Double-targeted antibody in new form and using it

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. What is presented is an antibody representing a neutralising VEGFR-2/KDR antibody with its hypervariable regions being identical to the hypervariable regions of TTAC 0001 of VEGFR-2/KDR antibody fused with a binding domain of angiopoietin 2 which is Tie-2 ligand for treating cancer by angiogenesis inhibition. A DNA coding the above antibody, an expression vector containing the above DNA, and a CHO host cell transformed by the above vector for preparing the antibody are also described. What is also presented is a method for preparing the antibody involving: host cell incubation, and the antibody recovery from a culture fluid of CHO cell. What is described is a pharmaceutical composition for treating an angiogenesis-related disease, containing an effective amount of the above antibody and at least one pharmaceutically acceptable carrier.

EFFECT: invention enables preparing the VEGFR-2/KDR antibody fused with the binding domain of angiopoietin 2 which may be used for effective treatment of a disease related to excessive angiogenesis.

13 cl, 10 dwg, 8 ex

 

The technical field

The present invention relates to the antibody with a dual focus in a new form containing a water-soluble ligand, fused with the N-end of the heavy chain or light chain antibodies, DNA that encodes the antibody with a dual focus, the recombinant expression vector containing such DNA, cell host transformed by a recombinant expression vector, a method for producing antibodies with a dual focus by culturing the specified host cell and pharmaceutical compositions containing the specified antibody with a dual focus.

The level of Technology

The term "angiogenesis" refers to the mechanism by which new blood vessels are formed from pre-existing blood vessels by growth, differentiation and migration of endothelial cells. It is known that angiogenesis plays an important role in the process of normal growth, including wound healing and the menstrual cycle in women (Risau, Nature, 386: 671, 1997), a abnormally increased angiogenesis plays a critical role in growth and metastasis of tumors and chronic diseases such as age-related macular degeneration (AMD), diabetic retinopathy, psoriasis, rheumatoid arthritis and chronic inflammation (Carmeliet and Jain, Nature, 407: 249,2000).

In 1971, etc. j. Folkman J. Folkman) proposed the hypothesis that the growth and meta is datirovanie tumors depend on angiogenesis, and, thus, the strategy of therapy directed against angiogenesis, may include a new therapeutic agent against solid cancers. Further, many researchers have shown growing interest in numerous studies related to the inhibition mechanism of angiogenesis (Ferrara and Kerbel, Nature, 435: 967, 2005). The character progression of angiogenesis is determined by the overall balance between factors that stimulate angiogenesis, and factors, any abscopal angiogenesis, and involves a complex and sequential multi-stage processes. In relation to this process it should be noted that, firstly, the various factors that stimulate angiogenesis, including the vascular endothelial growth (VEGF), secretory tissues with tumor or damage associated with their corresponding receptors on the periphery of existing vascular endothelium cells, activating cells of the vascular endothelium and thus increasing the permeability of vascular endothelial cells. In addition, the secretion of proteases, such as matrix metalloproteinase (MMP)provides a breakdown of the basement membrane and extracellular matrix around the cells of the vascular endothelium, resulting from endothelial cells of blood vessels out of an existing capillary vessel and migrate/proliferate towards the tissue, secreting factor, stimulirujughee. Migrating and proliferating cells of the vascular endothelium to form a tubular structure in the blood vessel, and as a result, when pericyte, which is the structural support for the cells of the vascular endothelium, fall into this tubular structure is formed stable and Mature blood vessel. In this case, it is known that the angiopoietin 1 (Ang1), the secretory cells of the vascular endothelium plays an important role in the flow of pericytes and stabilization of blood vessels by binding to its receptor Tie-2 (Suri et al., Cell, 87:1171,1996). At the same time, angiopoietin 2 (Ang2), which is known to inhibit the interaction between Ang1 and Tie-2 shows an affinity for Tie-2, close to the affinity Ang1, and, thus, Ang2 can be used for competitive inhibition of phosphorylation induced by Ang1 (Maisonpierre et al., Science, 277:55, 1997). However, it was reported that phosphorylation of Tie-2, Ang2 induced, depends on the shape of the cells and methods of the experiment (Kim et al., Oncogene, 19:4549, 2000). In addition, it was reported that blood vessels become fragile and cells of the vascular endothelial become sensitive to stimuli such as VEGF, resulting in the inhibition of the interaction between endothelial cells of blood vessels and parititon in the early stages of angiogenesis (Klagsbrun and Moses, Chem. Biol., 6:R217, 1999; Veikkola and Alitalo, Semin Cancer Biol., 9:211, 1999; Carmeliet and Jain, Nature, 407:249, 2000). In particular, Ang1 relatively widely expressi is : in normal tissues (Maisonpierre et al., Science, 277:55, 1997), but weakly expressed in tumor tissues (Hayes et al., Br. J. Cancer, 83:1154, 2000). On the other hand, from the fact that sverkhekspressiya Ang2 in cancer tissues with high angiogenic potential, or normal tissues such as the placenta, uterus and ovaries, have been active reconstruction of blood vessels (Kong et al., Cancer Res., 61:6248, 2001; Ahmad et al., Cancer, 92:1138, 2001), we can conclude that the onset of angiogenesis in the tumor occurs when the content of Ang2 exceeds the content of Ang1. Therefore suggest that Ang2 acts as an agonist in the signaling mechanism of the Tie-2. In General, since the signaling mechanisms of the angiopoietin and Tie-2 unambiguously not identified, to identify the signaling mechanisms require further investigation. However, I believe that Ang1 and Ang2 plays an important and distinct role in angiogenesis.

The angiopoietin contains N-terminal domain (N-domain), consisting of approximately 50 amino acids, bipedally domain (C-domain), consisting of 215 amino acids, and the fibrinogen-like domain (F-domain), consisting of approximately 215 amino acids. Among these, N - and C-domains associated with the polymerization of the angiopoietin, and F is the domain associated with the binding of the receptor Tie-2 (Davis et al., Nat. Strut. Biol., 10:38, 2003). Phosphorylation required for the induction of signals Tie-2, is achieved by receptor dimerization, as the other tyrosinekinase receptors. On the basis of the fact that the angiopoietin must be multimerization (Procopio et al., J. Biol. Chem., 274:30196, 1999; Schlessinger, Cell, 103:211, 2000), suggest that these ligands can be used as targets for developing new drugs that involve the inhibition of angiogenesis. In particular, Davis and others argued that according to measurements made using genetic engineering techniques, the minimum module angiopoietin for phosphorylation of Tie-2 is a tetramer and can be used as an antagonist, if this module consists of dimers (Davis et al., Nat. Strut. Biol., 10:38, 2003). In another study it was reported that dimeric variant Ang1 does not provide an effective signal transmission Tie-2 (Cho et al., Proc. Natl. Acad. Sci. U.S.A., 101:5547, 2004). Thus, despite the fact that was not reported on the role of Ang1 as antagonist, inhibition of signal transmission Tie-2 changes in the oligomeric structure of this molecule indicates the ability to use strategies to prevent multimerization of angiopoietin for suppression mechanism of signal transmission Tie-2. From the described structure related angiopoietin and Tie-2 shows that the binding site

Tie-2 is present in the angiopoietin and structure, providing a link between Ang1 and Tie-2, similar to the structure that provides the binding between Ang2 and Tie-2 (W.A. Barton et al., Nt. Struct. Biol., 13:524, 2006). Therefore, the authors of the present study is an attempt to effectively inhibit angiogenesis by creating antibodies dual directional, in which the binding site of the angiopoietin, in particular, Ang2, according to the present invention, Tie-2 merged with an existing antibody.

At the same time, in the present invention, attention was paid to the mechanism of signal transmission VEGF/VEGFR as another target for inhibiting angiogenesis. VEGF, which is known to have a significant impact on most of the stages of angiogenesis, widely secreted in the area of the hypoxic region of a tumor. In 1989 other Nperror etc. of a company Genentech identified VEGF by isolation and purification of the protein and cloning of cDNA (Leung et al., Science 246:1306, 1989). It is known that VEGF designated as VEGF-A, is represented by four isotypes (VEGF121, VEGF165, VEGF189 and VEGF206). Among them, it was reported that VEGF 165 is present in large amounts in all tissues except placenta (Tisher et al., J. Biol. Chem., 266:11947, 1991). It is known that VEGF binds to its receptors VEGFR-1 and VEGFR-2 with high affinity, but stimulates the mechanisms associated with angiogenesis, such as proliferation and migration of endothelial cells of blood vessels, by transmitting their signals through VEGFR-2. Therefore, VEGF and VEGFR-2, mainly used for the inhibition mechanisms of angiogenesis, inducer is by VEGF, what has been described in numerous articles (Ellis and Hicklin, Nature Rev. Cancer, 8:579, 2008; Youssoufian et al., Clin. Cancer Res., 13:5544s, 2007). For example, Avastin company Genentech is humanitariannet antibody to VEGF-A (Ferrara et al., Biochem. Biophy. Res. Comm., 333:328, 2005) and was approved by the Management under the control over products and medicines (FDA) for metastatic colon cancer in 2004, non-small cell lung cancer in 2006 and Her-2-negative metastatic breast cancer in 2008, respectively, and was released on the market. In recent years we have conducted extensive clinical research on a variety of solid tumors to expand the range of indications. In addition, Lucentis, manufactured by the same company, is an antibody obtained by separating the Fab-fragment of Avastin with the aim of increasing the permeability when injecting Lucentis in the retina to excessive inhibition of angiogenesis underneath the macula, which is a major violation in age-related macular degeneration (Eter et al., Biodrgus, 20:167, 2006). Lucentis was approved in 2006, the US FDA as a therapeutic agent for the treatment of the wet form of age-related macular degeneration (wet AMD). Other therapeutic antibody target is VEGF is VEGF-trap company Regeneron (Holash et al., PNAS, 99:11393, 2002). This soluble receptor-bait"obtained by merging the second immunoglobulin house is on VEGFR-1 and third immunoglobulin domain of VEGFR-2 with human Fc, still have not received approval from the U.S. FDA, and he currently undergoing phase III trials against metastatic breast cancer, metastatic lung cancer, metastatic colon cancer and hormonereceptor prostate cancer.

