Pcsk9 antagonists

The present invention antagonizes the activity of the extracellular precursor protein convertase subtilisinkesin type 9 (PCSK9), including its interaction with the low density lipoprotein (LDL) receptor (LDLR), such as complete antibodies. With respect to long antibodies or antigen-binding portions thereof, peptides, and aptamers. More specifically, the present invention relates to compositions comprising antagonistic PCSK9 antibodies, peptides, and / or aptamers, and methods of using these antibodies and / or peptides and / or aptamers as pharmaceuticals. Antagonistic PCSK9 antibodies, peptides, and aptamers can be used therapeutically to lower blood LDL-cholesterol levels, familial hypercholesterolemia, atherosclerosis, atherosclerosis. , And more generally, can be used for the prevention and / or treatment of cholesterol and lipoprotein metabolic disorders, including cardiovascular disease (CVD).

In the United States, millions of people are at risk for heart disease and the resulting heart events. CVD and underlying atherosclerosis are the leading causes of death common to all demographic groups, despite the availability of treatments for their multiple risk factors. Atherosclerosis is an arterial disease that is responsible for many death-related coronary heart diseases in developed countries. Currently, several risk factors for coronary heart disease have been identified: abnormal lipidemia, hypertension, diabetes, smoking, poor diet, inactivity and stress. The most clinically significant and common abnormal lipidemia is beta-lipoproteins (very low density lipoprotein (VLDL) and LDL) with hypercholesterolemia in the absence or presence of hypertriglyceridemia. Characterized by an increase in (Fredrickson et al., 1967, N Engl J Med., 276: 34-42, 94-103, 148-156, 215-225, and 273-281). Despite treatment with statins (the current standard for nursing atherosclerosis), 60-70% of cardiovascular events, heart attacks and strokes occur and have been significantly met for many years with respect to CVD. There is no request. In addition, new guidelines suggest that lower LDL levels should be achieved to protect high-risk patients from premature CVD [National Cholesterol Education Program (NCEP), 2004].

PCSK9, also known as NARC-1, has been identified as a protein with a gene mutation in some forms of familial hypercholesterolemia. PCSK9 is synthesized in the endoplasmic reticulum as a zymogen that undergoes autocatalytic processing in the motif LVFAQ. In a population study, some PCSK9 mutations are "acquired" and found in individuals with autosomal dominant hypercholesterolemia, while other "lost" (LOF) mutations It has been shown to be associated with lower plasma cholesterol. Morbidity and mortality studies in this group clearly demonstrated that reduced PCSK9 function significantly reduced the risk of cardiovascular disease.

Remarkably important in the treatment of CVD, LOF mutations can sensitize humans to statins, allowing efficacy at lower doses (and thus ameliorating the risks associated with safety and tolerance). , Potentially achieve lower plasma cholesterol levels than current treatments.

PCSK9 is secreted into plasma mainly by hepatocytes. Genetic modification of PCSK9 in mice confirmed its ability to regulate blood lipids in PCSK9, suggesting that it acts to downregulate hepatic LDLR protein levels.

The mechanism and site by which PCSK9 downregulates the LDLR protein has not been clearly established. When overexpressed, PCSK9 can act both intracellularly and as a secreted ligand for LDLR. There is strong evidence that extracellular PCSK9 binds to cell surface LDLR and promotes LDLR degradation at intracellular sites. However, it is possible that PCSK9 interacts with the LDLR as the two proteins are translated within the endoplasmic reticulum (ER) and transported through the endosome compartment to the cell membrane. According to Maxwell et al., 2005, Curr. Opin. Lipidol., 16: 167-172, PCSK9-mediated endocytosis and degradation of LDLR was not altered by proteasome inhibitors, and a variety of lysosomal and non-lysosomal proteases. It was shown that it was also not modulated by the class. Two naturally occurring mutations of familial hypercholesterolemia, S127R and D129G, are deficient in self-processing and secretion of these mutant proteins as levels is significantly reduced in the medium of the transfected cells. It has been reported that it was or was undetectable. Nevertheless, these mutants demonstrated an enhanced ability to downregulate LDLR, consistent with their identification in individuals with high plasma LDL (Homer et al., 2008, Atherosclerosis, 196: 659-666, Cameron). , 2006, Human Molecular Genetics, 15: 1551 ~ 1558, Lambert et al., 2006, TRENDS in Endocrinology and Metabolism, 17: 79 ~ 81. These mutants are clearly not secreted extracellularly, but still down LDLR. Due to its regulation, this strongly suggests that the site of action in the cell is physiologically important.

<p num="0007"> From the information available in the art, and prior to the present invention, it is highly likely that an antibody, peptide, or aptamer-based PCSK9 antagonist is introduced into the blood circulation to selectively antagonize extracellular PCSK9. It is unclear if it is effective in reducing the incidence of hypercholesterolemia and associated CVD, and if so, which properties of PCSK9 antagonists are required for such in vivo efficacy. It remained as it was.</p>

<p num="0008"> The present invention relates to antagonistic antibodies, peptides, and aptamers that selectively interact and inhibit the function of PCSK9. For the first time, certain PCSK9 antagonists have been demonstrated to be effective in vivo to lower blood cholesterol.</p><p num="0009"> In one embodiment, the invention is an antagonist of isolated PCSK9 comprising an antibody, peptide, or aptamer that interacts with PCSK9 and, when administered to the subject, lowers LDL-cholesterol levels in the subject's blood. I will provide a. The antagonist can be an antibody, eg, a monoclonal antibody or a human antibody, a humanized antibody, or a chimeric antibody.</p><p num="0010"> In another embodiment, the invention specifically binds to PCSK9 and is mediated by PCSK9 for LDLR levels as measured in vitro using the LDLR downregulation assay in Huh7 cells disclosed herein. Provided is an isolated anti-PCSK9 antibody, which is a complete antagonist of the effect.</p><p num="0011"> In yet another embodiment, the invention antagonizes the extracellular interaction of PCSK9 and LDLR as measured by in vitro binding of PCSK9 and LDLR and, when administered to the subject, in the blood of said subject. Provides isolated antibodies that lower LDL-cholesterol levels in the. Preferably, as described in Kwon et al., 2008, PNAS, 105: 1820-1825, the antibody overlaps more than about 75% of the surface on PCSK9 interacting with the EGF-like domain of LDLR on human PCSK9. Recognize epitopes.</p><p num="0012"> In yet another embodiment, the invention has been deposited in the American Type Culture Collection, 5A10 produced by the hybridoma cell line assigned accession number PTA-8986, the American Type Culture Collection. 4A5 produced by the hybridoma cell line assigned accession number PTA-8985, 6F6 produced by the hybridoma cell line assigned accession number PTA-8984, and American A number of PCSK9 that overlaps with a second epitope recognized by a monoclonal antibody selected from the group consisting of 7D4 produced by a hybridoma cell line that has been deposited in the Type Culture Collection and is assigned accession number PTA-8983. An antibody that recognizes one epitope is provided.</p><p num="0013"> In another embodiment, the invention recognizes epitopes on human PCSK9 containing amino acid residues 153-155, 194, 195, 197, 237-239, 367, 369, 374-379 and 381 of the PCSK9 amino acid sequence. , Provide antibodies against human PCSK9. Preferably, the antibody epitopes on human PCSK9 are amino acid residues 71, 72, 150-152, 187-192, 198-202, 212, 214-217, 220-226, 243, 255-258, 317, 318, Does not include one or more of 347-351, 372, 373, 380, 382, and 383.</p><p num="0014"> In yet another embodiment, the invention presents VH complementarity determining regions 1 (CDR1) with the amino acid sequence (SYYMH) set forth in SEQ ID NO: 8, VH CDR2 with the amino acid sequence (EISPFGGRTNNYNEKFKS) set forth in SEQ ID NO: 9, and /. Alternatively, it is specific to VH CDR3 having the amino acid sequence shown in SEQ ID NO: 10 (ERPLY ASDL), or PCSK9 containing a variant thereof having one or more conservative amino acid substitutions in the sequence of CDR1, CDR2, and / or CDR3. Provide an antibody that binds to, and the variant retains essentially the same binding specificity as the CDRs defined by the sequences. Preferably, the variant comprises up to about 10 amino acid substitutions, more preferably up to about 4 amino acid substitutions.</p><p num="0015"> Furthermore, the present invention has VL CDR1 having the amino acid sequence shown in SEQ ID NO: 11 (RASQGISSALA), CDR2 having the amino acid sequence shown in SEQ ID NO: 12 (SASYRYT), and / or the amino acid sequence shown in SEQ ID NO: 13 (QQRYSLWRT). Target antibodies that contain CDR3, or a variant of CDR1, CDR2, and / or a variant thereof having one or more conservative amino acid substitutions in the sequence of CDR3, the variant being CDR1 and essentially defined by the sequence. Retains the same binding specificity. Preferably, the variant comprises up to about 10 amino acid substitutions, more preferably up to about 4 amino acid substitutions.</p><p num="0016"> In another embodiment, the invention comprises a particular VL CDR1, CDR2, and / or CDR3 sequence, or a variant thereof having one or more conservative amino acid substitutions in CDR1, CDR2, and / or CDR3. , VH complementarity determining regions CDR1 having the amino acid sequence shown in SEQ ID NO: 59, 60, or 8, VH CDR2 having the amino acid sequence shown in SEQ ID NO: 61 or 9, and / or VH CDR3 having the amino acid sequence shown in SEQ ID NO: 10. , Or an antibody further comprising a variant thereof having one or more conservative amino acid substitutions in the sequence of CDR1, CDR2, and / or CDR3, wherein the variant is CDR1, CDR2 defined by the sequence. , And / or retain essentially the same binding specificity as CDR3. Preferably, the variant comprises up to about 20 amino acid substitutions, more preferably up to about 8 amino acid substitutions. In another preferred embodiment, the antibody of the invention has a variable heavy chain sequence comprising or consisting of SEQ ID NO: 54 and a variable light chain sequence comprising or consisting of SEQ ID NO: 53.</p><p num="0017"> The invention also comprises a polypeptide selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, or both SEQ ID NO: 14 and SEQ ID NO: 15, or one or more conservative amino acid substitutions in the sequence. A humanized antibody comprising the variant is also provided, the variant retains essentially the same binding specificity as the antibody defined by said sequence (s). It also includes antibodies lacking terminal lysine on the heavy chain, as this is usually lost in a certain percentage of the antibody during production.</p><p num="0018"> Preferably, the variant comprises up to about 20 amino acid substitutions, more preferably up to about 8 amino acid substitutions. Preferably, the antibody further comprises an immunologically inactive constant region and / or the antibody is IgG.₂, IgG₄, IgG_2Δa, IgG_4Δb, IgG_4Δc, IgG₄ S228P, IgG_4Δb S228P and IgG_4Δc It has an isotype selected from the group consisting of S228P. In another preferred embodiment, the constant region is deglycosylated Fc.</p><p num="0019"> In one embodiment, the invention comprises administering to a subject a therapeutically effective amount of an antagonist of the invention, LDL, LDL-cholesterol, in the blood, serum, or plasma of the subject in need thereof. Alternatively, it provides a method of lowering the level of total cholesterol.</p><p num="0020"> In one embodiment, the invention is a therapeutically effective amount of book for use in lowering levels of LDL, LDL-cholesterol, or total cholesterol in the blood, serum, or plasma of a subject in need thereof. Provided is an antagonist of the invention. In addition, the invention is a therapeutically effective amount of the invention in the manufacture of a medicament for lowering the level of LDL, LDL-cholesterol, or total cholesterol in the blood, serum, or plasma of a subject in need thereof. Provides the use of antagonists.</p><p num="0021"> In yet another embodiment, the invention a) provides a PCSK9-negative host animal, b) immunizes the PCSK9-negative host animal with PCSK9, and c) an antibody, antibody-producing cell, or said PCSK9. An antibody that specifically binds to PCSK9 is prepared, which comprises obtaining a nucleic acid encoding an antibody derived from a negative host animal and preparing the antibody from the antibody-producing cells or the nucleic acid encoding the antibody. Provide a method.</p><p num="0022"> The invention also includes methods of reducing the level of LDL in the blood of a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody prepared according to the invention. Subjects can be further treated by administering statins. In a preferred embodiment, the subject is a human subject.</p><p num="0023"> In one embodiment, the antibody has a pH in the range of about 5.0 to about 6.5, an antibody of about 1 mg / ml to about 200 mg / ml, a histidine buffer of about 1 ml to about 100 ml, a histidine buffer of about 0.01 mg / ml. It is administered in the form of a sterile aqueous solution containing ~ about 10 mg / ml of polysorbate 80, about 100 millimorer to about 400 millimorer of trehalose, and about 0.01 millimorer to about 1.0 millimorer of EDTA disodium dihydrate.</p><p num="0024"> In another embodiment, the invention provides a therapeutically effective amount of antibody prepared according to the invention for use in reducing the level of LDL in the blood of a subject in need thereof. In addition, the invention provides the use of therapeutically effective amounts of antibodies prepared according to the invention in the manufacture of pharmaceuticals to reduce the level of LDL in the blood of subjects in need thereof. The therapeutically effective amount can optionally be combined with the therapeutically effective amount of statin.</p><p num="0025"> In another embodiment, the invention provides a hybridoma cell line that produces an antibody specific for PCSK9 or an antigen-binding portion thereof. 4A5 with ATCC accession number PTA-8985, 5A10 with ATCC accession number PTA-8986, 6F6 with ATCC accession number PTA-8984, and 7D4 with ATCC accession number PTA-8983 Selected from the group consisting of.</p><p num="0026"> In another embodiment, the invention specifically binds to PCSK9 and has heavy chain variable region (VH) complementarity determining regions 1 (CDR1), SEQ ID NO:, having the amino acid sequence set forth in SEQ ID NO: 8, 59, or 60. VH CDR2 having the amino acid sequence shown in 9 or 61, and / or VH CDR3 having the amino acid sequence shown in SEQ ID NO: 10, or having one or more conservative amino acid substitutions in CDR1, CDR2, and / or CDR3. Light chain variable region (VL) CDR1s containing variants and / or having the amino acid sequence shown in SEQ ID NO: 11, VL CDR2 having the amino acid sequence shown in SEQ ID NO: 12, and / or having the amino acid sequence shown in SEQ ID NO: 13. Provided is a cell line that recombinantly produces antibodies containing VL CDR3, or variants thereof having one or more conservative amino acid substitutions in CDR1, CDR2, and / or CDR3. Preferably, the cell line recombinantly produces an antibody comprising SEQ ID NOs: 53 and / or 54, more preferably SEQ ID NOs: 14 and / or 15.</p>

