at the 3'-terminal nucleotide or in 2'-5'-linked oligonucleotides and the 5'position of 5'terminal nucleotide. Oligonucleotides instead pentofuranose sugar can also contain replicas of sugar, such as cyclobutylamine group. Representative United States patents that describe the receipt of such modified groups of sugars include as non-limiting examples of U.S. patents№: 4981957; 5118800; 5319080; 5359044; 5393878; 5446137; 5466786; 5514785; 5519134; 5567811; 5576427; 5591722; 5597909; 5610300; 5627053; 5639873; 5646265; 5658873; 5670633; 5792747 and 5700920, each of which is incorporated herein by reference in full.

Oligonucleotides can also include modification or replacement nucleic bases (often denoted in this area simply as "base"). As used herein, "unmodified" or "natural" n is kleinhovia grounds include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleic base include other synthetic and natural nucleic acid bases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, gipoksantin, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-through and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-tiomkin and 2-tocitizen, 5-halogenerator and cytosine, 5-PROPYNYL (-C≡C-CH3or-CH2-C≡CH) -uracil and cytosine and other alkyline derivatives of pyrimidine bases, 6-azauracil, cytosine and thymine, 5-uracil (pseudorutile), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenine and guanine, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted orally and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine 2-aminoadenine, 8-azaguanine and 8-azadani, 7-deazaguanine and 7-deazaadenosine and 3-deazaguanine and 3-deazaadenosine. Additional modified nucleic base include tricyclic pyrimidines such as phenoxybenzamine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), fenotiazinas (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazines (for example 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbacyclin (2H-pyrimido[4,5-b]indol-2-one), periodontitis (H-pyrido[3',':4,5]pyrrolo[2,3-d]pyrimidine-2-one). Modified nucleic base can also include nucleic bases, in which the purine or pyrimidine base is replaced with another heterocycle, for example, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Additional nucleic bases include nucleic bases described in U.S. patent No. 3687808, nucleic bases, described in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and nucleic bases described in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613. Some of these nucleic bases are particularly suitable for increasing the affinity of binding of the oligomeric compounds according to the invention. These include 5-substituted pyrimidines, 6-isoperimetry and N-2, N-6 and O-6 substituted purines, including 2-aminopropylene, 5-propenylboronic and 5-propylitized. It is shown that replacement of 5-methylcytosine increase the stability of the duplex nucleic acid is 0.6 to 1.2°C. (Sanghvi et al, Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are the preferred replacement of the bases, even more particularly when combined with 2'-O-methoxyethylamine modifications sugars. Representative United States patents that describe the synthesis of modified nucleic acid bases, include as non-limiting examples: Pat the t U.S. No. 3687808, as well as U.S. patents№: 4845205; 5130302; 5134066; 5175273; 5367066; 5432272; 5457187; 5459255; 5484908; 5502177; 5525711; 5552540; 5587469; 5594121, 5596091; 5614617; 5645985; 5830653; 5763588; 6005096; 5681941 and 5750692, each of which is incorporated herein by reference.

Another modification of antisense oligonucleotides is a chemical linking to the oligonucleotide one or more molecules or conjugates that enhance the activity of the oligonucleotide, its distribution in cells or capture cells. Compounds according to the invention may include groups for conjugation, covalently linked, functional groups such as primary or secondary hydroxyl group. Group for conjugation according to the invention include intercalators, reporter molecules, polyamine, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical groups for conjugation include cholesterol, lipids, cationic lipids, phospholipids, cationic phospholipids, Biotin, fenesin, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarins and dyes. In the context of this invention, groups that enhance the pharmacodynamic properties, include groups that improve the capture oligomer, HC is licevaya resistance to degradation and/or amplifying specific sequences for hybridization with RNA. In the context of this invention, groups that enhance the pharmacokinetic properties, include groups that improve the capture, distribution, metabolism or excretion of the oligomer. Group for conjugation include as non-limiting examples of lipid groups, such as cholesterol group (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioether, e.g., hexyl-S-tritylation (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), taholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, for example, dodecanediol or undecisive residues (Saison-Behmoaras et al., EMBO J. 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, pp. 49-54), a phospholipid, for example, dihexadecyl-rat-glycerol or 1,2-di-O-hexadecyl-rat-glycero-3-H-phosphonate of triethylamine (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), poliaminov or polietilenglikoli chain (Manoharan et al., Nucleosides &Nucleotides, 1995, 14, 969-973), or adamantanone acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmitinovuyu group (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237) or octadecylamino or paxilalertsbuypaxilonlinewi group. The oligonucleotides according to the invention can also be konjugierte with active drugs, such as aspirin, warfarin, phenylbutazone, IB is Profanum, supervenom, fenbufen, Ketoprofen, (S)-(+)-pranoprofen, carprofen, ancillarisation, 2,3,5-triiodobenzoic acid, flufenamic acid, folinovoy acid, benzothiadiazides, chlorthiazide, diazepino, indomethacin, a barbiturate, a cephalosporin, sulfate drug, antidiabetic agent, antibacterial agent or an antibiotic. Conjugates of oligonucleotide-drug and obtaining them are described in patent applications U.S. serial number 09/334130 (filed June 15, 1999) and U.S. patent№ 4828979; 4948882; 5218105; 5525465; 5541313; 5545730; 5552538; 5578717, 5580731; 5580731; 5591584; 5109124; 5118802; 5138045; 5414077; 5486603; 5512439; 5578718; 5608046; 4587044; 4605735; 4667025; 4762779; 4789737; 4824941; 4835263; 4876335; 4904582; 4958013; 5082830; 5112963; 5214136; 5082830; 5112963; 5214136; 5245022; 5254469; 5258506; 5262536; 5272250; 5292873; 5317098; 5371241, 5391723; 5416203, 5451463; 5510475; 5512667; 5514785; 5565552; 5567810; 5574142; 5585481: 5587371; 5595726; 5597696; 5599923; 5599928 and 5688941, each of which is incorporated herein by reference.

It is not necessary for all positions in this connection to be modified in the same way, and, in fact, in the same connection, and even in a single nucleotide in the oligonucleotide can be more than one of the above modifications. The present invention also relates to antimuslim compounds which are chimeric compounds. In the context of this invention the chimeric antimic the new compounds or "chimeras" are antisense compounds, specific oligonucleotides, which contain two or more chemically great areas, each consisting of at least one monomer unit, i.e nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region, where the oligonucleotide is modified so as to give the oligonucleotide increased resistance to degradation by nucleases, the enhanced ability to capture cells and/or increased affinity of binding to the nucleic acid target. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of splitting the hybrid RNA:DNA or RNA:RNA. As an example, Mcasa H is a cellular endonuclease which cleaves the chain RNA duplex RNA:DNA. Thus, activation of RNase leads to cleavage of the RNA target, thereby greatly increasing the efficiency of inhibition by the oligonucleotide gene expression. Thus, when using chimeric oligonucleotides can be obtained comparable results compared to phosphorothioate deoxyoligonucleotide, hybridization with the same area of the target. Chimeric antisense compounds according to the invention can be in the form of composite structures of two or more oligonucleotides, modifitsirovanniy the oligonucleotides, oligonucleosides and/or mimetics of oligonucleotides, as described above. Preferred chimeric antisense oligonucleotides include at least one modified 2' sugar residue (preferably 2'-O-(CH2)2-O-CH3) at the 3'end, to give stability to nucleases and region, with at least 4 consecutive residues of sugars, 2'-H to give the activity of RNase H. Such compounds also indicate in this field as hybrids or gapperi. Preferred gapperi be field modified at the 2' sugar residues (preferably 2'-O-(CH2)2-O-CH3) at the 3'end and the 5'-end, separated by at least one area of at least 4 consecutive residues of sugars, 2'-H, and preferably include phosphorothioate communication frame. Representative United States patents that describe the receipt of such hybrid structures include as non-limiting examples of U.S. patents№ 5013830; 5149797; 5220007; 5256775; 5366878; 5403711; 5491133; 5565350; 5623065; 5652355; 5652356 and 5700922, each of which is incorporated herein by reference in full.