Inhibiting angiogenesis antibody target is a growth factor receptor vascular endothelial VEGFR-2, include IMC-1121B (EP A) company Imclone, CDP-791 (PCT/GB02/04619) company UCB, TTAC-0001 (PCT7KR07/003077), developed by the authors of the present invention. IMC-1121B is a monoclonal antibody is identified by screening a complete library of Fab person, at the moment it passes the test of phase III in respect of metastatic breast cancer are held, and the conduct of phase III trials in the treatment of cancer of the stomach is planned in 2009. CDP-791 company UCB is humanitariannet antibody, and currently it is undergoing a phase II trial in respect of non-small cell lung cancer in the form of pegylated Fab double. Since this antibody does not contain plot Fc cannot ojidaet antibody-dependent cretaceouspaleogene cytotoxicity or complementability cytotoxicity. Finally, TTAC-0001, developed by the authors of the present invention and tested the pre-clinical stage, is a monoclonal antibody Ident is fitiavana by screening the complete library of human ScFv. This antibody is only showing reactivity against flk-1 (a homologue of VEGFR-2)derived from mice or rats, despite the fact that his target is VEGFR-2, which represents one of the important features that distinguish it from IMC-1121B company Imclone (PCT/KR07/003077). In particular, interspecies cross-reactivity of TTAC-0001 possible to conduct tests in the disease model animal, which facilitates the development of anti-cancer agent for the treatment of certain tumors in the future and the completion of related studies.

Thus, studies of VEGF and VEGFR-2 have advanced considerably over the last 5 years, and have developed a variety of therapeutic agents through market research and conducting clinical trials. In addition to the development of therapeutic antibodies, inhibiting angiogenesis, a variety of therapeutic agents on the basis of antibodies that have a single target for each disease, have been approved by the FDA and entered the market. For example, the main therapeutic agents based on antibodies, which are leaders in the global market for monoclonal antibodies include Eribitux (Imclone)whose target is the receptor for epidermal growth factor (EGFR) and which is positioned as a therapeutic agent for the treatment of metastatic colon cancer, Herceptin (Genentech)whose target is Her-2/nu, and which is positioned as a therapeutic agent for the treatment of metastatic breast cancer, and TM Rituxan whose target is CD 20 and which is positioned as a therapeutic agent for the treatment of non-Hodgkin's lymphoma.

Thus, according to the latest market trends antibodies, in addition to the development of antibodies that act on a single target, actively carried out extensive research on the development of the so-called antibodies dual directional (bespecifically antibodies or antibodies with multiple specificities (multispecific antibodies), which can have two or more targets simultaneously (Van Spriel et al., Immunol. Today, 21:391, 2000; Kufer et al., Trend in Biotechnol., 22:238, 2004; Marvin and Zhu, Curr. Opin. Drug Discovery Dev., 9:184, 2006). Among these antibodies belonging to this class, there are no antibodies, which would be approved by the FDA and would be produced on a commercial scale. However, these antibodies are constantly learning on laboratory and clinical levels due to the continuous interest in them and their potential. Antibodies belonging to this class, is divided mainly on (1) antibody-based ScFv, (2) antibody-based Fab and (3) antibody-based IgG etc.

First, in the case of multiple antibodies specificity on the basis of ScFv, this antibody is ditelo obtained by the combination of VL and VH different ScFv and have hybrid ScFv in the form of heterodimer (Holliger et al., Proc. Natl. Acad. Sci. U.S.A., 90:6444, 1993). However, such antibodies are lacking, allcauses in low stability due to the affinity of binding with heterodimers. In addition, published data on tandem ScFv obtained by linking different ScFv with each other (Kipriyanov et al., J. Mol. Biol., 293:41, 1999; Robinson et al., Brit. J. Cancer, 99:1415, 2008), heterodimeric ScFv obtained from the expression of Jun and Fos that have the potential to bind to the ends of the various ScFv (De Kruif and Logtenberg, J. Biol. Chem., 271:7630, 1996), heteromeric mini-antibodies, obtained by expression SN and CL from Fab all ScFv, respectively (Muller et al., FEBS lett., 432:45,1998), and method of constructing mini-antibodies in heteromerous the form of ScFv (Merchant et al., Nat. Biotechnol., 16:677, 1998). In this case, the method of constructing mini-antibodies include replacement of some amino acids CH3 domain, which is homodimers the Fc domain, to change the heterodimeric structure in the form of a "ledge " in the hollow" and, accordingly, the expression of these modified CH3 domains from the ends of the ScFv. In addition, the scientific literature has reported several analogues on the basis of ScFv (Kipriyanov and Le Gall, Curr. Opin. Drug Discovery Dev., 7:233, 2004); also in the scientific literature reported ScFv triple-specificity on the basis of Triatel and ScFv with quadruple specificity on the basis of tetrathele (Hudson and Kortt, J. Immunol. Methods, 231:177, 1999).

Secondly, multiple antibodies specificity on the basis of Fab, presented mainly in the form heterodimeric Fab obtained by a combination of a Fab-specific antigens that are associated with each other disul idname links or through the mediator (Brennan et al., Science, 229:81, 1985; Kostelny et al., J. Immunol., 148:1547, 1992). At the same time, there are messages about how to obtain antibodies with a dual focus, with a valency of two antigens by expression of ScFvs to different antigens on the basis of the end of the heavy chain or light chain-specific Fab (Schoonjans et al., J. Immunol., 165:7050, 2000; Lu et al., J. Immunol. Methods 267:213, 2002), and antibodies with a dual focus in the form of glycosilated with valence four antigens by embedding the hinge region between the Fab and ScFv (Coloma and Morrison, Nat. Biotechnol., 15:159, 1997). Also available in the scientific literature publications about getting Bitel dual directional, with three valences to antigens, by attaching ScFvs to different antigens to the ends of the light chain and heavy chain Fab, and receiving Bitel triple specificity, with three valences to antigens, by attaching different ScFvs to the ends of the light chain and heavy chain Fab (Schoonjans et al., J. Immunol., 165:7050, 2000). There are also data about the triple antibody specificity F(ab')3, which are obtained by chemical conjugation three different Fab (Tutt et al., J. hnmunol., 147:60,1991).

Thirdly, in the case of multiple antibodies specificity on the basis of IgG, the company Trion Pharma was obtained hybrid hybridoma producing antibody with a dual focus, the so-called quadroma by hybridization hybrid mice and rats. This company produces antibodies dual direction the particular Ertumaxomab (antigens: Her-2/neu, CD3), located on the second phase of the trials for the treatment of metastatic breast cancer (Kiewe and Thiel, Expert Opin. Investig. Drugs, 17:1553, 2008), and Catumaxomab (antigens: Arcam, CD3), located on the second phase of the trials for the treatment of stomach cancer and ovarian cancer, and III phase trials for the treatment of malignant ascites (Shen and Zhu, Curr. Opin. Mol. Ther., 10:273, 2008). However, these antibodies cannot avoid reactions of the antibody human mouse (HAMA response) or rat (HARA-response) antibodies, which occur during prolonged introduction. At the same time aware of the antibody with a dual focus, designed on the principle of "knobs-into-holes"obtained in the form of heterodimer by substitution of certain amino acids in one of homodimeric CH3 domains of the Fc to the relevant other heavy chains with identical domains of the light chain (Merchant et al., Nat. Biotechnol., 16:677, 1998). In addition to antibodies with a dual focus in the form of heterodimers, there is information about the antibody (ScFv)4-IgG (antigen: EGFR, IGF-1R), which is expressed in the form of glycosilated in the merger of two different ScFvs with constant and not variable domains of light and heavy chains of IgG (Lu et al., J. Biol. Chem., 279:2856, 2004). However, the disadvantage of this antibody is low productivity, but the scientists from the same research group has received di-ditelo with improved productivity to the same targets by addressing not the remainder of the antibody (ScFv)4-IgG, and confirmed its effectiveness (Lu et al., J. Biol. Chem., 280:19665, 2005). However, for such antibodies have not yet solved the problem of low stability, which is typical for deatil. Shen with colleagues from Imclone also received the antibody with a dual focus by joining the single variable domain of the α-receptor platelet-derived growth factor mouse (DERIVED-α) N-end of the light chain of the chimeric monoclonal antibody to human VEGFR-2, IMC-1C11, and has shown its effectiveness (Shen et A1., J. Biol. Chem., 281:10706, 2006; Shen et al., J. Immunol. Methods, 318:65, 2007). Recently, Rossi and colleagues had suggested that the antibody with multiple valency of CD20 obtained using the so-called method of "dock and lock (DNL)" ("dock and lock"), use "dimerise/host domain (dimerization and docking domain, DPD) regulatory subunit R protein kinase A (PKA) and zakariayi domain RKA (Rossi et al., Proc. Natl. Acad. Sci. U.S.A., 103:6841, 2006; Rossi et al., Cancer Res., 68:8384, 2008), this research group has reported a double antibody orientation obtained using this method (Chang et al., Clin. Cancer Res., 13:5586, 2007). It is known that antibodies obtained using the method of DNL, have several advantages, they are easy to use, they can be combined, as they are modular and they show good stability in vivo, but their disadvantage is that they can destroy the camping under the action of proteases in vivo, and also cause problems associated with immunogenicity.

In scientific literature there is a lot of information about the antibodies dual or multiple specificity, and these antibodies, from a functional point of view, have advantages and disadvantages depending on the morphological features due to the intended use. In particular, active research is ongoing, aimed at developing therapeutic antibodies dual or multiple specificity for cancer treatment. However, it is very important to choose the antigens that are targeted by antibodies obtained in the course of such research, so that antibodies to perform its function.

Description of the invention

Technical Task

Thus, to generate antibodies dual specificity for cancer treatment by inhibiting angiogenesis, the authors present invention has developed an antibody with a dual focus, able to neutralize two receptors, VEGFR-2 and Tie-2, is closely associated with the mechanism of angiogenesis, in a new form, which to date has not been mentioned in the literature, confirmed demonstrate this antibody anticancer effect in vivo and at the cellular level, comparable or superior to the effect of antibodies that are specific only to VEGFR-2 or Tie-2, which became the basis of the present invention.

Task nastojasih the invention is the provision of antibodies with a dual focus in a new form, containing a water-soluble ligand, fused with the N-end of the heavy chain or light chain of the antibody.

Another objective of the present invention is the provision of DNA that encodes the specified antibody with a dual focus.

Another object of the present invention is to provide a recombinant expression vector containing this DNA.

Another objective of the present invention is the provision of a host cell transformed with the indicated recombinant expression vector.

Another objective of the present invention is a method of obtaining antibodies with a dual focus by incubation of the specified host cell.

Another objective of the present invention is to provide pharmaceutical compositions that contain the antibody with a dual focus.

Technical Solution

In the present invention proposed a new form of antibodies dual directional containing a water-soluble ligand, fused with the N-end of the heavy chain or light chain of the antibody.

In the description of the present invention, the term "antibody" refers to a protein molecule that is produced by b-lymphocytes that are specific recognizes various types of antigens and used by lymphocytes as a receptor for antigen. This molecule has a Y-shaped and consists of a TLD is identical light chains and two identical heavy chains. All light chains and heavy chains contain variable and constant region. The four chains are fastened together by disulfide bonds in the flexible regions of the heavy chains, and the area referred to as the hinge region. Variable region heavy chains and all light chains are linked together to form two identical antigen-binding sites. The structure of the constant region of heavy chain antibodies distinguish five classes of antibodies: A(IgA), D(IgD), E(IgE), G(IgG) and M(IgM). Each class is called isotype and has unique structural characteristics and differs from other biological properties. The present invention includes antibodies of all isotypes, preferred for use is IgG.