<figref num="1">Effect of anti-PCSK9 antagonistic monoclonal antibodies 7D4.4, 4A5.G3, 6F6.G10.3 and 5A10.B8 on the ability of mouse PCSK9 (A) and human PCSK9 (B) to downregulate LDLR in cultured Huh7 cells. It is a figure which exemplifies. 6F6.G10.3 is a subclone of 6F6, 7D4.4 is a subclone of 7D4, 4A5.G3 is a subclone of 4A5, and 5A10.B8 is a subclone of 5A10.</figref><figref num="2">Anti-PCSK9 antagonistic monoclonal antibody 6F6.G10.3, 7D4. That blocks in vitro binding of recombinant biotin-labeled human PCSK9 (A) and mouse PCSK9 (B) to the immobilized recombinant LDLR extracellular domain. FIG. 5 illustrates the dose response of 4, 4A5.G3, 5A10.B8, negative control antibody 42H7, and PBS.</figref><figref num="3">Anti-PCSK9 monoclonal antagonist antibody 6F6, which blocks the binding of recombinant biotin-labeled human PCSK9 (30 nM) to europium-labeled recombinant LDLR extracellular domain (10 nM) in solution, at neutral pH, in vitro. It is a figure which illustrates the dose response of G10.3, 7D4.4, 4A5.G3 and 5A10.B8.</figref><figref num="4">It is a figure which illustrates the comparative epitope binding of an anti-PCSK9 antibody.</figref><figref num="5">FIG. 5 illustrates a Western blot of binding of anti-PCSK9 antibody to serum PCSK9 from various species.</figref><figref num="6">It is a figure which illustrates the effect of the anti-PCSK9 monoclonal antibody 7D4 on the blood cholesterol level in a mouse.</figref><figref num="7">FIG. 5 illustrates the effect of (A) the partial antagonist polyclonal anti-PCSK9 mAb CRN6 on downregulation of LDLR and (B) the lack of effect on cholesterol levels in mice.</figref><figref num="8">It is a figure which illustrates the time course of the cholesterol lowering effect obtained by using the anti-PCSK9 antagonist antibody 7D4 in a mouse.</figref><figref num="9">FIG. 5 illustrates the dose dependence of anti-PCSK9 antagonist mAb 7D4 on serum total cholesterol, HDL and LDL reduction in mice.</figref><figref num="10">It is a figure which illustrates the dose dependence of the cholesterol lowering effect of the anti-PCSK9 antagonist antibody 5A10 in a mouse.</figref><figref num="11">It is a figure which illustrates the dose dependence of the cholesterol lowering effect of anti-PCSK9 antagonist antibody (A) 4A5 and (B) 6F6 in mouse.</figref><figref num="12">It is a figure which shows the Western blot of the effect of the anti-PCSK9 antagonist antibody on the liver LDLR level.</figref><figref num="13">It is a figure which illustrates the lack of effect of the anti-PCSK9 antagonist antibody 4A5 in the LDLR-/-mouse model.</figref><figref num="14">FIG. 5 illustrates the effect of multiple doses of anti-PCSK9 antagonist antibody on total serum cholesterol over a longer period of time than seen at a single dose in mice.</figref><figref num="15">It is a figure which illustrates the time course of the effect of the anti-PCSK9 antagonist antibody 7D4 on the lipid parameter in a cynomolgus monkey model.</figref><figref num="16">FIG. 5 illustrates the dose and time response of anti-PCSK9 antagonist antibody 7D4 to serum cholesterol levels in cynomolgus monkeys.</figref><figref num="17">FIG. 5 illustrates a comparison of anti-PCSK9 antagonist antibodies 4A5, 5A10, 6F6 and 7D4 to serum cholesterol levels in cynomolgus monkeys.</figref><figref num="18">It is a figure which illustrates the time course of the effect of the anti-PCSK9 antagonist antibody 7D4 on the plasma cholesterol level of the cynomolgus monkey fed the fat diet of 33.4% kcal to which 0.1% cholesterol was added.</figref><figref num="19">It is a figure which illustrates the effect of L1L3 (humanized anti-PCSK9 monoclonal antibody) on the downregulation of LDLR in Huh7 cells.</figref><figref num="20">Recombinant biotin-labeled human PCSK9 (A and B) and mouse PCSK9 (C and D) with immobilized recombinant LDLR extracellular domain, in vitro, pH 7.5 (A and C) and pH 5.3 (B) It is a diagram illustrating the dose response of L1L3 humanized antibody, mouse precursor 5A10, and negative control antibody 42H7 in blocking binding at and D).</figref><figref num="21">It is a figure which illustrates the effect on serum cholesterol of the mouse treated with 10 mg / kg L1L3.</figref><figref num="22">FIG. 5 illustrates the effect of administration of 5A10 antibody or L1L3 to cynomolgus monkeys and the measurement of changes in serum HDL (A) and serum LDL (B) as a function of time.</figref><figref num="23A">It is a figure which shows the crystal structure of PCSK9 (display of a light gray surface) bound with L1L3 antibody (display of a black image).</figref><figref num="23B">FIG. 5 shows the crystal structure of PCSK9 (display of light gray surface) bound to the EGF-like domain of LDLR (display of black image) (Kwon et al., PNAS, 105, 1820-1825, 2008).</figref><figref num="23C">It is a figure which shows the surface area display of PCSK9, and the L1L3 epitope is shown in dark gray.</figref><figref num="23D">It is a figure which shows the surface area display of PCSK9, and the LDLR EGF-like domain epitope is shown in dark gray.</figref><figref num="24A-G">It is a table showing the substitutions made in the CDR of antibody 5A10 during the process of affinity maturation and optimization to achieve specific properties. PCSK9 binding associated with antibodies with these CDR substitutions is also shown. The number following each sequence is the sequence number specified for each sequence.</figref>

The present invention relates to antibodies, peptides, and aptamers that antagonize the function of extracellular PCSK9, including its interaction with the LDLR. More specifically, the present invention comprises methods for making antagonistic PCSK9 antibodies, peptides, and aptamers, compositions comprising these antibodies, peptides, and / or aptamers, and pharmaceuticals of these antibodies, peptides, and / or aptamers. Regarding how to use as. Antagonistic PCSK9 antibodies and peptides can be used to lower blood LDL-cholesterol levels, including familial hypercholesterolemia, atherosclerosis, atherosclerosis, and more generally CVD. It can also be used to prevent and / or treat cholesterol and lipoprotein metabolic disorders.

General technology Unless otherwise specified, the practice of the present invention uses conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology within the technical scope of the art. .. Such techniques include Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989), Cold Spring Harbor Press, Oligonucleotide Synthesis (MJGait ed., 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory. Notebook (JECellis ed., 1998), Academic Press, Animal Cell Culture (RI Freshney ed., 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998), Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, 1993-1998), J. Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (DM Weir and CC Blackwell), Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, 1987), Current Protocols in Molecular Biology (FM Ausubel et al., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., 1994), Current Protocols in Immunology (JEColigan et al., ed., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (CA Janeway and P. Travers, 1997), Antibodies (P.Finch, 1997), Antibodies: a practical approach (D. Catty, IRL Press, 1988-1989), Monoclonal antibodies : a practical approach (P. Shepherd and C. Dean, Oxford University Press, 2000), Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. It is fully described in literature such as Zanetti and JD Capra, Harwood Academic Publishers, 1995).

Definition An "antibody" is an immunoglobulin molecule capable of specifically binding to a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc. via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. .. As used herein, the term refers to intact polyclonal or monoclonal antibodies as well as fragments thereof (Fab, Fab', F (ab')).₂, Fv), single chain (ScFv) and domain antibodies, etc.), fusion proteins containing antibody moieties, and any other modified configuration of immunoglobulin molecules containing antigen recognition sites. Antibodies include antibodies of any class (or subclasses thereof) such as IgG, IgA, or IgM, and the antibodies need not be of any particular class. Immunoglobulins can be assigned to various classes depending on the antibody amino acid sequence of the constant domain of the heavy chain. There are five major immunoglobulin classes, namely IgA, IgD, IgE, IgG, and IgM, some of which are subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Can be further classified into. The heavy chain constant domains corresponding to the various immunoglobulin classes are called alpha, delta, epsilon, gamma, and mu, respectively. The structure and three-dimensional configuration of subunits of various immunoglobulin classes are well known.

As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population can be present in small amounts, as naturally as possible. It is the same except for the existing mutations. Monoclonal antibodies are highly specific because they are directed to a single antigenic site. Moreover, each monoclonal antibody is directed to a single determinant on the antigen, as opposed to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes). The modifier "monoclonal" indicates the characteristics of an antibody as being derived from a population of substantially homogeneous antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used in accordance with the present invention may first be made by the hybridoma method described by Kohler and Milstein, 1975, Nature, 256: 495, or recombinants such as those described in US Pat. No. 4,816,567. It may be prepared by the DNA method. Monoclonal antibodies may also be isolated from phage libraries prepared using, for example, the techniques described in McCafferty et al., 1990, Nature, 348: 552-554.

As used herein, a "humanized" antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (Fv, Fab, Fab', F) containing a minimal sequence derived from a non-human immunoglobulin. (ab')₂Alternatively, it refers to the form of a non-human (for example, murine) antibody which is another antigen-binding partial sequence of the antibody. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody), a mouse, rat in which residues from the recipient's complementarity determining regions (CDRs) have the desired specificity, affinity, and ability. , Or residues from CDRs of non-human species (donor antibodies) such as rabbits. In some examples, the Fv framework region (FR) residue of human immunoglobulin is replaced by the corresponding non-human residue. In addition, humanized antibodies may contain residues that are not found in the recipient antibody or in the imported CDR or framework sequence, but have been included to further refine and optimize the performance of the antibody. In general, humanized antibodies contain substantially all of at least one, typically two variable domains, and all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, the FR region. All or substantially all of them are of human immunoglobulin consensus sequences. Humanized antibodies also optimally include at least a portion of an immunoglobulin, typically a constant region or domain (Fc) of a human immunoglobulin. Antibodies with modified Fc regions as described in WO 99/58572 are preferred. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and / or CDR H3) that have been modified with respect to the original antibody. Is also referred to as one or more CDRs "derived" from one or more CDRs from the original antibody.

As used herein, a "human antibody" is an antibody having an amino acid sequence corresponding to an antibody that can be produced by a human and / or known to those of skill in the art or disclosed herein. It means an antibody prepared by using any of the techniques for producing a human antibody. This definition of human antibody includes an antibody comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising a murine light chain and a human heavy chain polypeptide. Human antibodies can be produced using a variety of techniques known in the art. In one embodiment, human antibodies are selected from a phage library expressing 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 are also produced by immunization of animals into which the human immunoglobulin locus has been introduced by gene transfer instead of the endogenous locus, for example, mice in which the endogenous immunoglobulin gene has been partially or completely inactivated. be able to. This technique is described in US Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016. Alternatively, human antibodies can be prepared by immortalizing human B lymphocytes that produce antibodies against the target antigen (such B lymphocytes can be recovered from the individual or immunized in vitro). For example, Cole et al., Monoclonal Antibodies and Cancer See Therapy, Alan R. Liss, p. 77, 1985, Boerner et al., 1991, J. Immunol., 147 (1): 86-95, and US Pat. No. 5,750,373.

The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, alone or in combination. As is known in the art, the heavy and light chain variable regions are from four framework regions (FRs) connected by three complementarity determining regions (CDRs), each containing a hypervariable region. Become. The CDRs in each strand are held together very proximally by FR and, together with the CDRs from the other strand, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) Interspecific sequence variability-based techniques (ie, Kabat et al., Sequences of Proteins of Immunological Interest, (5th Edition, 1991, National Institutes of Health, Bethesda). MD)), and (2) techniques based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol., 273: 927-948). The CDR used herein may be a CDR defined by either method or a combination of both methods.

The "constant region" of an antibody known in the art refers to the constant region of an antibody light chain or the constant region of an antibody heavy chain, alone or in combination.

As used herein, the term "PCSK9" refers to any form of PCSK9 and variants thereof that retain at least a portion of the activity of PCSK9. Unless otherwise indicated, such as by specifically referring to human PCSK9, PCSK9 includes all mammalian species of the native sequence PCSK9, such as humans, canines, felines, horses, and bovines. Includes sub-animals. One exemplary human PCSK9 is Uniprot Accession No. Q8NBP7 (SEQ ID NO:<u style="single">188 ()</u>).

