Pcsk9 antagonists

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology and biotechnology. There are presented: an isolated antibody or its variant specifically recognising PCSK9, and a based pharmaceutical composition for lowering LDL-cholesterol. There are described: using for lowering blood cholesterol and/or LDL; reducing an incidence rate and/or correcting abnormal cholesterol and/or LDL; using for preparing a drug for lowering blood cholesterol and/or LDL; reducing the incidence rate and/or correcting abnormal cholesterol and/or LDL. There are disclosed versions of cell lines producing PCSK9 antibody or its antigen-binding portion and deposited in the American typical culture collection (ATCC) under Nos. PTA-8986, ATCC PTA-8984, ATCC PTA-8983, respectively. What is described is a coding nucleic acid and a host cell for producing the based antibody.

EFFECT: invention provides PCSK9 agonist antibodies that can find application in medicine for lowering cholesterol.

18 cl, 24 dwg, 9 tbl, 9 ex

 

The area belongs to this invention

The present invention relates to antibodies, for example a full-sized antibodies or their antigennegative parts, peptides and aptamers that counteract the activity of extracellular probely convertase subtilisin Kexin type 9 (PCSK9), including its interaction with the receptor low-density lipoprotein (LDLR). More specifically, this invention relates to compositions containing antagonistic antibodies against PCSK9, peptides and/or aptamers, and to methods of using these antibodies and/or peptides, and/or aptamers as a drug. Antagonistic antibodies, peptides and aptamers against PCSK9 can be used for therapeutic purposes to reduce cholesterol levels-LDL in the blood and can be used for the prevention and/or treatment of disorders of the metabolism of cholesterol and lipoproteins, including familial hypercholesterolemia, atherogenic dyslipidemia, atherosclerosis, and, more generally, cardiovascular disease (CVD).

Prerequisites to the creation of inventions

Millions of people in the U.S. are at risk for developing heart disease and subsequent cardiac events. CDV and underlying atherosclerosis is the leading cause of death among all demographic groups, despite the availability of methodology the treatment, aimed at multiple risk factors. Atherosclerosis is a disease of the arteries and is responsible for coronary heart disease associated with numerous deaths in industrialized countries. Currently, some risk factors for coronary heart disease are installed: dyslipidemia, hypertension, diabetes, Smoking, poor nutrition, sedentary lifestyle and stress. The most clinically relevant and common dyslipidemia characterized by increased beta-lipoproteins (very low density lipoproteins (VLDL) and LDL) with hypercholesterolemia or hypertriglyceridemia (Fredrickson et al., 1967, N Engl J Med. 276:34-42, 94-103, 148-156, 215-225, and 273-281). There is a long felt a significant unmet need for treatment with CDV 60-70% of cardiovascular events, heart attacks and strokes, despite treatment with statins (modern standard treatment of atherosclerosis). Moreover, the new standards require that must be achieved even lower LDL in order to protect patients with high risk early CDV [National Cholesterol Education Program (NCEP), 2004].

PCSK9, also known as NARC-1, was identified in some forms of familial hypercholesterolemia as a protein with a mutation. PCSK9 synthetic which varies as Imogene, which in the endoplasmic reticulum exposed autocatalytically processing in motive LVFAQ. Population studies have shown that some mutations in PCSK9 are mutations "purchasing options" and found in individuals with autosomal dominant hypercholesterolemia, while other mutations with loss of function (LOF) is associated with reduced cholesterol in blood plasma. Research causes of morbidity and mortality in this group clearly demonstrate that reduction of PCSK9 function significantly reduces the risk of cardiovascular disease.

The result is important for the treatment of CDV LOF mutation may increase sensitivity to statins, ensuring efficiency at a lower dose (thereby reducing the risks associated with security and stability) and potentially reaching lower levels of cholesterol in the blood plasma than with conventional treatment methods.

PCSK9 is secreted into the plasma mainly by hepatocytes. Genetic modulation of PCSK9 in mice confirmed the ability of PCSK9 to regulate blood lipids and showed that its action is aimed at the suppression of hepatic LDLR protein levels.

The mechanism of suppression and place of application steps, which inhibits PCSK9 LDLR protein has not been clearly established. In the case of overexpression of PCSK9 can act is in hepatocytes, and in the form of a secreted ligand for the LDLR. There is clear evidence that extracellular PCSK9 binds to the cell surface LDLR and contributes to the degradation of the LDLR in the intracellular location. However, it is also possible that PCSK9 may interact with LDLR, when two proteins are translated inside the endoplasmic reticulum (ER) and move through endosomal compartments towards the cell membrane. Maxwell et al., 2005, Curr. Opin. Lipidol. 16:167-172, showed that PCSK9 mediated LDLR endocytosis and degradation did not change under the influence of inhibitors of the proteosome and did not modelirovanie different classes of lysosomal and alsoany proteases. Two natural mutation in the family hypercholesteraemia, S127R and D129G, as reported, were with defect autoprocessing and secretion, because the levels of these mutant proteins were greatly reduced or not was determined in the medium of transfected cells. These mutants showed an increased ability to suppress LDLR, based on their identification of individuals with high levels of LDL in the blood plasma (Homer et al., 2008, Atherosclerosis 196:659-666; Cameron et al., 2006 Human Molecular Genetics 15:1551-1558; Lambert et al., 2006, TRENDS in Endocrinology and Metabolism 17:79-81). Since these mutants are obviously not are secreted extracellular and yet suppress LDLR, this clearly shows that the intracellular location of the application on istia is physiologically important.

From the information available in the art prior to the present invention, it is unclear whether the introduction of PCSK9 antagonist based on the antibody, peptide or aptamer into the bloodstream to selectively counteract extracellular PCSK9 effective to reduce hypercholesterolemia and associated incident CDV and, if so, what properties of the PCSK9 antagonist required for such effectivenessin vivo.

The invention

This invention relates to the antagonistic antibodies, peptides and aptamers that selectively interact with PCSK9 and inhibit its function. For the first time demonstrated that some PCSK9 antagonists are effectivein vivoto reduce blood cholesterol.

In one embodiment, the present invention relates to isolated PCSK9 antagonist, which contains the antibody, peptide or aptamer, which vzaimodeistvie with PCSK9 and with the introduction of the patient lowers cholesterol-LDL in the blood of the patient. This antagonist can be an antibody such as a monoclonal antibody or a human antibody, humanitariannet or chimeric antibody.

In another embodiment, the invention relates to isolated anti-PCSK9 antibody, which specifically binds to PCSK9 and which is a full antagonist mediated by PCSK9 de the effects on the levels of LDLR in the measurement of in vitrothrough analysis of the suppression of the LDLR in Huh7 cells, as disclosed in the present description.

In another embodiment, this invention relates to an isolated antibody that counteracts the extracellular interaction of PCSK9 to LDLR, as determined by the binding of PCSK9 to LDLRin vitroand, when administered to the patient, reduces cholesterol-LDL in the blood of the patient. Preferably this antibody recognizes an epitope on human PCSK9, which overlaps with more than 75% of the surface of PCSK9, which interacts with the EGF-like domain of the LDLR, as described by Kwon et al., 2008, PNAS, 105:1820-1825.

In another embodiment, this invention relates to an antibody that recognizes a first epitope on PCSK9, which overlaps with the second epitom, which is recognized by a monoclonal antibody selected from the group consisting of 5A10, which is produced by hybridoma cell line deposited in the American type culture Collection and having registration number PTA-8986; 4A5, which is produced by hybridoma cell line deposited in the American type culture Collection and having registration number PTA-8985; 6F6, which is produced by hybridoma cell line deposited in the American type culture Collection and having registration number PTA-8984 and 7D4, which is produced by g is britaney cell line, deposited in the American type culture Collection and having registration number PTA-8983.

In another embodiment, this invention relates to a PCSK9 antibody, where the antibody recognizes an epitope on human PCSK9 containing amino acid residues 153 to 155, 194, 195, 197, 237-239, 367, 369, 374-379 and 381 amino acid sequence of PCSK9 sequence SEQ ID NO: 53. Preferably the epitope for antibodies to PCSK9 person does not contain one or more of the amino acid residues 71, 72, 150-152, 187-192, 198-202, 212, 214-217, 220-226, 243, 255-258, 317, 318, 347-351, 372, 373, 380, 382 and 383.

In another embodiment, this invention relates to an antibody that specifically binds to PCSK9, containing the first complementarity determining region (CDR1) VH, with the amino acid sequence presented in SEQ ID NO: 8 (SYYMH), VH CDR2 with the amino acid sequence presented in SEQ ID NO: 9 (EISPFGGRTNYNEKFKS), and/or VH CDR3 with the amino acid sequence presented in SEQ ID NO: 10 (ERPLYASDL), or a variant having one or more conservative amino acid substitutions in said sequences of CDR1, CDR2 and/or CDR3 where this option retains essentially the same specificity as CDR specified by the specified sequences. Preferably this option contains approximately ten amino acid substitutions andmore preferably approximately four amino acid substitutions.

The invention additionally aims to antibody containing a VL CDR1 with the amino acid sequence presented in SEQ ID NO: 11 (RASQGISSALA), CDR2 with the amino acid sequence presented in SEQ ID NO: 12 (SASYRYT), and/or CDR3 with the amino acid sequence presented in SEQ ID NO: 13 (QQRYSLWRT), or a variant having one or more conservative amino acid substitutions in said sequences of CDR1, CDR2 and/or CDR3, where this option retains essentially the same specificity as CDR1, specified by the specified sequences. Preferably this option contains approximately ten amino acid substitutions, and more preferably approximately four amino acid substitutions.

In another embodiment, this invention relates to the antibody containing the specific sequence of the VL CDR1, CDR2 and/or CDR3, or a variant having one or more conservative amino acid substitutions in said sequences of CDR1, CDR2 and/or CDR3, and additionally contains a complementarity determining region VH CDR1 with the amino acid sequence presented in SEQ ID nos: 59, 60, or 8, VH CDR2 with the amino acid sequence presented in SEQ ID NO: 61 or 9, and/or VH CDR3 with the amino acid sequence presented in SEQ ID NO: 10, or a variant having one Il is somewhat conservative amino acid substitutions in said sequences of CDR1, CDR2 and/or CDR3, where this option retains essentially the same binding specificity as CDR1, CDR2 and/or CDR3, certain specified sequences. Preferably this option contains approximately twenty amino acid substitutions, and more preferably approximately eight amino acid substitutions. In another preferred embodiment, the antibody according to this invention has a variable sequence of the heavy chain, containing or consisting of the sequence SEQ ID NO: 54, and the variable sequence of the light chain containing or consisting of the sequence SEQ ID NO: 53.

This invention also provides humanitariannet antibody containing polypeptides selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, or both of the sequences SEQ ID NO: 14 and SEQ ID NO: 15, or a variant having one or more conservative amino acid substitutions in said sequences, where this option retains essentially the same binding specificity as the antibody defined by a specified sequence (sequences). It also includes an antibody with a missing terminal lysine on the heavy chain, as it is usually lost in parts of antibodies in the production process.

Preferably this option contains approximately on the twenty amino acid substitutions, and more preferably approximately eight amino acid substitutions. Preferably the antibody further comprises immunologically inert constant region and/or the antibody has an isotype that is selected from the group consisting of IgG2, IgG4, IgG2∆a, IgG4∆b, IgG4∆c, IgG4S228P, IgG4∆bS228P and IgG4∆cS228P. In another preferred embodiment, this constant region is deglycosylated Fc.

In one embodiment, the invention provides a method of reducing LDL, cholesterol-LDL or total cholesterol in the blood, serum or plasma of a patient in need thereof, comprising introducing into the body of the patient a therapeutically effective amount of an antagonist according to this invention.

In one embodiment, the invention provides a therapeutically effective amount of the antagonist according to this invention for use in reducing LDL, cholesterol-LDL or total cholesterol in the blood, serum or plasma of a patient in need thereof. The invention additionally provides the use of a therapeutically effective amount of the antagonist of this invention in the manufacture of a medicine for lowering LDL, cholesterol-LDL or total cholesterol in the blood, serum or plasma of a patient in need this is.

In yet another embodiment, the invention provides a method of obtaining antibodies that specifically binds PCSK9, which includes: (a) provide PCSK9-negative animal host; (b) immunization specified PCSK9-negative animal host using PCSK9; and (c) obtaining antibody, antibody productive cells or nucleic acid that encodes the antibody from the specified PCSK9-negative animal host, and obtaining the antibody from the specified antibody productive cells or specified nucleic acid that encodes the antibody.

The invention also encompasses a method of reducing LDL levels in blood of a patient in need thereof, comprising introducing into the body of the patient a therapeutically effective amount of the antibodies obtained in accordance with this invention. The patient then may be subjected to treatment by administration of a statin. In the preferred embodiment, this patient is the man.

In one embodiment, the antibody used in the composition in the form of a sterile aqueous solution having a pH ranging from about 5.0 to about 6.5 and containing antibody from about 1 mg/ml to about 200 mg/ml his-tag buffer is from about 1 millimolar to about 100 millimolar the tion, Polysorbate 80 from about 0.01 mg/ml to about 10 mg/ml trehalose from about 100 mm to about 400 mm, and disodium EDTA dehydrate from about 0.01 millimolar to about 1.0 in millimolar.

In another embodiment, the invention provides a therapeutically effective amount of the antibodies obtained in accordance with this invention, for use in lowering LDL levels in blood of a patient in need thereof. The invention additionally provides the use of a therapeutically effective amount of this antibody, obtained in accordance with this invention, in the manufacture of a medicine for lowering LDL levels in blood of a patient in need thereof. This therapeutically effective amount may be combined with a therapeutically effective amount of the statin.

In another embodiment, the invention provides a hybridoma cell line which produces PCSK9-specific antibody, or antigennegative area where this hybridoma cell line selected from the group consisting of:

4A5, having ATCC registration number PTA-8985;

5A10, having ATCC registration number PTA-8986;

6F6, having ATCC registration number PTA-8984;

7D4, having ATCC registration number PTA-983.

In another embodiment, the invention provides a cell line that recombinante produces an antibody that specifically binds to PCSK9 and contains the first complementarity determining region (CDR1) variable regions of the heavy chain (VH)having the amino acid sequence represented in SEQ ID NO: 8, 59 or 60, VH CDR2 having the amino acid sequence represented in SEQ ID NO: 9 or 61, and/or VH CDR3 having the amino acid sequence represented in SEQ ID NO: 10, or a variant having one or more conservative amino acid substitutions in CDR1, CDR2 and/or CDR3, and/or contains variable regions CDR1 light chain (VL) amino acid sequence presented in SEQ ID NO: 11, VL CDR2 with the amino acid sequence presented in SEQ ID NO: 12, and/or VL CDR3 with the amino acid sequence presented in SEQ ID NO: 13, or a variant having one or more conservative amino acid substitutions in CDR1, CDR2 and/or CDR3. Preferably the cell line recombinante produces the antibody containing the sequence of SEQ ID NO: 53 and/or 54, and more preferably the sequence of SEQ ID NO: 14 and/or 15.

A brief description of the drawings/figures

The Figure 1 presents the effect of anti-PCSK9 antagonist monoclonal antibodies 7D4.4, 4A5.G3, 6F6.G10.3 and 5A10.B8 able to the th mouse PCSK9 (A) and human PCSK9 (B) to suppress the LDLR in cultured Huh7 cells. 6F6.G10.3 is subclan 6F6, 7D4.4 is subclan 7D4, 4A5.G3 is subclan 4A5, and 5A10.B8 is subclan 5A10.

The Figure 2 presents the effect of dose of anti-PCSK9 antagonist monoclonal antibodies 6F6.G10.3, 7D4.4, 4A5.G3, 5A10.B8, antibody negative control 42H7 and PBS to the blockade of the binding of recombinant biotinylated human PCSK9 (A) and mouse PCSK9 (B) with immobilized extracellular domain of recombinant LDLRin vitro.

The Figure 3 presents the effect of dose of anti-PCSK9 antagonist monoclonal antibodies 6F6.G10.3, 7D4.4, 4A5.G3 and 5A10.B8 to the blockade of the binding of recombinant biotinylated human PCSK9 (30 nm) to the extracellular domain of recombinant LDLR (10 nm)labeled with europium, in solution with a neutral pH value ofin vitro.

The Figure 4 presents the comparative binding of anti-PCSK9 antibody epitope.

The Figure 5 shows Western blots of communication anti-PCSK9 antibodies with serum PCSK9 from different species.

The Figure 6 presents the effect of anti-PCSK9 monoclonal antibody 7D4 on the levels of blood cholesterol in mice.

The Figure 7 shows (A) the effect of partially antagonistic polyclonal anti-PCSK9 mAb CRN6 on the generally hopeless regulation of LDLR and (B) no effect on cholesterol levels in mice.

The Figure 8 shows the dynamics of the effect of lowering cholesterol levels will reach the CSOs application of anti-PCSK9 antagonist antibodies 7D4 in mice.

The Figure 9 shows the dose-dependent effect of anti-PCSK9 antagonist mAb 7D4 at reducing total cholesterol, HDL and LDL in the serum of mice.

Figure 10 depicts the dose-dependent effect of anti-PCSK9 antagonist antibody 5A10 to reduce cholesterol levels in mice.

In the Figure 11 depicts the dose-dependent effect of anti-PCSK9 antagonist antibodies 4A5 (a) and 6F6 (b) for the reduction of cholesterol levels in mice.

The Figure 12 shows Western blots of action of anti-PCSK9 antagonist antibodies on the levels of LDLR in the liver.

The Figure 13 shows the lack of effect of anti-PCSK9 antagonist antibodies 4A5 on a murine model LDLR-/-.

The Figure 14 shows the effect on total serum cholesterol repeated injections of anti-PCSK9 antagonist antibodies in mice for a longer time than that observed after administration of a single dose.

The Figure 15 shows the dynamics of the effects of the anti-PCSK9 antagonist antibodies 7D4 on lipid parameters in the model for humanoid monkeys.

In the Figure 16 presents the effect of dose and dynamic response of anti-PCSK9 antagonist antibodies 7D4 on the levels of serum cholesterol in apes.

Figure 17 presents a comparison of the actions of anti-PCSK9 antagonist antibodies 4A5, 5A10, 6F6 and 7D4 on the levels of serum cholesterol in man is Breznik monkeys.

The Figure 18 shows the dynamics of the effect of anti-PCSK9 antagonist antibodies 7D4 on cholesterol levels in blood plasma from apes, fed the diet from 33.4% kcal fat, supplemented with 0.1% cholesterol.

The Figure 19 shows the effect L1L3 (humanitariannet anti-PCSK9 monoclonal antibody) on the generally hopeless regulation of LDLR in Huh7 cells.

The Figure 20 shows the effect of dose gumanitarnogo antibodies L1L3, mouse predecessor 5A10 and antibody negative control 42H7 to the blockade of the binding of recombinant biotinylated human PCSK9 (A and B) and mouse PCSK9 (C and D) with the immobilized extracellular domain of recombinant LDLRin vitroat pH 7.5 (A and C) and pH of 5.3 (B and D).

Figure 21 presents the effect on levels of blood cholesterol after treatment of mice L1L3 at a dose of 10 mg/kg

The Figure 22 shows the effect of introducing antibody 5A10 or L1L3 apes and measure changes in HDL serum (A) and LDL serum (B) as a function of time.

Figure 23A shows the crystal structure of PCSK9 (light gray surface)associated with the antibody L1L3 (image of black color). Figure 23B shows the crystal structure of PCSK9 (light gray surface)associated with EGF-like domain of the LDLR (images black) (Kwon et al., PNAS, 105, 1820-1825, 2008). Figure 23C shows an image of the surface of PCSK9 with what pitapa for L1L3, marked in dark gray. Figure 23D shows the image surface PCSK9 epitope for EGF-like domain of the LDLR, marked in dark gray.

In Figures 24A-G presents replacement, made in the CDR of the antibody 5A10 in the process of maturation and optimization of affinity and ensure certain properties. Also presents the binding of PCSK9 associated with antibodies having these substitutions in the CDR. The number after each sequence represents the SEQ ID NO assigned to each sequence.

Detailed description of the invention

The present invention relates to antibodies, peptides and aptamers that counteract the function of extracellular PCSK9, including its interaction with the LDLR. More specifically, this invention relates to methods for antagonistic antibodies, peptides and aptamers against PCSK9, compositions containing these antibodies, peptides and/or aptamers, as a medicinal product. Antagonistic antibodies and peptides against PCSK9 can be used to reduce cholesterol levels-LDL blood and can be used for the prevention and/or treatment of disorders of the metabolism of cholesterol and lipoproteins, including familial hypercholesterolemia, atherogenic dyslipidemia, atherosclerosis, and, more generally, CDV.

Common procedures

When practical OS is the implementation of the present invention will be involved, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), Microbiology, cell biology, biochemistry and immunology, which are among the skills in this area. Such technologies are described fully in the literature sources, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Definitions

"Antibody" is an immunoglobulin molecule able to specifically bind a target, such as carbohydrate, polynucleotide, lipid, polypeptide and the like, using at least one is the second portion of the recognition of the antigen, located in the variable region of the immunoglobulin molecules. The term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single-chain (ScFv) and domain antibodies fused proteins containing part of the antibodies, and any other modified configuration of the immunoglobulin molecules, which contains a plot of the recognition of the antigen. The antibody includes the antibody of any class such as IgG, IgA or IgM (or a subclass), and this antibody should not be of a particular class. Depending on or antibody-based test amino acid sequence of the constant domain of the heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The constant domains of the heavy chains, which correspond to the different classes of immunoglobulins are called alpha, Delta, Epsilon, gamma and mu, respectively. Structures of subunits and three-dimensional configurations of different classes of immunoglobulins are well known.

The term "monoclonal antibody" refers to an antibody obtained from a population essentially homogeneous antibodies, namely individual antibodies included : the tion, are identical except for possible naturally occurring mutations that can be introduced in small quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Moreover, in contrast to the preparations of polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The definition of "monoclonal" indicates the character of the antibody as obtained from the essentially homogeneous population of antibodies, and should not be construed as a requirement to receive this antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be obtained by hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be obtained by methods of recombinant DNA, such as described in U.S. patent No. 4816567. Monoclonal antibodies can also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example.

The term "humanitariannet" antibody refers to forms of non-human (in the example, murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2or other antigennegative subsequences of antibodies)which contain minimal sequence derived from the immunoglobulin other than human. Preferably humanized antibodies are human immunoglobulins (antibody-recipient), in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of species that are not human (antibody-donor), such as a mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases the remains of the frame Fv region (FR) of a human immunoglobulin replaced by residues that do not come from man. Moreover, humanitariannet antibody may contain residues that are not found either in the antibody-recipient, nor in the imported CDR or frame sequences, but are included for additional purification and optimization steps antibodies. In most cases humanitariannet antibody will contain essentially the whole of at least one, and typically two, variable domain, in which all or substantially all areas of the CDRs correspond to those of immunoglobulin other than human,and all or substantially all areas FR represent those of the consensus sequence of human immunoglobulin. Optimally this humanitariannet antibody will also contain at least part of a constant region or domain (Fc) of an immunoglobulin, usually one of a human immunoglobulin. Preferred are antibodies having Fc region modified as described in WO 99/58572. Other forms of humanized antibodies have one or more CDR (CDR L1, CDR L2, CDR-L3, CDR-H1, CDR H2, and/or CDR H3), that are modified with respect to the original antibody, which are also referred to as one or more "derived from" one or more CDRs of the source antibody.