Antisense compounds used in accordance with this invention can be conveniently and simply to get through a well-known method of solid-phase synthesis. Equipped with the e for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, Calif.). Additionally or alternatively possible to use any other means for such synthesis known in this field. It is well known, the application of such methods to obtain oligonucleotides, such as phosphorothioates and alkylated derivatives. Compounds according to the invention can also be mixed, to encapsulate, konjugierte or otherwise combined with other molecules, molecule structures or mixtures of compounds, such as liposomes bound receptor molecules, oral, rectal, topical or other formulations, for assisting the capture, distribution and/or absorption. Representative United States patents that describe the receipt of such contributing to the capture, distribution and/or absorption of compounds that include as non-limiting examples of U.S. patents№ 5108921; 5354844; 5416016; 5459127; 5521291; 5543158; 5547932; 5583020; 5591721; 4426330; 4534899; 5013556; 5108921; 5213804; 5227170; 5264221; 5356633; 5395619; 5416016; 5417978; 5462854; 5469854; 5512295; 5527528; 5534259; 5543152; 5556948; 5580575 and 5595756, each of which is incorporated herein by reference.

Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic groups, such as organic groups described in WO 90/10048, and the other is a group, such as poly(L-lysine), which increase the affinity of the oligonucleotide in relation to a target sequence of nucleic acid. In addition, for modification of the specificity of binding of antisense or sense oligonucleotide against nucleotide target sequence to semantic or antimuslim oligonucleotides can be attached intercalating agents such as ellipticine and alkylating funds or complexes with metals.

Antisense or sense oligonucleotides may be introduced into the cell containing the sequence of a target nucleic acid by any method of gene transfer, including, for example, mediated CaPO4transfection of DNA, electroporation, or by using transferring genes vectors such as Epstein-Barr. In a preferred method, the antisense or sense oligonucleotide is introduced into a suitable retroviral vector. Cells containing the sequence of the target nucleic acid is brought into contact with the recombinant retroviral vectorin vivoorex vivo. Suitable retroviral vectors include, as non-limiting examples of retroviral vectors derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or dvuhkabinnye vectors, denoted DCT5A, DCT5B and DCT5C (see WO 90/13641).

Also senses the e or antisense oligonucleotides can be introduced into the cell, containing the nucleotide sequence of the target by forming a conjugate with a ligand-binding molecule, as described in WO 91/04753. Suitable binding ligand molecules include as non-limiting examples of cell surface receptors, growth factors, other cytokines, or other ligands that bind to receptors on the cell surface. Preferably, the conjugation of a ligand-binding molecule does not substantially interferes with the ability of a ligand-binding molecule to contact the corresponding molecule or receptor, or does not block the penetration of sense or antisense of the oligonucleotide or its conjugated version into the cell.

Alternatively, the sense or antisense oligonucleotide can be introduced into the cell containing the sequence of a target nucleic acid by forming a complex of the oligonucleotide-lipid as described in WO 90/10448. Complex sense or antisense of the oligonucleotide-lipid preferably dissociates inside the cell under the action of endogenous lipase.

The length of the molecules, antisense or sense RNA or DNA, as a rule, is at least about 5 nucleotides, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990 or 1000 nucleotides, where the term "approximately" in this context means the length of the reference nucleotide sequence of plus or minus 10% from the specified length.

The probes can also be used in the methods of PCR to obtain a pool of sequences for identification of closely-related polypeptide coding sequences.

The nucleotide sequence encoding the polypeptide, can also be used to construct hybridization probes for mapping the gene encoding the polypeptide and for the genetic analysis of individuals with genetic diseases. Provided in this document nucleotide sequence can be mapped on the chromosome and specific regions of the chromosome using known methods, such as hybridization ofin situanalysis of the coupling relative to known chromosomal markers and hybridization screening libraries.

The polypeptide can be used in assays to identify the other proteins or molecules involved in the interaction with the polyp is predom through bundling. By such means it is possible to identify inhibitors of the interaction with the receptor/ligand by binding. For screening of peptide or low molecular weight inhibitors of the interaction by binding you can also use proteins involved in these interactions by binding. You can develop srinilaya tests to find the best compounds that mimic the biological activity of the natural polypeptide or the receptor of the polypeptide. Such srinilaya assays include assays suitable for high throughput screening of chemical libraries, making them particularly suitable for the identification of low-molecular candidates for the drug. Consider low-molecular compounds include synthetic organic or inorganic compounds. The analyses may be performed in a variety of formats, including analyses of binding of protein-protein, biochemical screening tests, immunological assays and cell-based assays, which are well characterized in this field.

Encoding the polypeptides, nucleic acids or their modified forms can also be used to obtain transgenic animals or animals with "knockout", which in turn is suitable for development and screening therapeutics and suitable reagents. Transgenic animal (e.g., mouse or rat) is an animal having cells that contain a transgene, where the transgene is introduced to the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A transgene is a DNA that is integrated into the genome of cells from which a transgenic animal develops. In one embodiment, the implementation of encoding a polypeptide cDNA can be used to clone encodes a polypeptide of genomic DNA in accordance with well known techniques and the genomic sequences used to obtain transgenic animals that contain cells which Express encoding a polypeptide DNA. Methods of producing transgenic animals, particularly animals such as mice or rats, have become common in this area and are described, for example, in U.S. patent No. 4736866 and 4870009. As a rule, in specific cells, the transgene polypeptide is administered with the use of tissue-specific enhancers. Transgenic animals containing a copy of encoding the transgene polypeptide introduced into the germ line of the animal at an embryonic stage can be used to verify the validity of the increased expression encodes a polypeptide DNA. Such animals can be used as test animals for reagents, which are believed to provide protection, for example, from p is tological conditions associated with its overexpression. In accordance with this aspect of the invention, the animals are treated with the reagent and reduced compared with untreated animals bearing the transgene, the frequency of pathological conditions will mean a potential therapeutic effect on the disease state.

The alternative, to construct an animal with a "knockout" gene, which is defective or modified encoding the polypeptide gene as a result of homologous recombination between the endogenous coding for the polypeptide gene and introduced into an embryonic stem cell of the animal encodes a polypeptide modified genomic DNA, it is possible to use non-human homologues of polypeptides. For example, encoding the polypeptide cDNA can be used to clone encodes a polypeptide of genomic DNA in accordance with conventional methods. The portion encodes a polypeptide genomic DNA can be deleted or replaced with another gene, such as the gene encoding a selective marker, which can be used to monitor integration. Typically, the vector comprises several thousand base pairs of unaltered flanking DNA (5'-and 3'-end) [e.g., see Thomas and Capecchi, Cell, 51:503 (1987) for a description of vectors for homologous recombination]. The vector is introduced into a line of embryonic stem cells (for example, what exploits electroporation and selected cells, in which the introduced DNA homologous recombinable with endogenous DNA [e.g., see Li et al., Cell 69:915 (1992)]. Then the selected cell is injected into a blastocyst of an animal (e.g. mouse or rat) with the formation of aggregation chimeras [see for example Bradley, inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), p. 113-152]. Then chimeric embryo can implant the appropriate pseudoelement the female animal, nurse and the embryo to grow to term with obtaining an animal with a "knockout". Offspring carrying in their gametes homologous nekombinirovannyh DNA can be identified by standard methods and use of breeding animals in which all cells of the animal contain homologous nekombinirovannyh DNA. Animals with knockout can be characterized by their ability to withstand certain pathological conditions and in respect of development in them due to the absence of the polypeptide of pathological conditions.

Encoding the polypeptides nucleic acid can also be used in gene therapy. In applications of gene therapy genes introduced into the cell to achieve the synthesis of a therapeutically effective genetic productin vivofor example, to replace the defective gene. "Gene therapy" includes traditional gene therapy, where long-term effect is reached by means of a single treatment is, and the introduction of gene therapies that includes single or repeated administration of a therapeutically effective DNA or mRNA. As therapeutic agents for blocking the expression of certain genesin vivoyou can use antisense RNA and DNA. Already shown that short antisense oligonucleotides can be introduced into the cell, where they act as inhibitors, despite their low intracellular concentrations, due to their limited capture of the cell membrane (Zamecnik et al., Proc. Natl. Acad. Sci. USA 83:4143-4146 [1986]). Oligonucleotides to increase their capture can be modified, for example, by replacing their negatively charged fosfolipidnyh groups uncharged groups.