As used herein, "PCSK9 antagonist" refers to PCSK9 mediated by PCSK9 signaling, including PCSK9-mediated down-regulation of LDLR and PCSK9-mediated reduction of LDL blood clearance. An antibody, peptide, or aptamer that can inhibit biological activity and / or downstream pathways (s). PCSK9 antagonistic antibodies block, antagonize, suppress or reduce PCSK9 biological activity, including downstream pathways mediated by PCSK9 signaling, such as LDLR interaction and / or induction of cellular responses to PCSK9 (significant degree). Antibodies are included (to any degree including). For the purposes of the present invention, the term "PCSK9 antagonistic antibody" includes all of the terms, subjects, and functional states and characteristics already identified, including PCSK9 itself, PCSK9 biological activity (but not limited to, LDLR). Interaction, LDLR downregulation, and its ability to mediate any aspect of reduced blood LDL clearance), or the consequences of biological activity are substantially nullified to any significant degree. It will be clearly understood that it will be reduced or neutralized. In some embodiments, the PCSK9 antagonist antibody binds to PCSK9 and prevents interaction with the LDLR. Examples of PCSK9 antagonist antibodies are provided herein.

As used herein, a "complete antagonist" is an antagonist that essentially completely blocks the measurable effect of PCSK9 at an effective concentration. A partial antagonist means an antagonist that can partially block measurable effects but is not a complete antagonist even at the highest concentrations. Essentially complete means that the measurable effect is blocked by at least about 80%, preferably at least about 90%, more preferably at least about 95%, and most preferably at least about 98% or 99%. .. Related "measurable effects" are described herein, down-regulation of LDLR with PCSK9 antagonists assayed in vitro in Huh7 cells, reduction of total cholesterol levels in blood (or plasma) in vivo, And includes in vivo reductions in LDL levels in blood (or plasma).

As used herein, the term "clinically meaningful" means a reduction of at least 15% in blood LDL-cholesterol levels in humans or a reduction in total blood cholesterol of at least 15% in mice. It is clear that plasma or serum measurements can act as a surrogate for blood level measurements.

As used herein, the terms "PCSK9 antagonistic peptide" or "PCSK9 antagonistic aptamer" include PCSK9 including downstream pathways mediated by PCSK9 signaling, such as LDLR interaction and / or induction of a cellular response to PCSK9. Includes any conventional peptide or polypeptide or aptamer that blocks, antagonizes, suppresses or reduces biological activity (up to any degree, including significant degrees). PCSK9 antagonistic peptides or polypeptides include Fc fusions containing LDLR and soluble moieties of LDLR, or mutants thereof that have a higher affinity for PCSK9.

The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein and of any length, preferably relatively short (eg 10-100). Amino acid chain of). The chain may be linear or branched, may contain modified amino acids, and / or may be interrupted by non-amino acids. The term also refers naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, eg, conjugation with a labeling component. Modified amino acid chains are also included. The definition also includes, for example, polypeptides containing one or more analogs of amino acids, such as unnatural amino acids, and other modifications known in the art. It should be understood that a polypeptide can exist as a single chain or an associated chain.

As is known in the art, "polynucleotide" or "nucleic acid" as used herein for compatibility means a nucleotide chain of any length, including DNA and RNA. .. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into the strand by DNA or RNA polymerase. Polynucleotides may include modified nucleotides such as methylated nucleotides and analogs thereof. Modifications to the nucleotide structure, if present, can be given before or after assembly of the strand. Sequences of nucleotides, can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps", replacing one or more of the naturally occurring nucleotides with analogs, internucleotide modifications, for example, uncharged linkages (eg, methyl phosphonate, etc.). Phosphate triesters, phosphoamidates, carbamates, etc.) and charged linkages (eg, phosphorothioates, phosphorodithioates, etc.), such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.) Etc.), those containing pendant portions such as, those using intervening substances (eg, aclysine, solarene, etc.), those containing chelating agents (eg, metals, radioactive metals, boron, oxidizing metals, etc.), alkylation Included are those containing agents, those using modified linkages (eg, alpha anomeric nucleic acids, etc.), and unmodified forms of polynucleotides (s). Any of the hydroxyl groups normally present in the sugar can be replaced, for example, with a phosphonic acid group, a phosphate group, protected by a standard protecting group, or activated to prepare additional linkages with additional nucleotides. , Or can be conjugated with a solid carrier. OH at the 5'and 3'ends It can be phosphorylated or replaced with an amine or an organic cap group moiety of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized into standard protecting groups. Polynucleotides include, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic analogs, alpha-or beta-anomeric sugars, Similarities to commonly known ribose or deoxyribose sugars, including epimer sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs and debase nucleoside analogs such as methylribosides. The body can also be contained. One or more phosphodiester bonds can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, phosphates P (O) S (thioate), P (S) S (dithioate), (O) NR.₂("Amidate"), P (O) R, P (O) OR', CO or CH₂("Form acetal") [In the formula, each R or R'independently contains an H, or ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or arargyl. A good, substituted or unsubstituted alkyl (1 to 20 Cs)] is included. Not all linkages in the polynucleotide need be identical. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

A "PCSK9 antagonistic aptamer" containing a nucleic acid or protein sequence is selected, for example, from a large pool of random sequences and specifically binds to PCSK9. Aptamer nucleic acids are double-stranded DNA or single-stranded RNA. Nucleic acid aptamers can include modified bases or functional groups, including but not limited to 2'-fluorine nucleotides and 2'-O-methyl nucleotides. Aptamers can include hydrophilic polymers such as polyethylene glycol. Aptamers are made by methods known in the art and can be selected for PCSK9 antagonistic activity by routine modification of the methods disclosed in the Examples.

As used herein, an antibody, peptide, or aptamer has an equilibrium dissociation constant of 20 nM or less, preferably less than about 6 nM, more preferably less than 20 nM, as measured by the method disclosed in Example 2 herein. PCSK9 "interacts" when less than about 1 nM, most preferably less than about 0.2 nM.

An epitope that "preferentially binds" or "specifically binds to" an antibody or polypeptide (used herein for compatibility) is a term well understood in the art. And methods for determining such specific or preferred binding are also well known in the art. A molecule is "specifically bound" or "specifically bound" or "when it reacts or associates with a particular cell or substance more often, faster, longer lasting and / or with higher affinity than another cell or substance. It is said to indicate "priority binding". An antibody "specifically binds" to a target if it binds to the target with a higher affinity than it binds to other substances, binding force, more easily, and / or longer lasting. "Preferentially join". For example, an antibody that specifically or preferentially binds to a PCSK9 epitope has a higher affinity, binding force, easier, and / or longer than it binds to other PCSK9 epitopes or non-PCSK9 epitopes. An antibody that persists and binds to this epitope. Also, by reading this definition, for example, an antibody (or partial or epitope) that specifically or preferentially binds to a first target may bind specifically or preferentially to a second target. It will be understood that it does not have to be. Therefore, a "specific bond" or "preferential bond" does not necessarily require (but can be included) an exclusive bond. In general, but not always, reference to a bond means a preferred bond.

As used herein, "substantially pure" means at least 50% pure (ie, free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure, and even more preferably. A substance that is at least 98% pure, most preferably at least 99% pure.

"Host cells" include individual cells or cell cultures that can or have been recipients of vectors (s) for uptake of polynucleotide inserts. A host cell contains the progeny of a single host cell, which is not necessarily completely with the original parent cell (in morphology or genomic DNA complementarity) due to natural, accidental, or intentional mutations. May not be identical. Host cells include cells transfected with the polynucleotides of the invention (s) in vivo.

As is known in the art, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a mutant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is usually defined as a stretch from the amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. The numbering of residues in the Fc region is that of the EU index similar to Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin generally contains two constant domains, CH2 and CH3.

As used in the art, "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. The preferred FcR is the native sequence human FcR. In addition, preferred FcRs are those that bind IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, allelic variants and alternative spliced forms of these receptors. Is included. FcγRII receptors include FcγRIIA (activated receptor) and FcγRIIB (inhibitory receptor), which have similar amino acid sequences, primarily with different cytoplasmic domains. FcRs include 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 transferring maternal IgG to the foetation (Guyer et al., 1976, J.Immunol., 117: 587, and Kim et al., 1994, J.Immunol., 24: 249).

As used herein with respect to an antibody, the term "competing" means that the first antibody or its antigen-binding portion binds to the first antibody and its cognate epitope in the absence of the second antibody. Means binding to an epitope in a manner sufficiently similar to the binding of the second antibody or its antigen-binding portion such that it is detectablely reduced in the presence of the second antibody compared to the binding of the first antibody. To do. An alternative, the binding of the second antibody to its epitope, is also detectable, but not necessarily, reduced in the presence of the first antibody. That is, the first antibody can inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its corresponding epitope. However, if each antibody detectably inhibits the binding of the other antibody to its cognate epitope or ligand, then to the same extent, to a greater or lesser extent, the antibody will have its corresponding epitope ( It is said that there is "cross-competition" with each other for the combination of (s). Both competing and cross-competitive antibodies are included by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (eg, steric hindrance, conformational change, or binding to a common epitope or portion thereof), one of ordinary skill in the art will be based on the teachings provided herein. It should be understood that such competing and / or cross-competitive antibodies may be included and may be useful to the methods disclosed herein.

Antibodies that have an epitope that "overlaps" the surface on PCSK9 that interacts with another (second) epitope or the EGF-like domain of the LDLR means that they share space with respect to the interacting PCSK9 residues. Percentage of overlap, eg, the surface area of PCSK9 buried in the complex with LDLR to calculate the percentage overlap between the PCSK9 epitope of the claimed antibody and the surface of PCSK9 interacting with the EGF-like domain of LDLR. Calculate per residue. The buried area is also calculated for these residues in the PCSK9: antibody complex. To prevent possible duplication beyond 100%, the surface area of residues with a larger buried surface area in the PCSK9: antibody complex than in the LDLR: PCSK9 complex, LDLR: PCSK9 complex (100%). Set as a value from. Percent surface overlap is calculated by summing all of the LDLR: PCSK9 interaction residues and weighted by the interaction area.

A "functional Fc region" possesses at least one effector function of a native sequence Fc region. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity, phagocytosis, and downregulation of cell surface receptors (eg, B cell receptors). Etc. are included. Such effector function generally requires the Fc region to be combined with a binding domain (eg, antibody variable domain), and various assays known in the art for assessing such antibody effector function. Can be evaluated using.

The "native sequence Fc region" includes the same amino acid sequence as the amino acid sequence of the naturally found Fc region. The "mutant Fc region" contains an amino acid sequence different from that of the native sequence Fc region due to at least one amino acid modification, but still retains at least one effector function of the native sequence Fc region. Preferably, the variant Fc region is at least one amino acid substitution compared to the native sequence Fc region or the Fc region of the parent polypeptide, eg, about 1 to about 1 to about in the native sequence Fc region or the Fc region of the parent polypeptide. It has 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions. The variant Fc region herein is preferably at least about 80% sequence identity with the native sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% sequence identity with it. More preferably, it possesses at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% sequence identity.

As used herein, "treatment" is a technique for obtaining beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical outcomes are, but are not limited to, increased LDL clearance and the incidence of abnormal cholesterol and / or lipoprotein levels resulting from metabolic and / or feeding disorders. Decreased or ameliorated, or includes one or more of familial hypercholesterolemia, atherosclerotic dyslipidemia, atherosclerosis, and more generally cardiovascular disease (CVD).

"Reduced incidence" means any of the reduced severity (which includes the need and / or amount of other drugs commonly used for this condition and / or treatment). Reduction of exposure) can be included. As will be appreciated by those skilled in the art, individuals may differ in their response to treatment, thus, for example, "methods of reducing incidence". It reflects the administration of a PCSK9 antagonist antibody, peptide, or aptamer based on the reasonable prediction that such administration may cause such a reduction in incidence in that particular individual.

"Resolving" means reducing or ameliorating one or more symptoms compared to not administering a PCSK9 antagonist antibody, peptide, or aptamer. "Relieving" also includes shortening or reducing the duration of symptoms.

The "effective dose" or "effective amount" of a drug, compound, or pharmaceutical composition as used herein is an amount sufficient to produce any one or more beneficial or desired results. .. For prophylactic use, beneficial or desired outcomes are the biochemical, histological and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presented during the development of the disease. This includes eliminating or reducing the risk, reducing the severity of the disease, or delaying its onset. For therapeutic use, beneficial or desired outcome, reducing one or more symptoms of hypercholesterolemia or abnormal lipidemia, atherosclerosis, CVD, or coronary heart disease, Clinical consequences include reducing the dose of other medications required to treat the disease, enhancing the efficacy of another medication, and / or delaying the progression of the patient's disease. Effective doses can be administered in one or more doses. For the purposes of the present invention, an effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As will be understood in the clinical context, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Therefore, an "effective dose" may be considered in the context of administering one or more therapeutic agents, where the desired results can or are achieved in combination with one or more other agents. May consider giving a single drug in effective doses.

The "individual" or "subject" is a mammal, more preferably a human. Mammals also include, but are not limited to, livestock, sports animals, pets, primates, horses, dogs, cats, mice and rats.

As used herein, "vector" means a construct capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA encapsulated in liposomes. Alternatively, it includes an RNA expression vector and specific eukaryotic cells such as producing cells.