The term "human antibody" means an antibody having the amino acid sequence corresponding to that of antibodies, which are produced in humans and/or which was produced using any of the techniques for making human antibodies known to the person skilled in the art, or disclosed here. This definition of human antibodies includes an antibody that contains at least one polypeptide heavy chains of a human or at least one polypeptide light chain of a human. One such example is an antibody containing light chain polypeptides mouse and heavy chains of a human. Human antibodies can be obtained using various techniques known in the art. In one embodiment, the human antibody selected from Pago the second library, where that phage library expresses human antibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, Proc. Natl. Acad. Sci. (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581). Human antibodies can also be obtained by immunization of animals who locus of human immunoglobulin was introduced in transgenic location endogenous locus, such as mice, in which genes of endogenous immunoglobulin have been partially or completely inactivated. This approach is described in U.S. patent№№ 5545807; 5545806; 5569825; 5625126; 5633425; and 5661016. Alternative human antibody can be obtained using immortalized In human lymphocytes that produce an antibody directed against a target antigen (such b lymphocytes can be obtained from the individual or can be immunizedin vitro). See, for example, Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., 1991, J. Immunol., 147 (1):86-95; and U.S. patent No. 5750373.

The term "variable region" refers to the variable region of the light chain of the antibody or variable region of the heavy chain of the antibody, either individually or in combination. As is known in the prior art, the variable regions of the heavy chain and light chain each consists of four frame regions (FR)connected by three complementarity determining regions (CDR), which contain the ISU is variabelny areas. Data CDRs in each chain are fastened together in close proximity by using data FR and data CDRs from the other chain, contributing to the formation of antigenspecific centers antibodies. There are at least two ways CDR: (1) the method based on interspecific variation sequences (namely Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD)); and (2) a method based on crystallographic studies of complexes of the antibody-antigen (Al-Iazikani et al, 1997, J. Molec. Biol. 273:927-948). As used here, the CDR can be attributed to the CDR, defined either by using a single method or a combination of both methods.

As is known in the prior art, the "constant region" of an antibody refers to the constant region of the light chain of the antibody, or the constant region of heavy chain antibodies, either individually or in combination.

The term "PCSK9" refers to any form of PCSK9 and its variants that retain at least partial activity of PCSK9. Unless otherwise noted, for example, through specific references to human PCSK9, PCSK9 includes native PCSK9 sequence of all mammalian species, e.g. human, dog, cat, horse and bull. One example of PCSK9 person found in the Uniprot data Bank with access number Q8NBP7 (SEQ ID NO:16).

The term "PCSK9 antagonist" refers to the antibody, peptide or aptamer, which can inhibit the biological activity of PCSK9 and/or to suppress the cascade(s) reactions, mediated signaling by PCSK9, including PCSK9-mediated generally hopeless regulation of LDLR, and PCSK9 mediated decrease clearance of LDL from the blood. Antagonistic antibody against PCSK9 encompasses antibodies that block, oppose, supression or reduce (to any degree, including significant) biological activity of PCSK9, including top-down metabolic pathway mediated signaling by PCSK9, such as interaction with LDLR and/or detecting a cellular response to PCSK9. For the purpose of the present invention will be clearly understood that the term "antagonistic antibody against PCSK9" encompasses all the previously identified terms, titles, and functional forms and characteristics, due to which he PCSK9 biological activity of PCSK9 (including, but not limited to its ability to mediate any aspect of its interaction with the LDLR, oppressive regulation of LDLR and decrease clearance of LDLR blood) or manifestations of biological activity essentially cancelled, reduced or neutralized in any significant degree. In some embodiments, implementation, antagonistic antibody against PCSK9 binds to PCSK9 and prevents interaction with the LDLR. There are examples of antagonistic antibodies against PCSK9.

Full antagonist" is an antagonist, which, when effective the Noy concentration, essentially, completely blocks measurable effect of PCSK9. Under partial antagonist understand antagonist, which could partially block measurable effect, but which, even at the highest concentration, not a full antagonist. Under essentially complete implies that measurable effect blocked at least about 80%, preferably at least about 90%, more preferably at least about 95% and most preferably at least about 98%, or 99%. Relevant measurable effects described here and include generally hopeless regulation of LDLR by PCSK9 antagonist, as tested on Huh7 cellsin vitro, lower levels of total cholesterol in the blood (or plasma)in vivoand lower levels of LDL in the blood (or plasma)in vivo.

The term "clinically significant" means, at least, a reduction of 15% of LDL-cholesterol in humans or, at least, a reduction of 15% of the levels of total blood cholesterol in mice. It is obvious that measurement of plasma or serum can serve as substitutes for measuring levels in the blood.

The term "antagonistic peptide against PCSK9" or "antagonistic aptamers against PCSK9" includes any common polypeptide or apt the EP, that inhibits, prevents, suppresses or reduces (to any degree, including significant) biological activity of PCSK9, including top-down metabolic pathway mediated signaling by PCSK9, such as interaction with LDLR and/or detecting a cellular response to PCSK9. Antagonistic peptides or polypeptides against PCSK9 include the merger of Fc containing LDLR and soluble part of the LDLR, or their mutations with higher affinity to PCSK9.

The terms "polypeptide", "Oligopeptide", "polypeptide" and "protein" are used herein interchangeably to denote chains of amino acids of any length, preferably relatively short (e.g., 10-100 amino acids). This chain may be linear or branched, and may contain modified amino acids, and/or may be interrupted by non-amino acids. These terms also include amino acid chain that has been modified naturally or by intervention, for example, disulfide bond formation, glycosylation, lipiduria, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with component label. Also in this definition are included, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids and t is th equivalent) as well as other modifications known in the prior art. Assume that the polypeptides may occur in a single circuit or in the form of integrated circuits.

As is known in the prior art, the terms "polynucleotide" or "nucleic acid", used herein interchangeably, refer to chains of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be built into the chain using DNA or RNA polymerase. Polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogues. If present, changes in the structure may be transferred before or after Assembly of this circuit. The nucleotide sequence can be interrupted dinucleotide components. Polynucleotide may be further modified after polymerization, for example, by conjugation with component label. Modifications of other types are, for example, "caps", substitution of one or more natural nucleotides and their analogs, mezhnukleotidnyh modifications such as, for example, modification by introduction of uncharged compounds (for example, methylphosphonate, phosphotriesters, phosphamidon, carbamates and the like) and charged the of soedinenii (for example, fosforito, phosphorodithioates and the like), modification containing side groups, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, and the like), modification, contain intercalating agents (e.g., acridine, psoralen, etc.), modification, containing hepatoblastoma agents (e.g., metals, radioactive metals, boron, metals, oxidizing agents, and the like), modification, containing alkylating agents, modification, containing modified compounds (e.g., alpha anomeric nucleic acid and the like), as well as unmodified forms of polynucleotide(s). In addition, any of the hydroxyl groups originally present, may be replaced by, for example, phosphonate groups, phosphate groups, protected by standard protective groups, or activated for more connections to additional nucleotides, or may be conjugated with a solid-phase substrate. 5' and 3' terminal OH group can be phosphorylated or replaced by amines or organic motifs blocking groups ranging in length from 1 to 20 carbon atoms. Other hydroxyl group can also be derivatization standard protective groups. Polynucleotide can also contain functionally similar forms of sugars ribose or de who exiliboa, which is well known in the prior art, including, for example, 2'-O-methyl, 2'-O-allyl, 2'-fluoro - or 2'-azido-ribose, carbocyclic analogues of sugars, alpha - or beta-anomeric sugars, epimeria sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugar, sedoheptulose, acyclic analogs and analogs abasically nucleosides, such as methylribose. One or more fosfodiesterazu can be replaced by alternative linker groups. These alternative linker groups include, but are not limited to, options for implementation, in which the phosphate is replaced by P(O)S ("tiat"), P(S)S ("ditial"), (O)NR2(amidate"), P(O)R, P(O)OR', CO or CH2(formatall"), in which each R or R' represents independently H, or substituted or unsubstituted alkyl (1-20 C)optionally containing either (-O-) connection, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldi. Not all communication polynucleotide must be identical. The above description applies to all used here polynucleotides, including RNA and DNA.

"Aptamer antagonist against PCSK9, which contains a nucleic acid or protein sequence, for example, selected from a large Bank of random sequences and specifically binds to PCSK9. Nucleic acid of this aptamer is dwuhtsepochechny the th DNA or single-stranded RNA. Aptamers consisting of a nucleic acid may include modified bases or functional group, including, but not limited to, 2'-fluorine-containing nucleotides and 2'-O-methyl nucleotides. Aptamers can include hydrophilic polymers, such as polyethylene glycol. Aptamers can be obtained by methods known in the art and selected for antagonistic action against PCSK9 in the routine modifications of the methods disclosed in the Examples.

The antibody, peptide or aptamer "interact with" PCSK9 in the case when the equilibrium dissociation constant equal to or less than 20 nm, preferably less than about 6 nm, more preferably less than about 1 nm, most preferably less than about 0.2 nm, as measured using the methods disclosed in Example 2.

The epitope, which preferably binds" or "specifically binds" (used here interchangeably) antibody or peptide, is a well studied in the prior art, the concept, and how to determine such specific or preferred network is also well known in the prior art. The molecule, as they say, has "specific binding" or "preferred binding"if it reacts or connects more often, more quickly, with more long-term the duration and/or with greater affinity with a particular cell or substance, than with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and/or more long-term than with other substances. For example, an antibody that specifically or preferentially binds to an epitope on PCSK9, is an antibody that binds to this epitope with greater affinity, avidity, more readily, and/or longer than it binds to other epitopes on PCSK9 or not-PCSK9-epitopes. After reading this definition is also clear that, for example, the antibody (or motif or epitope), which specifically or preferentially binds to the first target may specifically or preferentially to contact or may not contact with the second target. Essentially, a "specific binding" or "preferred binding" does not necessarily imply (although it may enable) exclusive binding. In most cases, but not necessarily, the reference to the linking means, the preferred binding.

The term "essentially pure" refers to a substance which at least 50% pure (i.e. contains no impurities), more preferably at least 90% pure, more preferably at least 95% pure, E. the e is more preferably at least 98% pure and most preferably at least 99% pure.

The term "a host cell" includes an individual cell or cell culture which can be or have been recipients for vectors (vectors) for embedding polynucleotide inserts. Cell hosts include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic complement of DNA) of the original parent cell due to natural, accidental, or deliberate mutation. The term a host cell includes cells, transfetsirovannyhin vivousing polynucleotide (polynucleotides) according to this invention.

As is known in the prior art, the term "Fc region" is used to denote the C-terminal region of the heavy chain of immunoglobulin. "Fc region" may be a natural sequence Fc region or variant Fc region. Despite the fact that the boundaries of the Fc region of the heavy chain of immunoglobulin may vary, Fc-region of the heavy chain of IgG person is usually determined by the segment from amino acid residue in position Cys226, or from Pro230, to its terminal carboxyl group. The numbering of the residues in the Fc-region corresponds to that in the index pointer of the EU, as in Kabat (Kabat). Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. Fc region of immunoglobulin usually contains two constant domain, and CH3 C2.

Used in the prior art, the term "Fc receptor" and "FcR" describes a receptor that binds to the Fc region of antibodies. Preferred FcR is a natural sequence FcR person. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the subclasses of the FcγRI, FcγRII and FcγRIII, including allelic variants and forms of alternative splicing of these receptors. The FcγRII receptors include FcγRIIA (an"activating receptor") and FcγRIIB (an"inhibiting receptor"), which have similar amino acid sequences that differ primarily by their cytoplasmic domains. Review of FcR are presented in Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, Immunomethods, 4:25-34; and de Haas et al., 1995, J. Lab. Clin. Med., 126:330-41. "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., 1976 J. Immunol., 117:587; and Kim et al., 1994, J. Immunol., 24:249).

The term "compete", as used here in relation to the antibody, means that the first antibody or the antigen-binding site binds to the epitope in a sense quite the same with the binding of the second antibody, or antigennegative plot, thus, the binding of the first antibody with its paired epitope significantly reduced in the presence of second antibody compared to what vyzyvaniem the first antibody in the absence of the second antibody. The alternative, whereby the binding of this second antibody to its epitope is also markedly reduced in the presence of this first antibody may be, but not necessarily. Thus, the first antibody can inhibit the binding of the second antibody to its epitope, without inhibition data of the second antibody bind this first antibody with its corresponding epitope. However, in the case where each antibody significantly inhibits the binding of another antibody with its paired epitope or ligand, either in the same, more or less, these antibodies are called "cross-competing" with each other for binding to their respective epitopes (epitopes). And competing and cross-competing antibodies covered by the present invention. Regardless of the mechanism of occurrence of such competition or cross-competition (e.g., steric mismatch, conformational change, or linking with the shared epitope or part of it), the person skilled in the art will take into account, based on the information provided here, that such competing and/or cross-competing antibodies are covered and can be used in the methods disclosed here.

Under an antibody epitope, which "overlaps" with other (secondary) epitome, or the surface is part of an on PCSK9, which interacts with the EGF-like domain on LDLR assume joint use area relative to the PCSK9 residues that interact. For the calculation of the percentage of the area of overlap, for example the percentage of the area of overlapping epitope on PCSK9 for the claimed antibodies with the surface of PCSK9, which interacts with the EGF-like domain on the LDLR, the surface area of PCSK9 is hidden when in complex with LDLR calculated based on the residue. This hidden surface are also calculated for these residues in the complex PCSK9:the antibody. To prevent more than 100% of the possible overlapping surface area for residues that have a more extensive hidden surface area in the complex PCSK9:antibody than in LDLR complex:PCSK9, was set to values of LDLR complex:PCSK9 (100%). The percentage area of overlap is calculated by summing all of the interacting residues LDLR:PCSK9 and weighted by the area of interaction.

"Functional Fc region has at least one effector function of native sequence Fc region. Examples of "effector functions" include C1q binding; complementability cytotoxicity; the binding of the Fc receptor; antibody-dependent cretaceouspaleogene cytotoxicity; phagocytosis; generally hopeless regulation of receptors on the cell surface (n is an example, receptor In cells) and the like. Such effector functions in most cases require a combination of Fc-region-binding domain (e.g., variable domain antibodies) and can be estimated using a variety of research, known in the prior art for the assessment of such effector functions of antibodies.

"Native sequence Fc region contains the amino acid sequence identical to the amino acid sequence of Fc-field, discovered in nature. "Variant Fc region" contains an amino acid sequence that differs from the native sequence Fc region of at least one amino acid modification, despite this saves at least one effector function of native sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or Fc region of the parent polypeptide, e.g. from about one to approximately ten amino acid substitutions, and preferably from approximately one to approximately five amino acid substitutions in a native sequence Fc region or Fc region of the parent polypeptide. Variant Fc region will preferably possess at least 80% of identichnost the Yu with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least 90% sequence identity with it, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity with it.

"Treatment" is a method of obtaining beneficial or desired clinical results. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: increased clearance of LDL and decrease the incidence or intensity reduction abberant levels of cholesterol and/or lipoproteins in the result of metabolic disorders and/or eating disorders, or including familial hypercholesterolemia, atherogenic dyslipidemia, atherosclerosis, and, more generally, cardiovascular disease (CDV).

"Reducing incidence" means reducing the severity, which may include reducing need for and/or quantity (e.g., exposure to) other drugs and/or therapies generally used for this condition. As is clear to a person skilled in the art, individuals may vary in their response to treatment, and, in fact, for example, "method of reducing incidence" displays the application antagonistical the th antibody peptide or aptamer against PCSK9 on the basis of reasonable expectation that such administration may likely cause such a reduction in incidence in that particular individual.

"Improvement" means the decrease in the intensity or positive dynamics of one or more symptoms compared with no use of antagonistic antibody, peptide or aptamer against PCSK9. "Improvement" also includes shortening or reducing the duration of the symptom.

"Effective dose" or "effective amount" of the drug, compound or pharmaceutical composition is an amount sufficient to achieve one or more beneficial or desired results. For preventive use is beneficial or desired results include the elimination or reduction of risk, reduction of the severity, or delay the manifestation of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes, defined during the development of the disease. For therapeutic applications beneficial or desired clinical results include results such as a reduction of hypercholesterolemia or one or more symptoms Dilip the of the Academy, atherosclerosis, CDV, or ischemic heart disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another drug, and/or delay progression of the disease in patients. Effective dose can be introduced into the body in one or more injections. For the purposes of this invention, an effective dose of the drug, compound or pharmaceutical composition is an amount sufficient to perform a prophylactic or therapeutic treatment, either directly or indirectly. As is clear in the clinical sense, an effective dose of the drug, compound or pharmaceutical composition may or may not be provided in combination with another drug, compound or pharmaceutical composition. Thus, the "effective dose" can be considered in the context of the use of one or more therapeutic agents, and the only therapeutic agent can be considered as introduced in an effective amount if, in conjunction with one or more other means, the desired result is achieved or can be achieved.

"Individual" or "patient" is a mammal, more preferably human. Mammals also on the hunger but not limited to, farm animals, animals-participants in sports, Pets, primates, horses, dogs, cats, mice and rats.

The term "vector" means a construct that is able to deliver, and, preferably, to Express one or more genes of interest or sequences in the cell host. Examples of vectors include, but are not limited to, viral vectors, vectors expressing naked DNA or RNA, plasmids, Comedy or phage vectors, vectors expressing the DNA or RNA associated with cationic condensing agents, vectors expressing the DNA or RNA encapsulated in liposomes, and some eukaryotic cells, such as cells-producers.

The term "sequence that controls the expression" means a nucleic acid sequence which controls transcription of the nucleic acid. Sequence controlling the expression may be a promoter such as a constitutive or inducible promoter or enhancer. The sequence controlling the expression of functionally associated with the transcribed sequence of the nucleic acid.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any substance which is toroe, when combined with the active ingredient, allows the ingredient to retain biological activity and is directionspanel with the immune system of the patient. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as phosphate buffered saline solution, water, emulsions, such as emulsion type water in oil, and various types of moisturizers. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or normal saline solution (0.9%). Compositions containing such media are developed by well known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing, 2000).

The term "kon" refers to the rate constant for the Association of the antibody with the antigen. In particular, the rate constant (konand koff) and equilibrium dissociation constants are measured using fragments of the antibody Fab (namely monovalent) and PCSK9.

The term "koff" refers to the rate constant for the dissociation of antibodies from complex antibody/antigen.

The term "KD" refers to the equilibrium dissociation constant of the interaction of antibody-antigen.

A. SPO is usually used for prevention or treatment of diseases, associated with hypercholesterolemia

In one aspect this invention relates to a method of treatment or prevention of hypercholesterolemia, and/or at least one symptom of dyslipidemia, atherosclerosis, CDV or coronary heart disease in an individual, comprising the introduction into the organism of a particular individual an effective amount of an antagonistic antibody or peptide or aptamer against PCSK9, opposing circulating PCSK9.

In an additional aspect, the invention provides an effective amount of an antagonistic antibody, peptide or aptamer against PCSK9, opposing circulating PCSK9, for use in the treatment or prevention of hypercholesterolemia, and/or at least one symptom of dyslipidemia, atherosclerosis, CDV or ischemic heart disease in the individual. The invention additionally provides the use of an effective amount of an antagonistic antibody, peptide or aptamer against PCSK9, opposing or extracellular circulating PCSK9, when the industrial production of pharmaceuticals for the treatment or prevention of hypercholesterolemia and/or at least one symptom of dyslipidemia, atherosclerosis, CDV or ischemic hearts from the individual.

Preferably therapeutic application of antibodies is, peptide or aptamer results in reduction of blood cholesterol and/or decreased LDL blood. Preferably the blood cholesterol and/or LDL blood at least about 10% or 15% lower than before the introduction. More preferably, the blood cholesterol and/or LDL blood at least about 20% lower than before the introduction of antibodies. Even more preferably the blood cholesterol and/or LDL blood at least about 30% lower than before the introduction of antibodies. Advantageously, if the blood cholesterol and/or LDL blood at least approximately 40% lower than before the introduction of antibodies. More advantageously, if the blood cholesterol and/or LDL blood at least approximately 50% lower than before the introduction of antibodies. Very preferably, if the blood cholesterol and/or LDL blood at least about 60% lower than before the introduction of antibodies. Most preferably, if the blood cholesterol and/or LDL blood at least about 70% lower than before the introduction of antibodies.

With respect to all methods described herein, reference antagonist antibody, peptide, or aptamer against PCSK9 also includes compositions containing one or more additional substances. These compositions can optionally contain suitable inert fillers, such as pharmaceutically acceptable excipients, including buffers, which the choir is known in the prior art. The present invention can be used alone or in combination with other conventional treatment methods.

Antagonistic antibody, peptide or aptamer against PCSK9, you can enter into the body of an individual via any suitable route of administration. Specialist in this field should be obvious that the examples described herein are not intended to limit, and to illustrate the available methods. Accordingly, in some embodiments, the implementation of an antagonistic antibody peptide or aptamer against PCSK9 is injected into the body of the individual in accordance with known methods, such as intravenous administration, e.g., in the form of a bolus or by continuous infusion over a period of time, intramuscularly, intraperitoneally, inside the spinal cord, percutaneous, subcutaneous, intraarticular, sublingual, intra-articular, via insufflation, podvoloshino, oral, by inhalation or local routes of administration. Use can be system, for example intravenously, or local. Effective for use are commercially available vaporizers for liquid compositions, including ejector and ultrasonic inhalers inhalers. Liquid compositions can be directly entered via inhalation, and the dried powder may be what led spray after recovery. Alternative antagonistic antibodies, peptides or aptamers against PCSK9 can be aerolithe using fluorocarbon compositions and dosage inhaler or Invalidovna in the form of a lyophilized and milled powder.

In one embodiment, antagonist antibody, peptide, or aptamer against PCSK9 is applied by using a site-specific technology or technologies targeted local delivery. Examples of site-specific technologies or technologies targeted local delivery include various implantable depot sources of the antagonistic antibody, peptide or aptamer against PCSK9, or catheters for local delivery, such as catheters for infusion, permanent catheter, or needle catheters, synthetic implants, adventitious cuff, shunts and stents or other implantable devices, site specific carriers, direct injection, or is directed introduction. See, e.g., PCT publication no WO 00/53211 and U.S. patent No. 5981568.

Various compositions antagonist antibody, peptide, or aptamer against PCSK9 can be used for injection. In some embodiments, the implementation can be entered pure antagonist antibody, peptide, or aptamer against PCSK9. In some embodiments, the implementation of an antagonistic antibody, peptide or uptime is against PCSK9 and pharmaceutically acceptable filler can be in various compositions. Pharmaceutically acceptable excipients are known in the prior art, and they are relatively Intertime substances that facilitate the introduction of a pharmaceutically effective substances. For example, the filler can give form or consistency or to act as a diluent. Suitable fillers include, but are not limited to, stabilizing substances, moisturizing or emulsifying agents, salts to change the osmolarity, encapsulating agents, buffers, and substances that enhance penetration through the skin. Fillers and compositions for parenteral and aparentally delivery drugs described in Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing (2000).

These agents can be combined with pharmaceutically acceptable carriers, such as saline, ringer's solution, dextrose and the like. The particular dosage regimen, that is, dose, time and repetition, will depend on the specific individual and medical history of a particular individual.