Preferably, the antibody according to the present invention includes antibody to antigens that are expressed in specific neoplastic cell, stromal cell cancer, a tumor-associated cell epithelium, the predecessor of the tumor-associated cells, circulating tumor-associated endothelial cell, circulating cell tumor stem cell cancer and so on, but is not limited to the aforementioned types.

More specifically, the antibodies according to the present invention include an antibody to a protein expressed on the cell surface such as growth factor receptor vascular endothelial (VEGFR-1), the growth factor receptor vascular endothelial (VEGFR-2), growth factor receptor vascular endothelial (VEGFR-3), FMS-like tyrosinekinase 3(FLT3)receptor c-FMS / colony-stimulating factor 1 (CSF1R), rearranged during transfection (RET), factor mesenchymal-epithelial transition (c-Met), the receptor for epidermal growth factor (EGFR), Her2/neu, HER3,, HER4, receptors of fibroblast growth factor (FGFR), receptors of the insulin-like growth factor (IGFR), receptors of platelet factor growth (PDGFRs), receptors of factors stem cells (C-KIT), the area of the gap cluster (BCR), integrin, matrix metalloproteinase(MMP) and so on, but is not limited to the foregoing.

According to the present invention, the antibody may be polyclonal or monoclonal" antibody, and more preferably a monoclonal antibody. Monoclonal antibody is the antibody obtained from an essentially homogeneous population of antibodies. That is, specific antibodies comprising such a population are identical except for possible naturally mutations that may be present in small numbers. Monoclonal antibody is highly specific for a single antigenic site. In addition, in contrast to polyclonal antibodies, including various antibodies to different epitopes, each monoclonal antibody targeted to another epitope presence is adequate for the antigen. This definition should not be understood in the sense that the monoclone necessarily produces antibody specific way. For example, suitable within the present invention the antibody can be obtained by the hybrid method described in Kohler et al., Nature, 256:495(1975), or by recombinant DNA (see U.S. Patent No. 4816567). Also monoclonal antibody can be, for example, selected from a library of phage antibodies by the method described in Clackson et al., Nature, 352:624-628(1991); Marks et al., J. Mol. Biol., 222:581-597(1991).

The antibody according to the present invention preferably is a "humanitariannet antibody". The term "humanitariannet antibody" refers to an amino acid sequence that is partially or fully derived from the germline of the person by replacing the sequence of the antibody containing hypervariable segment (CDR), the sequence belonging to the person. The implementation of the replacement is the easiest way to completely replace the mouse constant region constant region of human antibodies. Thus can be obtained chimeric human/mouse, which can significantly reduce immunogenicity, to a level acceptable for pharmaceutical use. However, it is preferable that the variable plot and even CDR region of the antibody were humanitarian one way, and is known in this field. Wireframe plot variable region is replaced by the corresponding frame section and section CDR, does not belong to man, leave essentially unchanged (intact) or replaced with the sequence derived from the human genome. Intact human antibody receive in genetically modified mice whose immune systems are modified to fit the human immune system.

In a particularly preferred case, the antibody according to the present invention is a "human antibody". The human antibody is an antibody containing the amino acid sequence corresponding to the amino acid sequence of the antibody obtained by any method providing antibodies produced by a human, or human antibodies. The human antibody can be obtained using various techniques known in this field. According to one implementation options conduct screening human antibodies from ragovoy library, which expressed human antibodies. Nature Biotechnology 14:309-314(1996): Sheets et al. PNAS (USA) 95:6157-6162 (1998); Hoogenboom and Winter. J. Mol. Biol. 227:381(1991); Marks et al. J. Mol. Biol. 222:581 (1991)). Human antibodies can be obtained by introducing immunoglobulin locus man transformed animal, nab the emer mouse endogenous immunoglobulin genes of which is partially or fully inactivated. During processing by antigen was found that the obtained antibody man was highly similar to the antibodies produced by the person, in all aspects of rebuilding, Assembly, and repertoire of antibodies. Such methods are described, for example, in U.S. Patents№5545807, 5545806, 5569825, 5625126, 5633425 and 5661016 and Marks et al. Biotechnology 10:779-783 (1992); Lonberg et al. Nature 368:856-859(1994); Morrison, Nature 368:812-13 (1994); Fishwild et al. Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995). Another way a human antibodies can be obtained by immortalization of human lymphocytes (for example, b-lymphocytes can be distinguished from the population or to immunize in vitro)that produce the antibody to the target antigen (Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R Liss, p.77 (1985); Boerner et al. J. Immunol., 147(1):86-95 (1991); and U.S. patent No. 5750373).

In the description of the present invention the term "water-soluble ligand" means a portion of a protein or protein as a whole, which is associated with specific receptor in the cell, in particular on the surface of the cell, and demonstrates the property of water solubility, and can therefore be dissolved in water. For example, water-soluble ligands include, without limitation: growth factor vascular endothelial (VEGF), growth factor epidermal (EGF), growth factor, placenta (PIGF), growth factor vibrola the tov (FGF), insulin-like growth factor, platelet-derived growth factor (PDGF), growth factor hepatocyte (HGF), angiopoietin, etc.

The antibody with a dual focus in a new form according to the present invention each part, the antibody and the ligand plays its unique role.

However, the antibody with a dual focus can suppress or amplify two signals simultaneously and, thus, may be more effective as compared to the case when inhibited or enhanced by only one signal. Compared with the case where each signal influences its inhibitor, in this case the injected dose can be reduced and the two signals will be ingibirovany or strengthened at the same time and in the same area.

According to the present invention it is preferable that the antibody was drained with a water-soluble ligand via a linker. According to the present invention, the term "linker" refers to a peptide fragment that connects two parts of the fused protein. According to the present invention is acceptable linkers include peptides containing from 5 to 25 amino acids, preferably from 10 to 20 amino acids, more preferably, from 10 to 15 amino acids.

To obtain antibodies with a dual focus in accordance with the present invention have the sequence of a nucleic acid encoding the antibody double napravlennos is I. This sequence of nucleic acid can be obtained by joining the 3'-end of nucleic acid sequence that encodes a soluble ligand, with the 5'-end of nucleic acid sequence that encodes a heavy chain or light chain antibodies. According to one aspect, the nucleic acid sequence encoding the antibody with a dual focus, fused via a linker, can be obtained by creating a nucleic acid sequence of the linker in the primer and the NDP.

The gene encoding this antibody dual directional, thus obtained, are ligated into the vector to obtain a recombinant expression plasmid, the plasmid is injected in a cage-owner with obtaining cell-transfectant or cell-transformant, cage-owner multiply, isolate and purify the antibody with a dual focus, resulting in a gain target antibody with a dual focus.

According to the present invention used for the expression of antibodies with a dual focus a host cell can be prokaryotic or eukaryotic. Usually use the cell-master in which you can enter a DNA molecule with high efficiency and in which the expression of the introduced DNA also occurs with high efficiency. Examples of such host cells include known the major eukaryotic and prokaryotic cells, such as E. coli, Pseudomonas spp., Bacillus spp., Streptomyces spp., bakerie and yeast cells such insects as Spodoptera Frugiperda 9 (SF9), animal cells such as cells of the Chinese hamster ovary (Cho) cells and mouse, COS1, COS7, liver cells of the embryo human 293 cells, African green monkeys, such as BSC 1, BSC 40, and BMT 10, and tissue culture of human cells.

According to the present invention for the expression of antibodies with a dual focus, you can use various combinations of host for expression / expression vector. For example, expression vectors, suitable eukaryotic hosts include SV40, a virus of a papilloma of cattle, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus. To expression vectors that can be applied in bacterial hosts include bacterial plasmids such as pBluescript, pGEX2T, pUC, pCR1, pBR322, pMB9 and their derivatives, plasmids such RP4 characterized by a wide list of possible owners, λgtl0 and λ11, phage DICK, presents various derivatives of phage lambda, such as NM989, and other phage DNA such as M13 and filamentous phage with odnozadachnoy DNA. Expression vectors that you can use with yeast cells include the 2µ plasmid and its derivatives. A vector that can be used in insect cells, is pVL941.

For transformation of cells is ozaena recombinant expression vector applicable, for example, transfection using DEAE-dextran, electroporate, transduction, calcium phosphate transfection, transfection using cationic lipids, "scrape-loading" (scraping-load) and infection.

According to the present invention the cell master can be incubated in a suitable nutrient medium under conditions allowing expression and secrete antibodies with a dual focus when using small-scale or large-scale fermenter during the incubation the flasks on the rocking chair in laboratory or industrial fermenter. Incubation is carried out in a suitable nutrient medium containing sources of carbon and nitrogen and inorganic salts, one of the known methods. A suitable environment is available commercially or can be prepared using the components and their specific compositions described in the catalogue of the American type culture Collection (ATSS).

The double antibody orientation can be selected from the culture fluid by methods known in this field. For example, an antibody with a dual focus can be selected from the culture fluid by standard methods, including, without limitation, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. Antibody with a dual focus can be dopolnitelnye using various technologies, known in this field, including chromatography (e.g. ion exchange, affinity, hydrophobic or exclusion), electrophoresis, separation (for example, precipitation with ammonium sulfate), LTO-page, or extraction.

The composition according to the present invention can be delivered to specific molecules in any suitable way. The composition according to the present invention can be applied in animals, including humans, either directly (for example, locally by injection, subcutaneous injection or by local injection directly into the tissue) or systematically (for example, parenterally or orally), by any appropriate means. If the composition according to the present invention, parenteral, for example intravenous, subcutaneous, ocular, intraperitoneal, intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral, intraspinal, intraventricular, intrathecal, intracerebral, intravesical, intranasal or by spraying, the composition preferably comprises part of an aqueous or physiologically compatible fluid suspension or solution. Accordingly, as the carrier or filler is physiologically available, it should not have a negative effect on the balance of electrolytes and/or fluids in patients in the delivery of patients to target is notizie.

The pharmaceutical composition containing the antibody with a dual focus in accordance with the present invention, can be prepared in a form for oral administration, such as powder, granules, tablets, capsules, suspension, emulsion, syrup or spray, sterile solution for injection, suppository and in the form of the drug for subcutaneous injection according to standard methods, and applied later. Carrier, excipient and diluent, which can be included in the composition, may contain: lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, aritra, ▫ maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, cut hydroxybenzoate, talc, magnesium stearate and mineral oil. The composition optionally may contain a diluent or filler, such as seal-thinning agent, binder, wetting agent, disintegrity agent or surfactant.