There are many available methods for introducing nucleic acids into living cells. The methods vary depending on, carry whether nucleic acid in cultured cellsin vitroor in the cells of the intended hostin vivo. Suitable for transfer of nucleic acid into mammalian cellsin vitrothe methods include the use of liposomes, electroporation, microinjection, merge cells, DEAE-dextran, the method of deposition of calcium phosphate, etc. are Preferred in the present methods of gene transferin vivoinclude transfection with viral (typically retrovi usname) vectors and mediated by liposomes with viral protein shell transfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]). In some situations it is desirable to provide a source of nucleic acid by means that are directed to the target cells, such as antibody specific for membrane protein on the cell surface or to the target cell, a ligand for a receptor on the target cell, etc. When using liposomes for directions and/or to facilitate capture it is possible to use proteins that are associated with membrane protein cell surface associated with endocytosis, e.g. capsid proteins or fragments thereof, genotype to a particular cell type, antibodies for proteins which undergo internalization in the cycle, proteins, aimed intracellular localization and increasing the time half-life within the cell. Way-mediated receptor endocytosis is described, for example, Wu et al., J. Biol. Chem. 262, 4429-4432 (1987) and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). For an overview of the protocols marking genes and gene therapy, see Anderson et al., Science 256, 808-813 (1992).

Molecules encoding the polypeptides, nucleic acids or fragments described herein are suitable for identification of chromosomes. In this respect there is a continuing need to identify new chromosomal markers, as currently available, relatively few of marker chromosomes reagents on the basis of actual data of the sequence is. Each molecule of nucleic acid according to the present invention can be used as a chromosome marker.

Polypeptides and nucleic acid molecules according to the invention can be used for the purpose of diagnosis for tissue typing, where the polypeptides can be otherwise expressed in one tissue compared to another, preferably in a diseased tissue compared to normal tissue of the same tissue type. The nucleic acid molecules can be used to obtain probes for PCR, Northern blotting, analysis, Southern analysis and Western blot testing.

This invention relates to methods of screening compounds to identify compounds that prevent the action of the polypeptide (antagonists). Developed srinilaya tested for antagonist drug candidates to identify compounds that bind or form a complex with the polypeptides encoded by the genes defined in this document, or otherwise hindering the interaction of the encoded polypeptides with other cellular proteins, including, for example, inhibition of expression of the polypeptide by the cell. Such srinilaya assays include assays suitable for high throughput screening of chemical libraries, making them particularly suitable DL the identification of low molecular weight drug candidates.

The analyses may be performed in a variety of formats, including analyses of binding of protein-protein, biochemical screening tests, immunological assays and cell-based assays, which are well characterized in this field.

All tests for antagonists are common in that they apply contact drug candidate with a polypeptide encoded identified in this document nucleic acid under conditions and for a time suitable to allow these two components interact.

In the analysis of binding can be divided or to determine the interaction of binding and complex formation. In a specific embodiment, the polypeptide or the drug candidate immobilized on the solid phase, for example, on the tablet for micrometrology through covalent or non-covalent attachment. Non-covalent joining, as a rule, carried out by coating the solid surface with a solution of polypeptide and drying. An alternative to attaching the polypeptide to a solid surface, you can use the immobilized antibody, e.g., a monoclonal antibody specific to the polypeptide for immobilization. The analysis is performed by binding neimmunizirovannah component that can be marked on aktywami label, to the immobilized component, for example, the coated surface containing the attached component. After completion of the reaction, unreacted components are removed, for example by washing and detects attached to a solid surface complexes. When the original neemalirovannym component carries detektiruya label, detection of immobilized label on the surface means the formation of the complex. When initially neemalirovannym component does not bear the label, the formation of the complex can be detected, for example, through the use of labeled antibodies that specifically bind to the immobilized complex.

If the connection candidate interacts, but is not associated with the polypeptide, its interaction with that polypeptide can be analyzed well-known methods for detecting protein-protein interactions. Such assays include traditional approaches, such as, for example, cross-linking, coimmunoprecipitate and joint cleaning on gradients or chromatographic columns. In addition, protein-protein interactions can be monitored using based on the yeast genetic system described by Fields with co-workers (Fields and Song, Nature (London), 340:245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)), as described by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991). Many activators of transcription, the same is as yeast GAL4, consist of two physically separate nodal domains, where one acts as a DNA-binding domain and the other functions as the activation domain of transcription. In the yeast expression system described in the above publications (generally referred to as "dvuhserijnaya system"), take advantage of this property and apply two hybrid protein, the one in which the protein target is merged with the DNA-binding domain of GAL4, and the other in which the activating protein-candidates merged with the activating domain. The expression of reporter gene GAL1-lacZunder the control of activated GAL4 promoter depends on the recovery of GAL4 activity due to protein-protein interactions. Colonies containing interacting polypeptides detected using a chromogenic substrate for β-galactosidase. Full set (MATCHMAKER™) for identifying protein-protein interactions between two specific proteins using twohybrid method commercially available from Clontech. This system can also be extended to the mapping of protein domains involved in specific protein interactions, as well as for detection of amino acid residues that are crucial for these interactions.

Compounds that prevent the interaction of the gene encoding identified on this dock is the COP polypeptide, and other intra - and extracellular components can be tested as follows: usually get the reaction mixture containing the product of the gene and the intra - or extracellular component under conditions and for a time, providing interaction and binding of the two products. To test the ability of the compound candidate to inhibit the binding reaction performed in the absence and in the presence of test compounds. In addition, in a third reaction mixture, you can add a placebo, which serves as a positive control. The binding (complex formation) between the test compound and the intra - or extracellular component present in the mixture control as described earlier in this document. The formation of a complex in the control reaction(s), but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction between the test compound and its partner's reaction.

For analysis on the antagonists of the polypeptide can be added to a cell along with the compound to be screened for a particular activity and the ability of the compounds to inhibit interest activity in the presence of the polypeptide indicates that the compound is an antagonist of the polypeptide. Alternatively, antagonists may be detected by whom is inromania polypeptide and a potential antagonist with membrane-bound receptors or encoded receptor polypeptide under conditions suitable for the analysis of competitive inhibition. The polypeptide can be labeled, for example, radioactively, so that the number of molecules of a polypeptide associated with the receptor, can be used to determine the effectiveness of the potential antagonist. Encoding the receptor gene can be identified by numerous methods known to experts in this field, for example, panning ligands and FACS sorting. Coligan et al., Current Protocols in Immun., 1(2): Chapter 5 (1991). Preferably, use cloning for expression, where polyadenylated RNA obtained from the cells responsible for the polypeptide and a cDNA library created from this RNA is divided into groups and used for transfection of COS cells or other cells that do not meet the polypeptide. Transfetsirovannyh cells grown on slides, exposed to labeled polypeptide. The polypeptide can be marked in a variety of ways, including iodination or the introduction of site recognition site-specific protein kinase. After fixation and incubation, the glass is subjected to autoradiographical analysis. Identify positive group and receive subgroups and again transferout a consistent process of dividing into subgroups and re-screening, which ultimately will lead to one clone encoding the presumed receptor.

As an alternative approach for receptor identification labeled polypeptide can photouplink to associate with the cell membrane or preparations of extracts expressing the receptor molecule. Cross-linked substance divided by PAGE and exhibit on x-ray film. The labeled complex containing the receptor, you can cut, split into peptide fragments, and subjected to protein microsequencing. Obtained after mikroekonomia amino acid sequence can be used to design a set of degenerate oligonucleotide probes to screen the cDNA library for identification of the gene encoding the presumed receptor.

In another analysis for antagonists, mammalian cells or a membrane preparation expressing the receptor, can be incubated with a labeled polypeptide in the presence of the connection candidate. Then you can measure the ability of the compound to enhance or block this interaction.

More specific examples of potential antagonists include an oligonucleotide that binds to the hybrid immunoglobulin with the polypeptide, and, specifically, antibodies, including as non-limiting examples, poly - and monoclonal antibodies and antibody fragments, single-chain antibodies, antiidiotypic antibodies and hemery is or humanized versions of such antibodies or fragments, as well as antibodies and antibody fragments of the person. Alternative potential antagonist may be a closely related protein, for example a mutant form of the polypeptide that recognizes the receptor but has no effect, thereby competitive inhibition of the activity of the polypeptide.