Antibodies against PCSK9 you can also enter through inhalation, as described herein. In most cases, the application of antibodies against PCSK9, possible primary dose may be approximately 2 mg/kg In the context of the present invention a typical daily dose may nahodites is in the range from about 3 μg/kg to about 30 μg/kg, up to 300 mcg/kg to 3 mg/kg, 30 mg/kg to 100 mg/kg or more, depending on the above factors. For example, you can apply a dose of approximately 1 mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 10 mg/kg and about 25 mg/kg For re-introductions for several days or more, depending on the condition, treatment continues until a desired suppression of symptoms, or until until you have achieved a satisfactory therapeutic levels, for example, to reduce the levels of LDL in the blood. An exemplary dosage regimen includes the introduction of the initial dose of about 2 mg/kg, with subsequent weekly supporting dose of antibodies against PCSK9 approximately 1 mg/kg, or followed by a dose of approximately 1 mg/kg every two weeks. However, you can apply a different dosage regimes, depending on the nature of the pharmacokinetic decay. For example, in some embodiments, the implementation provides a dose of from one to four times per week. In other embodiments, the implementation provided by the dose once a month or once in two months, or once in three months. The progress of this treatment can easily be tracked using conventional methods and analyses. The dosage regimen (including that of the antagonist(s) PCSK9) may ranged Atsa over time.

In the context of the present invention an adequate dose of antagonist antibody, peptide, or aptamer against PCSK9 will depend on the applicable antagonist antibody, peptide, or aptamer against PCSK9 (or their combinations), the type and severity at treatment of symptoms, whether the substance as a preventive or therapeutic purposes, previous therapy, the clinical history of the patient and response to this matter, PCSK9 levels of blood in a patient, the rate of synthesis and clearance of PCSK9 in a patient, the rate of clearance of injected substances the patient and judgment of the attending physician. Usually the doctor will prescribe an antagonistic antibody, peptide or aptamer against PCSK9 until then, until you reach the dose at which get the desired result. Dose and/or frequency may change during the course of treatment. Empirical evidence, such as half-life, as a rule, contribute to determining the dose. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to extend the time half-life of antibodies and prevent attacks on the antibody by the immune system of the host body. The frequency of injection can be determined and adjusted over the course of treatment, and, as a rule, but neo is satalino, based on treatment and/or suppression and/or facilitate and/or delay of symptoms, such as hypercholesterolemia. An alternative can be adequately slow continuous release compositions antibody antagonists against PCSK9. Various compositions and devices for achieving sustained release known in the prior art.

In one embodiment, dosages for antagonistic antibody, peptide or aptamer can be determined empirically in individuals who have had one or more injections antagonist antibody, peptide, or aptamer. Individuals enter gradually increasing doses of the antagonist antibody, peptide, or aptamer against PCSK9. To evaluate the effectiveness can be tracked indicator of this disease.

Introduction antagonistic antibody peptide or aptamer against PCSK9 in accordance with the method according to the present invention can be continuous or intermittent, depending, for example, on the physiological state of the recipient, whether the prophylactic or therapeutic, and other factors known to skilled practitioners. Introduction antagonist antibody, peptide, or aptamer against PCSK9 may be essentially continuous over a predetermined period of time or can the be series divided by time doses.

In some embodiments, the implementation can be more than one antagonist antibody, peptide, or aptamer. May be present at least one, at least two, at least three, at least four, at least five different, or more, antagonistic antibodies and/or peptides. Typically, these antagonistic antibodies or peptides against PCSK9 may have complementary activities that do not negatively impact each other. Antagonistic antibody, peptide or aptamer against PCSK9 can also be used in combination with other antagonists against PCSK9 or antagonists against the receptor and PCSK9. For example, you can use one or more of the following antagonists against PCSK9: antisense molecule directed to PCSK9 (including antisense molecule directed to a nucleic acid encoding PCSK9), a compound inhibiting PCSK9, and a structural analogue of PCSK9. Antagonistic antibody, peptide or aptamer against PCSK9 can also be used in combination with other substances, which are designed to enhance or Supplement the effectiveness of these agents.

Acceptable carriers, excipients or stabilizers are non-toxic in the doses and concentrations, and may include buffers such as phosphate, citrate and other organizations the practical acid; salts, such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (such as octadecylsilane ammonium chloride; chloride hexadecane; benzalconi chloride, benzene chloride; phenol, butyl or benzyl alcohol; alkylarene, such as methyl or propyl paraben; catechin; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; hepatoblastoma substances, such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; soleobrazutaya counterions such as sodium; metal complexes (e.g., complexes of Zn-protein); and/or nonionic surfactants such as TWEENTM, PLURONICSTMor polyethylene glycol (PEG).

Liposomes containing antagonistic antibody, peptide or aptamer against PCSK9 obtained by methods known in the prior art, such as described in Epstein, et al., 1985, Proc. Natl. Acad. Sci. USA, 82:3688; Hwang, et al., 1980, Proc. Natl Acad. Sci. USA, 77:4030; and U.S. patent No. 4485045 and 4544545. L is bosomy with increased circulation time are disclosed in U.S. patent No. 5013556. Extremely effective liposomes can be obtained by the method of reverse-phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivationally the phosphatidylethanolamine (PEG-PE). Liposomes extruded through filters with defined pore size to obtain liposomes with the desired diameter.

Active ingredients may also be enclosed in microcapsules obtained, for example by the method koatservatsii or by polymerization at the phase boundary, for example hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems tools (for example, liposomes, albumen microspheres, microemulsions, nanoparticles and nanocapsules), or in the microemulsions. Such methods are described in Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing (2000).

Can be obtained drugs with a slow release. Suitable examples of drugs with a slow release include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of slow release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), Paul is the lactides (U.S. patent No. 3773919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable copolymer of ethylene and vinyl acetate, degradable copolymers of lactic acid-glycolic acid such as LUPRON DEPOTTM(injectable microspheres composed of copolymer of lactic acid-glycolic acid and leuprolide acetate), isobutyrate sucrose acetate and poly-D-(-)-3-hydroxybutiric acid.

Compositions used to applyin vivomust be sterile. This requirement is easily performed, for example, by filtration through sterile filtration membranes. therapeutic composition antagonist antibody, peptide, or aptamer against PCSK9 is usually placed in a container having a sterile inlet opening, for example a bag of IV solution or bottle with stopper, produceesas with a needle for subcutaneous injection.

Suitable emulsions may be prepared using commercially available fat emulsions, such as IntralipidTM, LiposynTM, InfonutrolTM, LipofundinTMand LipiphysanTM. The active substance may be either dissolved in a pre-mixed emulsion composition, or alternatively may be diluted in the oil (e.g. soybean oil, safflower oil, oil from cotton seeds, sesame oil, corn oil, and almond oil) and em is lsia is prepared by mixing the phospholipid (for example, phospholipids eggs, soybean phospholipids or lecithin soybeans and water. You should take into account that there may be added other ingredients such as glycerol or glucose, to correct toychest this emulsion. Suitable emulsion will typically contain up to 20% oil, for example between 5 and 20%. The fat emulsion may contain droplets of fat from 0.1 to 1.0 μm, in particular from 0.1 to 0.5 μm, and have a pH value in the range from 5.5 to 8.0.

Data emulsion compositions can be of a composition obtained by mixing an antagonistic antibody, peptide or aptamer against PCSK9 with IntralipidTMor its components (soybean oil, phospholipids eggs, glycerin and water).

Compositions for inhalation or insufflation include solutions, suspense in pharmaceutically suitable aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions may contain appropriate pharmaceutically acceptable excipients, as described above. In some embodiments, the implementation of these songs are introduced orally or through the nasal respiratory route for local or systemic effect. The composition preferably in a sterile pharmaceutically acceptable solvents can be sprayed by using gases. Sprayed solutions you can inhale during the war of the spray device, or spray device can be attached to the face mask, tent or breathing apparatus for intermittent positive pressure. Composition of solutions, suspensions or powders can be introduced, mainly, oral or nasal, using devices that deliver this song properly.

B. Antagonists of PCSK9

In the methods according to this invention using antagonistic antibody, peptide or aptamer against PCSK9, which refer to any peptide or nucleic acid molecule that blocks, suppresses or reduces (including significantly reduces) the biological activity of PCSK9, including top-down metabolic pathway mediated signaling by PCSK9, such as the identification of the cellular response to PCSK9.

Antagonistic antibody, peptide or aptamer against PCSK9 should show one or more of the following properties: (a) contact PCSK9; (b) to block the interaction of PCSK9 to LDLR; (c) block or decrease PCSK9-mediated generally hopeless regulation of LDLR; (d) to inhibit PCSK9-mediated decreased clearance of LDL, (e) increase clearance of LDL in the medium by cultured hepatocytes, (f) increase clearance of LDL from the blood through the liverin vivo(g) to improve sensitivity to statins, and (h) to block the interaction of PCSK9 with the other, even identical and factors.

For the purposes of this invention, the antibody, peptide or aptamer, preferably reacts with PCSK9 thus, to inhibit signaling function PCSK9 and interaction with the LDLR. In some embodiments, the implementation of the antibody antagonist against PCSK9 specifically recognizes PCSK9 primates. In some embodiments, the implementation of the antibody antagonist against PCSK9 binds to PCSK9 primates and rodents.

Antibodies are effective in the present invention can encompass monoclonal antibodies, polyclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, Fv, Fc, etc.), hybrid antibodies, bespecifically antibodies, heteroconjugate antibodies, single chain (ScFv), mutants fused proteins, including antibodies (for example, domain antibodies), human antibodies, humanized antibodies, and any other modified configuration of the immunoglobulin molecules, which contains a plot of the recognition of antigen required specificity, including glycosylated variants of antibody variants amino acid sequences of antibodies, and covalently modified antibodies. Antibodies may be murine, rat, human, or any other origin (including hybrid or humanized antibodies).

In some embodiments, the implementation of the antibody antagonist against the PCSK9 submitted is a monoclonal antibody. Antibody antagonist against PCSK9 can also be humanized. In other embodiments, the implementation of this antibody is a human antibody.

In some embodiments, the implementation of this antibody contains a modified constant region, such as a constant region that is immunologically inert, that is, having a reduced potential to provoke an immune reaction. In some embodiments, the implementation of this constant region is modified, as described in Eur. J. Immunol., 1999, 29:2613-2624; PCT publication No. WO99/58572; and/or patent application in the UK No. 9809951.8. Fc can be an IgG2person or IgG4man. Fc can be an IgG2individuals containing the mutation A330P331 in S330S331 (IgG2∆a), in which amino acid residues are numbered according to the sequence of wild-type IgG2. Eur. J. Immunol., 1999, 29:2613-2624. In some embodiments, the implementation of this antibody contains a constant region of IgG4containing the following mutations (Armour et al., 2003, Molecular Immunology 40 585-593): E233F234L235 in P233V234A235 (IgG4∆c), numbered in accordance with IgG4 wild type. In another embodiment, Fc represents IgG4man E233F234L235 in P233V234A235 with deletion G236 (IgG4∆b). In another embodiment, Fc is a Fc of any IgG4human(IgG4, IgG4∆b 4∆c), which contains a stabilizing mutation of the hinge area of S228 in P228 (Aalberse et al., 2002, Immunology 105, 9-19). In another embodiment, this Fc can be deglycosylated Fc.

In some embodiments, the implementation of the constant region is deglycosylated in the modification of the remainder of the attachment of oligosaccharide (such as Asn297) and/or flanking residues that are part of a sequence of recognition of glycosylation in this constatnly area. In some embodiments, the implementation of this constant region is an enzymatic deglycosylation for N-linked glycosylation. The constant region can be deglycosylation for N-linked glycosylation enzyme or as a result of expression in the cell host with a lack of glycosylation.

The affinity of binding (KD) antibody antagonist against PCSK9 with PCSK9 (e.g., PCSK9 person) may be from about 0.002 to about 200 nm. In some embodiments, the implementation of the affinity of the binding may be any of approximately 200 nm, about 100 nm, about 50 nm, about 10 nm, about 1 nm, about 500 PM, about 100 PM, about 60 PM, about 50 PM, about 20 PM, about 15 PM, will bring the flax 10 PM about 5 PM or around 2 PM. In some embodiments, the implementation of the affinity of binding is less than about 250 nm, about 200 nm, about 100 nm, about 50 nm, about 10 nm, about 1 nm, about 500 PM, about 100 PM, about 50 PM, about 20 PM, about 10 PM, about 5 PM or around 2 PM.

One way to determine the affinity of binding of the antibody to PCSK9 is to determine the affinity of binding of monofunctional Fab fragments of this antibody. In order to get monofunctional Fab fragments, antibody (e.g., IgG) should be split using papain or downregulation recombinante. The affinity to PCSK9 Fab fragment antibodies can be determined using surface plasmon resonance system (surface-plasmon resonance (SPR) Biacore3000TM, Biacore, INC, Piscataway NJ), equipped with predisposizione streptavidin sensor chips (SA) using a rolling buffer HBS-EP (0,01M HEPES, pH of 7.4, and 0.15 NaCl, 3 mm EDTA, 0.005% of (vol/vol) surfactant P20). Biotinylated human PCSK9 (or any other PCSK9) can be diluted in buffer HBS-EP to a concentration of less than 0.5 μg/ml and injected through separate channels of the chip at different contact time, to achieve the two ranges of density is of Tigana, or 50-200 reaction units (RU) for detailed kinetic studies, or 800-1000 EN for screening analysis. Studies of regeneration showed that 25 mm NaOH in 25% (vol/vol) ethanol effectively removes the Association of the Fab, while retains the activity of PCSK9 on the chip for over 200 injections. Typically, serial dilutions (perekryvayushchimisya a concentration of 0.1-10× estimated KD) of purified Fab samples are injected for 1 min at 100 μl/min and allowed time dissociation up to 2 hours. The concentration of Fab proteins was determined by ELISA and/or electrophoresis in SDS-PAGE using Fab known concentration (as determined by amino acid analysis) as standard. The kinetic rate of Association (konand the rate of dissociation (koff) obtained simultaneously by fitting data to the model binding Langmuir 1:1 (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6. 99-110)using the BIAevaluation program. Values of the equilibrium dissociation constants (KDdefined as koff/kon. This Protocol is suitable for use in determining the affinity of binding of the antibody with any PCSK9, including human PCSK9, PCSK9 another mammal (e.g., PCSK9 mouse, rat PCSK9, PCSK9 primates), as well as various forms of PCSK9 (e.g., α and β forms). The affinity of the antibodies to bind and usually is measured at 25°C, but you can also measure at 37°C.

Antibody antagonists against PCSK9 can be obtained by any method known in the prior art, including the method shown in Example 1. The method and the immunization of an animal host typically correspond developed and generally accepted techniques to stimulate and generate antibodies, as described hereafter. General methods for the production of antibodies of human and mouse known in the prior art and/or described herein. Preferred in the present method of producing antibodies include immunization knock-out animals PCSK9-(PCSK9 -/-), as disclosed here.

Provides that any mammal, including man, or his antitelomerase cells can be processed in such a way as to serve as the basis for obtaining the hybridoma cell lines of a mammal, including man. Basically vaccination of an animal host performed intraperitoneally, intramuscularly, orally, subcutaneously, vnutripolostno and/or intradermally with the amount of immunogen, including, as described here.

Hybridoma can be obtained from lymphocytes and immortalized myeloma cells using conventional technology hybridization of somatic cells by Kohler, B. and Milstein, C, 1975, Nature 256:495-497, or modification by Buck, D. W., et al., 1982, In Vitro, 18:377-381. In this hybridization can what about the use of available line myeloma, including, but not limited to, X63-Ag8.653 and myeloma lines are available at the Salk Institute, Cell Distribution Center, San Diego, Calif., USA. Typically, this method involves the fusion of myeloma cells and lymphoid cells using substances that promote cell fusion, such as polyethylene glycol, or by using electrical methods, well known to the person skilled in the art. After fusion, the cells are separated from the environment in which it merged, and grown in a selective culture medium, such as the environment gipoksantin-aminopterin-thymidine (HAT), to eliminate dehybridization parent cells. Any environment described here, supplemented or not supplemented with serum, can be used for culturing hybridomas that secrete monoclonal antibodies. As another alternative technologies merge cells, you can use the EBV-immortalized cells for production of monoclonal antibodies against PCSK9 according to this invention. Hybridoma increased and sublunary, if desired, and supernatant examined for activity against the immunogen through the conventional procedures of immunological analysis (e.g., radioimmunoassay, enzyme-linked immunosorbent assay or fluorescence immunoassay).

Hybridoma that can be used as source of antibodies encompass all derivatives, glue the Ki-offspring parent hybridomas which produce specific monoclonal antibody against PCSK9, or part thereof.

Hybridoma that produce such antibodies, it is possible to growin vitroorin vivousing known procedures. Data monoclonal antibodies can be isolated from the culture medium or body fluids, using generally accepted procedures for the purification of immunoglobulins, such as fractionation with ammonium sulfate, gel electrophoresis, dialysis, chromatography and ultrafiltration, if necessary. Undesirable activity, if present, may be eliminated, for example, banishing the drug through the absorbent material produced from this immunogen attached to the solid phase, and elwira or releasing the required antibodies against the immunogen. Immunization of an animal host using human PCSK9 or a fragment containing the target amino acid sequence, conjugate with a protein that is immunogenic for species undergoing immunization, such as hemocyanin lymph snails, serum albumin, thyroglobulin bull, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, such as maleimides sulfosuccinimidyl ester (conjugation through cysteine residues), N-hydroxysuccinimide (by residue l is Zina), the glutaraldehyde, succinic anhydride, SOCl2or R1N=C=NR, where R and R1represent different alkyl groups, can lead to the production of a population of antibodies (e.g. monoclonal antibodies).

If necessary, can be defined sequence of interest antibody antagonist against PCSK9 (monoclonal or polyclonal), and the polynucleotide sequence may then be cloned into a vector for expression or reproduction. Sequence encoding an antibody, can be maintained in the vector in the cell host, and this cell-host you can then multiply and freeze for future use. Production of recombinant monoclonal antibodies in cell culture can be done by cloning antibody genes from b cells using known prior art methods. See, for example, Tiller et al., 2008, J. Immunol. Methods 329, 112; U.S. patent No. 7314622.

Alternative polynucleotide sequence can be used for genetic manipulation to "humanize" the antibody or to improve the affinity, or other characteristics of the antibody. For example, the constant region may be designed to be more like a constant region of a human, in order to avoid immune R. the shares, if the antibody used in clinical trials and the treatment of the person. It may be necessary to carry out genetic manipulation sequence of the antibody to obtain a greater affinity to PCSK9 and greater efficiency in the inhibition of PCSK9. A qualified specialist in this field will be obvious that you can change one or more polynucleotides in this antibody-antagonist against PCSK9 and still maintain its affinity for binding to PCSK9.

There are four main steps humanization of monoclonal antibodies. These are: (1) determination of the nucleotide precomputed and amino acid sequences of variable domains of light and heavy chains of the original antibody; (2) designing gumanitarnogo antibodies, i.e. deciding which frame region of the antibodies will be used in the humanization process; (3) the actual methods/technologies humanization; and (4) transfection and expression of gumanitarnogo antibodies. See, for example, U.S. patents№№ 4816567; 5807715; 5866692; 6331415; 5530101; 5693761; 5693762; 5585089; and 6180370.

Was described by a number of molecules "humanized" antibody containing the binding site of the antigen derived from a nonhuman immunoglobulin, including chimeric antibodies having a V region rodents, or modified V region rodents, and their corresponding CDR, if the s with constant domains of a human. See, for example, Winter et al., 1991, Nature 349:293-299; Lobuglio et al., 1989, Proc. Nat. Acad. Sci. USA 86:4220-4224; Shaw et al., 1987, J Immunol. 138:4534-4538; and Brown et al., 1987, Cancer Res. 47:3577-3583. Other reference materials described CDR rodents, embedded in the carrier frame area man (FR) before merging with the constant domain of a suitable human antibodies. See, for example, Riechmann et al.,1988, Nature 332:323-327; Verhoeyen et al., 1988, Science 239:1534-1536; and Jones et al., 1986, Nature 321:522-525. In another reference material described CDR rodents, reinforced recombinante designed frame areas rodents. See, for example, European patent publication No. 0519596. These "humanized" molecules designed to minimize unwanted immunological reactions against the molecules of anti-human antibodies rodents, which limits the duration and effectiveness of therapeutic use of these substances in the body-person. For example, the constant region of the antibodies can be designed to be inert (e.g., do not run the complement lysis). See, e.g., PCT publication No. WO99/58572; patent publication UK No. 9809951.8. Other methods of humanizing antibodies, which can be used are disclosed in Daugherty et al., 1991, Nucl. Acids Res. 19:2471-2476 and U.S. patents№№ 6180377; 6054297; 5997867; 5866692; 6210671; and 6350861; and in PCT publication no WO 01/27160.

In another od the om embodiment, fully human antibodies can be obtained, using commercially available strains of mice that were bred to Express specific human immunoglobulins. Transgenic animals bred for the formulation of more appropriate or a more active immune response, can also be used for the generation of humanized antibodies or human antibodies. Examples of such systems are XenomouseTMprovided by the company Abgenix, Inc. (Fremont, CA), HuMAb-Mouse® and TC MouseTMprovided by the company Medarex, Inc. (Princeton, NJ), and mouse Veloclmmune® company Regeneron Pharmaceuticals, Inc. (Tarrytown, NY).

Alternative antibodies can be obtained recombinante and expressed using any method known in the prior art. In another embodiment, antibodies can be obtained recombinante using phage display technology resulting in. See, for example, U.S. patent No. 5565332; 5580717; 5733743; and 6265150; and Winter et al., 1994, Annu. Rev. Immunol. 12:433-455. Alternatively, the technology of phage display (McCafferty et al., 1990, Nature 348:552-553) can be used for the production of human antibodies and fragments of antibodiesin vitrofrom different libraries of genes of variable (V) domain of immunoglobulin immunized donors. According to this technology, the genes of the domain V antibodies cloned inside reading frames in the gene either main or minor envelope protein of filamentous bacteriophage, such as M13 or fd, and reproducing the Xia as functional fragments of the antibodies on the surface ragovoy particles. Because filamentous particles contain a copy single-stranded DNA phage genome, selection on the basis of the functional properties of this antibody also resulted in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties In cells. Phage display can be implemented in various forms; see, e.g., Johnson, Kevin S. and Chiswell, David J., 1993, Current Opinion in Structural Biology 3:564-571. For phage display technique, you can use multiple sources of V-gene segments. Clackson et al., 1991, Nature 352:624-628 insulated heterogeneous group of antibodies against oxazolone from a small randomized combinatorial library of V genes derived from the spleens of immunized mice. Antigenic repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse group of antigens (including proteins) can be selected, in General, following the methods described in Mark et al., 1991, J. Mol. Biol. 222:581-597, or Griffith et al., 1993, EMBO J. 12:725-734. When the natural immune response genes of antibodies with high speed accumulate mutations (somatic spermatazoa). Some of the changes will ensure a higher affinity, and b-cells, reproducing high-affinity surface immunoglobulin, preferably replicated and differenciates is during subsequent sensitization antigen. This natural process can be simulated using a technology known as "shuffling circuits" (Marks et al., 1992, Bio/Technol. 10:779-783). In this method, the affinity of the primary antibody man, obtained using phage display can be enhanced through the consistent replacement of genes V regions of the heavy and light chains on the repertoire of naturally occurring variants (immune repertoire) genes V domains obtained from unimmunized donors. This method makes possible the production of antibodies and fragments of antibodies with affinity in the range of PM-nm. The strategy of obtaining a very large repertoire of phage antibodies (also known as "the founder of all libraries") was described by Waterhouse et al., 1993, Nucl. Acids Res. 21:2265-2266. The shuffling of genes can also be used for the extraction of human antibodies from antibodies rodents, where the antibody is similar affinity and specificity with the original rodent antibody. In accordance with this method, also called "imprinting antigenic determinants", genes V domain of a heavy or light chain antigen rodents obtained through phage display technology resulting substituted immune repertoire of V genes of the domains of the person receiving the hybrids rodent-human. The selection of the antigen results in the allocation of human variable regions, capable vosstanovlenie functional binding site to the antigen, that is, the epitope determines (detects) the choice of a partner. When the process is repeated in order to replace the remaining V-domain of a rodent, get a human antibody (see PCT publication no WO 93/06213). Unlike traditional humanization of rodent antibodies by CDR introduction, this method provides fully human antibodies that have no residues of the framework or CDR, derived from the rodent.