As used herein, the term "expression control sequence" means a nucleic acid sequence that directs transcription of a nucleic acid. The expression control sequence can be a promoter such as a constitutive or inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

As used herein, a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is a subject that, when combined with an active ingredient, allows the ingredient to retain biological activity. Includes any substance that is non-reactive with the immune system. Examples include, but are not limited to, phosphate buffered solutions, emulsions such as water, oil / water emulsions, and any of the standard pharmaceutical carriers such as various types of wetting agents. Is done. The preferred diluent for aerosol or parenteral administration is phosphate buffered solution (PBS) or normal (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (eg, Remington's Pharmaceutical Sciences, 18th Edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990, and Remington, The Science and See Practice of Pharmacy, 20th Edition, Mack Publishing, 2000).

The term "k" used herein._on"" Refers to the rate constant of association between the antibody and the antigen. Specifically, the rate constant (k_onAnd k_off) And equilibrium dissociation constants are measured using Fab antibody fragments (ie monovalent) and PCSK9.

The term "k" used herein._off"" Refers to the rate constant at which an antibody dissociates from an antibody / antigen complex.

The term "K" as used herein._D"" Refers to the equilibrium dissociation constant of antibody-antigen interaction.

A. How to prevent or treat disorders associated with hypercholesterolemia In one aspect, the invention comprises administering to an individual an effective amount of a PCSK9 antagonist antibody or peptide or aptamer that antagonizes circulating PCSK9, hypercholesterolemia and / or atherosclerosis in the individual. Provided is a method for treating or preventing at least one symptom of atherosclerosis, CVD or coronary heart disease.

In a further aspect, the invention is used in the treatment or prevention of hypercholesterolemia and / or at least one symptom of abnormal lipidemia, atherosclerosis, CVD or coronary heart disease in an individual. , Provide effective amounts of PCSK9 antagonistic antibodies, peptides, or aptamers that antagonize circulating PCSK9. Furthermore, the present invention relates to the manufacture of a medicament for treating or preventing hypercholesterolemia and / or at least one symptom of hyperlipidemia, atherosclerosis, CVD or coronary heart disease in an individual. Provides the use of an effective amount of PCSK9 antagonist antibody, peptide, or aptamer that antagonizes extracellular or circulating PCSK9.

Advantageously, therapeutic administration of antibodies, peptides, or aptamers results in lower blood cholesterol and / or lower blood LDL. Preferably, blood cholesterol and / or blood LDL is at least about 10% or 15% lower than before administration. More preferably, blood cholesterol and / or blood LDL is at least about 20% lower than before administration of the antibody. Even more preferably, blood cholesterol and / or blood LDL is at least 30% lower than before administration of the antibody. Advantageously, blood cholesterol and / or blood LDL is at least 40% lower than before administration of the antibody. More advantageously, blood cholesterol and / or blood LDL is at least 50% lower than before administration of the antibody. Very preferably, blood cholesterol and / or blood LDL is at least 60% lower than before administration of the antibody. Most preferably, blood cholesterol and / or blood LDL is at least 70% lower than before administration of the antibody.

For all methods described herein, references to PCSK9 antagonist antibodies, peptides, and aptamers also include compositions containing one or more additional agents. These compositions may further comprise suitable excipients such as pharmaceutically acceptable excipients, including buffers well known in the art. The present invention can be used alone or in combination with other conventional treatment methods.

PCSK9 antagonist antibodies, peptides, or aptamers can be administered to an individual via any suitable route. It will be apparent to those skilled in the art that the examples described herein are not intended to be limiting and are illustrations of available techniques. Thus, in some embodiments, the PCSK9 antagonist antibody, peptide, or aptamer is administered intravenously, eg, as a bolus or continuous infusion over a period of time, intramuscularly, intraperitoneally, intracephaly, transdermally, subcutaneously, intraarticularly. Administer to an individual according to a known method, such as sublingual, intramuscular, gas injection, intrasubarachnoid space, oral, inhalation or topical route. Administration can be systemic, eg intravenous, or topical. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. The liquid formulation can be directly atomized and the lyophilized powder can be atomized after reconstitution. Alternatively, the PCSK9 antagonist antibody, peptide, or aptamer can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and ground powder.

In one embodiment, the PCSK9 antagonist antibody, peptide, or aptamer is administered via site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of PCSK9 antagonist antibodies, peptides, or aptamers or local delivery catheters such as infusion catheters, indwelling catheters, or needle catheters, synthetic implants. Includes pieces, epithelial wraps, shunts and stents, or other implantable devices, site-specific carriers, direct injections, or direct applications. See, for example, PCT Publication WO 00/53211 and US Pat. No. 5,981,568.

Various formulations of PCSK9 antagonist antibodies, peptides, or aptamers can be used for administration. In some embodiments, the PCSK9 antagonist antibody, peptide, or aptamer may be administered undiluted. In some embodiments, PCSK9 antagonist antibodies, peptides, or aptamers and pharmaceutically acceptable excipients can also be in the form of various formulations. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that facilitate the administration of pharmacologically effective substances. For example, the excipient can give shape or consistency or act as a diluent. Suitable excipients include, but are not limited to, stabilizers, wetting agents and emulsifying agents, salts for varying osmotic pressure, encapsulants, buffers, and skin penetration enhancers. Excipients and formulations for parenteral and non-parental drug delivery are described in Remington, The Science and Practice of Pharmacy, 20th Edition, Mack Publishing (2000).

These agents can be combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution and the like. The specific dosing regimen, ie dose, timing and repetition, depends on the particular individual and the medical history of that individual.

The PCSK9 antibody can also be administered by inhalation as described herein. In general, the initial candidate dose can be about 2 mg / kg for administration of PCSK9 antibody. For the purposes of the present invention, typical daily doses are from about 3 μg / kg to 30 μg / kg, up to 300 μg / kg, up to 3 mg / kg, up to 30 mg / kg, 100 mg / kg, depending on the factors mentioned above. It can be in the range of any of up to kg or more. For example, doses of about 1 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 10 mg / kg, and about 25 mg / kg can be used. Repeated doses over several days or longer, depending on the condition, continue treatment until the desired suppression of symptoms occurs, or until, for example, a level of treatment sufficient to reduce blood LDL levels is achieved. Let me. An exemplary dosing regimen comprises administering an initial dose of about 2 mg / kg followed by a weekly maintenance dose of about 1 mg / kg of PCSK9 antibody, followed by a biweekly maintenance dose of about 1 mg / kg. .. However, other dosing regimens may be useful, depending on the pattern of pharmacokinetic disruption that the worker desires to achieve. For example, in some embodiments, dosing is intended 1 to 4 times a week. In other embodiments, dosing is intended once a month or every other month or once every three months. The progress of this treatment is easily monitored by conventional techniques and assays. The dosing regimen, which includes the PCSK9 antagonist used (s), may vary over time.

For the purposes of the present invention, the appropriate dose of PCSK9 antagonist antibody, peptide, or aptamer may be the PCSK9 antagonist antibody, peptide, or aptamer (or composition thereof) used, the type and severity of symptoms to be treated, the agent. Whether administered for prophylactic or therapeutic purposes, previous treatment, patient history and response to drug, patient's blood PCSK9 level, PCSK9 synthesis and clearance rate in patient, clearance rate of administered drug in patient , As well as the judgment of the attending physician. Typically, the clinician administers a PCSK9 antagonist antibody, peptide, or aptamer until the dose at which the desired result is achieved is reached. Dose and / or frequency may vary over the course of treatment. Empirical considerations such as half-life generally contribute to dose determination. For example, an antibody compatible with the human immune system, such as a humanized antibody or a fully human antibody, can be used to prolong the half-life of the antibody and prevent the antibody from being attacked by the host's immune system. The frequency of administration may be determined and adjusted over the course of treatment and is generally, but not necessarily, based on symptoms such as treatment and / or suppression and / or remission and / or delay of hypercholesterolemia. Alternatively, a continuous continuous release formulation of the PCSK9 antagonist antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

In one embodiment, the dose of the antagonist antibody, peptide, or aptamer can be empirically determined in an individual given one or more doses of the antagonist antibody, peptide, or aptamer. Individuals are given increasing doses of PCSK9 antagonist antibody, peptide, or aptamer. Disease indicators can be subsequently given to assess efficacy.

Administration of a PCSK9 antagonist antibody, peptide, or aptamer according to the methods of the invention is known, for example, to the physiological conditions of the recipient, whether the purpose of administration is therapeutic or prophylactic, and to those skilled in the art. It can be continuous or intermittent, depending on other factors. Administration of the PCSK9 antagonist antibody, peptide, or aptamer can be essentially continuous or at a series of spaced doses over a preselected period of time.

In some embodiments, there may be multiple antagonist antibodies, peptides, or aptamers. There can be at least one, at least two, at least three, at least four, at least five different or more antagonistic antibodies and / or peptides. In general, these PCSK9 antagonist antibodies or peptides may have complementary activity that does not adversely affect each other. PCSK9 antagonist antibodies, peptides, or aptamers can also be used in combination with other PCSK9 antagonists or PCSK9 receptor antagonists. For example, one or more of the following PCSK9 antagonists may be used: antisense molecules against PCSK9 (including antisense molecules against nucleic acids encoding PCSK9), PCSK9 inhibitory compounds, and PCSK9 structural similarities. body. PCSK9 antagonist antibodies, peptides, or aptamers can also be used in conjunction with other drugs that serve to enhance and / or complement the efficacy of the drug.

Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used, buffers such as phosphoric acid, citric acid, and other organic acids, salts such as sodium chloride, ascorbic acid. And antioxidants including methionine, preservatives (octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol , Cyclohexanol, 3-pentanol, and m-cresol, etc.), low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, or immunoglobulin, 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 dextrin, chelating agents such as EDTA, sucrose, mannitol, trehalose or sorbitol. Sugars such as, salt-forming counterions such as sodium, metal complexes (eg, Zn-protein complexes), and / or nonionic surfactants such as TWEEN , PLURONICS or polyethylene glycol (PEG). May include.

Liposomes containing PCSK9 antagonist antibodies, peptides, or aptamers include Epstein et al., 1985, Proc.Natl.Acad.Sci.USA, 82: 3688, Hwang et al., 1980, Proc.Natl Acad.Sci.USA, 77:4030. , And US Pat. Nos. 4,485,045 and 4,544,545, etc., prepared by methods known in the art. Liposomes with enhanced circulation time are disclosed in US Pat. No. 5,013,556. Particularly useful liposomes can be prepared by a reverse phase evaporation method using a lipid composition containing phosphatidylcholine, cholesterol and PEG derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter with a defined pore size to give liposomes with the desired diameter.

The active ingredient is also microcapsules prepared in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, eg, by coacervation techniques or interfacial polymerization. , For example, may be encapsulated in hydroxymethyl cellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively. Such techniques are disclosed in Remington, The Science and Practice of Pharmacy, 20th Edition, Mack Publishing (2000).

Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg poly (2-hydroxyethyl-methacrylate), or'poly (vinyl alcohol)), polylactic acid (US Pat. No. 3,773,919), L-glutamic acid and 7 ethyl-. Degradable lactic acid-glycolic acid copolymers such as copolymers with L-glutamate, non-degradable ethylene-vinyl acetate, LUPRON DEPOT (injectable microspheres consisting of lactic acid-glycolic acid copolymers and leuprolide acetate), sculose isobuchi acetate Includes rate, and poly-D- (-)-3-hydroxybutyric acid.

The formulation used for in vivo administration must be sterile. This is easily achieved, for example, by filtering through a sterile filtration membrane. Therapeutic PCSK9 antagonist antibodies, peptides, or aptamer compositions are generally placed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be perforated by a hypodermic needle.

Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid , Liposyn , Infonutrol , Lipofundin and Lipiphysan . The active ingredient may be dissolved in a premixed emulsion composition or in oil (eg, soybean oil, Benibana oil, cottonseed oil, sesame oil, corn oil or almond oil) and phospholipids (eg egg phosphorus). It may form an emulsion when mixed with lipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other components such as glycerol or glucose may be added to adjust the isotonicity of the emulsion. Suitable emulsions typically contain up to 20% oil, eg 5-20%. The fat emulsion can contain fat droplets of 0.1 to 1.0 μm, particularly 0.1 to 0.5 μm, and can have a pH in the range of 5.5 to 8.0.

The emulsion composition can be prepared by mixing a PCSK9 antagonist antibody, peptide, or aptamer with Intralipid or its components (soybean oil, egg phospholipids, glycerol and water).

Compositions for inhalation or gas injection include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, as well as powders. The liquid or solid composition may contain the appropriate pharmaceutically acceptable excipients described above. In some embodiments, the composition is administered by the oral or nasal respiratory route for local or systemic effects. The composition in a preferably sterile pharmaceutically acceptable solvent can be atomized by using a gas. The atomized solution may be breathed directly from the atomizer, or the atomizer may be attached to a face mask, tent or intermittent positive pressure ventilator. The composition of the solution, suspension or powder can be administered, preferably orally or nasally, from a device that delivers the formulation in a suitable manner.

B.PCSK9 antagonist The methods of the invention block, suppress or reduce (including significantly reducing) PCSK9 biological activity, including downstream pathways mediated by PCSK9 signaling, such as inducing a cellular response to PCSK9. Alternatively, a PCSK9 antagonist antibody, peptide, or aptamer, which refers to a nucleic acid molecule, is used.

A PCSK9 antagonist antibody, peptide, or aptamer should exhibit any one or more of the following characteristics: (a) binding to PCSK9, (b) blocking the interaction of PCSK9 with the LDLR. (C) Blocking or reducing PCSK9-mediated downregulation of LDLR, (d) Inhibiting PCSK9-mediated reduction in LDL blood clearance, (e) PCSK9-mediated reduction in medium Increasing LDL clearance, (f) increasing in vivo blood LDL clearance by the liver, (g) sensitizing statins, and (h) PCSK9 with other unidentified factors Blocking interactions.