Another potential antagonist is a structure antisense RNA or DNA, obtained with the method of the antisense nucleic acid, for example, when the molecule is antisense RNA or DNA acts, directly blocking the translation of mRNA by hybridizing with the mRNA target and preventing protein translation. The way antisense nucleic acids can be used to control gene expression through triple helix formation or through antisense DNA or RNA, where both methods are based on binding of polynucleotide with DNA or RNA. For example, encoding the 5'portion of the polynucleotide sequence, which encodes the Mature polypeptide of this paper, can be used for the design of antisense RNA oligonucleotide with a length from about 10 to 40 base pairs. Oligonucleotide DNA project so that it was complementary region of the gene involved in transcription (triple helix - see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan et al., Science,251:1360 (1991)), thereby preventing transcription and the production of the polypeptide. Antisense oligonucleotide RNA hybridized with mRNAin vivoand blocks translation of the mRNA molecule to the polypeptide (antisense nucleic acid - Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, FL, 1988). The above-described oligonucleotides can also be delivered to cells such thatin vivocan be expressed antisense RNA or DNA, inhibiting the formation of the polypeptide. When using antisense DNA, the preferred oligodeoxyribonucleotide obtained from the site of initiation of translation, for example, approximately between positions -10 and +10 on the nucleotide sequence of the target genes.

Potential antagonists include low molecular weight compounds that bind to the active site, the binding site of the receptor or growth factor or other relevant binding site of the polypeptide, thereby blocking the normal biological activity of the polypeptide. Examples of low molecular weight compounds include as non-limiting examples of small peptides or similar peptides molecules, preferably soluble peptides and synthetic ones organic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of kata is to siroute specific cleavage of RNA. Ribozymes act on the basis of sequence-specific hybridization with complementary RNA target, followed endonucleolytic splitting. Specific sites of cleavage of the ribozymes in the potential RNA target can be identified by known methods. For more details see, for example, Rossi, Current Biology, 4:469-471 (1994) and PCT publication no WO 97/33551 (published 18 September 1997).

Molecules of nucleic acid in the formation of triple helix used for inhibition of transcription should be single-stranded and can be deoxynucleotides. Part of the reason these oligonucleotides were designed to ensure the formation of a triple helix by the rules of the mating grounds Justina, which generally require relatively large areas of purines or pyrimidines on one of the circuits of the duplex. For more details see, for example, PCT publication no WO 97/33551, above.

These low molecular weight compounds can be identified by one or more of screenrush analyses discussed herein above, and/or any other screening methods, well known to specialists in this field.

The selected encoding the polypeptide nucleic acid can be used for recombinant get polypeptide using well known in the field of methods and how opisanoj this document. In turn, the resulting polypeptides can be used to generate antibodies using well-known in the field of methods and as described in this document.

Antibodies specifically binding a polypeptide identified according to the present document, as well as other molecules identified by screenrush analyses described in this document above, it is possible as pharmaceutical compositions to enter for the treatment of various disorders, including malignant tumor

If the polypeptide is intracellular and as inhibitors using whole antibodies, the preferred internalization of antibodies. However, to deliver the antibody or fragment of antibody in cells you can also use lipofectin or liposomes. When using fragments of antibodies, the preferred smallest inhibitory fragment that specifically binds with the binding domain of the protein target. For example, on the basis of sequences of the variable regions of the antibodies can be constructed peptide molecules that retain the ability to bind to the sequence of the protein target. Such peptides can be synthesized chemically and/or get through recombinant DNA technology. See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).

The composition according to the present d is the document may also contain more than one active connection in accordance with the need for specific indications, being treated, preferably such compositions with a custom action that do not affect each other adversely. Alternative or additionally, the composition may contain the means strengthening its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent. Accordingly, these molecules are present in combination in amounts that are effective for the intended purpose.

The following examples are offered only for purposes of illustration and are not intended to limit the scope of the present invention in any way.

EXAMPLES

Reagents

Chromogenic substance S2366 were obtained from DiaPharma Group, Inc. (West Chester, OH), Lys-plasminogen from Haematologic Technologies Inc. (Essex Junction, VT), and the tissue activator of plasminogen (t-PA) from Genentech, Inc. (South San Francisco, CA). Pro-HGF expressed in ovarian cells of the Chinese hamster (CHO) in the absence of serum and purified by chromatography on HiTrap SP Sepharose provided by David Kahn (Genentech). HGFA containing residues Val373-Arg407 expressed in baculovirus expressing the system and purified as described (34). Purified recombinant human FVII, expressed in human 293 cells, was obtained as a gift from Mark O'connell (Genentech) and described previously (42). To obtain PCPS vesicles used dioleoyl-1,2-diacyl-sn-is Litera-3-(phospho-L-serine) (PS) and oleoyl-1,2-diacyl-sn-glycero-3-phosphocholine (PC) (Avanti Polar Lipids Inc., Alabaster, AL) (molar ratio 7:3), essentially as described (43). The molecular weight markers represented the standards SeeBlue Plus2 and MultiMark (Invitrogen, Carlsbad, CA).

Expression and purification of hepsin

cDNA full size Hasina obtained from the Association I.M.A.G.E. (ATCC, Manassas, VA), were digested with restriction endonucleasesEcoRIandNot I(New England Biolabs Inc. Beverly, MA) was inserted into the eukaryotic expressing vector pRK5E. The secretory labeled cDNA of His Hasina designed by merging cDNA that encodes a signal sequence of human HGF (amino acids Met1-Gly31) with cDNA encoding the extracellular domain of human hepsin (Arg45-Leu417: the numbering system in accordance with Somoza et al., 2003 (5)). In addition, to the C-end after Leu417 added tag His8and the final design of the cDNA was inserted into the eukaryotic expressing vector pCMV.PD5. Hepsin expressed in the system transient expression in cells of the Chinese hamster ovary (CHO) and was purified by affinity chromatography with Nickel-nitrilotriacetic acid (Ni-NTA), essentially as described for obtaining sHAI-1B wild-type (34).

Expression and purification sHAI-1B mutant sHAI-1B and sHAI-2

A soluble form of HAI-1B (sHAI-1B), containing the entire extracellular domain, was received in the system transient expression in CHO cells and purified as described previously (34). Using SAI is-specific mutagenesis, the remains of the P1KD1 (Arg260) and Kd2 are used (Lys401) individually replaced with Ala and the resulting proteins, sHAI-1B(R260A) and sHAI-1B(K401A), expressed and purified as described (34).

Full HAI-2 was obtained from a cDNA library derived from RNA embryonic lung man (BD Biosciences Clontech, Palto Alto, CA) with the use of the site oligo-dT/Not I as a primer and adaptor with a plot forSal Ias the second circuit. cDNA was digested with the use ofSal IandNot I; cDNA, more than 2.8 TPN ligated in pRK5D. Single-stranded DNA library, cDNA easy person/pRK5D were obtained using standard methods of molecular biology. The reverse primer (5'-TTTCTTGAGGCACTCCTCCTTG-3') was annealed with a pool of single-stranded cDNA was increased with the use of DNA polymerase T7 or T4. The synthesized double-stranded DNA transformedE. coliand colonies were subjected to screening using the standard methods of hybridization on filters. The size of the inserts was analyzed by PCR and restriction cleavage by the endonuclease,XbaI. Identified full-size clones HAI-2 and confirmed by DNA sequencing. A soluble form of HAI-2 (sHAI-2) was obtained by constructing a cDNA encoding the extracellular domain (Ala28-Lys197; the numbering system in accordance with Kawaguchi et al., 1997 (39)) and HAI-2 and an additional C-terminal tag His8with a spacer in the form of Gly. Then received the th cDNA was inserted into the baculovirus expressing vector, derived from pVL1393 (BD Biosciences Pharmingen, San Diego, CA). sHAI-2 expressed in the baculovirus expressing the system and purified by affinity chromatography with Ni-NTA, essentially as described to obtain the β-chain of HGF (44). Protein concentrations were determined by analyses of quantitative determination of amino acids.