It is obvious that although the above discussion relates to humanized antibodies, discussed the General principles applicable in obtaining antibodies for use, for example, dogs, cats, primates, horses, and ruminants. Additionally it is obvious that one or more aspects described herein humanization of antibodies can be combined, for example the introduction of the CDR, the mutation of the framework and CDR mutation.

Antibodies can be obtained recombinante, first releasing antibodies and antitelomerase cells from animal hosts, getting the sequence of the gene and using this sequence of the gene for the expression of this antigen recombinante in the cells of the host (e.g., CHO cells). Another method that can be applied, is the expression of the sequences of antibodies in plants (e.g. tobacco) or transgenic milk. Methods the expression of antibodies recombinante in plants or mo is the eye have been described. See, e.g., Peeters, 2001, et al. Vaccine 19:2756; Lonberg, N. and D. Huszar, 1995, Int. Rev. Immunol 13:65; and Pollock, et al., 1999, J Immunol Methods 231:147. Methods of obtaining derivatives of antibodies, such as humanized, single-chain, and the like, known in the prior art.

Also for the selection of antibodies that are specific for PCSK9, you can use the methods of immunological analysis and methods of sorting using flow cytometry, such as cell sorting with excitation fluorescence (FACS).

Antibodies may be associated with different media. The media can be active and/or inert. Examples of well-known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylase, glass, natural or modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be either soluble or insoluble, depending on the purpose of the present invention. Qualified in this field specialist will be known to other carriers for binding antibodies, or he will be able to determine such, using routine experimental work. In some embodiments, the implementation of these media contain substances aimed at the heart.

DNA encoding the data monoclonal antibodies, it is easy to select and sequenced using conventional procedures (e.g., by using on gonucleotide probes, which can specifically bind to genes encoding the heavy and light chain data monoclonal antibodies). Hybridoma cells serve as a preferred source of such DNA. After DNA extraction can be placed in expressing vectors (such as expressing vectors disclosed in PCT publication no WO 87/04462), which then transferout in cell host, such as E. coli cells, COS cells monkeys, cells of the Chinese hamster ovary (CHO), or myeloma cells that do not otherwise produce immunoglobulin protein, to achieve the synthesis of monoclonal antibodies in the recombinant cell host. See, e.g., PCT publication no WO 87/04462. This DNA also may be modified, for example, by substituting the coding sequence of the constant domains of light and heavy chains of the person instead of the homologous sequences of the mouse, Morrison et al., 1984, Proc. Nat. Acad. Sci. 81:6851, or by covalent joining the coding sequence of the immunoglobulin whole or part of the coding sequence nimmanahaeminda polypeptide. In this case, the "chimeric" or "hybrid" antibodies that have the binding specificity of the monoclonal antibody against PCSK9.

Antibody antagonists against PCSK9 and polypeptides derived from antibodies can be identified or oher karitane using methods, known in the prior art, whereby reduction, improvement or neutralize the biological effect of PCSK9 is determined and/or measured. In some embodiments, the implementation of an antagonistic antibody or polypeptide against PCSK9 identify, incubare possible agent with PCSK9 and tracking linking and/or present the reduction or neutralization of the biological activity of PCSK9. The analysis of binding can be carried out with purified PCSK9 a polypeptide (or polypeptides) or with cells naturally secreting or transfitsirovannykh to Express the PCSK9 polypeptide(s). In one embodiment, the analysis of binding is an analysis of competitive binding, which assess the ability of potential antibody to compete with a PCSK9 antagonist for binding to PCSK9. This analysis can be done in various forms, including ELISA. In other embodiments, implementation of the antibody antagonist against PCSK9 was determined by incubation of possible agent with PCSK9 and track linking and accompanying inhibition of expression of LDLR and/or clearance of blood cholesterol.

After the initial identification, the activity of the candidate antibody antagonist against PCSK9 can be further validated and improved by biological tests that WPI is local to test the targeted biological activities. Alternatively, the bioanalysis can be used for direct screening of candidate substances. Some of the ways identify and describe the antagonistic antibodies, peptides and aptamers against PCSK9 described in detail in the Examples.

Antibody antagonists against PCSK9 can be characterized using methods well known in the prior art. For example, one way is to identify the epitope, which binds the antibody, or "mapping of epitopes". In the prior art there are many methods for the mapping and characterization of the location of epitopes on proteins, including the determination of the crystal structure of the complex of antibody-antigen, competitive analysis, analysis of the expression of gene fragments, synthetic-based analysis of peptides, as described, for example, Chapter 11 in Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999). In an additional example, the mapping of epitopes can be used to determine the sequence which binds the antibody antagonist against PCSK9. Mapping of epitopes available commercially from various sources, for example, the system Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitope can be a linear epitope, i.e. containing a single amino acid site, or conformational e is iTop, formed in the three-dimensional interactions of amino acids, which need not be contained in a single plot. The peptides of different lengths (for example, at least a length of 4-6 amino acids) can be isolated and synthesized (for example, recombinante) and used in the analysis of binding with the antibody is an antagonist against PCSK9. In another example, the epitope to which the antibody binds antagonist against PCSK9 can be determined in a systematic screening by using overlapping peptides derived from the sequence of PCSK9, and determining the binding with antibody antagonist against PCSK9. In accordance with the analysis of gene fragments of the gene open reading frame encoding PCSK9, fragmented either randomly or by using specific genetic structures and is determined by the reactivity of the expressed fragments of PCSK9 with the test antibody. Fragments of the gene can be obtained, for example, by PCR and then transcribed and translated into proteinin vitroin the presence of radioactive amino acids. The binding of this antibody with radioactively labeled fragments PCSK9 define then use thus and gel electrophoresis. Some epitopes may also be identified using large libraries occasionally the x peptide sequences, expressed on the surface of phage particles (phage library). Alternative, a library of overlapping peptide fragments can be tested for binding of the test antibody using a simple analysis of binding. In an additional example, mutagenesis antigennegative domain, experiments with exchange domains and alanine scanning mutagenesis can be performed to identify residues that are necessary, sufficient and/or required for binding epitope. For example, experiments on the exchange of domains can be implemented using mutant PCSK9, in which various fragments of this polypeptide PCSK9 were replaced (exchanged) PCSK9 sequences from other species, or closely related, but antigenically distinct protein (such as another member of the family proprotein convertase). Assessing the binding of an antibody with a given mutant PCSK9 can be used to determine the significance of a particular fragment of PCSK9 binding to the antibody.

Another method that can be used to describe antibodies of the antagonist against PCSK9, is the use of competitive analysis with other antibodies known to bind the same antigen, i.e. the various fragments on PCSK9, to determine binds to this antibody antagonist against PCSK9 that W is the epitope as with other antibodies. Competitive analysis is well known to a qualified specialist in this field.

To describe the antibody can also be used the crystal structure of the complex of antibody-antigen. Residues determined, calculating the difference between the accessible surface area between the crystal structure L1L3:PCSK9 and structure of one PCSK9. Residues of PCSK9, which demonstrate the hidden surface due to the formation of a complex with the antibody L1L3 included as part of the epitope. This is available for dissolving the surface of a protein is defined as the locus in the centre of a probe sphere (representing the molecule of solvent radius of 1.4 Å), because it turns the van-der-valesova surface protein. Available for dissolution of the surface is calculated by creating a surface point on the extended sphere around each atom (the distance from the center of the atom is equivalent to the sum of the radii of the given atom and probe) and excluding those that are inside the equivalent spheres associated with the neighboring atoms, as implemented in the program AREAIMOL (Briggs, P.J., 2000, CCP4 Newsletter No. 38, CCLRC, Daresbury).

Expressively vector can be used to direct expression of the antibody antagonist against PCSK9. The person skilled in the art are familiar with the application of expressing vectors to obtain the expression e is zogenix proteins in vivo. See, for example, U.S. patent No. 6436908; 6413942; and 6376471. Application expressing vectors include local or systemic administration, including injection, oral administration, gene gun or introduction through the catheter, and a local application. In another embodiment, this expressing the vector is injected directly into the sympathetic trunk or ganglion, or in the coronary artery, atrium, ventricle of the heart or the pericardium.

You can also use a targeted delivery of therapeutic compositions comprising expressing a vector or sebenarnya polynucleotide. Receptor-mediated delivery technology DNA is described, for example, Findeis et al., 1993, Trends Biotechnol. 11:202; Chiou et al., 1994, Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J.A. Wolff, ed.); Wu et al., 1988, J. Biol. Chem. 263:621; Wu et al., 1994, J. Biol. Chem. 269:542; Zenke et al., 1990, Proc. Natl. Acad. Sci. USA 87:3655; Wu et al., 1991, J. Biol. Chem. 266:338. Therapeutic compositions containing polynucleotide applied in the range from about 100 ng to about 200 mg of DNA for local administration in a gene therapy Protocol. Also in gene therapy Protocol, you can use a range of DNA concentrations from about 500 ng to about 50 mg, from about 1 μg to about 2 mg, from about 5 μg to about 500 μg and about 20 μg to about 100 μg. Therapeu the systematic polynucleotide and polypeptides can be delivered using the delivery systems of genes. This system of gene delivery can be viral or non-viral origin (see generally, Jolly, 1994, Cancer Gene Therapy 1:51; Kimura, 1994, Human Gene Therapy 5:845; Connelly, 1995, Human Gene Therapy 1:185; and Kaplitt, 1994, Nature Genetics 6:148). The expression of such coding sequences can be induced by using endogenous promoters for mammalian or heterologous promoters. The expression of the coding sequence may be constitutive or regulated.

The viral vectors of the basis for the delivery of necessary polynucleotide and expression in a desired cell is well known in the prior art. Examples of carriers on a viral basis include, but are not limited to, vectors based on recombinant retroviruses (see, e.g., PCT publication nos WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. patent No. 5219740 and 4777127; UK patent No. 2200651; and EP patent No. 0 345 242), the vectors on the basis of alpha viruses (for example, vectors of the virus Sindbis, forest virus Semliki (ATCC VR-67; ATCC VR-1247), virus Ross river (ATCC VR-373; ATCC VR-1246) and the virus Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and vectors based on adeno-associated virus (AAV) (see, e.g., PCT publication nos WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). You can also use the introduction of DNA related to killed adenovirus, as described in Curiel, 1992, Hum. Gene Ther. 3:147.

So is E. you can use non-viral delivery systems and methods including, but not limited to, polycation condensed DNA linked or not linked to killed adenovirus alone (see, e.g., Curiel, 1992, Hum. Gene Ther. 3:147); ligandclean DNA (see, for example, Wu, J., 1989, Biol. Chem. 264:16985); the delivery system of eukaryotic cells (see, for example, U.S. patent No. 5814482; PCT publication nos WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. You can also use naked DNA. Examples of methods of introducing naked DNA is described in PCT publication no WO 90/11092 and U.S. patent No. 5580859. Liposomes, which serve as the gene delivery system described in U.S. patent No. 5422120; PCT publication nos WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional approaches are described in Philip, 1994, Mol. Cell Biol., 14:2411 and Woffendin, 1994 Proc. Natl. Acad. Sci. 91:1581.

This invention encompasses compositions, including pharmaceutical compositions containing the antibodies described herein, or obtained described herein ways, and having the characteristics described here. Used herein, compositions comprise one or more antibodies, peptides or aptamers, which counteract the interaction of PCSK9 to LDLR, and/or one or more polynucleotide containing sequences encoding one or more of these antibodies or peptides. These compositions can dopolnitelnost suitable fillers, such as pharmaceutically acceptable excipients, including buffers, which are well known in the prior art.

Antagonistic antibodies and peptides against PCSK9 according to this invention is characterized by any (one or more) of the following characteristics: (a) communication with PCSK9; (b) blocking the interaction of PCSK9 to LDLR; (c) reduction in PCSK9-mediated down regulation of this LDLR; and (d) suppression of PCSK9-mediated inhibition of clearance of LDL from the blood. Preferably antibodies against PCSK9 have two or more of these properties. More preferably these antibodies have three or more of these properties. Most preferably these antibodies have all four properties.

Accordingly, the invention provides any of the following, or compositions (including pharmaceutical compositions)containing any antibody having a partial sequence of the light chain and a partial sequence of the heavy chain, as shown in Table 1. The underlined sequences represent the sequences of the CDR according to Kabat (Kabat), and bold - in accordance with Hatia (Chothia).

This invention also provides lots CDR antibody against PCSK9 (including CDR according to Kabat (Kabat), Hatia (Chothia)). The establishment of the CDR regions included in the net is about skills in this area. It is understood that in some embodiments, the implementation of the CDR can be a combination of CDR according to Kabat (Kabat) and Hotie (Chothia) (also called "combined CDR" or "extra long CDR"). In some embodiments, the implementation of the CDR data represent the CDR on Hatia (Chothia). In other words, in the variants of implementation with more than one CDR CDR data can be any, according to Kabat (Kabat), Hatia (Chothia), combined CDR or their combinations.

This invention also provides a method of obtaining any of these antibodies or polypeptides. Antibodies according to this invention can be generated using techniques known in the art. The polypeptides can be obtained by proteolytic or other degradation of the antibodies, and recombinant methods (namely separate or fused polypeptides), as described above, or by chemical synthesis. The polypeptides of antibodies, especially shorter polypeptides of approximately 50 amino acids, conveniently be obtained by chemical synthesis. The methods of chemical synthesis are well known in the prior art and available commercially. For example, the antibody can be obtained using an automated polypeptide synthesizer using solid-phase method. See also U.S. patent No. 5807715; 4816567 and 6331415.

In another embodiment, these antibodies and p is dipeptide you can get recombinante, using methods well known in the prior art. In one embodiment, polynucleotide contains a sequence encoding a variable region of the heavy chain and/or light chain antibody 4A5, 5A10, 6F6, 7D4 or L1L3. Sequence encoding an antibody, can be maintained in the vector in the cell host, and this cell host may be duplicated and frozen for future use. Vectors (including expressing vectors and cells of the hosts described hereafter.

This invention also covers scFv antibodies according to this invention. Single-chain fragments of variable regions receive by connecting the variable regions of the heavy and/or light chain, using a short linking peptide. Bird et al., 1988, Science 242:423-426. An example of a linking peptide is (GGGGS)3(SEQ ID NO: 24), which forms the joining of approximately 3.5 nm between the carboxyl end of one variable region and aminocom.com another variable regions. Can be designed and used linkers with other sequences. Bird et al., 1988, supra. Linkers should be short, flexible and polypeptides preferably contain less than about 20 amino acid residues. Linkers, in turn, can be modified to perform additional functions, as napierville medicines or fixation with rigid substrates. Single-chain variants can be obtained either recombinante or synthetically. For synthetic obtain scFv you can use the automated synthesizer. For recombinant obtain scFv suitable plasmid containing polynucleotide that encodes this scFv can be embedded in a suitable cell host, or eukaryotic, such as yeast, plants, insects or mammals, or prokaryotic, such as E. coli. Polynucleotide encoding interest scFv can be obtained using routine manipulations, such as ligation of polynucleotides. The obtained scFv can be isolated using standard methods of protein purification known in the prior art.

Also covered other forms of single-chain antibodies, such as diamela. Diately represent a divalent bespecifically antibodies, in which the domains VH and VL are expressed on a single polypeptide chain, but using a linker that is too short to provide education pairs between the two domains on the same chain, thereby forcing the data domains to pair with complementary domains of another chain and creating two binding site of antigen (see, e.g., Holliger, P., et al., 1993, Proc. Natl. Acad Sci. USA 90:6444-6448; Poljak, R.J., et al., 1994, Structure 2:1121-1123).

For example, bespecifically antibodies,monoclonal antibodies, which have binding specificity for at least two different antigens can be obtained using the disclosed here antibody. Methods of obtaining bespecifically antibodies are well known in the prior art (see, for example, Suresh et al., 1986, Methods in Enzymology 121:210). Traditionally, the recombinant getting bespecifically antibodies was based on simultaneous expression of two pairs of light chain-heavy chain immunoglobulin, where the two heavy chains have different specificities (Millstein and Cuello, 1983, Nature 305, 537-539).

In accordance with one method of obtaining bespecifically antibody variable domains of antibodies with the desired binding specificity (antigennegative active centers antibodies) merged with the sequences of the constant domains of immunoglobulin. This merger preferably with a constant domain of the heavy chain of immunoglobulin containing at least part of the hinge region, CH2 and CH3. It is preferable to have the first constant region of the heavy chain (SN), including site necessary for binding to the light chain, which is present at least in one of the mergers. DNA encoding the fusion heavy chain immunoglobulin and, if necessary, the light chain of the immunoglobulin, embedded in the individual expressing vectors and together transliterowany in a suitable organism, the host. This is considerable flexibility in the correction of the quantitative ratios of the three polypeptide fragments in the variants of implementation, in which unequal ratios of the three polypeptide chains used in the construction, optimum productivity. However, you can embed the coding sequences for two or all three polypeptide chains in one expressing vector when the expression of at least two polypeptide chains in equal ratios results in high productivity, or when the ratio does not have a specific value.

In the same way bespecifically antibodies comprise the heavy chain hybrid immunoglobulin with a first binding specificity in one arm and a pair of heavy chain-light chain hybrid immunoglobulin (providing a second binding specificity) in the other shoulder. This asymmetric structure, with a light chain immunoglobulin in only one half of this bespecifically molecules, facilitates the separation of the desired especifismo connections from unwanted combinations of immunoglobulin chains. This method is described in PCT publication no WO 94/04690.

Heteroconjugate antibodies, consisting of two covalently linked antibodies are also included in the range of the present invention. Such antibodies used for targeting cells of the immune system against unwanted cells (U.S. patent No. 4676980) and for the treatment of HIV infection (PCT of publicat and No. WO 91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies can be generated using any convenient ways of cross-stitching. Suitable cross-linking agents and methods of cross-stitching is well known in the prior art, and are described in U.S. patent No. 4676980.

Chimeric or hybrid antibodies can also be createdin vitrousing known methods in synthetic protein chemistry, including those that use cross-linking agents. For example, immunotoxins can be constructed using the exchange reaction of disulfides or through the formation of thioester linkages. Examples of suitable reagents for this purpose include aminothiols and methyl-4-mercaptopyrimidine.

Humanized antibodies containing one or more CDRs of the antibody 5A10 or 7D4, or one or more CDRs derived from an antibody 5A10 or 7D4, can be created, for example, using any method known in the prior art. For example, four General stages can be used for the humanization of monoclonal antibodies. These are: (1) determination of the nucleotide precomputed and amino acid sequences of variable domains of light and heavy chains of the original antibody; (2) designing gumanitarnogo antibodies, i.e. deciding which frame region of the antibodies will be used in the humanization process; (3) actionsee methods/technologies humanization; and (4) transfection and expression of gumanitarnogo antibodies. See, for example, U.S. patents№№ 4816567; 5807715; 5866692; 6331415; 5530101; 5693761; 5693762; 5585089 and 6180370.

In recombinant humanized antibodies plot Fc can be modified in order to avoid interaction with the receptor Fγ and the complement and immune systems. Methods of obtaining such antibodies are described in WO 99/58572. For example, the constant region can be constructed more like that of a constant region of cheloveka avoid the immune response, if the antibody used in clinical trials and the treatment of the person. See, for example, U.S. patent No. 5997867 and 5866692.

Humanized antibody containing the variable region of the light or heavy chain, or one or more CDRs of the antibody or its variants shown in Table 1, or one or more CDRs derived from this antibody or its variants shown in Table 2, can be created using any of the methods known in the art.

Humanized antibodies can be created by any method known in the art.

The invention includes modifications of the antibody and the polypeptide variants of the invention are presented in Table 1, including functionally equivalent antibodies which do not affect significantly their properties and variants which have enhanced the th or reduced activity and/or affinity. For example, amino acid sequence may be modified to obtain antibodies with the desired affinity for binding to PCSK9. Modification of polypeptides is an established technique in the prior art and requires no detailed description here. Modification of polypeptides illustrated in the Examples. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or insertions of amino acids that do not cause significant adverse changes in the functional activity or which is brought to maturity (increase) the affinity of a given polypeptide to its ligand, or the use of chemical analogues.

Insert amino acid sequences include amino - and/or carbkonate merge with a range of length from one residue to polypeptides containing a hundred or more residues, as well as insertions of one or multiple amino acid residues within the sequence. Examples of terminal insertions include an antibody with an N-terminal residue of methionine or the antibody fused to epitope tag. Other insertional variants of the antibody molecules include the fusion to the N - or C-end of antibodies, enzyme or polypeptide that enhances half-life of Unitel in the bloodstream.

Substituted variants have at least one UD is certain amino acid residue in the molecule antibodies and the remainder, inserted in its place. Areas, which are of great interest for substitutional mutagenesis include the hypervariable sites, but changes in FR also provided. Conservative substitutions are shown in Table 2 under the heading "conservative substitutions". If such substitutions result in the change in biological activity, then more substantial replacement indicated in Table 2 as "illustrative replacement or as described later in the application to classes of amino acids can be introduced and conducted the screening of the products obtained.

Significant changes in the biological properties of the antibody are accompanied by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, folded or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (C) the internal structure of the side chain. Naturally occurring residues are divided into groups based on common properties of the side chain:

(1) non-polar: Norleucine, Met, Ala, Val, Leu, Ile;

(2) uncharged polar: Cys, Ser, Thr, Asn, Gln;

(3) acidic (negatively charged): Asp, Glu;

(4) basic (positively charged): Lys, Arg;

(5) residues that influence the orientation of the chains, Gly, Pro; the

(6) aromatic: Trp, Tyr, Phe, His.

Nonconservative substitutions occur as a result of replacement of a member of one of these classes for another class.

Any cysteine residue not involved in maintaining the proper conformation of this antibody also may be substituted, usually to serine, to improve the oxidative stability of the molecule, and to prevent the modified cross-stitching. Conversely, cysteine bond (connection) can be introduced into the antibody to improve its stability, particularly when the antibody is an antibody fragment such as an Fv fragment.

Amino acid modifications can range from changes or modifications of one or more amino acids to complete changes in the field, for example, variable regions. Changes in variable regions can modify the affinity and/or specificity of binding. In some embodiments, the implementation of no more than one to five substitutions performed within the domain CDR. In other embodiments, the implementation of no more than one to three conservative amino acid substitutions performed within the domain CDR. In other embodiments, implementation of the domain CDR is a CDR H3 and/or CDR L3.

Modifications also include glycosylated and deglycosylated polypeptides, and polypeptides with other women posttranslation and modifications such as, for example, glycosylation with different sugars, acetylation and phosphorylation. Antibodies are glycosylated in a conservative positions in their constant regions (Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32). Oligosaccharide side chains of immunoglobulins have an effect on the function of a protein (Boyd et al., 1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem. 29:4175-4180) and intramolecular interactions between the parts of the glycoprotein, which may affect the conformation and is a three-dimensional surface of the glycoprotein (Jefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech. 7:409-416). Oligosaccharides may also contribute to the targeting of this glycoprotein on some molecules on the basis of specific recognition structures. It was also reported that glycosylation of the antibody effect on antibody-dependent cretaceouspaleogene cytotoxicity (ADCC). In particular, cells SNO controlled by the tetracycline expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase gene, catalyzes the formation of fissile bisected GlcNAc was reported to have improved ADCC activity (Umana et al., 1999, Nature Biotech. 17:176-180).