For the purposes of the present invention, antibodies, peptides, or aptamers preferably react with PCSK9 in a manner that inhibits PCSK9 signaling function and LDLR interactions. In some embodiments, the PCSK9 antagonist antibody specifically recognizes the primate PCSK9. In some embodiments, the PCSK9 antagonist antibody binds to primate and rodent PCSK9.

Antibodies useful in the present invention include monoclonal antibodies, polyclonal antibodies, antibody fragments (eg, Fab, Fab', F (ab') 2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugates. Antibodies, single chains (ScFv), variants thereof, fusion proteins containing antibody moieties (eg domain antibodies), human antibodies, humanized antibodies, and glycosylation variants of antibodies, amino acid sequence variants of antibodies, and sharing. Any other modified configuration of an immunoglobulin molecule containing an antigen recognition site of the required specificity, including a specifically modified antibody, can be included. Antibodies can be murine, rat, human, or any other source, including chimeric or humanized antibodies.

In some embodiments, the PCSK9 antagonist antibody is a monoclonal antibody. It is also possible to humanize PCSK9 antagonist antibodies. In other embodiments, the antibody is human.

In some embodiments, the antibody comprises a modified constant region, such as a constant region that is immunologically inactive, i.e., has a reduced potential to elicit an immune response. In some embodiments, the constant region is modified as described in Eur. J. Immunol., 1999, 29: 2613 ~ 2624, PCT Publication WO 99/58572, and / or UK Patent Application No. 9809951.8. Fc is human IgG₂Or human IgG₄Can be. Fc is a human IgG containing a mutation from A330P331 to S330S331₂(IgG_2Δa), And the amino acid residues are numbered with reference to the wild-type IgG2 sequence. Eur.J.Immunol., 1999, 29: 2613 ~ 2624. In some embodiments, the antibody comprises an IgG comprising a mutation from E233F234L235 to P233V234A235.₄Constant region (IgG)_4Δc) (Armour et al., 2003, Molecular Immunology, 40, 585-593), and the numbers refer to wild-type IgG4. In yet another embodiment, Fc is a human IgG.₄E233F234L235 to P233V234A235 with a deletion of G236 (IgG)_4Δb). In another embodiment, the Fc is any human IgG containing a hinge-stabilizing mutation from S228 to P228.₄ Fc (IgG)₄, IgG_4ΔbOr IgG_4Δc) (Aalberse et al., 2002, Immunology, 105, 9-19). In another embodiment, the Fc may be a deglycosylated Fc.

In some embodiments, the constant region is deglycosylated by mutating oligosaccharide-attached residues (such as Asn297) and / or adjacent residues that are part of the glycosylation recognition sequence of the constant region. In some embodiments, the constant region is enzymatically deglycosylated for N-linked glycosylation. The constant region can be deglycosylated for N-linked glycosylation, either enzymatically or by expression in a glycosylation-deficient host cell.

Binding affinity between PCSK9 antagonist antibody and PCSK9 (human PCSK9, etc.) (K_D) Can be from about 0.002 to about 200 nM. In some embodiments, the binding affinity is about 200nM, about 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM, about 60pM, about 50pM, about 20pM, about 15pM, about 10pM, about 5pM. , Or any one of about 2pM. In some embodiments, the binding affinity is 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 about. Less than any of 2pM.

One method of determining the binding affinity of an antibody for PCSK9 is by measuring the binding affinity of a monofunctional Fab fragment of the antibody. To obtain a monofunctional Fab fragment, the antibody (eg IgG) can be cleaved with papain or expressed by recombination. The affinity of the PCSK9 Fab fragment of the antibody is that of surface plasmon resonance (Biacore 3000 surface plasmon resonance (SPR) system, Biacore, INC, Piscataway) with a pre-fixed streptavidin sensor chip (SA). It can be determined by NJ) using HBS-EP running buffer (0.01 M HEPES, pH 7.4, 0.15 NaCl, 3 mM EDTA, 0.005% v / v surfactant P20). Biotin-labeled human PCSK9 (or any other PCSK9) diluted with HBS-EP buffer to a concentration of less than 0.5 μg / mL and injected over individual chip channels with variable contact time, two ranges The antigen density of, ie, 50-200 response units (RU) for detailed kinetic studies or 800-1,000 RU for screening assays can be achieved. Regenerative studies have shown that 25 mM NaOH in 25% v / v ethanol effectively removes bound Fab while retaining PCSK9 activity on the chip over more than 200 injections. Typically, a serial dilution of the purified Fab sample (0.1-10 x estimated K)_DInject for 1 minute with a dissociation time of 100 μL / min and allow up to 2 hours. Fab protein concentrations are determined by ELISA and / or SDS-PAGE electrophoresis using known concentrations of Fab (determined by amino acid analysis) as a standard. Kinetic meeting speed (k_on) And dissociation rate (k)_off) Uses the BIA evaluation program to translate the data into a 1: 1 Langmuir binding model (Karlsson, R., Roos, H., Fagerstam, L., Petersson, B., 1994., Methods Enzymology, 6.99-110). It can be obtained at the same time by applying it to the target. Equilibrium dissociation constant (K_D) Value is k_off/ k_onCalculate as. This protocol involves binding antibodies to human PCSK9, another mammalian PCSK9 (mouse PCSK9, rat PCSK9, primate PCSK9, etc.), and any PCSK9, including various forms of PCSK9 (alpha and β types, etc.). Suitable for use in determining affinity. Generally, the binding affinity of an antibody is measured at 25 ° C, but it can also be measured at 37 ° C.

PCSK9 antagonist antibodies can be made by any method known in the art, including the method provided in Example 1. The immunization pathways and schedules of host animals are generally consistent with established conventional techniques for stimulating and producing antibodies, as further described herein. General techniques for producing human and mouse antibodies are known in the art and / or described herein. Currently preferred methods for making antibodies are PCSK9 disclosed herein.^-Knockout (PCSK9-/-) Includes animal immunization.

It is contemplated that any mammalian subject, including humans, or antibody-producing cells derived from it can be engineered to serve as the basis for producing hybridoma cell lines in mammals, including humans. Typically, the host animal is inoculated with a certain amount of immunogen, including those described herein, intraperitoneally, intramuscularly, orally, subcutaneously, intrasole, and / or subcutaneously.

Hybridomas are modified by Kohler, B. and Milstein, C., 1975, Nature, 256: 495-497 general somatic cell hybridization techniques or Buck, DW et al., 1982, In Vitro, 18: 377-381. Can be prepared from lymphocytes and immortalized myeloma cells. Not limited to that, X63-Ag8.653 and Salk Institute, Cell Distribution Center, San Available myeloma systems can be used for hybridization, including those from Diego, Calif., USA. Generally, the technique involves fusing myeloma cells and lymphocyte cells using a fusion inducer such as polyethylene glycol or by electrical means well known to those of skill in the art. After fusion, cells are separated from the fusion medium and grown in selective growth medium such as hypoxanthine-aminopterin-thymidine (HAT) medium to eliminate non-hybridized parent cells. Any of the media described herein with or without serum can be used to culture hybridomas that secrete monoclonal antibodies. As another alternative to cell fusion techniques, EBV immortalized B cells can be used to produce the PCSK9 monoclonal antibody of the invention. If desired, the hybridoma is expanded and subcloned, and the supernatant is assayed for antiimmuneogenic activity by conventional immunoassay procedures (eg, radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

Hybridomas that can be used as antibody sources include all derivatives and progeny cells of parental hybridomas that produce PCSK9-specific monoclonal antibodies or portions thereof.

Hybridomas that produce such antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from culture medium or body fluids by routine immunoglobulin purification procedures such as ammonium sulfate salting out, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired. The presence of undesired activity is removed, for example, by flushing the preparation onto an adsorbent made from an immunogen attached to the solid phase and eluting or releasing the desired antibody from the immunogen to remove it. Can be done. Human PCSK9, or bifunctional or derivatizing agents, such as maleimide benzoyl sulfosuccinimide ester (conjugated via a cysteine residue), N-hydroxysuccinimide (via a lysine residue), glutaraldehyde, succinic anhydride. , SOCl₂Or R¹N = C = NR [in the formula, R and R¹Is a different alkyl group] and a target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemosianin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor. A population of antibodies (eg, monoclonal antibodies) can be obtained by immunizing a host animal with a fragment containing.

If desired, the PCSK9 antagonist antibody of interest (monoclonal or polyclonal) may be sequenced, after which the polynucleotide sequence can be cloned into a vector for expression or proliferation. The sequence encoding the antibody of interest may be maintained in the vector in the host cell, after which the host cell can be frozen for expansion and future use. Production of recombinant monoclonal antibodies in cell cultures can be carried out by means known in the art by cloning antibody genes from B cells. See, for example, Tiller et al., 2008, J.Immunol.Methods, 329,112, US Pat. No. 7,314,622.

In alternative methods, the polynucleotide sequence can be used for genetic engineering to "humanize" the antibody or to improve the affinity or other characteristics of the antibody. For example, the constant region can be manipulated to make it more similar to the human constant region in order to avoid an immune response when the antibody is used in clinical trials and treatments in humans. It may be desirable to genetically engineer an antibody sequence for higher affinity for PCSK9 and higher efficacy in inhibiting PCSK9. It will be apparent to those skilled in the art that one or more polynucleotide changes will be made to the PCSK9 antagonist antibody while still maintaining its binding capacity to PCSK9.

There are four general steps in humanizing a monoclonal antibody. They are (1) determining the nucleotide and expected amino acid sequences of the light and heavy chain variable domains of the initiating antibody, (2) designing the humanized antibody, that is, which antibody frame during the humanization process. The steps of deciding whether to use the work area, (3) the actual humanization method / technique steps, and (4) the transfection and humanized antibody expression steps. See, for example, U.S. Pat. Nos. 4,816,567, 5,807,715, 5,866,692, 6,331,415, 5,530,101, 5,693,761, 5,693,762, 5,585,089, and 6,180,370.

Several "humanizations" containing antigen binding sites derived from non-human immunoglobulins, including rodents fused to the human constant domain or chimeric antibodies with modified rodent V regions and their associated CDRs The antibody molecule is described. 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 See Res., 47: 3577 ~ 3583. Other references describe rodent CDRs implanted within the Human Support Framework Region (FR) prior to fusion with the appropriate human antibody constant domain. See, for example, Richmann et al., 1988, Nature, 332: 323 ~ 327, Verhoeyen et al., 1988, Science, 239: 1534 ~ 1536, and Jones et al., 1986, Nature, 321: 522 ~ 525. Another reference describes a rodent CDR supported by a recombinant engineered rodent framework region. See, for example, European Patent Publication No. 0519596. These "humanized" molecules are intended to minimize unwanted immunological responses to rodent anti-human antibody molecules that limit the duration and efficacy of therapeutic application of these parts in human recipients. Is designed for. For example, the antibody constant region can be engineered to be immunologically inactive (eg, does not initiate complement lysis). See, for example, PCT Publication WO 99/58572, UK Patent Application No. 9809951.8. Other methods of humanizing antibodies that are also available are Daugherty et al., 1991, Nucl. Acids Res., 19: 2471-2476 and US Pat. Nos. 6,180,377, 6,054,297, 5,997,867, 5,866,692, It is disclosed in Nos. 6,210,671 and 6,350,861 and in PCT Publication WO 01/27160.

In yet another alternative, fully human antibodies can be obtained using commercially available mice engineered to express a particular human immunoglobulin protein. Humanized or human antibodies can also be made using transgenic animals designed to produce a more desirable or more robust immune response. Examples of such techniques are Xenomouse from Abgenix, Inc. (Fremont, CA), HuMAb-Mouse and TC Mouse from Medarex, Inc. (Princeton, NJ), and Regeneron Pharmaceuticals. , Inc. (Tarrytown, NY) Veloc Immunone® mouse.

In an alternative method, the antibody can be recombinantly made and expressed using any method known in the art. Alternatively, the antibody can be recombinantly produced by phage display technology. See, for example, US Pat. Nos. 5,565,332, 5,580,717, 5,733,743, and 6,265,150, and Winter et al., 1994, Annu. Rev. Immunol., 12: 433-455. Alternatively, human antibodies and antibody fragments were used in phage display technology (McCafferty et al., 1990, Nature, 348: 552-553). It can be produced in vitro from the immunoglobulin variable (V) domain gene repertoire from non-immunized donors. According to this technique, the antibody V domain gene is cloned in-frame into either the major or sub-coated protein gene of filamentous bacteriophage such as M13 or fd and displayed as a functional antibody fragment on the surface of phage particles. .. Since filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of the gene encoding the antibody exhibiting these properties. Thus, phage mimics some of the properties of B cells. Phage display can be done in a variety of ways, for example Johnson, Kevin S. and Chiswell, David J., 1993, Current Opinion in Structural. See Biology, 3: 564 ~ 571. Several sources of V gene segments can be used for phage display. Clackson et al., 1991, Nature, 352: 624-628 isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleen of immunized mice. A repertoire of V genes from non-immunized human donors can be constructed, Mark et al., 1991, J. Mol. Biol., 222: 581-597 or Griffith et al., 1993, EMBO. Antibodies to diverse arrays of antigens, including self-antigens, can be isolated, essentially according to the techniques described by J., 12: 725-734. In the natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the introduced changes give high affinity, and B cells displaying high affinity surface immunoglobulins are preferentially replicated and differentiated during subsequent antigen immunization. This natural process can be mimicked by using a technique known as "chain shuffling". (Marks et al., 1992, Bio / Technol., 10: 779-783). In this method, the affinity of the "primary" human antibody obtained by phage display is that the heavy and light chain V region genes are of naturally occurring variants of the V domain gene obtained from non-immunized donors. It can be improved by sequentially replacing the repertoire (repertoire). This technique allows the production of antibodies and antibody fragments with affinities in the pM to nM range. Strategies for creating a very large phage antibody repertoire (also known as the "mother-of-all library"), Waterhouse et al., 1993, Nucl. Acids Res., 21: Described by 2265 to 2266. Gene shuffling can also be used to derive human antibodies from rodent antibodies, which have similar affinities and specificities to initiating rodent antibodies. This method, also known as "epitope imprinting," replaces the heavy or light chain V-domain genes of rodent antibodies obtained by phage display technology with a repertoire of human V-domain genes to replace rodent-humans. Make a chimera. Selection on the antigen results in the isolation of a human variable region capable of repairing a functional antigen binding site, i.e., the epitope governs (imprints) the partner's choice. Human antibodies are obtained when the process is repeated to replace the remaining rodent V domain (see PCT Publication WO 93/06213). Unlike traditional humanization of rodent antibodies by CDR transplantation, this technique provides fully human antibodies with no framework or CDR residues of rodent origin.