Analyses of the activation of FVII and plasminogen

FVII concentration of 0.11 mg/ml activated by 230 nm of hepsin in 30 mm Tris-HCl, pH 8,4, 30 mm imidazole, 200 mm NaCl (Tris buffer) in the presence of 0.5 mm of PCPS vesicles and 5 mm CaCl2at 37°C. Aliquots of the reaction mixture taken at different time points were analyzed by SDS-PAGE (reducing conditions) using 4-20% gradient gel (Invitrogen, Carlsbad, CA). Gels were stained with dye Simply Blue Safe Stain (Invitrogen).

The plasminogen concentration of 0.12 mg/ml were incubated with 40 nm of hepsin or 40 nm t-PA (positive control) in 20 mm Hepes pH 7.5, 150 mm NaCl (Hepes buffer) at 37°C. Aliquots of the reaction mixture taken at different time points were analyzed by SDS-PAGE as described for analysis of activation of FVII.

Activation of Pro-HGF by Hasina and HGFA

Pro-HGF (0.3 mg/ml) were incubated in Hepes buffer with 40 nm of hepsin or 40 nm HGFA for 4 h at 37°C and stored at -20°C until further use. Analyses of the split substances, HGFhepsinand HGFHGFAby SDS-PAGE showed that >95% of Pro-HGF converts the I in the double-stranded HGF.

Analyses of the activation of Pro-HGF and tagging of Pro-HGF125I have carried out as described (34, 45). In summary, labeled125I Pro-HGF at a concentration of 0.05 mg/ml in Hepes buffer were incubated with increasing concentrations (0,16-40 nm) hepsin or HGFA at 37°C. After 4 h was selected aliquots and analyzed by SDS-PAGE (4-20% gradient gel (Invitrogen Corp., Carlsbad, CA). For studies of inhibition of hepsin (15 nm) were incubated in Hepes buffer with 1 μm sHAI-1B, sHAI-1B(K401A), sHAI-1B(R260A) or sHAI-2 at 37°C. After 4 h, the samples were analyzed by SDS-PAGE and stained with the dye Simply Blue Safe Stain (Invitrogen).

Analysis of inhibition of enzymes

Testing conditions were similar to conditions described by Somoza et al. 2003 (5), using a chromogenic substrate S2366 (L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilide). Hepsin (final concentration of 0.4 nm) were incubated with increasing concentrations of inhibitors in Tris buffer for 30 min at room temperature. Added substrate S2366 and measured the change in optical density at 405 nm using kinetic microplate scanner (Molecular Devices, Sunnyvale CA). Concentration Hasina and S2366 in this final reaction mixture was 0.4 nm and 0.2 mm (some Km= 0.2 mm), respectively. Inhibitory activity was expressed as relative activity (vi/v0from activity neighborho the data of the enzyme. The concentration of inhibitor causing 50% inhibition (IC50), was calculated by entering the data in the program approximation curve regression with four parameters (Kaleidagraph, Synergy Software, Reading, PA). For each inhibitor were performed at least three independent experiments.

Analysis of cell proliferation and migration

The proliferation assays were performed on cell lines adenocarcinoma of the pancreas human BxPC3, obtained from the European Collection of Cell Cultures (CAMR Centre for Applied Microbiology and Research, Salisbury, Wiltshire, UK). Cells were grown in RPMI medium containing 10% ELS (Sigma, St. Louis, MO), 10 mm hepes, 2 mm glutamine, penicillin-streptomycin (Invitrogen, Carlsbad, CA) and 250 μg/ml G418 (Invitrogen). Confluently cell layers were washed in PBS, followed 10 mm EDTA/PBS and after incubation were detached by trypsin. Cells resuspendable in the environment for growth and sown (10000-15000 cells/well) in 96-well belladonnae tablets MT (Cultur Plate™, Packard/PerkinElmer, Boston, MA). After 24 h the medium for growth was replaced with RPMI-0,1% BSA. After an additional 24 h the medium was removed and added various concentrations of HGFhepsinand HGFHGFAin RPMI-0,1% BSA and the cells were allowed to grow for 72 hours Then conducted a quantitative analysis of cell proliferation by applying a set of CellTiter-Glo Luminescent Kit (Promega, Madison, WI) according to the manufacturer's instructions. Luminescence was measured on a luminometer for Mick is plansyou Tropix TR717 (Berthold 75323, Bad Wildbad, Germany). After subtracting background values (proliferation in the absence of HGF) HGF activityhepsinand HGFHGFAwere expressed as the percentage of proliferation of BxPC3 at 100 ng/ml control drug HGF (obtained from Dr. Ralph Schwall, Genentech).

Analysis of cell migration in cell lines breast cancer MDA-MB435 (HTB-129, ATCC, Manassas, VA) was performed as described (44). In brief, 0.2 ml of cell suspension in medium without serum (0,6-0,8 × 106cells/ml) was added to the upper chambers of 24-well plates transwell (pore size 8 μm) (HTS Multiwell™ Insert System, Falcon, Franklin Lakes, NJ) pre-coated with 10 μg/ml collagen type I from the tail of rats (Upstate, Lake Placid, NY). In the lower chamber of the added drugs HGF in the medium without serum. After incubation for 13-14 h cells on the apical side of the membrane was removed, and cells that migrated to the basal side were fixed in 4% paraformaldehyde followed by staining with 0.5% crystal violet solution. The cells were dissolved in 10% acetic acid and measured A560on a flatbed Molecular Devices. Premigration activity of HGF mutants were expressed as percentage of control drug HGF after subtracting basal migration in the absence of HGF.

Analysis of the phosphorylation of the Met receptor

Analysis of the activation of kinase receptor (KIRA) was performed as described (44). In brief, cells l the gotschna carcinoma A549 (CCL-185, ATCC, Manassas, VA) were sown in 96-well tablets at a density of 50,000 cells per well. After incubation over night at 37°C environment for the growth was removed and cells were kept in low serum for 30 to 60 min in medium containing 0.1% ELS. Added increasing concentrations of HGFhepsinand HGFHGFAin a medium containing 0.1% ELS. As control was used unsplit single-chain form of HGF (scHGF), in which the cleavage site mutated (Arg494Glu) (44). After 10 min incubation at 37°C the medium was removed and cells were literally in lyse buffer (Cell Signaling Technologies, Beverly, MA)supplemented with a mixture of protease inhibitors. Cell lysates were added to labeled BV-TAG antibody 4G10 antibody and biotinylated antibody to Met. After incubation for 1.5 to 2 h was added to the magnetic beads with streptavidin (Dynabeads, Bio Veris), and incubated for 45 minutes, the Beads with bound peroxidase substance (antibody to Met/Met/antibody to phosphotyrosine) was caught by an external magnet. After the stage of washing is obtained from the light source chemiluminescent signal was measured in relative fluorescent units on the device Bio Veris. For each experiment phosphorylation Met HGFhepsin, HGFHGFAor scHGF was expressed as percentage of the maximum signal obtained with the control drug HGF.

Results

Proteolytic PR is testirovanie Pro-HGF by Hasina

Soluble form of hepsin containing the entire extracellular domain (Arg45-Leu417; the numbering system in accordance with Somoza et al., 2003 (5)) and the C-terminal His label8expressed in CHO cells. During the cleaning process proferment of hepsin spontaneously transformed in its double-stranded form (Fig. 1A), most likely due to autoactivation (4). N-terminal sequencing proteasome domain mass of ≈ 30 kDa (163IVGGRDTSLGR173) confirmed the splitting of the expected peptide bond Arg162-Ile163, with the formation of active enzyme. Hepsin actively converted proferment FVII in double-stranded FVIIa (pigv), consistent with experiments, published Kazama et al., 1995 (9) using expressed on the cell surface of hepsin to determine the activation of FVII. Activity hepsin in respect of Pro-HGF was checked by measuring the conversion of labeled125I Pro-HGF in double-stranded HGF. The results showed that Pro-HGF was cut by Hasina, dependent on the concentration of way (figa). Activity Hasina was comparable with the activity of HGFA (pigv), where both enzymes was achieved full conversion of Pro-HGF at a concentration 4-13 nm. In the same system analysis activators of Pro-HGF factor XIa, factor XIIa and plasma kallickrein required for full conversion of Pro-HGF about 5-6 times larger concentrations (45). N-concavo the sequencing of the β-chain of HGF mass of ≈ 36 kDa and ~39 kDa gave identical sequences ( 495VVNGIPTRTNIG506by demonstrating that hepsin transformed Pro-HGF expected peptide bond Arg494-Val495. In contrast to factor XIa and plasma kallikrein hepsin not formed fragment α2-chain HGF (by splitting between Arg424-His425) (45), even after long periods of reaction. In addition, hepsin (40 nm) completely lack the ability to activate plasminogen within 5 h of reaction (Fig. 2C). In comparison with this efficient processing of plasminogen under the action of t-PA containing 50% of proferment, split already after 0.5 h (Fig. 2C).