Glycosylation of antibodies is typically either N-paired, or O-conjugate. N-conjugate refers to the attachment of a molecule of carbohydrate to the side chain of the TATKO asparagine. Tripeptide sequence asparagine-X-serine, asparagine-X-threonine and asparagine-X-cysteine, where X is any amino acid except Proline, are recognizable sequences for enzymatic attachment molecule of carbohydrate to the asparagine side chain. Thus, the presence of any of these Tripeptide sequences in the polypeptide creates a potential glycosylation site. O-conjugate of glycosylation refers to the attachment of one of the sugars N-atsetilgalaktozamin, galactose, or xylose to hydroxynicotinate, often to serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

Addition of glycosylation sites to the antibody is conveniently performed by changing the amino acid sequence such that it contains one or more of the above Tripeptide sequences (for N-conjugated sites of glycosylation). Change can also be made by adding or replacing one or more residues of serine or threonine in the sequence of the original antibody (for O-paired sites of glycosylation).

The nature of the glycosylation of antibodies can also be changed without changing the underlying nucleotide sequence. The character CH is Kazimirova largely depends on the host cell, used for the expression of antibodies. Because of the cell type used for expression of recombinant glycoproteins, such as antibodies as potential therapeutic drugs is rarely the native cell, variations in the nature of the glycosylation of antibodies can be expected (see, for example, Hse et al., 1997, J. Biol. Chem. 272:9062-9070).

In addition to the choice of host cell factors that affect glycosylation during recombinant generate antibodies include a method of growing, the composition of the culture medium, the saturation of the culture medium, oxygen saturation, pH, cleaning systems and the like. Various methods have been proposed to change the nature of glycosylation, executed in a specific organism, the host, including the introduction or overproduction of some enzymes involved in the production of oligosaccharides (U.S. patent No. 5047335; 5510261 and 5278299). Glycosylation, or some types of glycosylation, can be enzymatically removed from the glycoprotein, for example, using endoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1, endoglycosidase F2 and endoglycosidase F3. In addition, recombinant a host cell can be genetically engineered to be suitable for processing certain types of polysaccharides. These and similar methods well known is tiny technical level.

Other methods of modification include the use of methods of attaching known in the prior art, including, but not limited to, enzymatic methods, oxidative substitution and helatoobrazovateli. You can use the modification, for example, to attach labels for immunological analysis. Modified polypeptides receive, using generally accepted in the field methods, and screening can be performed using standard tests known in the prior art, some of which are described below in the Examples.

In some embodiments of this invention, the antibody contains a modified constant region, such as a constant region that is immunologically inert or partially inert, for example, does not trigger complement-mediated lysis, does not stimulate ADCC or does not activate the microglia; or have reduced activity compared to the unmodified antibody) in one or more of the following processes: run the complement-mediated lysis, stimulate ADCC or activation of microglia. Various modifications of the constant region can be used to achieve the optimal level and/or combinations of effector functions. See, e.g., Morgan et al., 1995, Immunology 86:319-324; Lund et al., 1996, J. Immunology 157:4963-9 157:4963-4969; Idusogie et al., 2000, J. Immunology 164:4178-184; Tao et al., 1989, J. Immunology 143:2595-2601; Jefferis et al., 1998, Immunological Reviews 163:59-76. In some embodiments, the implementation of the constant region is modified, as described in Eur. J. Immunol., 1999, 29:2613-2624; PCT publication No. WO99/58572; and/or patent application in the UK No. 9809951.8. In other embodiments, the implementation of this antibody contains a constant region of the heavy chain IgG2person, including the following mutations: A330P331 in S330S331 (numbering of amino acids according to the sequence of wild-type IgG2). Eur. J. Immunol., 1999, 29:2613-2624. In other embodiments, the implementation of this constant region is deglycosylation for N-linked glycosylation. In some embodiments, the implementation of this constant region is deglycosylation for N-linked glycosylation by changing glycosylases amino acid residues or flanking residues that are part of a recognizable sequence of N-glycosylamine in this constant region. For example, the site of N-glycosylation N297 can be changed in A, Q, K, or H. See Tao et al., 1989, J. Immunology 143:2595-2601; Jefferis et al., 1998, Immunological Reviews 163:59-76. In some embodiments, the implementation of this constant region is deglycosylation for N-linked glycosylation. This constant region can be deglycosylation for N-mated glycosylate the Oia enzymatic (e.g., removing the carbohydrate by the enzyme PNGase) or as a result of expression in the cell host with a deficit of glycosylation.

Other modifications of antibodies include antibodies that have been modified, as described in PCT publication no WO 99/58572. These antibodies contain, in addition to the binding domain targeting molecule is a target effector domain having the amino acid sequence essentially homologous to all or part of the constant domain of the heavy chain of human immunoglobulin. These antibodies are capable of binding molecule-target without running a significant complementarianism lysis, or cretaceouspaleogene destruction of this target. In some embodiments, the implementation of this effector domain is able to specifically bind FcRn and/or FcγRIIb. They are usually based on chimeric domains derived from two or more domains WithN2 heavy chain of human immunoglobulin. Antibodies, modified in this manner are extremely suitable for use in long-term treatment with antibodies, in order to avoid inflammation and other adverse reactions to conventional therapies antibodies.

The invention includes variants of bringing the affinity to maturation. For example, antibodies with a Mature affinity can be obtained by procedures known in the level of technology is key (Marks et al., 1992, Bio/Technology, 10:779-783; Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91:3809-3813; Schier et al., 1995, Gene, 169:147-155; Yelton et al., 1995, J. Immunol., 155:1994-2004; Jackson et al., 1995, J. Immunol., 154(7):3310-9; Hawkins et al., 1992, J. Mol. Biol., 226:889-896; and PCT publication number WO2004/058184).

The following methods can be used to regulate the affinity of antibodies for the characterization of the CDR. One way to describe characteristics of the CDR of the antibody and/or changes (for example, to improve the binding affinity of the polypeptide, such as an antibody, is called "scanning mutagenesis library. Typically, scanning mutagenesis library works as follows. Amino acids in one or more of the provisions in this CDR as opposed to two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids using methods recognized in the art. In the result, generated a small library of clones (in some embodiments, implement, one for each of the analyzed position of the amino acids), each with a complexity of two or more members (if two or more amino acids substituted at each position). Typically, this library also includes a clone containing the native (unsubstituted) amino acid. On a small number of clones, for example approximately 20-80 clones (depending on the complexity of the library), from each of the libraries screened for affinity to swazilan the Yu with polypeptide-target (or other target-binding), and identifies candidates with higher, same, lower affinity or without binding. Methods for determining the affinity of binding are well known in the prior art. The affinity of binding can be determined by analysis of surface plasmon resonance Biacore, which reveals a 2-fold or more difference in the affinity of binding. Biacore is extremely effective when the original antibody already binds with relatively high affinity, for example with KDapproximately 10 nm or below. Screening using surface plasmon resonance Biacore described herein in the Examples.

The affinity to swiatynia can be determined using Kinexa Biocensor, scintillation analysis of convergence, ELISA, immunoassay ORIGEN (IGEN), quenching of fluorescence, transfer fluorescence, and/or yeast display. Can also be screened affinity to bind with suitable biological analysis.

In some embodiments, the implementation of each amino acid position in the CDR replaced (in some versions of the implementation, one at a time) all 20 naturally occurring amino acids using mutagenesis recognized in the prior art (some of which are described here). In the result, generated a small library of clones (n is which versions of the implementation, one for each of the analyzed position of the amino acids), each with a complexity of two or more members (if all 20 amino acids substituted at each position).

In some embodiments, the implementation of this library for screening contains replacement in two or more provisions which may be in the same CDR, or in two or more CDR. Thus, the library can contain a substitution at two or more positions in one CDR. This library can contain a substitution at two or more positions in two or more CDR. This library can contain substitution in the 3, 4, 5 or more provisions, these provisions found in two, three, four, five, or six CDR. This substitution can be obtained using codons with low redundancy. See, for example, Table 2 in Balint et al., 1993, Gene, 137(1):109-18.

This CDR can be a CDRH3, and/or CDRL3. This CDR may be one or more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and/or CDRH3. This CDR's may be CDR according to Kabat (Kabat), CDR on Hatia (Chothia) or extended CDR.

You can spend sequencing of candidate compounds with improved binding and thus to identify mutational substitution in CDR, leading to enhanced affinity (also called "improved" replacement). Candidate connections, which are connected, can also be sequenced, who eat the identified replacement CDR, preserving the binding.

Can be done multiple cycles of screening. For example, candidate compounds (each contains an amino acid substitution at one or more positions in one or more CDR) with improved binding can also be useful for constructing a second library containing at least the original and the substituted amino acid in each superior position CDR (i.e. amino acid position in this CDR, in which the replacement mutation exhibits improved binding). Receiving, screening and selection of this library is discussed next.

Scanning mutagenesis library provides methods for characterization of the CDR, since the frequency of clones with improved binding to the same binding, reduced binding and no binding also provide information related to the importance of each amino acid position for the stability of the complex of antibody-antigen. For example, if the position of the CDR retains binding under all 20 amino acids, this position is defined as a position that is unlikely to need to bind antigen. On the other hand, if the position of the CDR retains binding only in a small percentage of substitutions, this position is recognized as important for the functioning of the CDR. Thus, the methods of scanning mutag the independent libraries provide information relevant to the provisions in the CDR, which can be changed on many different amino acids (including all 20 amino acids), and the provisions in the CDR, which cannot be changed or can be changed only some of the amino acids.

Candidate compounds that improve the affinity, can be combined into a second library, which includes an improved amino acid and may optionally include additional replacement in this position, depending on the desired complexity of the library, or acceptable use methods desired screening or selection. In addition, if necessary, and related amino acid position can be randomized to at least two or more amino acids. Randomization of adjacent amino acids can provide additional conformational flexibility in mutant CDR, which, in turn, can provide or facilitate the introduction of more enhancing mutations. This library can also contain substitutions in positions that do not show improved affinity in the first round of screening.

Screening or selection in this second library to identify members of the library with improved and/or altered affinity of binding, using any method known in the prior art, including screening using anal is for surface plasmon resonance Biacore, and the selection using any method of selection, known in the prior art, including phage display, yeast display, and ribosome display.

The invention also encompasses fused proteins containing one or more fragments or regions of the antibodies or polypeptides according to this invention. In one embodiment, is provided with a fused polypeptide, which consists of at least 10 nonessential amino acids of the variable region of the light chain, represented in SEQ ID NO: 53, 16, 17, 18 or 19, and/or at least 10 amino acids of the variable region of the heavy chain, represented in SEQ ID NO: 54, 20, 21, 22 or 23. In other embodiments, the implement is provided with fused polypeptide, which comprises at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 dispensable amino acids of the variable region of the light chain and/or at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 dispensable amino acids of the variable region of the heavy chain. In another embodiment, this fused polypeptide comprises a variable region light chain and/or variable region of the heavy chain, as not only is but in any of the pairs of sequences, selected from SEQ ID nos: 53 and 54, 16 and 20, 17 and 21, 18 and 22, and 19 and 23. In another embodiment, this fused polypeptide contains one or more CDR. In other embodiments, the implementation of this fused polypeptide contains the CDR H3 (VH CDR3) and/or CDR L3 (VL CDR3). For the purposes of this invention, the protein contains one or more antibodies and another amino acid sequence, with which there is no connection in the native molecule, for example, a heterologous sequence or a homologous sequence from another region. Examples of heterologous sequences include, but are not limited to, "tag"such as FLAG label or 6His tag. The label is well known in the prior art.

Fused polypeptide can be obtained by methods known in the prior art, for example, synthetically or recombinante. Basically fused protein according to this invention create more expression encoder their polynucleotide, using recombinant methods described herein, although they may be obtained by any other method known in the prior art, including, for example, by chemical synthesis.

This invention also provides compositions containing the antibodies or polypeptides conjugated (e.g., paired) with the agent, which facilitates contact with a solid substrate (such as Biotin or AB is Dean). For ease of reference will be mainly made for antibodies with the understanding that these methods apply to any binding to PCSK9 and/or antagonistic against PCSK9 variant implementation described here. Algae usually refers to the pairing of these components, as described here. Mate (which usually fixes these components in the immediate vicinity, at least for the introduction) can be achieved any number of ways. For example, the direct reaction between the agent and the antibody is possible when everyone has a Vice, capable of reacting with the other. For example, a nucleophilic group, such as amino - or sulfhydryl group on the same connection may be capable of reacting with a carbonyl-containing group such as anhydride or gelegenheid, or alkyl group containing easily leaving group (e.g. halogen) on another connection.

The antibody or polypeptide according to this invention can be associated with the agent-label such as a fluorescent molecule, a radioactive molecule or any others labels known in the prior art. In the prior art label, which in most cases provide (either directly or indirectly) signal.

This invention also provides compositions (including pharmaceutical com is osili) and sets containing, as clarifies the description of the invention, any or all of the antibodies and/or polypeptides described herein.

This invention also provides a dedicated polynucleotide encoding the antibodies and peptides of this invention, and vectors and cells of the host that contains polynucleotide.

Accordingly, this invention provides polynucleotides (or compositions, including pharmaceutical compositions), comprising polynucleotide encoding any of the following: antibodies 4A5, 5A10, 6F6, 7D4, L1L3, or any fragment or portion that has the ability to cause antagonism to PCSK9.

In another aspect, the invention provides polynucleotide encoding any of the antibodies (including antibody fragments and polypeptides described herein, such as antibodies and polypeptides that have impaired effector function. Polynucleotide can be obtained and expressed using procedures known in the art.

In another aspect, the invention provides compositions (such as pharmaceutical compositions)containing any of polynucleotides according to this invention. In some embodiments of implementation, the composition comprises expressing a vector containing polynucleotide encoding the antibody, as described herein. In another embodiment, this composition with the contains expressing vector, containing polynucleotide encoding any of the antibodies or polypeptides described herein. In other variants of implementation, the composition comprises either or both of polynucleotide presented in SEQ ID NO: 25 and SEQ ID NO: 26. Next described here expressing vectors and the use of polynucleotide compositions.

In another aspect, the invention provides a method of obtaining any of these polynucleotides.

The present invention covers polynucleotide, complementary to any such sequences. Polynucleotide can be single-stranded (coding or antimuslim) or dvuhtsepochnyj and may be DNA (genomic, cDNA or synthetic) or RNA. RNA molecules include molecules garns, which contain introns and are consistent with the DNA molecule of one-to-one, and the mRNA molecules that do not contain introns. Additional coding or non-coding sequences may, but need not, be present within polynucleotide of the present invention, and polynucleotide may, but is not required to be paired with other molecules and/or substrates.

Polynucleotide can contain native sequence (namely, an endogenous sequence that encodes the antibody or portion thereof) or may contain an option such posledovatel the property. Polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions, so that the immunoreactivity of the encoded polypeptide is not diminished, relative to the native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can in most cases be estimated as described here. Variants preferably exhibit at least 70% identity, more preferably at least about 80% identity, even more preferably about 90% identity, and most preferably about 95% identity with the polynucleotide sequence, which encodes the native antibody or part thereof.

Two polynucleotide or polypeptide sequences are called "identical"if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparison between the two sequences is usually carried out by comparing these sequences over a comparison window to identify and compare local regions of sequence similarity. "Comparison window", as used here, refers to the area of at least about 20 contiguous positions, usually about the 30 to about 75, or 40 to about 50, in which a sequence may be compared with the reference sequence with the same number of adjacent positions after optimal alignment of these two sequences.

Optimal alignment of sequences for comparison may be conducted using the Megalign program in the block Lasergene software for bioinformatics (DNASTAR, Inc., Madison, WI)using the default settings. This program combines several equalization schemes described in the following reference documents: Dayhoff, M.O., 1978, A model of evolutionary change in proteins " Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure (National Biomedical Research Foundation, Washington DC), Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, (Academic Press, Inc., San Diego, CA); Higgins, D.G. and Sharp, P.M., 1989, CABIOS 5:151-153; Myers, E.W. and W. Muller, 1988, CABIOS 4:11-17; Robinson, E.D., 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath, P.H.A. and Sokal, R. R., 1973, Numerical Taxonomy-the Principles and Practice of Numerical Taxonomy (Freeman Press, San Francisco, CA); Wilbur, W.J. and Lipman, D.J., 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, where the plot polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e. Asrava) 20 percent or less, usually from 5 to 15 percent, or 10 to 12 percent compared to the reference sequence (which does not include additions or deletions) for optimal alignment of these two sequences. The percentage is calculated by determining the number of positions in which the identical nucleic acid bases or amino acid residues occur in both sequences to get the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the results by 100 to obtain the percentage identity of the sequence.

Variants may also, or alternatively, to be essentially homologous to a native gene, or part thereof, or the complement. Such polynucleotide variants are capable of hybridizing to moderately stringent conditions with natural DNA sequence that encodes a native antibody (or a complementary sequence).

Appropriate "moderately stringent conditions" include pre-washing in a solution of 5× SSC, 0,5% SDS, 1.0 mm EDTA (pH 8.0), hybridization at 50°C-65°C, 5× SSC, overnight; followed by washing twice at 65°C, for 20 minutes each time, using a 2×, and 0.5× and 0.2× SSC containing 0.1% SDS.

"Very strict conditions" or the conditions of high stringency" are those, in which: (1) low ionic strength and high temperature for washing, for example of 0.015 M sodium chloride/0,0015 M sodium citrate/0.1% sodium dodecyl sulphate at 50°C; (2) during the hybridization is used denaturing substance, such as formamide, for example 50% (volume/volume) formamide with 0.1% bovine serum albumin/0.1% ficoll/0.1% polyvinylpyrrolidone/50 mm sodium phosphate buffer at pH 6.5 with 750 mm sodium chloride, 75 mm sodium citrate at 42°C; or (3) is used 50% of formamide, 5× SSC (0,75 M NaCl, of 0.075 M sodium citrate), 50 mm sodium phosphate (pH of 6.8), 0.1% sodium pyrophosphate, 5× denhardt's solution, voiced by ultrasound DNA from salmon sperm (50 µg/ml), 0.1% of SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2× SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by washing at 55°C in conditions of high stringency with the use of 0.1× SSC containing EDTA. A qualified person skilled in the art will understand how to select the temperature, ionic strength and the like, as necessary, to take account of factors such as the length of the sample, and the like.

A person of ordinary skill in this field will be clear that as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides are minimally the Yu gomologichnosti nucleotide sequence of any native gene. However, the present invention in particular provides polynucleotide, which differ due to differences in the use of codons. Additionally, alleles of such genes containing the polynucleotide sequences provided here, are in the range of the present invention. Alleles are endogenous genes that changed as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The obtained mRNA and protein can, but not necessarily, have a modified structure or function. Alleles can be identified using standard procedures (such as hybridization, amplification and/or comparison of database sequences).

Polynucleotide according to this invention can be obtained through chemical synthesis, recombinant methods or PCR. The methods of chemical synthesis are well known in the prior art and need no detailed description here. A qualified specialist in this field can use the sequence provided here, and a commercial DNA synthesizers to obtain the necessary DNA sequence.

To obtain polynucleotides using recombinant methods, polynucleotide containing the desired sequence may be embedded into a suitable vector, and this vector, the ocher is ü, can be introduced into a suitable cell host for replication and reproduction, as discussed later here. Polynucleotide can be embedded in the cells of the host by any means known in the art. Cells can be transformed by the introduction of exogenous polynucleotide through direct uptake, endocytosis, transfection, F-mating or electroporation. After injection of exogenous polynucleotide can be maintained in the cell as at vector (such as plasmid or integrated into the genome of the host cell. Amplificatory thus polynucleotide can be isolated from the host cell by methods well known in the prior art. See, for example, Sambrook et al., 1989, supra.

Alternative PCR makes possible the reproduction of DNA sequences. PCR is well known in the prior art and are described in U.S. patent No. 4683195, 4800159, 4754065 and 4683202, as well as in PCR: The Polymerase Chain Reaction, MuIMs et al., 1994, eds. (Birkauswer Press, Boston, MA).

RNA can be obtained using the selected DNA into an appropriate vector and embedding it in a suitable cell host. After cell replication and transcription of DNA into RNA, this RNA can then be isolated using methods well known to the person skilled in the art, as described, for example, Sambrook et al., 1989, supra.

Suitable vectors to clone the simulation can be designed in accordance with standard methods, or can be selected from a large number of vectors for cloning, available in the prior art. Despite the fact that the vectors for cloning may vary depending on the host cell, suitable vectors for cloning basically have the ability to reproduce itself, can have only one target for a specific restriction enzyme and/or may carry genes token, which can be used in the selection of clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and Shuttle vectors such as pSA3 and pAT28. These and many other vectors for cloning are available from commercial suppliers such as BioRad, Strategene and Invitrogen.

Expressing the vectors generally are replicable polynucleotide constructs that contain polynucleotide according to this invention. This means that expressing vector must be replicable in cells of the host or as episome, or as an integral part of the chromosomal DNA. Suitable expressing vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, Comedy and expressing vector(s), disclosed in PCT publication no WO 87/04462. Vector components can, as a rule the ILO to include, but not limited to, one or more of the following: a signal sequence, the start of replication, one or more marker genes; appropriate elements controlling transcription (such as promoters, enhancers and terminators). For expression (i.e. broadcast) also typically requires one or more elements controlling transcription, such as the sites of binding of ribosomes, the sites of translation initiation and stop codons.

Vectors containing interest polynucleotide, can be built into the cell host any of the many appropriate ways, including through electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; bombing microparticles; lipofectin; and infection (for example, when the vector is an infectious agent, such as vaccinia virus). The selection of vectors for transformation or polynucleotides often will depend on the properties of the host cell.

The invention also provides cell host containing any of polynucleotides described here. Any cell-hosts capable of overproductivity heterologous DNA can be used for the purpose of selection of genes coding for this interest is an antibody, the floor of the peptide or protein. Non-limiting examples of host cells of mammals include, but are not limited to, COS cells, HeLa, NSO and CHO. See also PCT publication no WO 87/04462. Suitable cell host, is not derived from mammals include prokaryotes (e.g., E. coli orB.subtillis) and yeast (such asS.cerevisae, S.pombe;orK.lactis). Preferably, these cells are the owners of Express cDNA level by approximately 5-fold excess, more preferably 10-fold excess of, even more preferably 20-fold higher than the level of the corresponding endogenous interest antibodies or protein, if present in the data cells of the host. Screening data cells of their hosts on the subject of specific binding to PCSK9 or a domain of PCSK9, is carried out by immunoassay or FACS. Can be specified cell, overproducers of interest is an antibody or a protein.

C. Composition

Compositions used in the methods according to this invention include an effective amount of an antagonistic antibody against PCSK9, a polypeptide derived from an antagonistic antibody against PCSK9, or other antagonists of PCSK9 described here. Examples of such compositions and method of their preparation are also described in the earlier section and the next. In one embodiment, this composition is I further comprises an antagonist against PCSK9. In another embodiment, the composition comprises one or more antibody antagonists against PCSK9. In other embodiments, the implementation of this antibody antagonist against PCSK9 recognizes human PCSK9. In other embodiments, the implementation of this antibody antagonist against PCSK9 is humanized. In other embodiments, the implementation of this antibody antagonist against PCSK9 contains a constant region that does not trigger an unwanted or unintended immune responses, such as antitelomerase lysis or ADCC. In other embodiments, the implementation of this antibody antagonist against PCSK9 contains one or more CDRs of a given antibody (such as one, two, three, four, five, or in some embodiments, the implementation of all six CDR). In some embodiments, the implementation of this antibody antagonist against PCSK9 is human.