According to one aspect, the antibody dual directional according to the present invention can be prepared in the form of a solid preparation for oral administration. To solid preparations for oral administration include tablet, pill, powder, granule, capsule, etc. Solid preparation for oral call is to be placed can be prepared by mixing at least one filler, for example, starch, calcium carbonate, sucrose, lactose or gelatin, with the extract. In addition to the simple filler can also be used lubricants such as magnesium stearate and talc.

According to another aspect, the pharmaceutical composition comprising the antibody with a dual focus in accordance with the present invention can also be prepared in the form of a liquid for oral administration. Liquid forms for oral administration include suspensions, liquid for internal use, emulsion, syrup, etc. Such liquid preparations may in addition to the basic inert solvent (e.g. distilled water, ethanol, the liquid paraffin) contain various fillers, such as, for example, a wetting agent, a sweetener, a flavouring agent, preservative, etc.

According to another aspect, the pharmaceutical composition comprising the antibody with a dual focus in accordance with the present invention can also be prepared in a form for parenteral administration, preferably for intraperitoneal administration. Forms for parenteral administration include sterile aqueous solution, anhydrous solvent, suspension, emulsion, freeze-dried preparation, a suppository. To a sterile aqueous solution which may be used in this case include: the solution of Hank, Rast is the PR ringer or a suitable buffer solution, such as physiological buffer solution, and the suspension, which is suitable for use anhydrous solvent may be a vegetable oil such as propylene glycol, polyethylene glycol or olive oil or ester, suitable for injection, such as etiloleat. If necessary, you can use a preservative, a stabilizing agent, wetting or salts or buffers for regulation of osmotic pressure. At the same time, in the case of a suppository, you can use common basis, such as Witepsol, Macrogol, tween 61, cacao butter, lauric oil or treated with glycerol gelatin.

Antibody dual directional according to the present invention most preferably is a DIG 0001, obtained according to one of the options for implementing the present invention.

Based TTS described in published international application no PCT/KR 07/003077, obtaining antibodies dual directional DIG 000 according to the present invention is made by combining (merging) the binding domain Ang2, which binds to Tie-2, through specific linker with N-terminal site of the light chain of the antibody (Examples 1 and 2).

Thus obtained antibody dual directional DIG 0001 tested for immobilization by means of the LTO-SDS page and protein blotting, and p is the following a more detailed analysis (Example 3) was used antibody which was cleared at least to the level of 95%, the method of liquid Express chromatographie proteins (IAHB) using a column with affinity protein A, column of SP-sepharose columns exclusion chromatography.

The ability of antibodies dual directional DIG 0001, purified as described above for binding to VEGFR-2 from D1 to D3-Fc and Tie-2-Fc was confirmed by the analysis of binding using methods enzyme-linked immunosorbent assay (ELISA). Also carried out the analysis of competitive binding to VEGF165 and Ang2 method ELISA to confirm the functionality of the antibodies as antibodies dual specificity.

According to the present invention, the evaluation of the viability of the cells in primary culture cells of the umbilical vein endothelium (HUVEC) confirmed that the antibody dual directional DIG 0001 able to suppress the viability of HUVEC cells induced separately VEGF or Ang1, and can effectively suppress the viability of HUVEC cells induced together VEGF and Ang1 (Example 5).

By assessing cell migration was confirmed that the antibody dual directional DIG 0001 according to the present invention are able to suppress the motility of HUVEC cells induced separately VEGF or Ang1, and can effectively suppress the motility of HUVEC cells induced together VEGF and Ang1 (Example 6).

Analysis methods the Ohm Western blotting confirmed the antibody dual directional DIG 0001 according to the present invention is able to inhibit the signaling mechanism of Tie-2, induced by Ang1, and the signaling mechanism of VEGFR-2, induced by VEGF. Analysis by the method of Western blot also confirmed that the antibody dual directional DIG 0001 able to simultaneously inhibit signaling mechanisms induced by Ang1 and VEGF (Example 7).

More specifically, confirmed that the introduction in the model of the finding of the animal antibodies dual directional DIG 0001 according to the present invention, the tumor significantly decreased in volume (Example 8).

The above antibody was prepared as follows. First DNA sequence encoding a domain Ang2, responsible for binding to Tie2, which can act antagonist of Tie-2, amplified by PCR. In this case, the antibody is constructed so that the linker DNA was linked to the 3'end of the amplified DNA fragment. Obtaining antibodies with a dual focus that can specific contact VEGFR-2 and Tie-2, was carried out by hybridization (merger) of this DNA fragment to the 5'-end sequence of the light chain DNA TTAS containing DNA linker.

Antibody dual directional DIG 0001 according to the present invention can be used for the treatment of angiogenesis-mediated of Zabol is to work through inhibition of angiogenesis.

According to the present invention, the term "associated with angiogenesis disease" includes diseases, but are not limited to: cancer, age-related macular degeneration, diabetic retinopathy, psoriasis, rheumatoid arthritis and chronic inflammation.

According to the present invention, the cancer includes, but is not limited to: gastric cancer, liver cancer, lung cancer, thyroid cancer, breast cancer, cervical cancer, colon cancer, pancreatic cancer, rectal cancer, colorectal cancer, prostate cancer, kidney cancer, melanoma, metastatic lesion of bones, prostate cancer, ovarian cancer and cancer of the blood.

Antibody dual directional according to the present invention can be introduced in a quantity sufficient to prevent, suppress or reduce the progression of a tumor, such as growth, invasion, metastasis and/or recurrence of the tumor, with the purpose of therapeutic treatment of patients with cancer. To achieve this goal, determine the appropriate number, which is called a therapeutically effective amount. An effective amount in the framework of this proposal will depend on the severity of the disease and the General condition of your own immune system of the patient.

The preferred number, according to the present from which bretania, is in the range from 0.01 mg/kg to 100 mg/kg and, more preferably, from 0.1 mg/m2 to 10 mg/m2.

However, the optimal dose varies depending on the disease will be cured, and the presence of side effects, and can be determined using known techniques. Introduction antibodies can be made in the form of periodic injections of diluted solid dosage forms or continuous intravenous or intraperitoneal injection of external storage containers (for example, a bag for injection into a vein) or internal storage containers (e.g., biodegradable implant). In addition, protein antibodies according to the present invention can also be entered in combination with different biologically active molecules. In any case, the optimal combination of protein antibodies and other molecules, route of administration and dosage can be determined by ordinary experimentation available to the average experts in this field.

The composition according to the present invention can be used in combination with other therapeutic agents or be mixed with other therapeutic agents.

When tumors including tumors in humans, treating the antibody with a dual focus, according to the present invention in combination with chemotherapeutic agents, radiation or additional antagonists receptively their combination, can be achieved synergies. In other words, the inhibition of tumor growth provided by the double antibody orientation according to the present invention may suddenly increase when used in conjunction with a chemotherapeutic agent, radiation or additional receptor antagonists or their combination. For example, a synergistic effect can be manifested in the form of higher suppression of tumor growth when treated in combination, than can be expected when treating the antibody with a dual focus in accordance with the present invention and a chemotherapeutic agent, radiation or additional receptor antagonist, or a combination. Preferably, the synergistic effect is manifested in the reduction of cancer for which you did not expect that he will be weakened in the treatment of the antibody with a dual focus in accordance with the present invention in conjunction with a chemotherapeutic agent or additional receptor antagonist.

Antibody dual directional according to the present invention is administered prior to chemotherapy or radiotherapy, after the commencement of these treatments, and before and during their combinations, i.e. chemotherapy and/or radiotherapy before and after these treatments, during and after the commencement of these treatments and the and to, during and after these treatments. For example, the antibody DIG 0001 usually administered for 1-30 days, preferably 3-20 days and more preferably 5-12 days prior to radiotherapy and/or chemotherapy.

Thus, the antibody according to the present invention can be applied in vivo and in vitro with the purpose of carrying out scientific research, prevention or treatment is widely known in this field. Of course, it is obvious that disclosed in the present application the invention may be changed and modified by the person skilled in the art, and such changes or modifications may not go beyond the scope of the present invention.

Useful results

In the present invention proposed a double antibody orientation on the basis of monoclonal human antibodies, which can effectively inhibit associated with angiogenesis signaling mechanism due to the simultaneous neutralization of the receptors VEGFR-2 and Tie-2, associated with angiogenesis, and the proposed composition comprising the antibody with a dual focus, for inhibiting angiogenesis and treatment of cancer. Antibody dual directional according to the present invention exhibits excellent efficiency of neutralization of receptors and is also effective in the treatment of cancer, when compared with currently-existing antibodies with a single is Oh specificity, due to the simultaneous neutralization of the two targets associated with angiogenesis. In the case of two targets, each of which is associated with another, when designing antibodies with a dual focus in a new form, created by the authors of the present invention, it is possible to expect a more favorable effects in comparison with real benefits when treating antibodies with a single specificity.

Description of the Drawings

These and other features, aspects and advantages of preferred embodiments of the present invention will be more fully described in the following detailed description of the invention accompanied by a graphic materials. For graphics:

Figure 1 shows the DNA sequence (SEQ ID NO:6) and gene functions that were built into the vector pIgGLD-mAng2-TTAC0001 lgt.

Figure 2 shows schematically the method of constructing vector pIgGLD-mAng2-TTAC0001 lgt according to the present invention.

Figure 3 presents the results obtained when analyzing the expression vector according to the present invention in cells CHO-DG44 and the determination of the level of production of antibodies with a dual focus method Western blotting.

Figure 4 presents the results of screening clones with high productivity during reprocessing MTX (up to 700 nm) to obtain a cell line with vysokoproduktivnogo according to the present invention.

Figure 5 presents data LTO-SDS page analysis of purified DIG 0001.

Figure 6 presents the results of the competitive analysis of VEGF and Ang-2-Fc with DIG 0001 using ELISA method.

Figure 7 presents the results of the survival analysis, demonstrating the viability of HUVEC cells when processing DIG 0001 according to the present invention.

On FIG presents the results of the analysis of migration, showing the motility of HUVEC cells under the action of DIG 0001 according to the present invention.

Figure 9 presents results demonstrating the activity of inhibiting the phosphorylation of VEGFR-2 and ERK, called VEGF, under the action of antibodies dual directional DIG 0001 according to the present invention, and the activity of inhibiting the phosphorylation of Tie-2, ERK and ACT caused Ang1, under the action of the indicated antibodies.

Figure 10 presents the effect of DIG 0001 according to the present invention on the inhibition of tumor growth in models of glioblastoma in mice.

An example implementation of the invention

The following Examples are presented solely for a more detailed disclosure of the present invention, and experts in the field to which the present invention will be apparent that the purpose of the present invention, the essence of the invention is not limited to the Examples.