Although the above description relates to humanized antibodies, it is clear that the general principles discussed are applicable, for example, to customizing antibodies for use in dogs, cats, primates, horses and bovinae. Will. Furthermore, it will be apparent that one or more aspects of antibody humanization described herein can be combined with, for example, CDR transplantation, framework mutations and CDR mutations.

Antibodies are recombinantly produced by first isolating the antibody and antibody-producing cells from the host animal, obtaining the gene sequence, and recombinantly expressing the antibody in the host cell (eg, CHO cell) using the gene sequence. obtain. Another method that can be used is to express the antibody sequence in a plant (eg, tobacco) or transgenic milk. Methods for recombinant expression of antibodies in plants or milk are disclosed. See, for example, 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. I want to. For example, methods for producing derivatives of antibodies such as humans and single chains are known in the art.

In addition, PCSK9-specific antibodies can also be isolated using flow cytometric fractionation techniques such as immunoassay and fluorescence activated cell fractionation (FACS).

The antibody can bind to many different carriers. The carrier can be active and / or inactive. Examples of well-known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylases, glass, natural and modified celluloses, polyacrylamide, agarose and magnetite. For the purposes of the present invention, the nature of the carrier can be either soluble or insoluble. One of ordinary skill in the art knows of other suitable carriers for the binding antibody, or can confirm such using routine experiments. In some embodiments, the carrier comprises a portion that targets the myocardium.

DNA encoding a monoclonal antibody uses conventional procedures (eg, by using an oligonucleotide probe that can specifically bind to the genes encoding the heavy and light chains of the monoclonal antibody). It can be easily isolated and sequenced. Hybridoma cells serve as the preferred source of such DNA. After isolation, the DNA is placed in an expression vector (such as the expression vector disclosed in PCT Publication WO 87/04462), which is then placed in E. coli, which otherwise does not produce immunoglobulin proteins. ) Cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells can be transfected into host cells to obtain monoclonal antibody synthesis in recombinant host cells. See, for example, PCT Publication WO 87/04462. Also, DNA is replaced by homologous murine sequences, for example, by substituting the coding sequences of human heavy and light chain constant domains, Morrison et al., 1984, Proc.Nat.Acad.Sci., 81: 6851, Alternatively, the immunoglobulin coding sequence can also be modified by covalently binding to all or part of the coding sequence of a non-immunoglobulin polypeptide. In this way, a "chimeric" or "hybrid" antibody with binding specificity for the PCSK9 monoclonal antibody herein is prepared.

PCSK9 antagonistic antibodies and antibodies-derived polypeptides can be identified or characterized using methods known in the art, and reduced, ameliorated, or neutralized PCSK9 biological activity is detected and / or measured. .. In some embodiments, the PCSK9 antagonist antibody or polypeptide is identified by incubating the candidate agent with PCSK9 and monitoring the binding and / or concomitant reduction or neutralization of PCSK9 bioactivity. The binding assay can be performed using purified PCSK9 polypeptide (s) or cells that naturally express or have been transfected to express the PCSK9 polypeptide (s). In one embodiment, the binding assay is a competitive binding assay that assesses the ability of a candidate antibody to compete with a known PCSK9 antagonist for PCSK9 binding. Assays can be performed in a variety of formats, including ELISA formats. In other embodiments, PCSK9 antagonist antibodies are identified by incubating a candidate agent with PCSK9 and monitoring binding and concomitant LDLR expression and / or inhibition of blood cholesterol clearance.

After initial identification, the activity of candidate PCSK9 antagonist antibodies can be further confirmed and refined by bioassays known to test targeted biological activity. Alternatively, bioassays can be used to screen candidates directly. Some of the methods for identifying and characterizing PCSK9 antagonist antibodies, peptides, or aptamers will be detailed in the examples.

PCSK9 antagonist antibodies can be characterized using methods well known in the art. For example, one method is to identify the epitope to which it binds, i.e., "epitope mapping." Numerous methods are known in the art for mapping and characterizing the location of epitopes on proteins, such as Harlow and Lane, Using Antibodies, a Laboratory Manual, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). , 1999), including analysis of the crystal structure of antibody-antigen complexes, competitive assays, gene fragment expression assays, and synthetic peptide-based assays. In a further example, epitope mapping can be used to determine the sequence to which the PCSK9 antagonist antibody binds. Epitope mapping is available from various suppliers, such as Pepscan Systems (Edelhertweg 15, 8219 PH). Commercially available from Lelystad (Netherlands). The epitope is a linear epitope, a conformational epitope formed by a three-dimensional interaction of amino acids that may or may not be contained in a single amino acid stretch. Can be. Peptides of various lengths (eg, at least 4-6 amino acid lengths) can be isolated or synthesized (eg, by recombination) and used in binding assays with PCSK9 antagonist antibodies. In another example, the epitope to which the PCSK9 antagonist binds can be determined by systematic screening by using a duplicate peptide derived from the PCSK9 sequence and determining the binding by the PCSK9 antagonist. According to the gene fragment expression assay, the open reading frame encoding PCSK9 is fragmented randomly or by specific gene construction to determine the reactivity of the expressed PCSK9 fragment with the antibody to be tested. Gene fragments are produced, for example, by PCR and then in In vitro, it can be transcribed and translated into proteins in the presence of radioactive amino acids. The binding of the antibody to the radiolabeled PCSK9 fragment is then determined by immunoprecipitation and gel electrophoresis. Specific epitopes can also be identified by using a large library of random peptide sequences displayed on the surface of phage particles (phage library). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in a simple binding assay. In a further example, antigen binding domain mutagenesis, domain exchange experiments and alanine scanning mutagenesis can be performed to identify sufficient and / or required residues required for epitope binding. For example, in domain exchange experiments, various fragments of the PCSK9 polypeptide are PCSK9 from another species, or proteins that are closely related but have distinctly different antigenicity (such as another member of the precursor protein convertase family). It can be done using the mutant PCSK9 that has been replaced (exchanged) with the sequence from). By assessing the binding of the antibody to the mutant PCSK9, the importance of a particular PCSK9 fragment in antibody binding can be assessed.

Yet another method that can be used to characterize PCSK9 antagonist antibodies is to use competitive assays with other antibodies known to bind the same antigen, namely various fragments on PCSK9. To determine if a PCSK9 antagonist antibody binds to the same epitope as other antibodies. Competitive assays are well known to those of skill in the art.

Antibodies and antibody: Antigens can also be characterized using the crystal structure of the complex. Residues are identified by calculating the difference in accessible surface area between the crystal structure of L1L3: PCSK9 and the PCSK9 structure alone. PCSK9 residues, which indicate the surface area buried during complex formation with the L1L3 antibody, are included as part of the epitope. The solvent accessible surface of a protein is defined as the position of the center of a probe sphere (representing a solvent molecule with a radius of 1.4 Å) as it rolls over the van der Waals surface of the protein. The solvent accessible surface area is run by the program AREAIMOL to create surface points on the expanding sphere around each atom (at a distance from the atomic center equal to the sum of the atoms and probe radii) and associate with adjacent atoms. Calculated by excluding those in the equivalent spheres (Briggs, PJ, 2000, CCP4 Newsletter, No. 38, CCLRC, Daresbury).

Expression vectors can be used to direct the expression of PCSK9 antagonist antibodies. Those skilled in the art are familiar with administering expression vectors to obtain expression of foreign proteins in vivo. See, for example, US Pat. Nos. 6,436,908, 6,413,942, and 6,376,471. Administration of the expression vector includes injection, oral administration, topical or systemic administration, including particle gun or catheter administration, and topical administration. In another embodiment, the expression vector is administered directly to the sympathetic trunk or ganglion, or intracoronary artery, atrium, ventricle, or pericardium.

Targeted delivery of expression vectors, or therapeutic compositions containing subgenome polynucleotides, can also be used. Receptor-mediated DNA delivery techniques include, for example, Findeis et al., 1993, Trends Biotechnol., 11:202, Chiou et al., 1994, Gene Therapeutics: Methods And Applications Of Direct Gene. Transfer (JAWolff edition), 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 polynucleotides are administered with DNA in the range of about 100 ng to about 200 mg for topical administration in gene therapy protocols. Also, DNA concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg can be used in the gene therapy protocol. Therapeutic polynucleotides and polypeptides can be delivered using a gene delivery vehicle. Gene delivery vehicles can be of viral or non-viral origin (generally Jolly, 1994, Cancer Gene Therapy, 1:51, Kimura, 1994, Human Gene). See Therapy, 5: 845, Connelly, 1995, Human Gene Therapy, 1: 185, and Kaplitt, 1994, Nature Genetics, 6: 148). Expression of such coding sequences can be induced using endogenous mammals or heterologous promoters. Expression of the coding sequence can be either constitutive or regulatory.

Viral vectors for delivering the desired polynucleotide and expressing it in the desired cell are well known in the art. Exemplary viral vehicles include, but are not limited to, recombinant retroviruses (eg, PCT Publications WO90 / 07936, WO94 / 03622, WO93 / 25698, WO93 / 25234, WO93 / 11230, WO93). / 10218, WO 91/02805, US Pat. No. 5,219,740, No. 4,777,127, GB Pat. No. 2,200,651, and EP Pat. No. 0 345 242), Alpha Viral Vectors (eg Sindbis Virus Vector, Semuliki) Forest virus (ATCC VR-67, ATCC VR-1247), Ross River virus (ATCC VR-373, ATCC VR-1246), Venezuelan encephalitis virus (ATCC VR-923, ATCC VR-1250, ATCC VR1249, ATCC See VR-532))) and adeno-associated virus (AAV) vectors (eg, PCT Publications WO94 / 12649, WO93 / 03769, WO93 / 19191, WO94 / 28938, WO95 / 11984 and WO95 / 00655. ) Is included. Administration of DNA linked to a dead adenovirus as described in Curiel, 1992, Hum.Gene Ther., 3: 147 can also be used.

Also, but not limited to, polycationic condensed DNA linked or unlinked with dead adenovirus alone (eg Curiel, 1992, Hum.Gene). Ther., see 3: 147), ligand-linked DNA (see, eg, Wu, J., 1989, Biol. Chem., 264: 16985), eukaryotic cell delivery vehicle cells (see, eg, US Pat. No. 5,814,482, PCT). Non-virus delivery vehicles and methods can also be used, including publication WO 95/07994, WO 96/17072, WO 95/30763, and WO 97/42338) and nuclear charge neutralization or fusion with cell membranes. Naked DNA can also be used. An exemplary method of introducing naked DNA is described in PCT Publication WO 90/11092 and US Pat. No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in US Pat. No. 5,422,120, PCT Publication WO 95/13796, WO 94/23697, WO 91/14445, and EP 0524968. Further techniques are described in Philip, 1994, Mol.Cell Biol., 14:2411 and Woffendin, 1994, Proc.Natl.Acad.Sci., 91: 1581.

The present invention includes compositions, including pharmaceutical compositions, comprising the antibodies described herein or antibodies made by the methods described herein and having the characteristics thereof. As used herein, a composition encodes one or more antibodies, peptides, or aptamers that antagonize the interaction of PCSK9 with LDLR, and / or one or more of these antibodies or peptides. Contains one or more polynucleotides containing the sequences that are present. These compositions may further comprise suitable excipients such as pharmaceutically acceptable excipients, including buffers well known in the art.

The PCSK9 antagonistic antibodies and peptides of the invention are characterized by any (s) of the following characteristics: (a) binding to PCSK9, (b) interaction of PCSK9 with LDLR. To block, (c) reduce PCSK9-mediated down-regulation of LDLR, and (d) inhibit PCSK9-mediated inhibition of LDL blood clearance. Preferably, the PCSK9 antibody has two or more of these features. More preferably, the antibody has three or more of these features. Most preferably, the antibody has all four characteristics.

Accordingly, the present invention provides a composition (including a pharmaceutical composition) comprising any of the following, or any antibody having a partial light chain sequence and a partial heavy chain sequence found in Table 1. To do. The underlined sequence is the CDR sequence according to Kabat, and the bold type is according to Cotia.