It is implied that these compositions can contain more than one antibody is an antagonist against PCSK9 (e.g., a mixture of antibody antagonists against PCSK9, which recognize different epitopes on PCSK9). Other illustrative compositions contain more than one antibody is an antagonist against PCSK9, which recognize the same epitope(s), or different types of antibody antagonists against PCSK9, which are associated with different epitopes on PCSK9.

The composition is used which may, in the present invention, may optionally contain pharmaceutically acceptable carriers, fillers or stabilizing agents (Remington: The Science and Practice of Pharmacy 20th Ed., 2000, Lippincott Williams and Wilkins, Ed. K. E. Hoover), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers substances are nontoxic to recipients at the dosages and concentrations, and may include buffers such as phosphate, citrate, or other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecylsilane ammonium chloride, chloride hexadecane; benzalkonium chloride, chloride benzene; phenol, butyl or benzyl alcohol; alkylarene, such as methyl or propyl paraben; catechin; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; hepatoblastoma agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; soleobrazutaya counterions such as sodium; complexes m is for metal (for example, the complexes Zn-protein); and/or nonionic surfactants such as TWEENTM, PLURONICSTMor polyethylene glycol (PEG). Pharmaceutically acceptable excipients are additionally described here.

In one embodiment, the antibody is introduced into the organism in the composition in the form of a sterile aqueous solution having a pH value in the range from about 5.0 to about 6.5 and containing antibody from about 1 mg/ml to about 200 mg/ml his-tag buffer is from about 1 millimolar to about 100 millimolar of Polysorbate 80 from about 0.01 mg/ml to about 10 mg/ml trehalose from about 100 millimolar to about 400 millimolar and disodium EDTA dehydrate approximately from 0.01 millimolar to approximately 1.0 millimolar.

The antibody-antagonist against PCSK9 and its composition can also be used in combination with other agents that are designed to enhance and/or complement the effectiveness of these substances.

D. Sets

The invention also provides kits for use in the Express system. Kits according to this invention include one or more containers containing the antibody antagonist against PCSK9 (e.g., humanitariannet antibody or a peptide described herein, and instructions for use in compliance and with all methods according to this invention, as described here. Typically, these instructions contain a description of the application of antagonistic antibody, peptide or aptamer against PCSK9 to the above-described therapeutic effects.

In some embodiments, the implementation of this antibody is humanitariannet antibody. In some embodiments, the implementation of this antibody is a human antibody. In other embodiments, the implementation of this antibody is a monoclonal antibody. Instructions for use the antibody is an antagonist against PCSK9, typically include information on dosage, dosage regimen, route of administration for the intended treatment. These containers can contain a single dose, mnogorazovye packaging (e.g. packaging with multiple doses) or subtrate dose. The instructions provided with the kits according to this invention, in most cases, are the written instructions on the label or the leaflet insert in packaging (such as the paper form included in the set), but is also suitable recognized machine instructions (such as instructions on a magnetic disk or optical disk memory).

Kits according to this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed packages splanky Mylar or plastic bags), and the like. Also provides packaging for use in combination with a special device such as an inhaler, a device for nasal administration (e.g., sprinklers) or device for infusion, such as minipump. The set may have a sterile access hole (for example, the container may be a bag for solution for intravenous or vial having a stopper protegeme needle for subcutaneous injection). The container also may have a sterile access hole (for example, the container may be a package solution for intravenous or bottle with stopper, sharp sterile needle for subcutaneous injection). At least one active substance in the composition is an antibody antagonist against PCSK9. The container (for example, pre-filled syringe or autoinjector) may further comprise a second pharmaceutically active substance.

The sets can be optionally provided with additional components such as buffers, and information for interpretation. Typically, the kit includes a container and a label or sheet(sheets)-liner on the container, or attached to the container.

Mutation and modification

For expression of antibody against PCSK9 according to the present invention, the DNA fragments encoding is vlasti V Hand VLfirst can be obtained using any method described above. Various modifications, such as mutations, deletions and/or additions can also be introduced into the DNA sequence using standard methods known to the person skilled in the art. For instance, mutagenesis can be carried out using standard methods, such as PCR-mediated mutagenesis, in which the mutated nucleotides introduced into the primers for PCR, so that the PCR contains the desired mutation or site-directed mutagenesis.

One type of replacement, for example, which can be done is to replace one or more cysteines in the antibody, which may be chemically reactive to another residue, such as, without limitation, alanine or serine. For example, it may be replaced with non-canonical cysteine. Replacement can be carried out in a CDR or framework region of the variable domain, or in the constant domain of the antibody. In some embodiments, the implementation of cysteine is canonical.

Antibodies can also be modified, for example, in the variable domains of the heavy and/or light chains, for example, to change the binding properties of this antibody. For example, a mutation may be made in one or more of the CDR regions to increase or decrease KDthis and the Titel for PCSK9, to increase or decrease koffor to change the binding specificity of this antibody. Methods site-directed mutagenesis is well known in the prior art. See, for example, Sambrook et al. and Ausubel et al., above.

Modifications or mutations can also be carried out in the framework region or constant domain to increase the time half-life of antibodies against PCSK9. See, e.g., PCT publication no WO 00/09560. The mutation in the frame region or constant domain may also be made to modify the immunogenicity of this antibody, to provide a site for covalent or non-covalent binding to another molecule or to change such properties as complement fixation, the FcR binding and antibody-dependent cretaceouspaleogene cytotoxicity. In accordance with this invention a single antibody may contain mutations in any one or more CDR or frame regions of the variable domain, or in the constant domain.

In a process called "the process of returning to the embryonic genotype" ("germlining"), some amino acids in sequences of VHand VLcan be changed to coincide with those found in nature in sequences of VHand VLthe germ line. In particular, the amino acid sequence of frame the regions of sequences V Hand VLcan be modified to match the sequences of the germline, to reduce the risk of immunogenicity of the introduction of this antibody. The DNA sequence of a germ-line genes for VHand VLthe man known in the prior art (see, for example, a database of sequences germ lines man "Vbase"; see also Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH publication No. 91-3242; Tomlinson et al., 1992, J. Mol. Biol. 227:776-798; and Cox et al., 1994, Eur. J. Immunol. 24:827-836.

Another type of amino acid substitutions that can be made is the removal of potential proteolytic sites in the antibody. Such sites can meet in a CDR or framework region of the variable domain or in the constant domain of the antibody. Replacement of cysteine residues and removal of proteolytic sites can reduce the risk of heterogeneity in product antibodies and thus increase its homogeneity. Another type of amino acid substitution eliminates a pair of l-asparagine-glycine, which form potential areas desametasone, as a result of changing one or both of these residues. In another example, can be split With lysine-end of the heavy chain of the antibody against PCSK9 according to this invention. In various embodiments of this invention, the heavy and Le is the cue chain antibodies against PCSK9 may optionally include a signal sequence.

After receiving the DNA fragments coding segments VHand VLaccording to the present invention, these DNA fragments can be further processed using standard methods of recombinant DNA, for example, to convert gene variable region genes in a full-size chain antibodies, genes Fab fragment or a scFv gene. During these manipulations, the DNA fragment encoding the VLor VHfunctionally linked to another DNA fragment, encoding another protein, such as protein a constant region of the antibody or a flexible linker. Used herein, the term "functionally linked" means that two fragments of DNA are connected so that the amino acid sequences encoded by the two DNA fragments remain in the frame.

The selected DNA encoding the V regionHcan be converted into a gene of a full-sized heavy chains through functional binding of the DNA that encodes the VHon the other DNA molecule that encodes a constant region of the heavy chain (CH1, CH2 and CH3). The gene sequence of the constant region of the heavy chain of the man known in the prior art (see, e.g., Kabat, E. A., et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publ. No. 91-3242)and DNA fragments containing this region, can be obtained by standard PCR amplification Constant region of the heavy chain may be a constant region IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD, but the most preferred is a constant region IgG1 or IgG2. The sequence of the constant region of IgG may be any of many alleles or allotypes encountered among different individuals, such as Gm(1), Gm(2)Gm(3) Gm(17). These allotype are representatives of the naturally occurring amino acid substitutions in the constant regions of IgG1. For gene heavy chain of the Fab fragment of DNA encoding the VHmay be functionally linked to another DNA molecule encoding only the constant region SN heavy chain. The constant region of the heavy chain SN can be obtained from any heavy chain genes.

The selected DNA encoding the V regionLcan be converted to full gene light chain (as well as in gene Fab light chain) using the functional binding of the DNA that encodes the VLon the other DNA molecule that encodes a constant region of light chain, CL. The gene sequence of the constant region of the light chain of a man known in the prior art (see, e.g., Kabat, E. A., et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publ. No. 91-3242)and DNA fragments containing this region, can be obtained by standard PCR amplification. The constant region of the light chain may be a constant region of the Kappa iliamna. The constant region of Kappa can be any of the different alleles found in different individuals, for example, Inv(1), Inv(2) and Inv(3). The constant region of the lambda can be obtained from any of the three genes lambda.

To create a scFv gene, the DNA fragments encoding the VHand VLfunctionally linked to another fragment encoding a flexible linker, such as encoding the amino acid sequence (Gly4-Ser)3so that the data sequence VHand VLcan be expressed in the form of a conjugate protein with single-stranded regions VLand VHconnected to the data by a flexible linker (see, for example, Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554). This single-chain antibody can be monovalent, if you use only a single VHand VL, bivalent, if used two VHand VLor polyvalent, if used more than two VHand VL. Can be created bespecifically or polyvalent antibodies that specifically bind to PCSK9 and with another molecule.

In another embodiment, can be obtained fused antibody or immunoadhesin that contains all or part of an antibody against PCSK9 according to the present invention associated with another polypeptide. In another embodiment, Khujand the exercise of only the variable domains of antibodies against PCSK9 is associated with a given polypeptide. In another embodiment, the domain VHantibodies against PCSK9 is associated with the first polypeptide, while the domain of VLantibodies against PCSK9 is associated with a second polypeptide, which is connected with the first polypeptide in such a way that the domains VHand VLcan interact with each other for forming the binding site of the antigen. In another preferred embodiment, the domain VHseparated from the domain VLlinker, so that the data domains of the VHand VLcan interact with each other. Titulo VH-linker-VLthen combined with the interest of the polypeptide. In addition, can be created and merged antibodies in which two (or more) single-chain antibodies are connected to each other. This is used if there is a desire to create a divalent or polyvalent antibody on a single polypeptide chain, or if there is a desire to create bespecifically antibody.

In other embodiments, the implementation of other modified antibodies may be obtained using nucleic acid molecules encoding the antibody against PCSK9. For example, "Kappa body" (Ill et al., 1997, Protein Eng. 10:949-57), "Minitel" (Martin et al., 1994, EMBO J. 13:5303-9), "diately" (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448) or Janusins" (Traunecker et al., 1991, EMBO J. 10:3655-3659 and Traunecker et al., 1992, Int. J. Cancer (Suppl.) 7:51-52) can be perceived by the s using standard methods of molecular biology, following the ideas given in the description.

Bespecifically antibodies or antihistamie fragments, can be obtained in various ways, including the fusion of hybridomas or linking of Fab fragments'. See, for example, Songsivilai & Lachmann, 1990, Clin. Exp. Immunol. 79:315-321, Kostelny et al., 1992, J. Immunol. 148:1547-1553. In addition, bespecifically antibodies may be formed as "diately" or "Janusins". In some embodiments, the implementation bespecifically antibodies bind to two different epitopes on PCSK9. In some embodiments, the implementation of data the modified antibodies described above are obtained using one or more variable domains or CDR regions of a human antibody against PCSK9 provided here.

Obtaining antigen-specific antibodies

More than 500 polyclonal and monoclonal antibodies generated against recombinant full-PCSK9 human, recombinant full-sized mouse PCSK9, and assessed various synthetic peptides for their ability to down regulation of protein LDLR in cultured liver cells human Huh7. Among these antibodies was a group of antibodies that are grown to and reactive with a group of polypeptides of 12-20 amino acid residues, which, as was predicted based on the structure of PCSK9, cover most of the surface is rnost protein. At the highest concentration of the best antibodies showed only approximately 60% blocking activity.

Thus, there was used an alternative and, to date, has not been studied approach, namely the development of monoclonal antibodies using PCSK9-zero mice immunized with the recombinant full-length protein PCSK9. This method of obtaining antibodies were generated antibodies, antagonists that demonstrate blocking the binding of PCSK9 to LDLR, full blocking PCSK9-mediated reduction of LDLR levels in Huh7 cells and reduction of LDLc in vivo, including mice, to levels comparable with those observed in mice PCSK9 -/-, as shown in Example 7.

Representative antibodies (hybridoma) according to the present invention were deposited in the American type culture collection (ATSS) February 28, 2008, and assigned them access numbers presented in Table 3. Were placed on the storage hybridoma for antibodies A, A, 6F6 and 7D4.

Table 3
Standard antibodyno access to ATS
AMOUTH-8985
AMOUTH-8986
6F6MOUTH-8984
7D4MOUTH-8983

Examples

Example 1: Generation and screening of antibody antagonists against PCSK9

General procedures for immunization of animals for the production of monoclonal antibodies:

Mice Balb/c or 129/bl6 pcsk9 -/- took an injection with 100 μg of antigen 5 times during the 13-day plan immunization. PCSK9 -/- (which are zero - or knockout mice) can be obtained from, or as described in, Rashid et al., 2005, Proc Natl Acad Sci USA 102:5374. See also U.S. patent No. 7300754. For the first 4 injections of the antigen was prepared by mixing recombinant protein with adjuvant, the immunogen was introduced through an injection in the back of the neck, in the sole of the foot or intraperitoneally, approximately every 3 days during the 11-day course, with the last booster administered intravenously without adjuvant. On day 13, mice painlessly were killed, and took their spleen. Lymphocytes were immortality by merging with the developed cell line to create clones of hybridomas using standard hybridoma technology, distributed in 96-well plates. Clones left for growth, then were selected by screening using ELISA, using the immunizing antigen, as shown below.

ELISA screening antic the l:

Organized the screening of supernatants from growing clones of hybridoma individually on their ability to bind recombinant human PCSK9 or recombinant mouse PCSK9. These studies were performed in 96-well tablets, coated overnight with 100 μl of 1 μg/ml of one of the antigens. After each stage the tablets were washed from excess reagents by using PBS containing 0.05% Tween-20. Then the tablets were blocked using PBS containing 0.05% BSA. The supernatant was added to these plates and incubated at room temperature for 2 hours. Added anti-mouse Fc goat conjugated with horseradish peroxidase (HRP), for binding to murine antibodies, United with the given antigen. Then added tetramethyl benzidine as a substrate for HRP, in order to determine the number of mouse antibodies in the supernatant. The reaction was stopped and the relative amount of antibodies was calculated by reading the absorption at 450 nm. Clones of hybridoma that secretively antibodies capable of binding with either murine or human PCSK9 were selected for further analysis.

PCSK9-mediated oppressive regulation of LDLR in Huh7 cells:

Clones of hybridoma, secreting antibodies that bind human or murine PCSK9 were multiplied and supernatant were collected. About the s IgG were purified from approximately 10 ml of the supernatant using beads with protein A, detalizirovan in PBS buffer, and the final volume was reduced to obtain solutions from 0.7-1 mg/ml antibody. Purified antibodies were then used to test their ability to inhibit the ability of PCSK9 to mediate the generally hopeless regulation of LDLR in Huh7 cells. The Huh7 cells were planted in 96-well plates and left to grow until reaching 80% of confluently in RPMI medium containing 10% FBS, 4 mm glutamine, penicillin and streptavidin. This medium was replaced by another containing 10% delipidation FBS, 8-16 hours for the induction of expression of LDLR. Then cells were incubated for 8-16 hours with 40 μl/well medium for expression 293, supplemented with 6 μg/ml human (preferably) or murine PCSK9, with or without 70-100 μg/ml of the tested antibodies. At the end of the incubation remove the medium containing PCSK9 and the antibody, and the cells were literally with 17 ál lyse buffer by shaking for one hour at 4°C. This lytic buffer consists of 50 mm glycerol phosphate, 10 mm HEPES pH 7.4, 1% Triton X-100, 20 mm NaCl and a mixture of protease inhibitors (Roche). Cell lysates were collected and tested for levels of LDLR protein after staining of Western blots after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Clones of hybridomas that produce antibodies that can partially or completely to maintain the level of LDLR were selected for the far is his analysis. Under the "analysis oppressive regulation of LDLR" mean the above analysis using Huh7 cells.

The Figure 1 shows the effect of antagonistic monoclonal antibodies 7D4.4, 4A5.G3, 6F6.G10.3 and 5A10.B8 against PCSK9 on the ability of human and murine PCSK9 to suppress LDLR in cultured Huh7 cells. Used 100 nm mouse or human recombinant PCSK9, and serial dilution 25-800 nm antibody. A) mouse PCSK9. B) human PCSK9. On the figures presented, Western blotting, demonstrating that these antibodies, in most cases, are more effective at blocking function PCSK9 human than mouse PCSK9. Several antibodies have generally similar affinity to human PCSK9, but differ in their affiniscape to mouse PCSK9.

Example 2: Determining binding affinity of antibodies

Affinity antibodies to PCSK9 were determined using surface plasmon resonance on the biosensor Biacore 3000, equipped with a sensor chip research quality using a rolling buffer HBS-EP (Biacore AB, Uppsala, Sweden - now GE Healthcare). Rabbit polyclonal anti-Ms IgG were aminosidine saturated with a chip using standard chemicals N-hydroxysuccinimide/ethyldimethylamine carbodiimide (NHS/EDC). The buffer was replaced with HBS-EP+1 mg/ml BSA+1 mg/ml of CM-dextran. Full-size IgG against PCSK9 was diluted closer is Ino to 15 µg/ml and was captured at 1 min at 5 μl/min to obtain levels of approximately 500 RU on flow cell, leaving one blank as a reference channel. to 3.73-302 nm hPCSK9 or 2,54-206 nm mPCSK9 was injected in the form of a 5-membered 3-fold series for 1 min at 100 μl/min Dissociation was monitored for 5 min Chip was restored after the last injection of each title with two 30-second stimulus 100 mm phosphoric acid. Buffer cycles provided forms for double compare with the reference data, which then were generally approximated in a simple binding model using the software Biaevaluation v.4.1. Affinity were derived from the ratio of the kinetic rate constants of (KD=koff/kon). The results of Example 2 are presented in Table 4. These results demonstrate that these antibodies have excellent activity against PCSK9 mouse or human PCSK9, as shown.

Table 4
mAbThe ligandInhibitor communication LDLR-PCSK9Konfor PCSK9 (1/s)Kofffor PCSK9 (1/s)KDfor PCSK9 (nm)
4A5people0.4 nmof 6.66×104 1,89×10-42,8
5A10people0.4 nmof 8.47×1048,55×10-51
6F6people1.5 nmto 9.15×104of 5.84×10-46,4
7D4people1.5 nm1,25×1057,94×10-46,4
4A5mouse3 nmof 1.41×105of 7.2×10-45,1
5A10mouse3 nmof 1.27×1054,89×10-4a 3.9
6F6mouse10 nmis 1.11×105of 1.97×10-317,7
7D4mouse1 nmto 3.92×1045,23×10-41,3

Example 3: Analysis of the effect of antibodies against PCSK9 to the interaction of PCSK9-LDLR

It was shown that PCSK9 binds to LDLR with the affinity of 180 nm at neutral pH (Cunningham et al., 2007, Nat Struct Mol Biol, 14(5):413-9). Recombinant protein PCSK9 human or mouse was biotinylated using reagents Pierce, following the instructions of the manufacturer. Tablets for ELISA (Corning Mixisorb) were covered with a solution of 1 μg/ml of the extracellular domain of recombinant LDLR (R&D Systems) in each well at 4°C overnight, blocked with 2% BSA+PBS for 2 hours at room temperature, and then washed 5 times with buffer for washing (1× PBS+0,05% Tween-20). The wells were incubated with 50 ál of biotinylated PCSK9 protein selected concentration for 1 h at room temperature. Communication LDLR-PCSK9 can be stabilized by adding 50 μl solution of 4% FDH+4% sucrose+PBS and incubation for 5 minutes, the Wells were washed 5 times with buffer for washing, incubated with 1:2000 dilution of HRP, conjugated with streptavidin (Invitrogen) for 1 h at room temperature, washed 5 times with buffer for washing. The substrate TMB was added to the wells, the solution which was inkubirovali from 20 to 30 min at room temperature, and the reaction was stopped by adding 1 M phosphoric acid. Signals were read at 450 nm.

The Figure 2 shows the effect of dose antagonistic against PCSK9 monoclonal antibodies 6F6.G10.3, 7D4.4, 4A5.G3, 5A10.B8, antibody negative control 42H7 and PBS to block binding of recombinant biotinylated human PCSK9 and PCSK9 mouse with immobilized extracellular domain of recombinant LDLRin vitro. In part (A) demonstrated the binding of PCSK9 person with the extracellular domain of LDLR person and that 7D4, 4A5, 5A10 and 6F6 are effective in blocking the binding, whereas 42H7 and PBS are not. In part (B) demonstrated the binding of PCSK9 mouse extracellular domain of LDLR man.

This interaction can also be demonstrated in free solution at a neutral pH value. The Figure 3 presents the effect of dose antagonistic monoclonal antibodies 6F6.G10.3, 7D4.4, 4A5.G3 and 5A10.B8 against PCSK9 to block binding of recombinant biotinylated human PCSK9 (30 nm) labeled with europium extracellular domain of recombinant LDLR (10 nm) in solution with a neutral pH value ofin vitro. In this assay measures the binding in free solution at a neutral pH value.

Example 4: Mapping of epitopes/binding antibodies using the crystal structure of the complex 1L3:PCSK9, Biacore and mutagenesis

a. Crystal structure of the complex L1L3:PCSK9. The remains were identified as a result of calculation of the difference in accessible surface area between the crystal structure L1L3:PCSK9 and structure of one PCSK9. Residues of PCSK9, which show a hidden surface area in the formation of a complex with the antibody L1L3, were taken into account as part of the epitope. This is available for dissolving the surface of a protein is defined as the locus in the centre of a probe sphere (representing the molecule of solvent radius of 1.4 Å), because it turns the van-der-valesova surface protein. Available for dissolution of the surface is calculated by creating a surface point on the extended sphere around each atom (the distance from the center of the atom is equivalent to the sum of the radii of the given atom and probe) and excluding those that are inside the equivalent spheres associated with the neighboring atoms, as implemented in the program AREAIMOL (Briggs, P.J., 2000, CCP4 Newsletter No. 38, CCLRC, Daresbury).