Example 1: construction of the expression vectorable get DIG 0001 DNA Fragment, corresponding to the domain Ang2, responsible for binding to Tie-2, amplified by PCR. To do this, Dr. Dimitar B. Nikolov (Dimitar Century Nikolov) from Memorial cancer center Sloan-Kettering, USA kindly provided by the cell line NECK producing human Ang2-RBD (Barton et al., Structure, 13:825, 2005), which was allocated genome DNA, which was used as matrix. For amplification binding domain Ang2 (F281-F496) based on the selected DNA PCR was performed as follows: One cycle -94°C for 4 min, 30 cycles (94°C for 45 seconds / 50°C for 45 seconds and 72°C for 1 minute), one cycle of 72°C for 7 min and 4°C before extraction of the samples. For the reaction used a mixture of the following reagents: 2 μl (10 pmol/ál) primer F-ksw001 containing a restriction site recognized by the restriction enzyme BstXI (5'-CAC TSS AGC GGT GTG GGT TCC TTC AGA GAC TGT GCT GAA GTA TTC, SEQ ID NO:1), 2 ál (10 pmol/ál) reverse primer R'-ksw001 (5'-act ACC TCC GCC TCC TGA GAA ATC TGC TGG TCG GAT CAT CAT GGT TG, SEQ ID NO:2), 1 ál (100 ng/ál) genomic DNA used as DNA template and 2.5 units of i-Max™Taq II (Intron#25261, Korea), used as a polymerase, 5 ál 10X buffer, 2 μl (each base plate 2.5 mm) mixture dNTP and 37.5 μl of distilled water. For the resulting panorama of the product is carried out by electrophoresis in 1%agarose gel and then isolated weak strip, Nicodemus is below 700 BP, using the kit for DNA extraction Product Pcrow HiYield™ Gel/PCR DNA extraction kit (RBC Bioscience #YDF300, Taiwan). They were re-PCR using selected DNA fragment as template, resulting in a received fragment binding domain Ang2, containing merged with him linker (designated as "mAng2"). Used the same PCR conditions as in the previous case, except that as the reverse primer used R-ksw001 (5'-GGA GCC TCC TCC GCC ACT ACC TCC GCC TCC TGA GAA ATC TGC TGG TCG GAT CAT CAT GGT TG, SEQ ID NO:3).

Simultaneously amplified plot of the light chain TAS, for subsequent linkage with Ang2 binding domain by PCR under the following conditions: One cycle of 94°C for 4 min, 30 cycles (94°C for 30 seconds / 50°C for 30 seconds / 72°C for 30 seconds, one cycle of 72°C for 5 minutes and 4°C before extraction of the samples. For PCR used a mixture of the following reagents: 2 μl (10 pmol/ál) primer F-ksw002, which was sewn on one of the options of the linker (5'-AGT GGC GGA GGA GGC TCC GGT TCC AAT TTT ATG CTG ACT CAG, SEQ ID NO:4), 2 μl (10 pmol/ál) primer R-ksw002 containing a restriction site recognized by the restriction enzyme BstXI (5'-CAG ATC TTTCCA CGA GGC TGGJTS JTS, SEQ ID NO:5), 10 ng pIgGLD-TTAC0001 Lgt (PCT/KR07/003077)used as DNA template and 2.5 units of i-Max™ Taq II (Intron #25261, Korea), used as a polymerase, 5 µl of 10X buffer, 2 µl of the mixture dNTP (each core is of 2.5 mm) and 37.5 μl of distilled water. The resulting PCR product were analyzed by electrophoresis in 1%agarose gel and then isolated fragment of the light chain TTAS, corresponding to approximately 350 BP, using the set for DNA-PCR products HiYield™ Gel/PCR DNA extraction kit (RBC Bioscience #YDF300, Taiwan)(labeled "TAS lgt").

To associate the obtained PCR area light chain TTAS (0001_lgt) with a fragment of the region Ang2 (mAng2) conducted a panorama with the overlapping areas (SOE-PCR). For PCR used the following conditions: One cycle of 94°C for 4 min, 30 cycles (94°C for 45 seconds / 50°C for 45 seconds and 72°C for 1 minute), one cycle -72°C for 7 min and 4°C before extraction of the samples. Also in this case, the NDP used a mixture of the following reagents: 2 μl (10 pmol/ál) primer F-ksw001 containing a restriction site recognized by the restriction enzyme BstXI, 2 μl (10 pmol/ál) primer R-ksw002, 10 ng of each fragment mAng2 and TAS Lgt used as DNA matrix, 2,5% i-MAXTM Taq II (Intron #25261, Korea), used as a polymerase, 5 µl of 10X buffer, 2 µl of the mixture dNTP (each base plate 2.5 mm) and 37.5 ál of distilled water. For the resulting PCR product electrophoresis was performed in 1%agarose gel and then isolated PCR Product merge mAng2-TTAC0001 lgt, corresponding to approximately 1 T. is., using the kit for DNA extraction Product Pcrow HiYield™ Gel/PCR DNA extraction kit (RBC Bioscience #YDF300, Taiwan) (labeled "mAng2-TTAC0001 lgt"). Selected PCR product was built into the T-vector using a kit for cloning TOPcloner TA cloning kit (Enzymonics #EZ111, Korea) and transformed by this vector Escherichia coli DH5α. Then the transformed E. coli was subjected to the procedure Miniprep and was treated with the restriction enzyme BstXI. After that, the vector containing the insert having a size of approximately 1 so called, were isolated and sequenced to verify its DNA sequence (Figure 1 and SEQ ID NO:6).

Designed expression vector containing the light chain for the expression of antibodies dual directional DIG 0001, in the following way (FIG. 2). First available to researchers expression vector pIgGLD-TTAC0001 Lgt containing light chain TTAS, cut through restrictase BstXI, and using electrophoretic separation of a 1%agarose gel was isolated fragment that can be used as a vector. In parallel, using restrictase BstXI cut T-vector containing the insert mAng2-TTAC0001 lgt, and using electrophoretic separation of a 1%agarose gel was allocated cut fragment. After, to obtain a single intact vector, the selected fragments kept in the presence of T4 DNA ligase (Errzynomics #M00S, Korea) at 4°C for approximately 12 hours. Then, the constructed vector transformed E. coli was subjected to the procedure Miniprep, and then, to confirm what happened inserting mAng2-TTAC0001 lgt constructed in the vector, cut it using restrictase BstXI. Recombinant vector, which was confirmed by the insert, designated as "pIgGLD-mAng2-TTAC0001 Lgt".

Example 2: Obtaining and identification DIG 0001

Cells CHO-DG44 (dhfr - deficient cells SNO) was transducible jointly constructed a vector for the expression of light chain, pIgGLD-mAng2-TTAC0001, and existing vector for the expression of the heavy chain, pIgGHD-TTAC0001 Hvy (PCT/KR07/003077), for the induction of spontaneous expression. Expression was confirmed using LTO-page and Western blotting. Transduction was performed using lipofectamine 2000 (lipofectamine™) (Invitrogen #11668-019, USA) according to manufacturer's instructions. Briefly: 5×105cells CHO-DG44 were seeded into each well of a 6-hole tablet containing nutrient medium α (Welgene, Korea), and incubated at high density as long as the density of cells reached 80-90%, at 37°C for 24 hours, at concentrations of CO2(5%)incubator with moisture. 3 μg of recombinant vector (1.5 mcg pIgGHD-TTAC0001 Hvy and 1.5 µg pIgGLD-mAng2-TTAC0001 Lgt) and 6 ál of lipofectamine 2000 (lipofectamin™) was separately dissolved in 250 µl be the serum nutrient medium α and kept for 5 minutes at room temperature. The diluted DNA and divorced lipofectamine 2000 (lipofectamin™) was mixed and left for 20 minutes at room temperature to complete the reaction, during which the formed complex DNA-lipofectamine 2000 (lipofectamin™). From the cultivated cells were removed the old culture medium was brought to 500 μl of a solution of complex DNA-lipofectamine 2000 (lipofectamin™) and 500 μl of serum-free nutrient medium α and incubated at 37°C for 6 hours in an incubator with CO2. Added 1 ml of medium α containing 20% detalizirovannoi fetal bovine serum, and incubated for further 48 to 72 hours. After separating the supernatant and to check for the expression of antibodies using methods LTO-page and Western blotting. 3). LTO-page and Western blotting were performed according to the methods commonly used in this field, using the following materials: 12% of the LTO-polyacrylamide gel, PVDF-membrane (Millipore #EPVH00010, USA)conjugated with HRP antibody goat to IgG (Kappa) man and conjugated with HRP antibody goat for IgG (Fc) man (Pierce, USA).

Example 3: obtaining a cell line for producing DIG 0001 and the isolation and purification of antibodies

Cell line for producing DIG 0001 received cell-based CHO-DG44 (dhfr-deficient Cho). Transduction to obtain recombinant expressing antibodies cell lines based on the CHO-DG4 conducted in the same manner as explained above. For the screening of transduced cells CHO-DG44 (dhfr-positive phenotype) used nutrient medium of amem, not containing gipoksantina and thymidine, as selective markers in the primary screening of transduced cells CHO-DG44 used 500 μg/ml G418 (Sigma-aldrich, USA) and 400 μg/ml of zeocin (Invitrogen, USA). To obtain monoclonal colonies for expression of recombinant antibodies cells isolated during primary screening were diluted to a density of 10 cells/ml and seeded in 96-well plate (Nunc, USA). Then the diluted cells were incubated for 2 weeks and was isolated single colonies derived from a single cell, and received from them the parent cell clones. To obtain cell lines with high expression level of the parent cell clone was consistently perseval from 3 to 5 times in a medium containing methotrexat (MT) in different concentrations (40 nm, 80 nm, 160 nm, 320 nm and 700 nm), and then assessed the level of expression using ELISA. With this purpose, 96-well plate made of 100 μl of 2 μg/ml of primary antibodies, antibodies goats for IgG (Fc) man (Pierce, USA)and kept at 4°C for 12 hours to form a coating of antibodies. Then from each well was removed, remaining solution was added 200 μl of a blocking solution containing 2% skim milk in 1x FBI, and videri the Ali at 37°C for 1 hour. Each well three times washed with wash buffer containing 0.05% tween-20 in 1x FBI, and brought the culture fluid from the cell line CHO-DG44, expressing the antibody, the reaction was carried out at room temperature for 1 hour. Each well again three times washed with wash buffer and then flush the buffer was diluted in the ratio 1:5000 secondary antibody conjugated with HRP antibody goat to IgG (Kappa) man, to the reaction was added 100 μl of the resulting solution of the antibody, the reaction was carried out at room temperature for 1 hour. Each well again three times washed with wash buffer and brought to 100 ál of TMB substrate (BD biosciences, USA), the reaction was carried out for 5-10 minutes. Then, to stop the chromogenic reaction was added to 50 μl of 2N sulfuric acid (H2SO4). We measured the optical density (OD) (in the range of from 450 to 650 nm using microplate reader (ASAP, Switzerland). To confirm the reliability of the results was performed ELISA analysis on a similar scheme as described above, using VEGFR-2 and Tie-2 as the primary antibody and conjugated with HRP antibody goat to IgG (Kappa) as secondary antibodies. The resulting clone, which showed high level of expression at a concentration of MTX 700 nm, the separation is as if the cell line with high expression level (FIGURE 4). Incubation of cell lines with high levels of expression were carried out in a nutrient medium α (Welgene, Korea)containing 10% detalizirovannoi fetal bovine serum (KDR, Korea), 100 units/ml penicillin (Hyclone, USA) and 100 μg/ml streptomycin (Hyclone, USA), cultivation was performed in an incubator at 37°C. in the atmosphere OBLASTNOI air mixture containing 5% CO2.