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The invention also provides a CDR portion of an antibody against PCSK9 (including Cotia and Kabat CDRs). Determining the CDR regions is well within the skill of one of ordinary skill in the art. It should be understood that in some embodiments, the CDRs can be a combination of Kabat and Cotia CDRs (also referred to as "combination CDRs" or "extended CDRs"). In some embodiments, the CDR is a Kabat CDR. In other embodiments, the CDR is a Cotia CDR. In other words, in embodiments with multiple CDRs, the CDRs can be Kabat, Cotia, combination CDRs, or any combination thereof.

The present invention also provides a method of making any of these antibodies or polypeptides. The antibody of the present invention can be prepared by a procedure known in the art. The polypeptide can be produced by proteolysis or other degradation of the antibody, the recombinant method described above (ie, single or fused polypeptide) or chemical synthesis. Antibody polypeptides, especially shorter polypeptides of up to about 50 amino acids, are conveniently made by chemical synthesis. Chemical synthesis methods are known in the art and are commercially available. For example, antibodies can be produced by an automated polypeptide synthesizer using the solid phase method. See also U.S. Pat. Nos. 5,807,715, 4,816,567, and 6,331,415.

In another alternative method, antibodies and peptides can be made recombinantly using procedures well known in the art. In one embodiment, the polynucleotide comprises a sequence encoding the heavy and / or light chain variable region of antibody 4A5, 5A10, 6F6, 7D4 or L1L3. The sequence encoding the antibody of interest can be maintained in the vector in the host cell and then the host cell can be frozen for expansion and future use. Vectors (including expression vectors) and host cells are further described herein.

The present invention also includes scFv of the antibody of the present invention. Single chain variable region fragments are made by linking light and / or heavy chain variable regions using short linking peptides. Bird et al., 1988, Science, 242: 423 ~ 426. An example of a ligated peptide is (GGGGS)₃(SEQ ID NO: 24), which crosslinks approximately 3.5 nm between the carboxy terminus of one variable region and the amino terminus of the other variable region. Linkers of other sequences are designed and used. Bird et al., 1988, above. The linker should be a short, flexible polypeptide, preferably consisting of less than about 20 amino acid residues. Instead, the linker can be modified for additional functions such as attachment of the drug or attachment to a solid support. Single chain variants can be produced either recombinantly or synthetically. An automatic synthesizer can be used to synthesize and generate scFv. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding scFv is placed in either a eukaryote such as yeast, plant, insect or mammalian cell, or a prokaryote such as E. coli. Can be introduced into a suitable host cell. The polynucleotide encoding the scFv of interest can be made by routine operations such as ligation of the polynucleotide. The resulting scFv can be isolated using standard protein purification techniques known in the art.

Other forms of single chain antibodies such as diabody are also included. Diabodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, allowing pairing between two domains on the same chain. Forcing a domain to pair with a complementary domain on another strand to give rise to two antigen-binding sites by using a linker that is too short for (eg, Holliger, P. et al., 1993, Proc. See Natl.Acad Sci.USA, 90: 6444-6448, Poljak, RJ et al., 1994, Structure, 2: 1121-1123).

For example, bispecific antibodies, monoclonal antibodies with binding specificity to at least two different antigens, can be prepared using the antibodies disclosed herein. Methods for making bispecific antibodies are known in the art (see, eg, Suresh et al., 1986, Methods in Enzymology, 121: 210). Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello,). 1983, Nature, 305, 537 ~ 539).

According to one technique for producing bispecific antibodies, an antibody variable domain with the desired binding specificity (antibody-antigen binding site) is fused with an immunoglobulin constant domain sequence. Preferably, the fusion is with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, CH2 and CH3 regions. It is preferred that the first heavy chain constant region (CH1) containing the site required for light chain binding is present in at least one of the fusions. The immunoglobulin heavy chain fusion and, if desired, the DNA encoding the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into the appropriate host organism. This provides great flexibility in adjusting the reciprocal proportions of the three polypeptide fragments in embodiments where the unequal ratio of the three polypeptide chains used in the construction provides the optimum yield. However, if expression of at least two polypeptide chains of equal ratio results in high yields, or if the ratio is not particularly important, insert the coding sequences of all two or all three polypeptide chains into one expression vector. It is possible to do.

In one approach, the bispecific antibody is a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair in the other arm (second). (Contains binding specificity). This asymmetric structure, which has an immunoglobulin light chain on only one of the bispecific molecules, facilitates the separation of the desired bispecific compound from a combination of unwanted immunoglobulin chains. This technique is described in PCT Publication WO 94/04690.

Heteroconjugated antibodies, including two covalently bound antibodies, are also within the scope of the invention. Such antibodies are used to target immune system cells against unwanted cells (US Pat. No. 4,676,980) and to treat HIV infections (PCT Publications WO91 / 00360 and WO92 / 200373, EP03089). No.) is used. Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable crosslinkers and techniques are well known in the art and are described in US Pat. No. 4,676,980.

Chimeric or hybrid antibodies may also be prepared in vitro using known methods of synthetic protein chemistry, including those involved in cross-linking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutylimidase.

Humanized antibodies containing one or more CDRs of antibody 5A10 or 7D4 or one or more CDRs derived from antibody 5A10 or 7D4 shall be made, for example, using any method known in the art. Can be done. For example, a monoclonal antibody can be humanized using four common steps. They are (1) determining the nucleotide and expected amino acid sequences of the light and heavy chain variable domains of the initiating antibody, (2) designing the humanized antibody, that is, which antibody frame during the humanization process. The steps of deciding whether to use the work area, (3) using the actual humanization method / technique, and (4) transfecting to express the humanized antibody. See, for example, U.S. Pat. Nos. 4,816,567, 5,807,715, 5,866,692, 6,331,415, 5,530,101, 5,693,761, 5,693,762, 5,585,089, and 6,180,370.

In recombinant humanized antibodies, the Fc moiety can be modified to avoid interaction with the Fcγ receptor and complement and the immune system. Techniques for preparing such antibodies are described in WO 99/58572. For example, the constant region can be manipulated to make it more similar to the human constant region in order to avoid an immune response when the antibody is used in clinical trials and treatments in humans. See, for example, US Pat. Nos. 5,997,867 and 5,866,692.

A humanized antibody comprising a light chain or heavy chain variable region or one or more CDRs or variants thereof of the antibody shown in Table 1, or one or more CDRs or variants thereof derived from the antibody shown in Table 2. , Can be made using any method known in the art.

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

The invention includes antibodies and polypeptides of the invention shown in Table 1, including functionally equivalent antibodies that do not significantly affect their properties and variants with increased or decreased activity and / or affinity. Modifications to variants are included. For example, the amino acid sequence can be mutated to obtain an antibody with the desired binding affinity for PCSK9. Modification of the polypeptide is a routine practice in the art and does not need to be detailed herein. Modifications of the polypeptide are illustrated in the examples. Examples of modified polypeptides include conservative substitutions of amino acid residues, one or more amino acids that do not significantly adversely alter functional activity or mature (enhance) the polypeptide's affinity for its ligand. Polypeptides with deletions or additions, or the use of chemical analogs are included.

Amino acid sequence inserts include amino and / or carboxyl-terminal fusions ranging in length from one residue to a polypeptide containing 100 or more residues, as well as sequences of single or multiple amino acid residues. Includes internal inserts. Examples of terminal inserts include antibodies with N-terminal methionyl residues or antibodies fused to epitope tags. Other insertion variants of the antibody molecule include fusion of the antibody to the N- or C-terminus of the enzyme or polypeptide that increases the half-life of the antibody in the blood circulation.

Substitution variants have at least one amino acid residue removed from the antibody molecule and a different residue inserted in their place. The most interesting sites for substitution mutagenesis include hypervariable regions, but FR alterations are also conceivable. Conservative substitutions are shown in Table 2 under the heading "Conservative substitutions". If such substitutions result in changes in biological activity, introduce more substantive changes, shown in Table 2 as "exemplary substitutions", or further described below with reference to the amino acid class, to introduce the product. Can be screened.

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Substantial alterations in the biological properties of antibodies include (a) the structure of the polypeptide backbone in the substitution region, eg, as a sheet or helix conformation, and (b) the charge or hydrophobicity of the molecule at the target site. , Or (c) its effect on the maintenance of side chain bulk is achieved by choosing substitutions that are significantly different. Naturally occurring residues are grouped based on common side chain properties: (1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile, (2) Uncharged polarity: Cys, Ser, Thr, Asn, Gln, (3) Acid (load power): Asp, Glu, (4) Basic (positive charge): Lys, Arg, (5) Residues affecting chain orientation: Gly, Pro, and (6) Aromatic: Trp, Tyr, Phe, His.

Non-conservative replacement is done by exchanging a member of one of these classes for one of another class.

Any cysteine residue that is not involved in maintaining the correct conformation of the antibody can generally be replaced with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine binding (s) can be added to the antibody to improve its stability, especially 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 a complete redesign of a region, such as a variable region. Changes in the variable region can alter binding affinity and / or specificity. In some embodiments, no more than 1-5 conservative amino acid substitutions are made within the CDR domain. In other embodiments, no more than 1 to 3 conservative amino acid substitutions are made within the CDR domain. In yet another embodiment, the CDR domains are CDR H3 and / or CDR L3.

Modifications also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications such as glycosylation, acetylation, and phosphorylation with various sugars. Antibodies are glycosylated at conserved positions in their constant region (Jefferis and Lund, 1997, Chem.Immunol., 65: 111-128, Wright and Morrison, 1997, TibTECH, 15: 26-32). The oligosaccharide side chains of immunoglobulins are the function of the protein (Boyd et al., 1996, Mol. Immunol., 32: 1311-1318, Wittwe and Howard, 1990, Biochem., 29: 4175-4180) and a portion of the glycoprotein. May affect the intramolecular interactions of glycoproteins and the conformation of glycoproteins and the three-dimensional surfaces presented (Jefferis and Lund, supra, Wyss and Wagner, 1996, Current). Opin.Biotech., 7: 409 ~ 416). Oligosaccharides can also play a role in targeting a given glycoprotein to a specific molecule based on a specific recognition structure. It has also been reported that antibody glycosylation affects antibody-dependent cellular cytotoxicity (ADCC). Specifically, CHO cells with expression of β (1,4) -N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes the formation of tetracycline-regulated dichotomous GlcNAc, were improved. It has been reported to have ADCC activity (Umana et al., 1999, Nature Biotech., 17: 176-180).

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of carbohydrate moieties to the side chains of asparagine residues. Tripeptide sequences Asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine [where X is any amino acid other than proline], but the enzymatic attachment of the carbohydrate moiety to the asparagine side chain It is a recognition sequence. Therefore, the presence of any of these tripeptide sequences in a polypeptide results in a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine. Can also be used.

Adding a glycosylation site to an antibody is conveniently achieved by modifying the amino acid sequence to contain one or more of the tripeptide sequences described above (for N-linked glycosylation sites). .. Modifications can also be made by adding or substituting one or more serine or threonine residues in the original antibody sequence (for O-linked glycosylation sites).

Also, the glycosylation pattern of an antibody can be altered without altering the underlying nucleotide sequence. Glycosylation is highly dependent on the host cell used to express the antibody. Recombinant glycoproteins as potential therapeutic agents, such as cell types used to express antibodies, are rarely native cells, so variations in antibody glycosylation patterns can be predicted ( See, for example, Hse et al., 1997, J. Biol. Chem., 272: 9062 ~ 9070).

In addition to host cell selection, factors that influence glycosylation during recombinant production of antibodies include growth mode, medium formulation, culture density, oxygen supply, pH, purification scheme, and the like. Various methods have been proposed for altering the glycosylation pattern achieved in a particular host organism, including the introduction or overexpression of certain enzymes involved in the production of oligosaccharides (US Pat. No. 5,047,335). , Nos. 5,510,261 and 5,278,299). Glycosylation, or certain types of glycosylation, is enzymatically removed from glycoproteins using, for example, Endo H, N-glycosidase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3. be able to. In addition, recombinant host cells can be genetically engineered to be deficient in the processing of certain types of polysaccharides. These and similar techniques are well known in the art.

Other modification methods include, but are not limited to, the use of coupling techniques known in the art, including but not limited to enzymatic means, oxidative substitution and chelation. Modifications can be used, for example, to attach labels for immunoassays. Modified polypeptides can be performed using procedures established in the art and screened using standard assays known in the art, some of which are described below and in the Examples. ing.

In some embodiments of the invention, antibodies containing modified constant regions, such as immunologically inactive or partially inactive constant regions, do not initiate complement-mediated lysis, for example. Has reduced activity (modified) in any one or more of ADCC-unstimulated, microglial-inactivated, or complement-mediated lysis initiation, ADCC stimulation, or microglial activation. Compared to non-antibodies). Optimal levels and / or combinations of effector function can be achieved using various modifications of the constant region. For example, 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 ~ 4184, Tao et al., 1989, J. Immunology, 143: 2595-2601, and Jefferis et al., 1998, Immunological See Reviews, 163: 59-76. In some embodiments, the constant region is modified as described in Eur. J. Immunol., 1999, 29: 2613 ~ 2624, PCT Publication WO 99/58572, and / or UK Patent Application No. 9809951.8. In another embodiment, the antibody comprises a human heavy chain IgG2 constant region containing a mutation from A330P331 to S330S331 (amino acid numbering refers to wild-type IgG2 sequence). Eur.J.Immunol., 1999, 29: 2613 ~ 2624. In yet another embodiment, the constant region is deglycosylated for N-linked glycosylation. In some embodiments, the constant region is for N-linked glycosylation by mutating glycosylated amino acid residues or flanking residues that are part of the N-glycosylation recognition sequence in the constant region. Is deglycosylated. For example, the N-glycosylation site N297 can be mutated to A, Q, K, or H. Tao et al., 1989, J. Immunology, 143: 2595-2601, and Jefferis et al., 1998, Immunological See Reviews, 163: 59-76. In some embodiments, the constant region is deglycosylated for N-linked glycosylation. The constant region can be deglycosylated either enzymatically for N-linked glycosylation (such as removing carbohydrates with the enzyme PNGase) or by expression in glycosylation-deficient host cells.