The results of the analysis of the crystal structure presented on Figure 23. In Figure 23A presents the crystal structure of PCSK9 (light gray image surface)associated with the antibody L1L3 (black image). The epitope for binding L1L3 with PCSK9 includes residues 153 to 155, 194, 197, 237-239, 367, 369, 374-379 and 381 amino acid sequence of PCSK9 (SEQ ID NO: 3). For comparison, the epitope binding domain of the LDLR EGF with PCSK9 includes residues 153 to 155, 194, 238, 367, 369, 372, 374-375 and 377-381 (Kwon et al., 2008, PNAS 105:1820-1825).

b. Group antibodies and epitopes, based on competition for binding to PCSK9. Full-size IgG were amino-connected touch chip SM (three on the chip, the end approximately 7000 RU)using EDC/NHS-mediated standard chemistry of amino compounds. One flow cell was left unmodified to provide a reference channel. PCSK9 human (100 nm) was pre-mixed with a mixture of IgG (500 nm final), and these complexes were injected into the chip through a 1-min injections of 10 ál/min Antibodies that are associated with competing epitopes, will block the binding of PCSK9 with the antibody immobilized on the chip. An alternative used the classic sandwich method, first injecting human PCSK9 at 50 nm for 1 min at a rate of 10 μl/min (for tying it by IgG on the chip) and then linking the mixture of IgG (final 500 nm each) for 2 min each. Fixed IgG was recovered using a weak acid (soft buffer for elution Pierce+1M NaCl). Antibodies directed against a variety of known epitopes, were used as positive controls for education sandwich in this analysis.

c. Structurally-induced mutagenesis to map titulovannaya epitopes. Based on the crystal structure of PCSK9 and the possible involvement D374 in the LDLR binding (Cunningham et al., 2007, Nat Struct MoI Biol, 14(5):413-419), nineteen mutant surface residues of PCSK9 (F379A, I369A, R194A, D374Y, D238R, T377R, K222A, R199A, F216A, R218A, R237A, D192R, D367R, R165A, R167A, A443T, A53V, I474V, H449A), close to or away from the position D374, were selected for the changes to mapping antielastase epitopes.

d. The mutant and the formation of antibodies. Data 19 point mutations were generated from the previously described design DNA of the wild type (Cunningham et al., 2007, above)using standard DNA technology. Mutant proteins were expressed using unstable transfection in HEK293T cells, secreted into the cell medium. Data mutant proteins were purified using high throughput system AKTA Xpress (GE Healthcare) via Ni2+gel chromatography, using conditions similar to that described previously. Protein concentration was determined using the LabChip instrument (Bio-Rad). Blocking murine PCSK9 antibodies 4A5, 7D4, 5A10 and 6F6 were expressed by unstable transfection in HEK293F cells, purified using a column of protein G, suirvey buffer 0.1 M glycine at pH 2.8 and neutralized in 1.0 M Tris at a pH of 9.0.

e. The field of PCSK9 that come into contact with a monoclonal antibody 5A10 and 7D4 (getting described below) were determined by imaging the tree (Sidec AB, Stockholm, Sweden). Loops provisions 186-200, 371-379, 176-181, 278-283, 449-453, 402-406 and 236-245 this PCSK9 were close to amino acid residues of this antibody. The sequence corresponding to the hinge, shown in Table 5, and in the preferred embodiment, antagonists according to this invention is contacted with one or more of these sequences in PSCK9.

f. Biacore binding mutants to immobilized LDLR. Protein is the extracellular domain of recombinant LDLR was recorded on the Biacore chip SA. Each mutant protein was injected into the Biacore-3000 M) in two copies from 25 mm to 0.012 mm and at five concentrations ranging from 1°C, with a movable buffer 50 mm Tris pH 7.5, 2 mm CaCl2, 200 mm NaCl, 0.02% of P20 and 1 mg/ml BSA. All results correspond exactly to the kinetic model binding is 1:1. In accordance with the expected mutation of residues direct contact with domain EGF-A (F379A, R194A, I369A, T377R, D238R) significantly weakens (10-100-fold) binding to LDLR. Moreover, three mutations, not in contact with EGF-A (R199A, R218A, K222A) showed weaker binding (5-15-fold). These new data indicate that they are involved in binding other domains of LDLR. In General, these experiments confirm the integrity and activity data mutants for subsequent experiments on mapping epitopes.

g. Binding mutants with immobilis Rovaniemi antibodies 4A5, 7D4, 5A10 and 6F6. Biotinylated antibodies against PCSK9 were immobilizovana on chips SA using standard methods. Experiments on binding mutants were performed using Biacore 3000 at 25°C with a movable buffer 50 mm Tris-HCl pH 7.5, 150 mm NaCl and 0.02% P20. The specimens were tested at concentrations of 333 nm or 111 nm in duplicate with those that give weak binding compared with wild type, due to the residues involved in the binding of mAb (listed below).

mAbbinding residues in the descending order of mutant effects
4A5R237, F379, 369, R194, R199 & D238
5A10R194, R237, I369, D238, R199
6F6R237, R194, F379, D238, I369, T377, R199
7D4R237, R194, F379, I369, R199

Example 5: Cloning and sequencing of antibodies

One million cells hybridoma homogenized using a spin-QIAshredder columns, and total RNA was isolated using the RNAeasy kit Micro company QIAGEN. Using a set of Superscript III RT manufactured by Invitrogen, synthesized cDNA. Variable regions of antibodies against PCSK9 cloned and with the use of sets of mouse IgG-Primer Sets firm Novagen, which contain degenerate primers for cloning genes of the heavy chains of mouse IgG and Kappa and lambda light chains of mouse IgG. The PCR conditions were: 1 cycle at 92°C for 2 min; two cycles at 94°C for 30 s, 44°C for 30 s and 72°C for 2 min; two cycles at 94°C for 30 s, 46°C for 30 s and 72°C for 2 min; two cycles at 94°C for 30 s, 48°C for 30 IC, and 72°C for 2 min; two cycles when 94°C for 30 s, 50°C for 30 s and 72°C for 2 min; two cycles at 94°C for 30 s, 52°C for 30 s and 72°C for 2 min; followed by 35 cycles at 94°C for 30 s, 54°C for 30 s and 72°C for 45 C. the Obtained PCR products were cloned into a vector for cloning Topo-TA, firms Invitrogen, and sequenced. The sequence of the cloned antibodies were confirmed by N-terminal sequencing of the first 10 amino acids of the original antibody derived from ascites.

Example 6: Preparation of antigens for immunization

Recombinant protein of human PCSK9 were obtained as described by Cunningham et al., 2007, Nat Struct Mol Biol, 14(5):413-9. To obtain recombinant protein mouse PCSK9 cDNA of murine PCSK9 was cloned in expressing vector PRK5 mammal with tag 6-His-end, using techniques known in the prior art, unstable transliterowany and expressed in HEK293 cells. Recombinant protein was purified from the private balcony is nizovannoj environment using Ni-column.

Surface peptides of human and mouse PCSK9 were selected on the basis of the structure of the protein PCSK9, and synthesized using Elim Biopharmaceuticals.

Example 7: PCSK9-specific antibodies as antagonists against PCSK9

1. Identification of PCSK9-specific antibody antagonists

a. Identification of antibodies, blocking PCSK9

Mouse antibodies to human and/or murine PCSK9 were developed as a result of immunization of mice with synthetic peptides of human PCSK9 and PCSK9 mouse, obtained as described in Example 6, or recombinant proteins, and organized the screening of antibodies by ELISA analysis using recombinant protein PCSK9 human and/or mouse as antigens, as described in Example 1 and other standard hybridoma methods. More than 500 positive clones were obtained and grown to confluently 6-hole tablets in 10 ml of medium. Collected supernatant, and total immunoglobulins in the conditioned medium were purified using mAb Select (Pierce). The ability of purified and concentrated mouse IgG to inhibit the function of mouse and human PCSK9 was tested in Huh7 cells using the methods described in Example 1. Clones of hybridoma expressing IgG, which exhibit some degree of blockade, were copied and re-tested. 60 promising clones were subclan is arranged, copied and injected either Balb/c or Nude mice, for the formation of ascites. Antibodies purified from ascitic fluids were re-tested for their ability to inhibit the generally hopeless regulation of LDLR human or murine PCSK9 in Huh7 cells. Four hybridoma clone 4A5, 5A10, 6F6 and 7D4, were identified as possessing the ability to inhibit the function of PCSK9 person, and at least partially inhibit the function of PCSK9 mouse. To determine the IC50for each of these blocking antibodies, a serial dilution of IgG used in this study, starting with 100 μg/ml to of 3.125 μg/ml, with a concentration in human and mouse PCSK9 constantly equal to 6 mg/ml

b. The effect of antagonists of PCSK9 on the link PCSK9-LDLR

It was shown that PCSK9 is localized together with LDLR at the cell compartments (Lagace et al., 2006, J Clin Inv, 116(11):2995-3005). Recombinant PCSK9 protein also binds to the extracellular domain of LDLRin vitro(Fisher et al., 2007, JBC, 282(28):20502-12). To determine a relationship between inhibition of PCSK9-mediated down regulation LDLR and inhibition due PCSK9-LDLR antibodies, the authors of this invention was tested for antibodies against PCSK9, which partially or completely block the function of PCSK9 on LDLR, and samples of antibodies that do not block. All partially antagonistic antibodies partially inhibited with Azania extracellular domain of LDLR with PCSK9, with the exception of one. Antibody antagonists, which completely block the function of PCSK9, namely 4A5, 5A10, 6F6 and 7D4, also completely blocked the binding of the extracellular domain of LDLR with PCSK9 (table 5). The values of the IC50for these four antibodies correlate with their affinity to bind to PCSK9.

c. The definition of epitopes data blocking antibodies

The Figure 4 shows the binding of anti-PSCK9 antibodies with epitopes. In part (A) presents information about the epitope of anti-PCSK9 mAb, specific binding of synthetic 13-18-dimensional peptide or binding epitope by Biacore. In part (B) displayed by Biacore analysis, the ability of immobilized antibodies 6F6, 5A10 and 4A5 to bind to human PCSK9, pre-mixed with mAb indicated on the y-axis.

Another monoclonal antibody against PCSK9, called 6G7, binds to recombinant PCSK9 mouse, but not human PCSK9. See Table 6. 6G7, 4A5, 5A10, 6F6 and 7D4 mutually exclude the ability of each other to bind to mouse PCSK9. Hybrid analysis between mouse and human PCSK9 showed that binding 6G7 with PCSK9 necessary catalytic domain. See Table 6. Thus, the binding sites on 4A5, 5A10, 6F6 and 7D4 block the catalytic site and/or the epitope associated with 6G7.

Table is CA 6
Recombinant protein6G7 binding
PCSK9 manNo
The human protein+catalytic site man+To-end mouseNo
The human protein+catalytic site mouse+s-end mouseYes
Protein mouse+catalytic site man+With the end of manNo
Protein mouse+catalytic site mouse+-the end of manYes
PCSK9 mouseYes

d. Determination of species-specific sequences of an antibody against PCSK9

To determine withspecific of antibodies against PCSK9 antibodies were incubated with plasma, obtained from different species, and the resulting complexes were purified and tested with independent antibody against PCSK9 using Western blotting. Antibodies 4A5, 5A10, 6F6 and 7D4 recognize PCSK9 human, simian, mouse, and rat. See Figure 5. Antelo 6G7 recognized only murine PCSK9, and independent control antibody 42H7 didn't recognize none of the tested PCSK9. Id.

e. Identify the bookmark sequences, antibodies, antagonists against PCSK9

Amino acid sequences of variable domains of PCSK9 antibodies 4A5, 5A10, 6F6 and 7D4 were determined using the method described in Example 5. Sequence data indicate that these antibodies are related but distinct from each other. Table 1 presents the amino acid sequence of the variable regions of each antibody. Table 7 shows the sequence of the CDR of the light chain and heavy chains from Table 1, as determined by the methods of Kabat (Kabat), Hatia (Chotia).

Anti-PCSK9 IgG 4A5, 5A10 and 6F6 using amine were combined with the Biacore chip. hPCSK9 (100 nm) was mixed with 500 nm 4A5, 5A10, 6F6 or 7D4 in different ratios and injected for 1 min at a rate of 10 μl/min, these four antibodies mutually block each other regardless investigated orientation, which indicates that they all are associated with competing epitopes. In contrast, they are able to form sandwich complexes with other partially blocking antibodies, which were mapped in specific areas, using synthetic peptides.

2. The effect of PCSK9-specific antibodies as antagonist against PCSK9in vivo

a. Antibody antagonists against PCSK9 to reduce cholesterol in the blood serum of mice

To determine that is about, can a monoclonal antibody antagonists against PCSK9 to influence cholesterol levelsin vivoby inhibiting the function of extracellular PCSK9, action 7D4 was tested against murine PCSK9in vitroon cholesterol blood serum after injection mice. Male mice C57/bl6 aged 6-7 weeks were kept under a 12 h Swettenham cycle, on day -7 allowed the blood to collect approximately 70 μl of serum. Antibody antagonist 7D4 against PCSK9 and control, matched isotype monoclonal antibody was administered to 7-week old male mice C57/bl6 by intraperitoneal (I.P. Pavlova.) injection at day 0, 1, 2 and 3. Mice painlessly were killed on day 4 without prior food restrictions and collected samples of blood serum. All frozen samples of blood serum were sent to IDEXX laboratories for measurements of total cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol. In Figure 6 it is shown that 7D4 reduced the level of cholesterol in blood serum at 48%, while the control antibody had no significant effect. And the number and percentage reductions were similar to those that have been reported for mice PCSK9-/- (knockout mouse PCSK9), which indicates that it is possible to achieve complete or nearly complete inhibition of PCSK9 function, blocking only the extracellular PCSK9, and that intracellular PCSK9 plays a small role, which does not play any, in down regulation of LDLR in normal physiological conditions. As expected, hepatic LDLR levels were stimulated in animals, which was introduced 7D4, compared to animals, which was a control antibody (Figure 6).

b. Partially blocking antibodies do not affect cholesterol levels in the blood

In Figure 7 it is shown that partially antagonistic polyclonal anti-PCSK9 mAb CRN6 does not affect cholesterol levels in mice. Two groups of 8-week-old mice C57/bl6 (n=10 mice/group) were allowed blood and perform the test cholesterol levels on day -7; mice were administered a dose of 15 mg/kg/day of antibodies CRN6 or control antibodies through the/in the day 0, 1, 2, and 3; and then drawing blood and checking the cholesterol levels 24 hours after the last dose. Figure 7A shows that the antibody CRN6 partially blocking PCSK9 mediated generally hopeless regulation of LDLR in Huh7 cellsin vitro. Figure 7B shows that the introduction of antibodies CRN6 does not affect cholesterol levels in the serum of mice.

c. Prolonged effect of mAb-antagonist against PCSK9 levels of serum cholesterol in mice.

The study period was carried out to determine the start time and duration to reduce cholesterol actions antibody antagonists against PCSK9 in mice. mAb 7D4 or physiological control Astor was introduced by/injection dose of 10 mg/kg or 3 ml/kg 48 6-week old mice C57/bl6. Eight mice from each of the treated groups were painlessly euthanized at day 1, 2, 4, 7, 14 and 21 after injection. Single injection 7D4 caused a rapid and sustained effect of reducing the levels of serum cholesterol. The 25% reduction in cholesterol in the serum was observed at 24 h after injection. See Figure 8. The maximum reduction of serum cholesterol were observed on day 7. At day 21 the reduction of cholesterol is no longer statistically significant. In part (B) presents the levels of HDL-cholesterol. LDL-cholesterol was very low.

The Figure 9 shows that the antibody antagonist against PCSK9 mAb 7D4 dose-dependent decreases the levels of total cholesterol, HDL and LDL in serum in mice. 8-week old mice C57/bl6 of the six groups (n=8/group) were allowed blood and perform the test basal levels of cholesterol per day -7 and were administered the indicated doses of antibodies or saline on day 0, 1, 2 and 3 by intraperitoneal (I.P. Pavlova.) bolus injection. Samples of serum were collected and were used to define levels of cholesterol 24 hours after the last dose. Figure 9A presents the levels of total cholesterol, which decreased to less than 60% of the control after the introduction of from 3 to 30 mg/kg/day. The maximum effect on total cholesterol was observed at the dose of 10 mg/kg, and a statistically significant decrease at 1 mg/kg per Figures who 9B presents the levels of HDL, which reduced to less than 70% after the introduction of from 3 to 30 mg/kg/day. Figure 9C presents the levels of LDL, which is reduced to almost zero at all tested doses of 0.3 mg/kg/day and above.

d. The effect of dose of antibody antagonists specific to PCSK9 in mice

The Figure 10 shows that the anti-PCSK9 antagonist antibody A dose-dependently reduces cholesterol levels in mice. Figure 10A presents six groups of 8-week-old mice C57/bl6 (n=8/group)that had introduced the dose of the antibody or saline daily at day 0, 1, 2 and 3, by/bolus injections. Samples of serum were collected and were used to define levels of cholesterol 24 hours after the last dose, and was shown a stepped decrease with increasing doses of the antibody. Figure 10B presents five groups of 8-week-old mice C57/bl6 (n=8/group)that had introduced the dose of the antibody or saline on day 0 by intraperitoneal (I.P. Pavlova.) bolus injection. Collected samples of blood serum and determined the levels of cholesterol on day 7, and also showed a stepwise decrease with increasing doses of the antibody.

The Figure 11 shows that the anti-PCSK9 antagonist antibodies 4A5 and 6F6 reduce cholesterol levels in mice in a dose-dependent manner. 8-week old mice C57/bl6 (n=8/group) were administered the indicated doses of an antibody or a physiologically the RCM solution on day 0 by intraperitoneal (I.P. Pavlova.) bolus injection. Collected samples of blood serum and determined the levels of cholesterol on the 7th day. In Figure 11A antibody A shows a stepwise decrease in total serum cholesterol with increasing doses of the antibody. In Figure 11B antibody 6F6 shows the reduction of total serum cholesterol at a dose of 10 mg/kg/day.

Anti-PCSK9 antibody antagonists 4A5, 5A10, 6F6 and 7D4 increase hepatic LDLR levels in mice, as shown in the Western blot. See Figure 12. For 4A5, 5A10 and 6F6, 8-week old mice C57/bl6 were administered 10 mg/kg of antibody or saline on day 0 through/bolus injection on day 7 animals painlessly cut, and the lysate of whole liver from 3 separate animals were analyzed for levels of LDLR protein and GAPDH by Western blotting. For antibodies 7D4, 8-week old mice Bl6/c57 was administered 10 mg/kg of antibody on day 0, 1, 2 and 3 by intraperitoneal (I.P. Pavlova.) bolus injection, animals painlessly were killed on day 4, and the lysate of whole liver from 3 separate animals were analyzed for levels of LDLR protein and GAPDH by Western blotting. All mice that received injections of antibodies, revealed high levels of LDLR compared with control mice treated with PBS.

The Figure 13 shows that the anti-PCSK9 antibody antagonist has no effect on mice LDLR-/-. 8-week old mice LDLR-/- (mouse LDLR KO) was administered 10 mg/kg 4A5 or fisiologicas the second solution at day 0, through intraperitoneal (I.P. Pavlova.) bolus injection. Sample serum (n=9-10 mice) were collected and were used to define levels of cholesterol on day 7. The introduction of this antibody did not modify significantly the levels of total cholesterol, HDL or LDL in serum.

The Figure 14 shows that a comprehensive treatment of mice with anti-PCSK9 antibody antagonists can significantly reduce total serum cholesterol. 8-week old mice C57/bl6 were administered the indicated doses of antibodies or PBS at day 0, 7, 14 and 21 through/bolus injections. At day 28 serum samples (n=5-11 mice) were collected and were used to define levels of cholesterol.

Example 8: Antibody antagonists against PCSK9 to reduce the level of LDL in blood serum in primates that do not belong to the human race

To determine the effect ofin vivoantibodies against PCSK9 antibody 7D4 was tested on the humanoid monkeys. Four 3-4-year-old apes at day 0 were injected with the vehicle (PBS+0.01% of Tween 20) and antibody 7D4 10 mg/kg / day 7. The lipid profile of blood plasma were analyzed day 0, 2, 7, 9, 11, 14, 21 and 28, on an empty stomach. A single injection of 10 mg/kg antibody 7D4 caused a sharp decrease in LDL in the blood plasma (60%) (Figure 15A) and the number of LDL particles (Figure 15D) all 4 animals, while had minimal effect on their levels of HDL (Figure 15B) and the number of HDL particles (Figure 15E). Total cholesterol is Rin (Figure 15C) was also reduced after the introduction of 7D4, while the level of triglycerides (Figure 15F) was not significantly changed. Also measured the levels of total 7D4 (G) and levels of total PCSK9 (H).

Figure 16 illustrates the effect of dose of anti-PCSK9 antibodies 7D4 on the levels of serum cholesterol in apes. Two males and two females apes, ages 3-5 years, in each group by/bolus injections were administered a certain dose of antibodies 7D4 (day 7) and an equivalent volume of saline on day 0. The plasma samples were obtained at the indicated time points and measured the levels of LDL in the blood plasma.

The Figure 17 shows a comparison of the effects of anti-PCSK9 antibodies 4A5, 5A10, 6F6 in 7D4 on the levels of blood cholesterol from apes. Two males and two females apes, aged 3-6 in each group by/bolus injections were administered 1 mg/kg of the indicated antibodies at day 0. The plasma samples were obtained at the indicated time points, we measured the levels of LDL in blood plasma and normalized to those in day-2.

Figure 18 presents the effect of anti-PCSK9 antibodies of antagonist 7D4 on cholesterol levels in blood plasma from apes, whose diet contained 33,4% kcal fat with addition of 0.1% cholesterol. Within 16 weeks of the diet of six 3-5 years of apes was with a high fat content. In particular the military term three monkeys were injected antibody 7D4 at a dose of 10 mg/kg, and three monkeys were injected with saline. The levels of LDL in individual monkeys were measured and normalized with those on the day of processing.

Example 9: Humanitariannet anti-PCSK9 antibody

Mouse monoclonal antibody A was humanitarian and his affinity brought to maturity, to provide antibodies L1L3. The affinity of antibodies L1L3 to mouse PCSK9 is 200 PM, and the affinity to human PCSK9 is 100 PM, when measured by Biacore. L1L3 completely inhibits PCSK9-mediated generally hopeless regulation of LDLR in cultured Huh7 cells, incubation with 100 nm human or mouse antibodies against PCSK9. See Figure 19.

The Figure 20 shows the effect of dose L1L3, mouse predecessor 5A10 and antibody negative control 42H7 to the blockade of the binding of recombinant biotinylated human PCSK9 and PCSK9 mouse with immobilized extracellular domain of recombinant LDLRin vitro. Figure 20A shows PCSK9 person associated with the extracellular domain of LDLR person at pH 7.5. In the Figure 20B presents human PCSK9 associated with the extracellular domain of LDLR person at a pH of 5.3. Figure 20C shows the mouse PCSK9 associated with the extracellular domain of LDLR person at pH 7.5. Figure 20D shows the mouse PCSK9 associated with the extracellular domain of LDLR person at a pH of 5.3.

Figure 21 presents the effect of 10 mg/kg L1L3 the level of serum cholesterol in mice. 8-week old mice C57/bl6 in two groups (n=8/group) on day 0 by intraperitoneal (I.P. Pavlova.) the injection was administered 10 mg/kg antibody L1L3 or an equivalent amount of saline. Serum samples blood was collected at day 2, 4 and 7, and determined the levels of cholesterol. L1L3 reduced total serum cholesterol by approximately 40% at day 2 and 4. In another study, when 10 mg/kg L1L3 was administered as a single intraperitoneal dose (IP) to C57BL/6 mice fed a normal diet (n=10), the levels of serum cholesterol were reduced by 47% compared with the group treated with saline on day 4 after treatment. When testing the effect of dose L1L3 was administered as a single IP dose of 0, 0,1, 1, 10 and 80 mg/kg (n=6/group) male rats Sprague-Dawley receiving a normal diet, cholesterol levels were dose-dependently reduced, with the maximum effect 50% at doses of 10 and 80 mg/kg at 48 hours after injection. The duration of suppression of cholesterol was also dose-dependent in the range from 1 to 21 days.