The biological activity obtained by splitting the HGF Hasina

Unlabeled Pro-HGF (0.3 mg/ml) was completely converted (>95%) in HGF 40 nm hepsin or 40 nm HGFA (figa) with the formation of HGFhepsinand HGFHGFA, respectively. In the analysis of the activation of kinase receptor (KIRA) on the cells of A549 lung carcinoma, both drugs HGF induced similar dependent on the concentration increase in the phosphorylation of Met, reaching maximum activity at 250 ng/ml (pigv). As shown previously (44), narasima single-chain form of HGF (scHGF) with a modified cleavage site (R494E) inactive (pigv). Control experiments showed that hepsin or HGFA separately have no effect (data not shown). In addition, HGFhepsineffectively stimulates the proliferation of the notches pancreatic cancer BxPC3. The activity was comparable with the activity of HGFHGFAin the investigated range of 5-100 ng/ml (figa). Similar results were obtained in the analysis of cell migration in cells MDA-MB435 using coated with collagen transwell migration system. As revealed in the analysis of cell proliferation, premigration effects of HGFhepsinwere dependent on the concentration and indistinguishable from the activity of HGFHGFA(pigv).

Inhibition of the enzymatic activity of hepsin by sHAI-1B and sHAI-2

Initial screening of 26 commercially available chromogenic substances showed that hepsin with the highest speed (data not shown) hydrolyzes S2366, the substrate provided Somoza et al., 2003 (5). Using S2366 as substrate was measured inhibitory activity of highly purified, soluble HAI-1B (sHAI-1B) and HAI-2 (sHAI-2) of the wild type. In addition, the authors received two mutant sHAI-1B: sHAI-1B(R260A) and sHAI-1B (K401A), in which individual domains Konitza were inactivated by substitution of residues of P1(Arg260 in KD1 and Lys401 in Kd2 are used) by alanine (34). The results showed that sHAI-1B, and sHAI-2 effectively inhibit the enzymatic activity of hepsin with values IC50the size of 21.1 ± 2,7 nm and 1.3 ± 0.3 nm, respectively (figure 5). In addition, mutant sHAI-1B(K401A)containing non-functional Kd2 are used, was as effective as sHAI-1B wild-type, while sHAI-1B(R260A) possessed a >47 times reduced intercourse is the want to make (figure 5). The obtained values IC50are summarized in table 1.

Table 1.
Inhibition of hepsin inhibitors domain Konitza
InhibitorsIC50(nm)
sHAI-1B wt21,1 ± 2,7
SHAI-1B(K401A)18,2 ± 3,7
SHAI-1B(R260A)>1000
SHAI-21,3 ± 0,3

The inhibition mediated by Hasina activation of Pro-HGF

The ability sHAI-1B and sHAI-2 to inhibit the processing of macromolecular substrate was measured in the analysis of activation125I-Pro-HGF. The obtained results were in accordance with their expected any abscopal activities defined in amylolyticus analyses. At concentrations of sHAI-2, sHAI-1B wild-type and sHAI-1B(K401A) in the amount of 1 μm, there was a complete inhibition of the cleavage of Pro-HGF (6). In contrast, 1 μm sHAI-1B(R260A) demonstrated a lack of inhibition and activation of Pro-HGF continued until complete conversion (6).

Discussion

The transmission signal HGF/Met plays an important role in human Fiziol the GII and pathology, including invasion and metastasis of the tumor. The local availability of active HGF is controlled chymotrypsin-like serine-proteases and related inhibitors that regulate the conversion of the inactive Pro-HGF in the extracellular environment. Thus, the violation of this "upper" path convertase Pro-HGF in malignant tumors can stimulate tumor growth by accelerating the processing of Pro-HGF. In this paper the authors demonstrate that hepsin, serine protease, is strongly activated in prostate cancer and ovarian cancer, is an effective activator of Pro-HGF.

Thus, it is likely that hepsin plays an important role in the implementation tumor growth through activation of the transmission signal HGF/Met, which is involved in prostate cancer (46-48), and ovarian cancer (49, 50).

Hepsin proteoliticeski were digested Pro-HGF by peptide bonds Arg494-Val495 without any additional cleavage by Arg424-His425 in the domain of Kringle 4, the site recognized by factor XIa and plasma kallicrein (45). Formed by Hasina double-stranded HGF was fully functional, including the phosphorylation of the Met receptor and stimulation of cell proliferation and migration activity, comparable with HGF formed by the HGFA. The molecular mechanism underlying mediated GE the Shin conversion of the inactive Pro-HGF in active growth factor, similar to the proteolytic conversion of proferment in enzyme similar to chymotrypsin serine proteases. This is confirmed by recent studies on the structural consequences of the activation of Pro-HGF, demonstrating that the splitting Arg494-Val495 leads to conformational changes in the protease β-chain of HGF and the full maturation of the binding site of the receptor Met (44, 51). This binding site Met, which is in the "active center" and "activation domain" HGF, has significant similarity to a region of the processing substrate of serine proteases (44, 51). Thus, the cleavage of Pro-HGF by Hasina leads to structural changes in the β-chain of HGF, which allows to form the current complex signal HGF/Met. The proteolytic activity of hepsin in respect of Pro-HGF is highly specific, as it does not break the plasminogen, the closest structural homologue of Pro-HGF. Hepsin does not have proteolytic activity against other substrates of serine proteases, such as prothrombin, protein C, factor X and factor IX (9).

Studies in mice with null mutation of HGF demonstrate that the path of HGF/Met is important for normal embryonic development and viability (52, 53). In contrast, mice with deficiency of gene Hasina develop normally, indicating that malverian is, that hepsin is the main activator of HGF during embryogenesis. Similar to Hasina, the disadvantages of other known Mac Pro-HGF, matriptase (54), factor XI (55), prekallikrein (56) and u-PA (57) are not lethal to embryos. Due to its importance in embryogenesis and postnatal physiology activity of HGF may be regulated in a consistent way across multiple systems (Mac Pro-HGF. If so, then the combined deficit of genes Mac Pro-HGF should result in defects similar to mice with null mutation of HGF. Alternatively, the Mac Pro-HGF governing the processing of HGF during embryogenesis, may not yet be identified.

HAI-1B, HAI-1 and HAI-2 are inhibitors on the surface of epithelial cells and is expressed in many normal tissues and tumors (34, 58-63). Essentially they are ideally located for the regulation of the enzymatic activity expressed by the epithelial cells of the TTSP and possibly other associated with cell surface serine proteases. Indeed, variants of splicing HAI-1, HAI-1 and HAI-1B effectively inhibit matriptase TTSP (MT-SP1), and complexes of HAI-1 matriptase found in human breast milk (38). In this paper, the authors showed that sHAI-1B and sHAI-2 are also effective inhibitors of the enzymatic activity of hepsin. In addition, experiments on spec is specific for the remainder of P 1mutagenesis showed that inhibition of hepsin fully mediated KD1 sHAI-B, as mutant sHAI-1B (R260A) was inactive in the analysis of Pro-HGF and possessed < 1%the activity of wild-type and mutant Kd2 are used in amiloliticheskoi analysis. Thus, hepsin, matriptase and HGFA not only have comparable activities in relation to the conversion of Pro-HGF (34), but also inhibited by sHAI-1B with the same efficiency (16-30 nm) specific KD1 way (34, 64). Variants of splicing HAI-1 and HAI-1B differ only by the absence or presence of 16 amino acids located at the C-end of KD1. Profile their expression in tissues, including malignant tumors of the prostate and ovarian cancer, identical to the present time, no significant differences in efficacy and safety profiles of protease target. Thus, the authors believe that the function of the two splicing variantsin vivoequivalent. The role of domains Konitza HAI-2 in the study of the inventors was not specifically investigated. For most of its enzymes are targets HAI-2 uses both N-and C-terminal domains of Konitza (41, 65).