Antibody dual directional DIG 0001, obtained by incubation of cell lines with high expression level, were subjected to the purification method JAHB using affinity column of protein A, column SP-separate columns exclusion chromatography and thus was purified to at least 95% and used in further studies (FIG. 5). First, the culture fluid was centrifuged in order to share the environment and the remains of the cells and then DIG 0001 contained in the separated nutrient medium was concentrated using a UF-membrane (Millipore, USA) to limit the bandwidth of 10,000 Da or less. Filtered through a UF-membrane environment was pre-purified by the method of affinal chromatography using protein A. a Brief description of this procedure is shown below. Filtered through UF-membrane environment was made on a column of protein a containing 0.1 M NaCl, stable 20 mm sodium phosphate buffer (pH 7.0), the same buffer enjoyed the Wali unbound protein. Then a protein that is not specific contacted with the resin containing protein a, washed 20 mm sodium phosphate buffer solution (pH 7.0)containing 0.5 M NaCl. A protein that is specific contacted with the protein And was suirable 0.1 M glycine-CL buffer (pH 3.5)containing 0.1 M NaCl, after which the selected sample was neutralized 1 M solution of Tris to a pH of 6.0. To prevent contamination of the sample DNA, endotoxin, protein And that may remain in the sample, selected by passing through the affinity column were cation-exchange chromatographic purification according to the following scheme. First, the sample suirvey from the column with protein A, was mixed with an equal volume of 20 mm sodium phosphate buffer (pH 6.0). Then a column of SP-separate (5 ml, GE healthcare) stabilized 10 mm sodium phosphate buffer (pH 6.0)containing 50 mm NaCl, and made her a sample and thus washed away unbound DNA and endotoxins. Bound peroxidase resin molecule antibodies were suirable using the modified values of pH and salt gradient (50 mm sodium phosphate buffer (pH 7.0), 1 M NaCl). Finally, to remove a multimeric antibodies, the sample was deposited on a column of Superdex 200 (16 mm × 60 cm, GE healthcare), stable FBI, and spent it cleaning method exclusion chromatography. Such antibodies, treated, were used to conduct analyses on cells and in vivo.

Primer; Competitive analysis DIG 0001

Competitive analysis by ELISA used to test whether competes antibody dual directional DIG 0001 with VEGF and Ang2 for binding to VEGFR-2 and Tie-2. To do this separately VEGF 165 and Ang2-RBD was distributed to 200 ng the wells of 96-hole tablet and for the formation of a coating of antibody 96-well plate was left at room temperature for a day. Then reaction was performed with 2% skim milk in the FBI at 37°C for 2 hours. After completion of the reaction 96-well plate were washed FBI and in wells with immobilized VEGF165 was made of a mixture of group a (mixture of this group were prepared by mixing various concentrations of DIG 0001 (0-250 nm) with VEGFR-2(ECD1-3))containing 100 ng Fc), which are pre-incubated at room temperature for 1 hour the reaction was carried out at room temperature for 2 hours. Then a mixture of group (a mixture of this group were prepared by mixing various concentrations of DIG 0001 (0-250 nm) with 500 ng Tie-2-Fc), which are pre-incubated at room temperature for 1 hour, was made in the wells with immobilized Ang2-RBD in the same way as described above, the reaction was carried out at room temperature for 2 hours. After two hours of reaction 96-well plate were washed FBI and to each well containing VEGF165, contributed 5 µg/ml of primary antibodies,mouse antibodies to VEGFR-2 (Reliatech, Germany), the reaction was carried out at 37°C for 1 hour. After washing the wells containing Ang2-RBD, phosphate buffer in each of the holes made 5 µg/ml of primary antibodies, mouse antibodies to Tie-2 (Abeam, UK), the reaction was carried out at 37°C for 1 hour in the same way as described above. After that, all wells containing VEGF165 and Ang2-RBD, made secondary antibody, diluted to 1:5000 conjugated with HRP antibody goat to mouse IgG (Abeam, UK), the reaction was carried out at 37°C for 1 hour. Then to initiate the chromogenic reaction was used reagent TMB substrate (BD biosciences, USA) and for stopping the reaction was added to 50 μl of 2N sulfuric acid (H2SO4). Evaluation of the passage of the chromogenic reaction was performed according to the spectral capacity of absorption at 450 nm and 650 nm using microplate reader (Tecan, Switzerland) (6).

Example 5: survival Analysis of HUVEC cells after exposure to DIG 0001

To determine changes in cell viability of umbilical vein endothelium human (HUVEC) after exposure to DIG 0001 assessed their survival. Incubation of HUVEC cells was carried out in a nutrient medium M199 (Invitrogen, USA)containing no phenol red and containing 20% fetal bovine serum (Hyclone, USA), 100 units/ml penicillin (Hyclone, USA), 100 μg/ml streptomycin (Hyclone, USA), 3 n who/ml fibroblast growth factor (pstate Biotechnology, USA) and 5 units/ml heparin (Sigma-Aldrich, USA), cultivation was performed in an incubator at 37°C. in the atmosphere OBLASTNOI air mixture containing 5% CO2. To assess the survival of endothelial cells of blood vessels these cells were made in a 24-well plate and incubated for 24 hours until the cell density reached 2×10 cells per well. After this 24-well plate was twice washed in culture medium M199 and over the next 6 hours, the cells were incubated in a nutrient medium M low serum (1% fetal bovine serum (Hyclone, USA)). Cells are first treated with various concentrations of the antibody for 30 minutes and then treated with 10 ng/ml VEGF (R&D systems, USA) and 100 ng/ml Ang1 (R&D systems, USA). After incubation for 48 hours, the cells within 2 hours was treated with WST-8 (Dojindo, Japan) and measured the absorbance at a wavelength of 450 nm. Then compare the obtained data about the viability of cells under different conditions (FIG.7).

Example 6: analysis of the migration of HUVEC cells after exposure to DIG 0001

To determine the inhibition of motility (chemotaxis) of HUVEC Cells after exposure to DIG 0001 assessing their migration. To assess the migration of HUVEC cells were used Transwell - polycarbonate liners filters in tablets with a pore size of 8 μm (Coming, USA). Before using the bottom of the filter covers the 10 mg of gelatin and dried. 10 ng/ml VEGF and 100 ng/ml Ang1 contributed to the lower hole containing a nutrient medium M containing 1% fetal bovine serum. Cells of the vascular endothelium that were pre-incubated in a nutrient medium containing low levels of serum for 6 hours, was divided by trypsin and diluted in culture medium M containing 1% fetal bovine serum to a density of cells 1×10 cells/ml Cells to the vascular endothelium pre-treated with different concentrations of antibody for 30 minutes, was distributed in 100 μl on the surface of the liner Transwell and incubated at 37°C for 3.5 hours in a cell incubator. Cultured cells were stained with hematoxylin and eosin (Sigma, SIEVES) or crystal violet (Sigma, USA), cells that did not migrate and remained on the outer surface of the filter were removed with a brush, cells that had migrated and was on the inner side of the filter, left. To compare the number of migrated cells these cells were photographed at 100-fold magnification with a microscope (Olympus, H, Japan) with a digital camera, calculation and analysis was performed on the results of 10 images obtained for each variant conditions analysis (FIG).

Example 7: analysis of the inhibition of the phosphorylation of VEGFR-2 and Tie-2 in the cells using methods immunoprobe the purpose and Western blotting

To assess the inhibition of phosphorylation of VEGFR-2 and Tie-2 when exposed to DIG 0001 conducted the analysis thus and Western blotting. Cells of the vascular endothelium after cultivation for 24 hours were incubated for 6 hours in a nutrient medium M199 containing 1% fetal bovine serum, and then subjected to pre-treatment antibody DIG 0001 26,7 mg/ml for 30 minutes. After that, the cells of the vascular endothelium was treated with VEGF at a concentration of 10 ng/ml Ang1 and at a concentration of 100 ng/ml for 15 minutes. For analysis thus cells of the vascular endothelial washed cold FBI and was treated with 500 μl of buffer solution for thus (1% Triton X-100, And 0.5% Nonidet P-40, 50 mm Tris/HCl (pH of 7.4), 150 mm NaCl, 2 mm argewandte sodium, 2 mm EGTA, 2 mm EDTA, 1 mm phenylmethylsulfonyl and 1 mm sodium fluoride). The solution was collected with a scraper, passed several times through a syringe (calibre needle 26), has achieved a sufficient degree of homogenization and centrifuged for 10 minutes at 12000g, followed by collecting the supernatant. To 300 g of solution was added to 2 μg immunoprecipitates of antibodies against Tie-2 (R&D systems, USA), the reaction was conducted for 8 hours. Then was added immobilized on agarose protein A/G (Santa Cruz Biotechnology, USA), which was associated with immunoprecipitation antibody. The resulting immune the complex, associated with immobilized onto agarose, protein A/G, centrifuged and washed 3-5 times with the buffer. Then, to remove agarose precipitates were added to the sample buffer on the basis of LTOs, boiled and centrifuged.

For analysis Western blotting cells of the vascular endothelium was treated with lytic buffer solution (1% (wt./about.) LTOs, 10 mm Tris (pH of 7.4), 2 mm argewandte sodium, 2 mm EGTA, 2 mm EDTA, 1 mm phenylmethylsulfonyl and 1 mm sodium fluoride) to obtain the solution to remove insoluble precipitates, boiled and centrifuged at 4°C at 10000 g for 5 minutes. The supernatant was mixed with sample buffer based on LTOs and boiled for 10 minutes. LTO-page and Western blotting were performed according to the methods commonly used in this field, using the following materials: 12% of the LTO-polyacrylamide gel, PVDF-membrane (Millipore #IPVH00010, USA), antibody to Tie-2 (abeam, UK) as primary antibodies for analysis of activity of inhibiting the phosphorylation of Tie-2 antibody to posterino (Upstate Biotechnology, USA); antibody to R/42 (Cell Signaling technology, USA) and antibody to phospho R/42 (Cell Signaling technology, USA); antibody to the ACT (Cell Signaling technology, USA) and antibody to phospho-ACT (Cell Signaling technology, USA); antibody to VEGFR-2 (Cell Signaling technology, USA) as primary antibodies for analysis of activity inhibin is of VEGFR-2 antibody to phospho-VEGFR-2 (Cell Signaling technology, USA); antibody to ACT (Cell Signaling technology, USA) and antibody to phospho-ACT (Cell Signaling technology, USA); and as a secondary antibody to bind with the primary antibodies for chemiluminescence conjugated with HRP antibody goat to mouse IgG (Santa Cruze Biotechnology, USA) and conjugated with HRP antibody goat to rabbit IgG (Santa Cruze Biotechnology, USA) 9).