Amino acid sequence fully humanized heavy chain L1L3 (SEQ ID NO: 15) shown in Table 8. The sequence of the variable region is underlined (SEQ ID NO: 54).

Amino acid sequence fully humanized light chain L1L3 (SEQ ID NO: 14) are presented in Table 9, the Sequence of the variable region is underlined (SEQ ID NO: 53).

Table 9
diqmtqspsslsasvgdrvtitcrasqgissalawyqqkpgkapklliysasyrytgvps60
rfsgsgsgtdftftisslqpediatyycqqryslwrtfgqgtkleikrtv aapsvfifpp 120
sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnSq esvteqdskd styslsstlt 180
lskadyekhk vyacevthqg lsspvtksfn rgec 214

Figure 22 presents the effect of intravenous administration of the effective dose (3 mg/kg) of antibody 5A10 (black circles in the graph) or antibodies L1L3 (black squares) for each of the four great apes in day zero. The change in HDL serum (Figure 22A) and LDL serum (Figure 22B) was measured in the interval from -2 to +28 days. The introduction of both antibodies has resulted in more than 70% reduction of LDL levels in the serum of approximately seven days, an effect that was largely remained approximately six days in animals that received L1L3. All animals were observed normal function of the liver and kidneys and almost normal hematocrit.

L1L3 dose-dependently reduced LDL-C, with the maximum effect observed in the group receiving 10 mg/kg, which was supported by 70% reduction of LDL-C to 21 days after administration of the dose, and fully recovered for 31 days. Introduction L1L3 did not affect the levels of HDL-C in all dosed groups. The animal is in a group with a dose of 3 mg/kg (n=4) was also introduced two additional/dose 3 mg/kg of antibody L1L3 at day 42 and 56 (every 2 weeks). These two additional doses again lowered LDL-C and maintained the levels of LDL-C to below 50% within 4 weeks. LDL-C was back to normal in two weeks. Levels of HDL-C serum remained unchanged throughout the study.

Were investigated the effectiveness L1L3 in patients with hypercholesterolemia primates, non-human, and pharmacodynamic interaction between L1L3 and statins, any abscopal HMG-CoA reductase. Before this study, for more than 18 months, a group of apes (n=12) were given a diet containing 35% fat (weight/weight) and 600 parts per million (ppm) cholesterol than raised levels of LDL-C in average up to 120 mg/DL compared with a normal average levels of 50 mg/DL. Surprisingly, there was no effect on total cholesterol or LDL-C serum after daily injection of the average dose (10 mg/animal) Crestor® (rosuvastatin calcium) for 6 weeks, and after subsequent daily high doses (20 mg/kg) for 2 weeks. A single injection of 3 mg/kg L1L3 with the use of Crestor® or media within 2 weeks effectively reduced LDL-C serum to 56% on day 5 after the treatment, and the levels of LDL-C was gradually recovered within 2.5-3 weeks, while no effect on levels of HDL-C. After transferring animals on a daily skin is of 50 mg/kg Zocor® (simvastatin), their LDL-C achieved a maximum reduction of 43% on day 5 and stabilized afterwards. After 3 weeks of introduction Zocor® 50 mg/kg/day, these animals were injected with a single dose of 3 mg/kg L1L3, while they still continued to enter Zocor® 50 mg/kg/day. Introduction L1L3 has resulted in an additional 65% decrease in LDL-C, in addition to 43% reduction by Zocor®, on day 5, and levels returned to the original within 2 weeks.

Other amino acid substitutions in the CDR were carried out in A in the process of humanization and bringing the affinity to maturity and to obtain specific properties. Sequence modified CDR and the ability to bind to PCSK9 for these antibodies containing these modified CDR shown in Figures 24A-g Numbers for each sequence in Figures 24A-G represent the SEQ ID NO for the following sequence.

Descriptions of all the cited reference material included here by reference.

1. The isolated antibody or variant, where the antibody or variant specifically bind PCSK9 and are full PCSK9 antagonist-mediated effect on LDLR levels, as measured in vitro in Huh7 cells, where the antibody contains the first complementarity determining region (CDR1) variable regions of the heavy chain (VH)having the amino acid sequence, p is redstavlennye in SEQ ID NO:8, 59 or 60, VH CDR2 having the amino acid sequence represented in SEQ ID NO:9 or 61, and VH CDR3 having the amino acid sequence represented in SEQ ID NO:10, and a CDR1 variable region of the light chain (VL)having the amino acid sequence represented in SEQ ID NO:11, VL CDR2 having the amino acid sequence represented in SEQ ID NO:12 and a VL CDR3 having the amino acid sequence represented in SEQ ID NO:13, and its variant contains a VH CDR1, VH CDR2, CDR3 VH, VL CDR1, VL CDR2 and VL CDR3, where the variant differs from the antibody that has one, two or three conservative amino acid substitutions in the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and/or CDR3 VL.

2. The antibody according to claim 1, where the variant VH CDR1 contains conservative amino acid substitution in position 8 in SEQ ID NO:59, a variant of the VH CDR2 contains conservative amino acid substitution in positions 3, 4, 5, 6 or 7 in SEQ ID NO:9, and/or VH CDR3 contains a conservative substitution in one or both of amino acid positions 7 and 9 in SEQ ID NO:10.

3. The antibody according to claim 1, where the VH region includes SEQ ID NO:54 and a VL region includes SEQ ID NO:53.

4. Humanitariannet antibody containing light chain having SEQ ID NO:14 and a heavy chain having SEQ ID NO:15, c, or without C-terminal lysine in SEQ ID NO:15, where the antibody specifically binds PCSK9.

5. The antibody according to claim 1, where the antibody further comprises immunologically inert constant region.

6. EN is Italo according to claim 5, where the antibody has an isotype that is selected from the group consisting of IgG2, IgG4, IgG2∆a, IgG4∆c, IgG4S228P, IgG4∆bS228P and IgG4∆cS228P.

7. The antibody according to claim 6, where the constant region is deglycosylated Fc.

8. Pharmaceutical composition for reducing cholesterol-LDL in the blood, and the pharmaceutical composition contains a therapeutically effective amount of the antibody according to any one of the preceding paragraphs.

9. The pharmaceutical composition of claim 8, further containing a therapeutically effective amount of the statin.

10. An isolated nucleic acid encoding the antibody according to claim 1.

11. A host cell that produces the antibody according to any one of claims 1 to 7, containing the nucleic acid of claim 10.

12. The way to lower cholesterol-LDL in the blood need in this patient, comprising the administration to a patient a therapeutically effective amount of the antibody according to any one of claims 1 to 7, or pharmaceutical composition of claim 8 or 9.

13. Cell line 4A5, having no access ATCC PTA-8985, which recombinante produces antibody against PCSK9 or antigennegative part.

14. Cell line 5A10, having no access ATCC PTA-8986, which recombinante produces antibody against PCSK9 or antigennegative part.

15. Cell line 6F6, having no access ATCC PTA-8984, which is th recombinante produces antibody against PCSK9 or antigennegative part.

16. Cell line 7D4, having no access ATCC PTA-8983, which recombinante produces antibody against PCSK9 or antigennegative part.

17. The use of antibodies according to any one of claims 1 to 7, or pharmaceutical composition of claim 8 or 9, to reduce levels of blood cholesterol and/or levels of low-density lipoprotein (LDL) blood and/or reducing morbidity or repair of damaged cholesterol and/or LDL levels caused by impaired metabolism of cholesterol and/or lipoproteins, including familial hypercholesterolemia, atherogenic dyslipidemia, atherosclerosis and cardiovascular disease.

18. The use of antibodies according to any one of claims 1 to 7, or pharmaceutical composition of claim 8 or 9, when receiving drugs to reduce levels of blood cholesterol and/or levels of low-density lipoprotein (LDL) blood and/or reducing morbidity or repair of damaged cholesterol levels and/or lipoprotein levels caused by impaired metabolism of cholesterol and/or lipoproteins, including familial hypercholesterolemia, atherogenic dyslipidemia, atherosclerosis and cardiovascular disease.



 

Same patents:

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: medicine.

SUBSTANCE: what is described is a hybrid cultured cell strain of the animals Mus museums Sp2/0Ag14-SpBcG/APC-15/A3 that is a produced of a monoclonal antibody specific to human protein C (to hPROC). The strain is deposited in the Russian Collection of Vertebrata Cell Culture of the Institute of Cytology of the Russian Academy of Sciences, No. 733(D). What is described is a monoclonal antibody prepared of the strain, specific to hPROC and showing the conformational properties. It binds hPROC in the presence of calcium ions and does not bind it in the presence of chelating agents. What is presented is an immunosorbent on the basis of said antibody.

EFFECT: applicability of the strain for preparing the MCA to hPROC, and producing on its basis an immune-affine sorbent for hPROC purification and concentration.

3 cl, 2 dwg, 3 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention discloses variants of a monoclonal antibody which identifies epitope in β-amyloid peptide with a VHHQK sequence. Described is a hybridome which produces the said antibody (ECACC 06030101), as well as versions of compositions based on the antibody. Disclosed are versions of the method for in vitro diagnosis based on the antibodies and versions of using the antibody for in vitro diagnosis of Alzheimer's disease using the monoclonal antibody and compositions based thereon. Binding of the antibody with amino acids 12-16 β-amyloid peptide - specifically detects neuritic plaques which are characteristic features of Alzheimer's disease, without detecting diffusion plaques, which are not features of the disease. The monoclonal antibody can be used to detect a subgroup among neuritic plaques, whose composition is characterised by deposits of isoforms of the β-amyloid peptide associated with the progression phase of the disease. The antibody can bind with isoforms of the β-amyloid peptide in a biological fluid such as urine, plasma, blood or spinal fluid.

EFFECT: said properties can be used during in vitro diagnosis of Alzheimer's disease when determining the progression phase of the disease.

50 cl, 7 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: claimed are versions of separated monoclonal antibody, specific to INNAR-1. Described are: bispecific molecule, immunoconjugate and compositions for treatment of IFNAR-1-mediated diseases and disorders based on monoclonal antibody. Also described are methods of inhibiting biological activity of type I interferons, method of treating diseases and disorders, mediated by type I interferon with application of antibody. Claimed are nucleic acid, which codes antibody, vector for antibody expression, cell, transformed by vector, as well as method of obtaining antibodies and antibody-producing hybridoma.

EFFECT: application of invention provides novel IFNAR-1 inhibiting antibodies, which block IFNAR-1 and bind its other epitope, in comparison with known antibody 64G12.

29 cl, 15 dwg, 6 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of a monoclonal antibody specific to GPVI polypeptide, peptide or its naturally occurred version. A based antithrombotic composition and a method for preparing thereof are described. The versions of methods for inhibition and treatment of various thrombocyte aggregation mediated diseases are disclosed. An antithrombotic set and hybridoma for producing the monoclonal antibody are described.

EFFECT: use of the invention provides the antibodies inhibiting thrombocyte aggregation that can find the further application in medicine for treating various thromboses.

13 cl, 16 dwg, 9 tbl, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to immunology and biotechnology. Claimed are versions of antibodies or their functional fragments, which are bound with receptor of human insulin-like growth factor I IGF-IR, and/or natural binding of its ligands IGF1 and/or IGF2 and/or are capable of specific inhibition of tyrosine kinase activity of said IGF-IR. Antibodies contain respective CDR sections of light and heavy chains. Described is mouse hybridoma I-3193 for production of antibodies. Composition for prevention or treatment of cancer, based on antibody application. Described is application of antibodies and/or composition for obtaining respective medication. Claimed is method of diagnostics in vitro of diseases, caused by over-expression or insufficient expression of receptor IGF-I based on antibodies..

EFFECT: invention application ensures antibodies able to bind with isophorms A and B insulin, insulin/ IGF-1 of hybrid receptors which can be applied in medicine for tumour treatment.

15 cl, 8 dwg, 2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: there are offered specific antibodies linked at least with KIR2DL1, KIR2DL2, KIR2DL3 human receptor, neutralise KIR-mediated NK cytolergy inhibition in relation to Cw3+ or Cw4+ target-cells. There are described: B-lymphocyte hybrid cell for producing the antibodies, versions of the method for producing the antibody, as well as a method for detecting a NK-cell, a method for purifying the NK-cells with the use of the antibody and versions of the pharmaceutical antibody composition. Using the antibody for preparing a medicinal agent is offered.

EFFECT: use of the invention provides producing the antibody which controls NK cytolergy of various types, intensifies cytotoxicity, increases NK cytolergy or cytotoxicity in individuals.

63 cl, 13 dwg, 3 tbl, 8 ex

FIELD: medicine; pharmacology.

SUBSTANCE: allocated human monoclonal antibodies which specifically bind a receptor of the epidermal growth factor (EGFR), and also corresponding compositions on the basis of antibodies and a biospecific molecule are described. Human antibodies can be received with use of the transgenic mouse capable to formation of set of isotypes of human monoclonal antibodies by recombination V-D-J and switching of isotypes. The pharmaceutical compositions containing human antibodies for treatment or prevention of diseases, mediated by expression EGFR, the transgenic animals distinct from a human, the specified expressing antibodies, hybridomes and transfectomes which produce human antibodies are also presented. Ways of therapy and diagnostics of the diseases mediated by expression EGFR, with use of human antibodies or their antigen-binding of fragments, and also methods of growth suppression of the cells expressing EGFR, and an induction of cytolysis of the specified cells are described.

EFFECT: invention allows obtaining therapeutic and diagnostic preparations of antibodies with improved properties.

53 cl, 22 dwg, 4 tbl, 11 ex

FIELD: biotechnology, immunology, medicine, oncology.

SUBSTANCE: strain of hybrid cultured mammalian cells Mus musculus VKPM H-98 is prepared by the hybridoma technology method. This strain is a producer of monoclonal antibodies possessing individual specificity to hypoglycosidated and deglycosidated isoforms of tumor-associated human antigen Muc I. Productivity of the strain and specificity of produced antibodies is estimated based on the immunoenzyme assay using some markers of specificity: natural purified antigen Muc I isolated from human milk; VNTR22-polypeptide; synthetic monomeric polypeptide (TR1); deglycosidated antigen Muc I (de-Muc I) prepared by chemical oxidation of natural Muc I; hypoglycosidated antigen Muc I (o-Muc I) prepared by periodate oxidation of natural Muc I. Monoclonal antibodies produced by the claimed strain recognize clinically significant isoforms of antigen Muc I and allows assaying its concentrations in human serum blood in carrying out the early diagnosis of tumors. Invention can be used in preparing monoclonal antibodies to tumor-associated human antigen Muc I.

EFFECT: valuable properties of strain.

2 dwg, 3 ex

FIELD: biotechnology, immunology, medicine, oncology.

SUBSTANCE: strain of hybrid cultured mammalian cells Mus musculus VKPM H-97 is prepared by the hybridoma technology method. This strain represents a producer of monoclonal antibodies possessing specificity to conformation-dependent of tumor-associated human antigen Muc I. Productivity of the strain and specificity of produced antibodies is estimated based on immunoenzyme assay using some markers of specificity: natural purified antigen Muc I isolated from human milk; VNTR22-polypeptide; synthetic monomeric polypeptide (TR1); deglycosidated antigen Muc I (de-Muc I) prepared by chemical oxidation of natural Muc I; hypoglycosidated antigen Muc I (o-Muc I) prepared by periodate oxidation of natural Muc I. Monoclonal antibodies produced by the claimed strain recognize clinically significant isoforms of Muc I antigen and allows assaying its concentration in human serum blood in carrying out early diagnosis. Invention can be used for preparing monoclonal antibodies to tumor-associated human antigen Muc I.

EFFECT: valuable properties of strain.

2 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biotechnology and immunology. Described are versions of antibodies, binding the GRM molecule, as well as their antigen-binding fragments, amino acid sequences of variable parts of which are presented in the claim materials. Nucleic acid, coding the said antibodies, is presented. Claimed is a method of obtaining the RGM-binding protein, which includes cultivation of a host cell in a culture medium under conditions suitable for obtaining the binding protein, capable of binding with RGM, where the host cell contains an expression vector, containing the separated nucleic acid, coding the said antibody. Described is a pharmaceutical composition for treating a disease, in which the SGM A activity produces a negative impact, which contains a therapeutically efficient quantity of the said antibody and a pharmaceutically acceptable carrier. Claimed is an application of the said antibody for obtaining a medication, used for a) reduction of hRGM A binding with a patient's Neogenin receptor; or b) for reduction of hRGM A binding with BMP-2 and BMP-4 in the patient.

EFFECT: invention makes it possible to obtain antibodies against GRM, which are used for treating diseases, associated with excessive interaction of RGM with the Neogenin receptor, BMP-2 and BMP-4.

13 cl, 16 dwg, 10 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to biotechnology and represents a polypeptide construction for treatment, prevention and relief of disorders, associated with an adhesion of platelets and platelet-mediated aggregation or its dysfunction, which includes one or more single-domain antibodies, aimed against the von Willebrand factor (vWF), and one or more single-domain antibodies aimed against serum albumen (SA). The invention also relates to nucleic acid, coding such polypeptide construction, to compositions, containing the said construction, and to its application for obtaining medications for prevention, treatment and relief of the said disorders.

EFFECT: claimed invention makes it possible to extend an assortment of medications for treatment, prevention and relief of disorders, associated with the platelet adhesion and platelet-associated aggregation or its dysfunction.

15 cl, 30 dwg, 32 tbl, 69 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology and biotechnology. There are presented versions of nucleic acids each of which codes a heavy-chain amino acid sequence of immunoglobulin IgG1. The above chain contains glycine-lysine dipeptide coded by ggaaaa, ggcaaa or gggaaa codon at the C terminal of the CH3 domain. There are described: a plasmid coding a heavy chain of immunoglobulin; version cells providing immunoglobulin IgG1 expression; a method for producing immunoglobulin in mammalian cells; a method for improving immunoglobulin expression in the mammalian cells; - using the versions of a nucleic acid.

EFFECT: using the invention provides preventing the by-product expression of weight 80 kDa that can find application in producing immunoglubulins.

18 cl, 7 dwg, 3 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to recovered monoclonal antibodies, particularly CDR-grafted humanised antibodies binding to an epitope of human RAGE molecule, and particularly possess an ability to inhibit RAGE binding to various ligands. The invention also refers to a method for preparing the above antibodies, a recovered nucleic acid coding them, an expression vector, a host cell and a pharmaceutical composition.

EFFECT: invention provides treating the diseases or disorders associated with advanced glycation end product (RAGE) receptor, including Alzheimer's disease effectively.

16 cl, 13 dwg, 10 tbl, 19 ex

FIELD: medicine.

SUBSTANCE: present invention refers to immunology and biotechnology. What is presented is an IL-1β-binding antibody or its IL-1β-binding fragment containing V heavy and light chain regions. The above antibody binds to human IL-1β with dissociation constant less than 1pM. Versions of the antibody are described. There are disclosed corresponding coding nucleic acids (NA), as well as: a NA passage vector to a host cell, the host cell producing a coded polypeptide. What is described is using the antibody for preparing the other format of the above antibody: "camel-like", VHH antibody, nanobody. What is disclosed is a pharmaceutical composition for treating or preventing an IL-1β-related disease in a mammal on the basis of the antibody, as well as a method of treating or preventing the IL-1β-related disease in a mammal.

EFFECT: using the invention provides the novel IL-1β-specific antibodies with high IL-1β affinity that can find application in medicine for preventing, treating the diseases mediated by IL-1β activity.

39 cl, 20 dwg, 6 tbl, 14 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of immunology. Claimed is a version of Fc polypeptide of human IgG with substitutions 2591 and 308F, where numeration of positions is given in accordance with EU Kabat index. Described is a version of the said polypeptide, including one or several substitutions of the following: 428L, 434S, 307Q, 319L, 250I in addition to the said ones. Disclosed are: a nucleic acid, coding the said versions, a host cell for production of the said versions of polypeptide, which contains the coding nucleic acid, a method of obtaining the said versions of polypeptide, including application of the cell expressing the said polypeptide and containing the nucleic acid, which codes the said polypeptide.

EFFECT: application of the invention provides polypeptide, demonstrating higher affinity with human FcRn, which can be applied in therapy of different diseases.

11 cl, 32 dwg, 14 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to immunology and biotechnology. Claimed are versions of an isolated monoclonal antibody, specific to hGM-CSF, where each version is characterised by a heavy and light chain. Each of the versions is characterised by the fact that it contains six appropriate CDR. Described are: a pharmaceutical composition, and a set, representing medication, based on the antibody application. Disclosed are: a coding isolated nucleic acid, an expression vector, containing it, and a vector-carrying host cell, used for the antibody obtaining. Described is a method of obtaining the antibody with the cell application.

EFFECT: claimed inventions can be applied for treating disease or disorder, associated with superexpression of hGM-CSF.

25 cl, 9 dwg, 14 tbl, 15 ex

FIELD: medicine.

SUBSTANCE: present invention refers to biotechnology and medicine. What is presented is a method for generating an antibody and its functional fragments against a tumour antigen expressed on the tumour surface resistant to at least one anti-tumour compound by applying ground homogenate, and/or suspension, and/or cell lysate originated from the same tumour for immunisation. There are also disclosed using the method according to the invention for producing the monoclonal antibodies and their functional fragments, the monoclonal antibodies produced by the method, nucleic acids coding them, an expression vector, a host cell, and a method for preparing the antibody with using them, as well as hybridomes secreting these antibodies and their functional fragments for preparing a drug, the anti-tumour composition and using it as a drug.

EFFECT: invention can find further application in therapy of resistant tumours.

42 cl, 7 dwg, 4 ex, 6 tbl

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

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers immunology and medicine. What is presented is an antibody for the recovery of the central nervous system, comprising an antigen-binding site that specifically binds to human Nogo A polypeptide or human NiG described by (SEQ ID NO: 2 and 3, respectively, presented in the description), wherein the antigen-binding site comprises: CDR-H1-6A3 (SEQ ID NO:8), CDR-H2-6A3 (SEQ ID NO:9) and CDR-H3-6A3 (SEQ ID NO:10); and CDR-L1-6A3 (SEQ ID NO:11), CDR-L2-6A3 (SEQ ID NO:12) and CDR-L3-6A3 (SEQ ID NO:13). There are also described a polynucleotide coding the above antibody; an expression vector comprising the above polynucleotide; and a host cell specified in bacterium, yeast or mammalian cell line comprising myeloma, hybridoma, or immortalised B-cell for producing the antibody according to the present invention. A pharmaceutical composition for the CNS recovery comprising an effective amount of the above antibody mixed with at least one acceptable carrier or solvent is also described. Using the polynucleotide, the expression vector or the host cell for the above pharmaceutical composition is also described. The invention enables producing the human Nogo A or NiG antibody effective in treating CNS injuries.

EFFECT: what is presented is a method for producing the above antibody involving the polynucleotide or vector expression in the host cell.

16 cl, 11 dwg, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology and medicine. What is presented is a method for preventing or treating an ocular condition related to high expression or activity of a complement factor D involving administering an antibody or its antigen-binding fragment into an individual. There are presented an anti-factor D 20D12 antibody and its antigen-binding fragment to be used to prepare a therapeutic agent, as well as containing the above antibody or its antigen-binding fragment, a kit for treating the ocular condition related to high activity or expression of factor D.

EFFECT: invention can find further application in therapy of the complement system related diseases.

14 cl, 6 dwg, 3 tbl

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