Functional relationship of hepsin with HAI-1 and HAI-2in vitrotogether with their localization on the surfaces of epithelial cellsin vivosuggests that they are physiologically meaningful system of enzyme-inhibitor. For example, two variants of splice the ha HAI-1 and HAI-2 are expressed in the lines of normal prostate cells and prostate cancer cells (34, 59, 61), and antigen HAI-1 is localized in a layer of secretory cells of glandular epithelium of the prostate (59). Intriguing that the expression of hepsin in prostate tumors localized in the same epithelial compartment (19, 20), supporting the idea that HAI-1 and possibly HAI-2 are associated with Hasinain vivo. Although the expression of hepsin greatly increased prostate cancer (17-22), HAI-1 and HAI-2 increased only slightly (approximately 1.5 times) in accordance with the results of gene expression, published by Welsh et al., 2001 (17). As a result, the enzymatic activity of hepsin in tumors can be controlled adequately and can lead to increased processing of Pro-HGF and progression of tumors. A similar imbalance systems (Mac/inhibitors of Pro-HGF described for matriptase/HAI-1 in ovarian cancer (62, 63), HGFA/HAI-1 in colorectal cancer (66, 67) and HGFA/HAI-1 in renal cell carcinoma (68). Increased expression of hepsin in some of these malignancies suggests that certain systems (Mac/inhibitors include several enzymes and, consequently, increased the ratio of enzyme/inhibitor can increase the value for zlokacestvennoe.

In conclusion, the presented results demonstrate that hepsin effectively activates Pro-HGF, thus the m revealing the functional relationship between Hasina on the epithelial surface of the tumor and the extracellular matrix, contains the inactive precursor of the growth factor. The discovery that HAI-1 and HAI-2 are effective inhibitors of hepsin, which is activated in prostate cancer and ovarian cancer, provides new approaches for the treatment of malignant tumors. For example, the functional domains of Konitza HAI-1 or HAI-2 can serve as frameworks for more specific and/or more effective inhibitors of enzymes using the phage display technique, which was successfully applied to other frames on the basis of domain Konitza (69, 70).

Footnotes/Abbreviations

1Factor VIIa, FVIIa; Hepes buffer, 20 mm Hepes pH 7.5, 150 mm NaCl; buffer Tris, 30 mm Tris-HCl, pH 8,4, 30 mm imidazole, 200 mm NaCl; Pro-HGF, single-stranded growth factor hepatocytes, HGF, double-stranded growth factor hepatocyte; HGFA activator growth factor hepatocyte; HAI-1, activator inhibitor growth factor hepatocyte-1; HAI-1B variant splicing activator inhibitor growth factor hepatocyte-1; HAI-2, activator inhibitor growth factor hepatocyte-2; KD1 and Kd2 are used, the N - and C-terminal domains of Konitza HAI-1B; sHAI-1B, soluble form of HAI-1B, which includes the extracellular domain; sHAI-2, a soluble form of HAI-2, which includes the extracellular domain; HGFhepsin, HGF is produced by activation of Pro-HGF by Hasina; HGFHGFA, HGF is produced by activation of Pro-HGF HGFA; scHGF, non-digestible odnosima echny HGF mutant cleavage site (Arg494Glu); u-PA, plasminogen activator urokinase type; t-PA, tissue plasminogen activator. Ni-NTA, Nickel-nitrilotriacetic acid.

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1. A method for the identification of inhibitory substances of the candidate, which inhibits the activation of HGF by Hasina, where the method includes (a) bringing the matter candidate in contact with the first sample containing pepsin and substrate Pro-HGF, and (b) comparison of the degree of activation of the substrate Pro-HGF in a sample with the degree of activation of the substrate Pro-HGF in the reference sample containing equal to the first sample number of hepsin and substrate Pro-HGF, but not in contact with the specified substance candidate, according to which the decrease in the degree of activation of the substrate Pro-HGF in the first sample compared with a reference sample indicates that the substance-the candidate is able to inhibit the activation of single-chain HGF (Pro-HGF) Hasina.

2. The method according to claim 1, where pepsin in the sample is effective for activation indicated the data Pro-HGF number.

3. The method according to claim 1, where the substrate Pro-HGF is a polypeptide containing HGF or its fragment that contains the form peptide bonds R494-V495 wild-type.

4. The method according to claim 1, where the substrate Pro-HGF contains the cleavage site of human HGF corresponding to the consensus cleavage site of the protease, where the cleavage site contains a basic residue at position P1and two hydrophobic amino acid residue in positions P1' and R2'.

5. The method according to claim 1, where the substance binds hepsin or Pro-HGF.

6. Means for inhibiting the interaction of hepsin and growth factor hepatocyte, where the tool comprises a sequence domain of Konitza, which is able to inhibit Pro-HGF activation under the action of hepsin, where the sequence domain Konitza (KD) is:
(i) sequence KD-1 domain Konitza inhibitor-1 activator growth factor of human hepatocytes (HAI-1), inhibitor-1 activator growth factor of human hepatocytes (HAI-1B); or (ii) one or both domains of Konitza inhibitor-2 activator growth factor of human hepatocytes (HAI-2).



 

Same patents:

FIELD: medicine.

SUBSTANCE: according to the following stages, flow cytometry is used to detect living cells, damaged cells, VNC cells and dead cells of a microorganism in a tested sample: a) the stage of processing the tested sample with enzyme chosen from lipolytic enzymes and protease with activity to destruct the cells differing from those of the microorganism, colloid protein particles or lipids found in the analysed sample; b) the stage of processing the tested sample with topoisomerase inhibitor and/or DNA-gyrase inhibitor; e) the stage of processing the tested sample been processed at the stages a) and b) with a kernel-dyeing agent, and d) the stage of detecting microorganisms in the tested sample processed by the kernel-dyeing agent with using flow cytometry.

EFFECT: convenient and fast detecting of live microorganisms and identification of the damaged and dead cells in foodstuff and clinical samples.

19 dwg, 8 tbl, 8 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to a method of identifying γ-secretase and its inhibitors and can be used in medicine when searching for active compounds for treating Alzheimer's disease. A genetic structure is formed, which codes fused protein, which contains a signal peptide and amino acid sequence GAIIGLMVGGVVIATVIVITLVML. In the obtained fused protein, except the GAIIGLMVGGVVIATVIVITLVML sequence, all sites acting as a signal for endo- or exocytosis, and/or protease splitting site are excluded.

EFFECT: invention allows for highly effective identification of γ-secretase or substances which inhibit its activity by reducing background signal and increasing specificity of the signal.

41 cl, 4 dwg, 17 ex

FIELD: production methods.

SUBSTANCE: method is based on the capability of defibrotide to increase the fermentation activity of plasmin and foresee the stages: a) making the contact in reactional area defibrotide, plasmin and substrate specific for plasmin which, because of reaction, provides the defined product b) the definition of the amount of obtained product in temporary points.

EFFECT: invention allows to define the biological activity of defibrotide in comparison with standard etalon with height accuracy and big repeatability.

9 cl, 6 dwg, 4 tbl, 1 ex

FIELD: medicine, oncology, molecular pharmacology.

SUBSTANCE: invention relates to a method and set for identifying the individual subjected to risk for arising in it the vascular and cancer disease. Method involves stages for the quantitative determination of the analyte concentration, i. e. pepsinogen I (PGI), in serum sample taken in the personal individual; comparison of the analyte concentration determined by the proposed method with a method-specific boundary value for this analyte; determination of the homocysteine concentration in a serum sample taken in this individual. The set comprises the combination of separate components that are necessary for the quantitative determination of the PGI concentration. Method provides the early detection of the possibility for arising the vascular and cancer disease in the patient.