Example 8: Inhibition of tumor growth under the action of DIG-0001 model of glioblastoma in an animal

As carriers of orthotopic glioblastoma used male mice of Balb/c-nu (Japan SLC), free from specific pathogens. According to known methods conducted intracerebral transplantation glioblastoma U-87MG (2×105cells from the American type culture Collection). The next day after intracerebral transplantation glioblastomas mice were divided into two groups (n=4) animals were injected with the following: (a) intraperitoneal injection of the FBI and (b) intraperitoneal injection of 0.5 mg/kg DIG-0001. All injections were repeated 5 times (15, 18, 21, 24 and 27, the day after making neoplastic cells). On the 29th day after the transplantation of neoplastic cells, mice were killed, brains were removed and did the front sections. One piece was fixed in a buffered solution of 10%formalin and were fixed in formalin, the remaining sections were concluded in the optimal environment for frozen sections (OST). Volume apocalyptically by measuring the volume of the fragment, most affected by the tumor, and calculating the volume using the following formula: Width2× Length × 0.5 in (FIG 10).

Industrial Applicability

The composition according to the present invention can be used to treat associated with angiogenesis diseases, in particular cancer.

Although it has been illustrated the preferred embodiment of the present invention, the experts in this field will understand that there may be changes and modifications, which will not go beyond the scope of the present invention in its broadest aspects. Various features of the present invention are listed in the following claims.

1. The antibody, which is a neutralizing antibody against VEGFR-2/KDR, hypervariable parts which are identical hypervariable portions of antibodies TTAC 0001 against VEGFR-2/KDR, fused with the binding domain of angiopoietin 2, which is the ligand of Tie-2, for the treatment of cancer by inhibiting angiogenesis.

2. The antibody according to claim 1, characterized in that said antibody comprises a neutralizing antibody, TAS 0001 against VEGFR-2/KDR.

3. The antibody according to claim 1, characterized in that the binding domain of angiopoietin 2 fused to the N-end of the heavy chain or light chain of the antibody via a linker.

4. DNA encoding the antibody according to any one of claims 1, 2 or 3.

5. The DNA according to claim 4, characterized in that said antibody is a neutralizing antibody against VEGFR-2/KDR, hypervariable parts which are identical hypervariable portions of antibodies TTAC 0001 against VEGFR-2/KDR, and light chain of the specified antibodies against VEGFR-2/KDR and binding domain of angiopoietin 2 encodes the sequence of bases of SEQ ID NO:6.

6. The recombinant expression vector containing the DNA according to claim 4.

7. The recombinant expression vector according to claim 6, containing pIgGLD-mAng2-TAS Lgt, map the cleavage of which is presented in figure 2.

8. A host cell which is a cell Chinese hamster ovary (CHO)transformed with recombinant expression vector according to claim 6 for obtaining antibodies.

9. A method of obtaining antibodies, including:
incubation host cell of claim 8; and
selection of antibodies from the culture fluid of the specified CHO cells.

10. The method according to claim 9, according to which the specified antibody additionally cleaned using liquid Express chromatography of proteins (IAHB), using affinity column of protein A, column SP-separate and exclusive chromatography.

11. Pharmaceutical composition comprising an effective amount of the antibody according to any one of claims 1, 2 or 3 and at least one pharmaceutically acceptable excipient, and found farmazevticheskay composition used to treat the associated with angiogenesis diseases.

12. The pharmaceutical composition according to claim 11, characterized in that the specified associated with angiogenesis disease selected from the group consisting of cancer, age-related macular degeneration, rheumatoid arthritis, diabetic retinopathy, psoriasis and chronic inflammation.

13. The pharmaceutical composition according to item 12, wherein the cancer is selected from the group consisting of stomach cancer, liver cancer, lung cancer, thyroid cancer, breast cancer, cervical cancer, colon cancer, pancreatic cancer, rectal cancer, colorectal cancer, prostate cancer, kidney cancer, melanoma, metastatic bone cancer, prostate cancer, ovarian cancer and cancer of the blood.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: compounds can be applied for treatment of oncologic and autoimmune diseases. Invention also characterises method of obtaining conjugates, pharmaceutical composition and medication, which contains modified proteins. In general formulae 1 or 2 , R1 is selected from the group representing (CH3)2N-,

R2 is selected from the group representing where R3 as terminal substituent represents -NH2, or and R4 represents H or C1-C3alkyl.

EFFECT: novel compounds possess affinity for CD16a receptor.

18 cl, 20 dwg, 3 tbl, 19 ex

FIELD: biotechnologies.

SUBSTANCE: chimeric monoclonal antibody is described, which specifically connects to human erythropoietin, characterised by the following criteria: a) Kd=2.4×10-9 M, isoelectric point in the range pH 7.5-8.0; b) sequence of the heavy chain SEQ ID NO:12; c) sequence of the light chain SEQ ID NO:14. A mouse hybridome strain is proposed, which is a producent of a monoclonal antibody to human erythropoietin, deposited in the Russian Academy of Agricultural Sciences, Specialised Collection of Cell Cultures of Farm and Game Animals under the No.84. Also a mouse anticlonal antibody is described, which specifically connects to human erythropoietin, produced by the specified hybridome and characterised by the following criteria: a) Kd=0.95×10-9 M, molecular weight = 160 kD, isopoint in the range pH 6.8-7.1; b) sequence of variable area of light chain SEQ ID NO:1; c) sequence of variable area of heavy chain SEQ ID NO:2; d) sequence of areas that define antibody complementarity: CDRH-1 - SEQ ID NO:5, CDRH-2 - SEQ ID NO:6, CDRH-3 - SEQ ID NO:7, CDRL-1 - SEQ ID NO:8, CDRL-2 - SEQ ID NO:9, CDRL-3 - SEQ ID NO:10.

EFFECT: invention makes it possible to expand arsenal of mouse antibodies against human erythropoietin.

3 cl, 3 dwg, 5 ex, 2 tbl

Antibodies to her // 2504553

FIELD: biotechnologies.

SUBSTANCE: invention describes versions of bispecific antibodies specifically bound to EGFR and HER3, which contain amino-acid sequences of variable regions of heavy and light chains respectively, SEQ ID NO: 30 and 29; or SEQ ID NO: 28 and 27; or SEQ ID NO: 28 and 29; or contain complementary regions CDR of heavy and light chains of the above sequences of variable regions. The invention describes nucleic acid coding a versions antibody, and a host cell containing the above nucleic acid and used for expression of the anitbody. Immunoconjugate containing antibody versions and cytotoxic agent used for treatment of cancer containing cells that express EGFR and HER3 are presented. A method for obtaining a bispecific antibody, which involves cultivation of a host cell so that an antibody is generated, is described. The invention describes a pharmaceutical composition for treatment of cancer containing EGFR- and HER3-expressing cells, which contains effective amount of bispecific antibody and pharmaceutically acceptable carrier. The invention proposes a treatment method of cancer containing EGFR- and HER3-expressing cells and an inhibition method of biological activity of EGFR and/or HER3 of a specimen, which involve introduction of effective amount of a bispecific antibody. Use of the above antibody in production of a remedy for treatment of cancer, the cells of which express EGFR and HER3, is described.

EFFECT: invention allows obtaining bispecific antibodies binding EGFR and HER3, which are not conjugates of two antibodies.

22 cl, 33 dwg, 4 tbl, 19 ex

FIELD: biotechnologies.

SUBSTANCE: two antibodies against IL-21 of a human being are presented. The first antibody includes a variable region of a heavy chain, which includes SEQ ID NO: 31, 33 and 35, and a variable region of a light chain, which includes SEQ ID NO: 39, 41 and 43. The second antibody includes a variable region of heavy chain, which includes SEQ ID NO: 47, 49 and 51, and variable region of light chain, which includes SEQ ID NO: 55, 57 and 59. Besides, the invention describes hybridomes producing the first and the second antibodies against IL-21 of a human being and deposited in the collection of cultures "American Type Culture Collection" and have numbers "ATCC Patent Deposit Designation PTA-8790" and "ATCC Patent Deposit Designation PTA-8786" respectively.

EFFECT: invention allows obtaining antibodies to IL-21 of a human being.

48 cl, 4 dwg, 16 tbl, 23 ex

FIELD: biotechnologies.

SUBSTANCE: method involves introduction to a plant, some part of the plant or a plant cell of nucleotide sequence for 80-100% of identical nucleotide sequence determined in SEQ ID NO: 17, and coding a composite protein containing a cytoplasmic end segment, a transmembrane domain, a steam area (CTS domain) of N-acetylglucosaminyl transferase (GNT1), which is merged with catalytic domain of beta-1,4-galactosyl transferase (GalT); with that, the above first nucleotide sequence is functionally connected to the first regulatory area being active in the plant; and the second nucleotide sequence for coding of a target protein; with that, the above second nucleotide sequence is functionally connected to the second regulatory area being active in the plant, as well as transient co-expression of the first and the second nucleotide sequences with synthesis of the target protein containing glycans, with reduced xylosylation, reduced fucosylation or their combination at comparison to the same target protein obtained from a wild plant. The invention described nucleic acid coding the protein that modifies glycosylation of target protein, a composite protein for modification of glycosylation of target protein; nucleic acid that codes it, as well as a plant, a plant cell and a seed, which contain the above nucleic acid or the above composite protein.

EFFECT: invention allows effective production of a target protein with reduced xylosylation, reduced fucosylation or their combination.

20 cl, 7 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes variable domains of heavy (VH) and light (VL) chains of murine antibody against tumour necrosis factor alpha (TNF-α) of a human being, as well as antigen-binding fragment Fab, which are selectively bound to TNF-α of the human being and neutralise it.

EFFECT: invention can be further used in development of medicines for therapy of TNF-α-mediated diseases and for diagnostics of such diseases.