EFFECT: improved method for assay.

4 cl

The invention relates to medicine, in particular to the creation of a new pharmaceutical compositions containing collagenase microbial origin

The invention relates to biotechnology, medical Microbiology, concerns the detection hydrolytically active enzyme, in particular aspartic protease in a sample or preparation

The invention relates to biotechnology and can be used for the development of anti-NPC therapeutic agents

The invention relates to the field of Bioorganic chemistry, namely to a new 6-(moselhotel-L-prolyl - L-arginyl)aminonaphthalene-1-isobutylamino formula:

< / BR>
as a substrate for the determination of thrombin

The invention relates to physico-chemical biology and biotechnology, namely, biological chemistry, molecular biology, Bioorganicheskaya chemistry, and can be used by scientific institutions in the study of the properties of proteins and enzymes

FIELD: medicine, oncology, molecular pharmacology.

SUBSTANCE: invention relates to a method and set for identifying the individual subjected to risk for arising in it the vascular and cancer disease. Method involves stages for the quantitative determination of the analyte concentration, i. e. pepsinogen I (PGI), in serum sample taken in the personal individual; comparison of the analyte concentration determined by the proposed method with a method-specific boundary value for this analyte; determination of the homocysteine concentration in a serum sample taken in this individual. The set comprises the combination of separate components that are necessary for the quantitative determination of the PGI concentration. Method provides the early detection of the possibility for arising the vascular and cancer disease in the patient.

EFFECT: improved method for assay.

4 cl

FIELD: production methods.

SUBSTANCE: method is based on the capability of defibrotide to increase the fermentation activity of plasmin and foresee the stages: a) making the contact in reactional area defibrotide, plasmin and substrate specific for plasmin which, because of reaction, provides the defined product b) the definition of the amount of obtained product in temporary points.

EFFECT: invention allows to define the biological activity of defibrotide in comparison with standard etalon with height accuracy and big repeatability.

9 cl, 6 dwg, 4 tbl, 1 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to a method of identifying γ-secretase and its inhibitors and can be used in medicine when searching for active compounds for treating Alzheimer's disease. A genetic structure is formed, which codes fused protein, which contains a signal peptide and amino acid sequence GAIIGLMVGGVVIATVIVITLVML. In the obtained fused protein, except the GAIIGLMVGGVVIATVIVITLVML sequence, all sites acting as a signal for endo- or exocytosis, and/or protease splitting site are excluded.

EFFECT: invention allows for highly effective identification of γ-secretase or substances which inhibit its activity by reducing background signal and increasing specificity of the signal.

41 cl, 4 dwg, 17 ex

FIELD: medicine.

SUBSTANCE: according to the following stages, flow cytometry is used to detect living cells, damaged cells, VNC cells and dead cells of a microorganism in a tested sample: a) the stage of processing the tested sample with enzyme chosen from lipolytic enzymes and protease with activity to destruct the cells differing from those of the microorganism, colloid protein particles or lipids found in the analysed sample; b) the stage of processing the tested sample with topoisomerase inhibitor and/or DNA-gyrase inhibitor; e) the stage of processing the tested sample been processed at the stages a) and b) with a kernel-dyeing agent, and d) the stage of detecting microorganisms in the tested sample processed by the kernel-dyeing agent with using flow cytometry.

EFFECT: convenient and fast detecting of live microorganisms and identification of the damaged and dead cells in foodstuff and clinical samples.

19 dwg, 8 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: there is offered a method for identification of an inhibiting candidate substance which enables inhibits hepsin-activated HGF. The method is based on comparing the degree of activation of pro-HGF substratum in a sample containing hepsin, pro-HGF and the analysed candidate substance, with the degree of activation of pro-HGF substratum in the reference sample who not containing the candidate substance. Also there is offered an agent for inhibiting hepsin and hepatocyte growth factor reaction containing a sequence of Kunitz domain which represents a KD-1 sequence of Kunitz domain HAI-1 or HAI-1B, or one or both Kunitz domains HAI-2.

EFFECT: invention allows identifying physiological hepsin modulators.

6 cl, 14 dwg, 1 tbl, 1 ex

FIELD: food industry.

SUBSTANCE: quantitative food proteins content determination method involves the following operations, sequentially performed: mixing test samples of the substrate and a fermentative substance coupled with a stabilising solution, incubation of the produced mixture at a temperature of 37°C, centrifugation of the fermentative-and-substrate complex and determination of quantitative food proteins content by way of calculation. The fermentative substance is represented by pancreatic juice preliminarily diluted with a stabilising solution down to 50% concentration, the ratio of the solution to the substrate test sample weight equal to 1:10. The prepared mixture incubation is performed during 5-15 minutes. Before quantitative food proteins content determination by way of calculation, the pure liquid fraction volume produced as a result of centrifugation is diluted with the stabilising solution at a ratio of 1:100-200. The food protein quantity is determined as equal to percentage expenditure of protease enzymes in comparison with a reference sample of pancreatic juice solution.

EFFECT: enhanced accuracy of determination of the quantitative food proteins content in food products.

3 tbl

FIELD: medicine.

SUBSTANCE: method of predicting efficiency of complex treatment of patients with nasopharyngeal cancer, which includes carrying out two-step course of radial therapy at the background of automyelochemical therapy, lies in the following: before treatment individual activity of enzyme kallikrein is determined in patient's blood plasma, and then on the 1-2 day after the end of the 1 step of complex treatment activity of enzyme kallikrein is determined in patient's blood plasma, and in case of its 2 and more fold reduction with respect to initial individual activity, efficiency of treatment is predicted, and in case if it reduces by less than 2 times, absence of clinical effect is predicted.

EFFECT: method ensures possibility of objective assessment of individual response to complex treatment and, in case of necessity, timely change of therapeutic mode.

3 ex

FIELD: medicine.

SUBSTANCE: there are presented a method and a kit for enzyme-linked thrombin-binding assay on C1 inhibitor functional activity. The method implies thrombin sorption in microplate wells, introduction of test samples containing the functionally active C1 inhibitor to be analysed, incubation that involves C1 inhibitor binding to thrombin, and measurement of the C1 inhibitor-bound amount by an enzyme conjugate with antibodies and a substrate of said enzyme. The kit comprises a flat-bottomed microplate with the sorbed preparation of thrombin, the enzyme conjugate with the human C1 inhibitor antibodies, a substrate buffer and a reference for measuring the active C1 inhibitor.

EFFECT: group of inventions provides the novel method and kit for enzyme-linked thrombin-binding assay on C1 inhibitor functional activity.

2 cl, 2 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to molecular biology and genetics. Disclosed is a method for qualitative and quantitative analysis of lipids that are strongly bound with genomic DNA, which comprises steps of isolating genomic DNA bound with lipids from cells using a detergent method, hydrolysis of DNA with a hydrolysing enzyme, separating lipids from the chloroform-methanol-water mixture at 37°C, evaporating the solvent, stabilising lipids with 2,6-di-tert-butyl-p-cresol, mixing the lipids with TMSH, chromatography with a gas chromatograph, followed by mass spectrometry.

EFFECT: method can be used to decode the lipid code of genomic DNA, ie, to determine the area of localisation of lipids that are strongly bound with genomic DNA for research and therapeutic purposes.

5 dwg

FIELD: measuring equipment.

SUBSTANCE: presented group of inventions refers to medicine. There are presented method and kit for the immune-enzyme assay of functional activity of human complement component C3. The method implies thrombin sorption in microplate wells, introduction of a test sample containing the human complement component C3 with unknown activity, incubation in the presence of ethylene diamine tetraacetate (EDTA), pouring out of the well contents, introduction of an enzyme conjugate with human complement component C3 antibodies and a substrate of the above enzyme. The component C3 activity is evaluated by the amount of the prepared product of enzymatic reaction. The kit comprises the flat-bottomed microplate with sorbed thrombin, conjugated enzyme and human complement component C3 antibodies, substrate buffer and donor blood serum with known activity C3 as a reference.

EFFECT: presented group of inventions enables evaluating the functional activity of human complement component C3 without the need of activation of the whole complement system, and possesses a good result reproducibility.

2 cl, 1 dwg, 2 ex

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