3 cl, 5 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to a molecule of nucleic acid, which is a cyclic or a linear vector fit for expression, of at least one target polypeptide in cells of mammals, including (a) at least one expressing cassette (POI) for expression of the target polypeptide; (b) an expressing cassette (MSM), including a gene of a selective marker of mammals; (c) an expressing cassette (MASM), including an amplificated gene of a selective marker of mammals; besides, the expressing cassette (POI) is flanked in direction 5' by the expression cassette (MASM), the expression cassette (MSM) is localised in direction 3' from the expression cassette (POI) and in which the expression cassettes (MASM), (POI) and (MSM) are arranged in the same orientation from 5' to 3'. Also the method is disclosed to produce the specified molecule of nucleic acid of the vector, as well as a cell of a host mammal, containing the specified molecule of nucleic acid of the vector, the method to produce a host cell containing the specified molecule of nucleic acid of the vector, and also the method to produce the target polypeptide, using the specified host cell.

EFFECT: invention makes it possible to efficiently produce a target polypeptide in mammal cells.

24 cl, 2 dwg, 4 tbl, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. There are presented: a method for tumour cell growth inhibition in an individual and a method for immune response enhancement in an individual involving the introduction of a PD-1 monoclonal antibody and a CTLA-4 10D1 monoclonal antibody to the individual. The PD-1 monoclonal antibody has the following properties: it binds to human PD-1 having the value KD equal to 1×10-8 M or less; however it binds neither to human CD28, nor to CTLA-4, nor to ICOS; it is able to enhance the T-cell proliferation in the mixed lymphocyte reaction (MLR) analysis; it is able to enhance the gamma interferon production in the MLR analysis; it is able to enhance the interleukine-2 (IL-2) secretion in the MLR analysis.

EFFECT: invention provides a synergic effect when using the above antibodies in a combination.

4 cl, 54 dwg, 7 tbl, 25 ex

Novel antibodies // 2490277

FIELD: chemistry.

SUBSTANCE: present invention relates to immunology. Disclosed is an anti-α5β1 antibody, which is described through amino acid sequences of six hypervariable regions and an antigen-binding moiety thereof. Described are conjugates of the disclosed antibodies with a medicinal agent or a label, a pharmaceutical composition, use of the disclosed antibodies to prepare a medicinal agent, methods and an industrial product for inhibiting angiogenesis and/or vascular permeability in a subject, and for treating cancer, an ophthalmic disease and an autoimmune disease in a subject. The invention describes an isolated nucleic acid, an expression vector, a cell and a method of producing an antibody or an antigen-binding moiety thereof, as well as a method of detecting α5β1 protein in a sample.

EFFECT: present invention can find further use in therapy and diagnosis of α5β1-mediated diseases.

52 cl, 11 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: disclosed are versions of human IL13 specific antibodies and a producing hybridoma cell line deposited in ATCC under number PTA-5657. Described are: versions of encoding polynucleotides; an antibody expression vector; a host cell for antibody expression, as well as versions of a method of producing an antibody using a vector, polynucleotide, hybridoma or host cell. The invention discloses a pharmaceutical composition for treating IL13-mediated diseases and methods of treating allergic, inflammatory and other diseases, which employ an anti-IL13 antibody.

EFFECT: providing antibodies which do not bind with target IL13 and neutralise activity of human IL13.

39 cl, 29 dwg, 11 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used in agriculture and food industry. Constructed are recombinant nucleotide sequences which encode polypeptides (DGLA-synthase) which consist of delta-9-elongase and delta-8-desaturase, independently and separately exhibiting inherent enzymatic activity.

EFFECT: disclosed are genetic constructs containing said nucleotide sequences and host cells transformed by said constructs, preferably cells of oil plants and yeast, which express the active DGLA-synthase enzyme and can be used to obtain in said cells long-chain polyunsaturated fatty acids, particularly industrially significant dihomo-gamma-linolenic acid and eicosatetraenoic acid.

18 cl, 119 dwg, 36 tbl, 62 ex

FIELD: biotechnology.

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

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

4 cl, 4 dwg, 7 tbl, 9 ex

FIELD: biotechnology.

SUBSTANCE: hybrid proteins GFN80 and GFN100 are formed based on recombinant human interferon alpha-2 fused on the N-terminus with the amino acid sequence of polypeptide S(G4S)16 or S(G4S)20, respectively. The strains of producer Saccharomyces cerevisiae RNCIM Y-3927 and Saccharomyces cerevisiae RNCIM Y-3928 are produced by recombinant method. The strains are used in the method of production of the hybrid protein GFN80 and GFN100, which comprises culturing under suitable conditions of yeast cells transformed by the expression vector, which contains the region of replication initiation of endogenous 2-micron plasmid of yeast Saccharomyces cerevisiae, and the promoter of yeast GAL1 controlling the expression of the gene comprising the DNA sequence SEQ ID NO:1 or SEQ ID NO:2, respectively, followed by isolation of the hybrid protein from the culture fluid.

EFFECT: invention enables to produce the hybrid recombinant human interferon alpha-2 with the prolonged action in the body of animals.

5 cl, 7 tbl, 15 ex

FIELD: medicine.

SUBSTANCE: there are presented recombinant chimeric polypeptides rmBmpA-frp83, rmOspA-frp83, rmDbpB-rmOspA, rmFlaA-frFlaB and rmOspCBg-rmOspCBa, prepared on the basis of gene expression amplified by PCR on the DNA of Borrelia garinii 20047T Western-Siberian isolate or in case of the protein rmOspCBa, on the DNA of Borrelia afzelii isolate.

EFFECT: invention extends the range of recombinant polypeptides applicable for the serum diagnosis of ixodic tick-borne borreliosis, providing higher specificity and sensitivity of the ITBB, including the differential diagnosis of the early stage and the stage of a disseminated infection in the territories of Borrelia burgdorferi s1 Western-Siberian isolates.

8 cl, 2 dwg, 3 tbl, 5 ex

Fused rage proteins // 2513695

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biochemistry. Claimed is fused protein for treating diseases, mediated by advanced glycation end products (AGE), consisting of a fragment of a version of human receptor of advanced glycation end products (RAGE), which has two point mutations H217R and R221H, and a fragment of constant domain of human immunoglobulin IgG4, joined with linker if necessary. In addition, considered are: nucleic acid and recombinant host cell for obtaining fused protein, as well as pharmaceutical composition for treatment of AGE-mediated diseases, which contain fused protein.

EFFECT: invention ensures lower aggregation of fused protein.

13 cl, 19 dwg, 3 ex, 9 tbl

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology, gene and protein engineering and specifically to recombinant plasmid DNA pG1-Rm7, which facilitates synthesis of hybrid protein G1-Rm7 in Escherichia coli cells, which is capable of biding the tumour necrosis factor and has bioluminescence of luciferase Renilla muelleri, where said plasmid DNA includes the nucleotide sequence SEQ ID NO: 1 and can be in medicine. The invention also relates to the protein pG1-Rm7 having molecular weight of 65.4 kDa, consisting of a single-strand anti tumour necrosis factor antibody, a GGSGGS peptide and modified luciferase Renalla muelleri and characterised by SEQ ID NO: 2.

EFFECT: invention enables to obtain a highly sensitive reporter for detecting a tumour necrosis factor via bioluminescent analysis.

2 cl, 4 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, in particular to genetic engineering Claimed is artificial gene, which codes chimeric protein of human angiogenin Recombinant plasmid pJZZ-A, representing vector pJexpress414 and containing said gene, embedded on sites NdeI/XhoI, is described Also claimed is recombinant strain of E coli BL21(DE3)/pJZZS-A.

EFFECT: invention makes it possible to increase efficiency of hybrid gene expression and increase production of recombinant chimeric protein of human angiogenin

3 cl, 3 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: present group of inventions relates to biotechnology. What is presented is a humanised anti-CD79b antibody and its antigen-binding fragment produced of murine antibody MA79b and CD79b having a substantially analogous binding affinity thereto. A polynucleotide, a vector, a host cell and a method for producing the anti-CD79b antibody according to the invention; immunoconjugates, compositions and methods for cell growth inhibition, a method of treating an individual suffering cancer, a method of treating a proliferative disease and tumour in a mammal, a method for B-cell proliferation inhibition; a method for detecting the presence of CD79b in a sample and method for binding the antibody to the CD79b expressing cell are also disclosed.

EFFECT: given invention can find further application in therapy of the CD79b associated diseases.

86 cl, 20 tbl, 9 ex, 51 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine. What is presented is a vaccine against pneumonia caused by Streptococcus pneumoniae, on the basis of a hybrid protein described by SEQ ID NO:1, containing fragments of the proteins Streptococcus pneumoniae PspA, Sprl 895, PsaA, as well as the ingredients of flagellin as an adjuvant, connected by flexible bridges.

EFFECT: invention provides the effective prevention and therapy of pneumonia ensured by the fact that the vaccine hybrid protein is composed of various immunogenic epitopes eliciting a specific immune response with formed immunological memory.

4 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to genetic engineering, specifically to creation of fibroblast growth factor receptor (FGFR) muteins, and can be used in medicine. The polypeptide of the FGFR4 receptor extracellular domain (ECD) acidic region mutein is an FGFR4 ECD chimera or a FGFR4 long acid box version and has more acid residues in the D1-D2 linker region than the wild-type FGFR4 ECD. The muteins may include a point mutation that inhibits glycosylation. The mutein is used to treat a disease associated with one or more FGFR ligands, e.g., proliferative diseases, including various types of cancer, angiogenic disorders and macular degeneration.

EFFECT: invention enables to obtain an FGFR4 ECD acidic region mutein, having low capacity to bind with tissue, by increasing the number of amino acid residues within the D1-D2 linker region.

32 cl, 22 dwg, 11 tbl, 18 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to immunology and biotechnology. Claimed is binding protein for binding one or more targets, which contains four polypeptide chains forming four functional antigen-binding sites. Four polypeptide chains contain VD1-(X1)n-VD2-C-(X2)n. VD1 stands for first variable domain of heavy chain, VD2 stands for second variable domain of heavy chain, C stands for CH1 domain, X1 stands for polypeptide linker, on condition that it is not constant domain, and X2 stands for Fc-region, and n equals 0 or 1. Two polypeptide chains contain VD1-(X1)n-VD2-C. VD1 stands for first variable domain of light chain, VD2 stands for second variable domain of light chain, C stands for CL domain, X1 stands for linker, on condition that it is not constant domain; and n equals 0 or 1. Conjugate of binding protein with visualising detecting cytotoxic or therapeutic agent is described. Disclosed are: nucleic acids (NA), coding polypeptide chains, as well as expressing vectors, vectors for replication, host cells which contain them, and method of obtaining antibody applying cells. Described is pharmaceutical composition for treatment or preventing target-associated disease or disorder based on binding protein. Method of treatment by introduction of binding protein is described.

EFFECT: application of invention provides new format (DVD-Ig) of antigen-binding molecules, which in the same dosage possess higher activity with respect to target than respective full-size antibodies, which can be applied in medicine for prevention and treatment of various diseases.

45 cl, 27 tbl, 5 ex

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