Other antibody modifications include antibodies modified as described in PCT Publication WO 99/58572. These antibodies include, in addition to the binding domain to the target molecule, an effector domain having an amino acid sequence that is substantially homologous to all or part of the constant domain of the human immunoglobulin heavy chain. These antibodies can bind to the target molecule without initiating significant complement-dependent lysis or cell-mediated destruction of the target. In some embodiments, the effector domain can specifically bind to FcRn and / or FcγRIIb. These are typically two or more human immunoglobulin heavy chains C._HBased on a chimeric domain derived from two domains. Antibodies modified in this manner are particularly suitable for use in chronic antibody therapy to avoid inflammation and other adverse reactions to conventional antibody therapy.

The present invention includes embodiments that have matured affinity. For example, affinity matured antibodies can be produced by procedures known in the art (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 WO 2004/058184).

The following methods can be used to regulate antibody affinity and characterize CDRs. One method of characterizing the CDR of an antibody and / or altering the binding affinity of a polypeptide such as an antibody (such as improvement) is called "library scanning mutagenesis". In general, library scanning mutagenesis works as follows. Two or more amino acid positions in the CDR using methods recognized in the art (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) , 15, 16, 17, 18, 19, or 20 amino acids, etc.). This results in a small library of clones (in some embodiments, one for each amino acid position analyzed), each of which has a complexity of two or more members (two or more at each position). If you replace the amino acids in). In general, the library also contains clones containing native (unsubstituted) amino acids. A small number of clones from each library, such as about 20-80 clones (depending on the complexity of the library), were screened for binding affinity for the target polypeptide (or other binding target) and increased. Identify the same, reduced or non-binding candidates. Methods for determining binding affinity are well known in the art. Binding affinities can be determined using Biacore surface plasmon resonance analysis, which detects differences in binding affinities of about 2-fold or greater. Biacore has a relatively high affinity for the starting antibody, for example K of about 10 nM or less._DIt is especially useful when combining with. Screening with Biacore surface plasmon resonance is described in the examples herein.

Binding affinity can be determined using Kinexa Biocensor, scintillation proximity assay, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, and / or yeast display. The binding affinity may also be screened using an appropriate bioassay.

In some embodiments, all amino acid positions in the CDRs are all 20 naturally occurring amino acids using mutagenesis methods recognized in the art, some of which are described herein. Replace with (one at a time in some embodiments). This results in a small library of clones (in some embodiments, one for each amino acid position analyzed), each of which has a complexity of 20 members (20 amino acids at each position). If you replace everything).

In some embodiments, the library to be screened comprises substitutions at more than one position, which can be in the same CDR or in more than one CDR. Therefore, the library can contain substitutions at more than one position in a CDR. The library can contain substitutions at two or more positions in two or more CDRs. The library may contain substitutions at 3, 4, 5, or more positions found in 2, 3, 4, 5 or 6 CDRs. Substitutions can be prepared using codons with low redundancy. See, for example, Table 2 of Balint et al., 1993, Gene, 137 (1): 109-18).

The CDRs can be CDRH3 and / or CDRL3. The CDR can be one or more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and / or CDRH3. The CDR can be a Kabat CDR, a Cotia CDR, or an extended CDR.

Candidates with improved binding can be sequenced, thereby identifying CDR-substituted mutants that result in improved affinity (also called "improved" substitutions). Candidates for binding can be sequenced, thereby identifying CDR substitutions that retain binding.

Multiple screenings can be performed. For example, a candidate with improved binding (each containing an amino acid substitution at one or more positions in one or more CDRs) will have an improved each improved CDR position (ie, the substitution mutant). It is also useful in the design of a second library containing at least the original and substituted amino acids at the amino acid position in the CDR that showed the binding. The preparation, screening, and selection of this library is further described below.

Also, the frequency of improved binding, same binding, reduced binding or non-binding clones is a library scan mutation as long as it provides information on the importance of each amino acid position in the stability of the antibody-antigen complex. Induction also provides a means of characterizing CDRs. For example, if a CDR position is changed to all 20 amino acids and the bond is retained, that position is identified as a position that is unlikely to be required for antigen binding. Conversely, if a CDR position retains binding only with a small percentage of substitutions, that position is identified as an important position for CDR function. Therefore, the library scanning mutagenesis method changes only to positions in the CDR that can be changed to many different amino acids (including all 20 amino acids), and to only a few amino acids that cannot be changed. Produces information about the location in the CDR that can be made.

Candidates with improved affinities may be matched in a second library, including improved amino acids, original amino acids, as well as desired or desired screening or selection methods. Depending on the complexity of the library allowed to be used, additional substitutions at that location may be included. In addition, adjacent amino acid positions can be randomized to at least two or more amino acids, if desired. Randomization of adjacent amino acids may allow additional conformational flexibility in mutant CDRs, which in turn may allow or facilitate the introduction of a larger number of improving mutations. The library may also include substitutions at positions that did not show improved affinity in the first screening.

The second library includes screening using Biacore surface plasmon resonance analysis and selection using any selection method known in the art, including phage display, yeast display, and ribosome display. Any method known in the art is used to screen or select library members with improved and / or altered binding affinities.

The invention also includes fusion proteins comprising one or more fragments or regions from the antibodies or polypeptides of the invention. In one embodiment, at least 10 contiguous amino acids in the variable light chain region set forth in SEQ ID NO: 53, 16, 17, 18, or 19 and / or the variable set forth in SEQ ID NO: 54, 20, 21, 22, or 23. A fusion polypeptide containing at least 10 amino acids in the heavy chain region is provided. In other embodiments, at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids in the variable light chain region and / or at least about 10, at least about 10 in the variable heavy chain region. A fusion polypeptide comprising about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids is provided. In another embodiment, the fusion polypeptide is shown in any of the sequence pairs selected from SEQ ID NOs: 53 and 54, 16 and 20, 17 and 21, 18 and 22, and 19 and 23. Includes region and / or heavy chain variable region. In another embodiment, the fusion polypeptide comprises one or more CDRs. In yet another embodiment, the fusion polypeptide comprises CDR H3 (VH CDR3) and / or CDR L3 (VL CDR3). For the purposes of the present invention, fusion proteins contain one or more antibodies and another amino acid sequence that is not attached to it in the native molecule, eg, a heterologous or homologous sequence from another region. Exemplary heterologous sequences include, but are not limited to, "tags" such as FLAG tags or 6His tags. Tags are well known in the art.

Fusion polypeptides can be made by methods known in the art, such as synthesis or recombination. Typically, the fusion proteins of the invention are made by preparing and expressing the polynucleotides encoding them using the recombinant methods described herein, including, for example, chemical synthesis. It can also be prepared by other means known in the art.

The present invention also provides a composition comprising an antibody or polypeptide conjugated (eg, linked) to an agent that facilitates coupling with a solid carrier (such as biotin or avidin). For simplicity, antibodies are generally referred to with the understanding that these methods apply to any of the PCSK9 binding and / or antagonist embodiments described herein. In general, conjugation refers to concatenating these components as described herein. Coupling (at least fixing these components at adjacent associations for administration) can be achieved in a number of ways. For example, if each has a substituent capable of reacting with the other, a direct reaction between the drug and the antibody is possible. For example, a nucleophile such as an amino or sulfhydryl group on one side is a carbonyl-containing group such as an acid anhydride or acid halide on the other side, or an alkyl group containing a good leaving group (eg, a halide). May react with.

Antibodies or polypeptides of the invention can be linked to fluorescent molecules, radioactive molecules or labeling agents such as any other labeling known in the art. Labels are known in the art and generally provide signals (directly or indirectly).

The invention also comprises compositions (including pharmaceutical compositions) and kits comprising any or all of the antibodies and / or polypeptides described herein as revealed herein. Also provided.

The present invention also provides isolated polynucleotides encoding the antibodies and peptides of the invention, as well as vectors and host cells containing the polynucleotides.

Accordingly, the present invention comprises a polynucleotide encoding any of the antibodies 4A5, 5A10, 6F6, 7D4, L1L3, or any fragment or portion thereof capable of antagonizing PCSK9. Alternatively, a composition including a pharmaceutical composition) is provided.

In another aspect, the invention encodes any of the antibodies (including antibody fragments) and polypeptides described herein, such as antibodies and polypeptides with impaired effector function. Provide a polynucleotide that is. Polynucleotides can be made and expressed by procedures known in the art.

In another aspect, the invention provides a composition (such as a pharmaceutical composition) comprising any of the polynucleotides of the invention. In some embodiments, the composition comprises an expression vector comprising a polynucleotide encoding an antibody described herein. In other embodiments, the composition comprises an expression vector comprising a polynucleotide encoding any of the antibodies or polypeptides described herein. In yet another embodiment, the composition comprises either or both of the polynucleotides set forth in SEQ ID NO: 25 and SEQ ID NO: 26. Administration of expression vectors and polynucleotide compositions is further described herein.

In another aspect, the invention provides a method of making any of the polynucleotides described herein.

Polynucleotides complementary to any such sequence are also included by the present invention. The polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genome, cDNA or synthesis) or RNA molecule. RNA molecules include HnRNA molecules that contain introns and correspond to DNA molecules in a one-to-one fashion, and mRNA molecules that do not contain introns. Additional coding or non-coding sequences may be present within the polynucleotides of the invention, but not necessarily, and the polynucleotides may be linked to other molecules and / or supporting materials, but are not necessarily linked. You don't have to.

A polynucleotide may include a native sequence (ie, an endogenous sequence encoding an antibody or a portion thereof) or a variant of such a sequence. Polynucleotide variants undergo one or more substitutions, additions, deletions and / or insertions so that the immunoreactivity of the encoded polypeptide is not diminished compared to the native immunoreactive molecule. contains. The effect of the encoded polypeptide on immunoreactivity can generally be evaluated as described herein. Preferably, the variant has at least about 70% identity, more preferably at least about 80% identity, and even more preferably at least about 90% identity with the polynucleotide sequence encoding the native antibody or portion thereof. It exhibits sex, most preferably at least about 95% identity.

Two polynucleotide or polypeptide sequences are said to be "identical" if the sequences of the nucleotides or amino acids in the two sequences are the same when aligned for maximum match as described below. Comparisons between two sequences are typically made by comparing the sequences across a comparison window to identify and compare local regions of sequence similarity. As used herein, "comparison window" refers to at least about 20 contiguous positions, typically 30 to about 75, or 40 to about 50 segments, with the same number of contiguous sequences. The reference sequence at the appropriate position can be compared after the two sequences are optimally aligned.

Optimal alignment of sequences for comparison can be performed using the default parameters using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI). This program integrates several alignment schemes described in the references below: Dayhoff, MO, 1978, A model of evolutionary change in proteins-Matrices for detecting distant relationships., Dayhoff, MO (ed.), Atlas of Protein Sequence and Structure (National Biomedical Research Foundation, Washington DC), Volume 5, Addendum 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, DG and Sharp, PM, 1989, CABIOS, 5: 151-153, Myers, EW and Muller W., 1988, CABIOS, 4: 11 ~ 17, Robinson, ED, 1971, Comb.Theor., 11:105, Santou, N., Nes, M., 1987, Mol.Biol.Evol., 4: 406 ~ 425, Sneath, PHA and Sokal, RR, 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy (Freeman Press, San Francisco, CA), Wilbur, WJ and Lipman, DJ, 1983, Proc.Natl.Acad.Sci.USA, 80: 726 ~ 730 ..

Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences across a comparison window at at least 20 positions, and a portion of the polynucleotide or polypeptide sequence in the comparison window is No more than 20 percent, usually 5 to 15 percent, or 10 to 12 percent of additions or deletions (ie gaps) compared to the reference sequence (without additions or deletions) for optimal alignment of the two sequences. Can include. The percentage determines the number of positions where the same nucleobase or amino acid residue is present in both sequences to obtain the number of matching positions, and the number of matching positions is the total number of positions in the reference sequence ( That is, it is calculated by dividing by the size of the window) and multiplying the result by 100 to obtain the percentage of sequence identity.

The variant can be substantially homologous to the native gene or a portion or complement thereof, or instead. Such polynucleotide variants can hybridize to naturally occurring DNA sequences (or complementary sequences) encoding native antibodies under moderately stringent conditions.

Appropriate "moderately stringent conditions" include pre-washing in a solution of 5 x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), 50 ° C-65 ° C, 5 Includes x SSC, overnight hybridization, followed by two washes at 65 ° C for 20 minutes with each of 2 x, 0.5 x and 0.2 x SSC containing 0.1% SDS.

As used herein, "highly stringent conditions" or "high stringency conditions" are: (1) low ion intensity and high temperature for cleaning, eg 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate used at 50 ° C, (2) modifiers such as formamide, eg 50% (v / v) formamide, during hybridization, 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer, pH 6.5, 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C, or (3) 50% formamide, 5 x SSC ( 0.75M NaCl, 0.075M Sodium Citrate), 50 mM Sodium Phosphate (pH 6.8), 0.1% Sodium Pyrophosphate, 5x Denhart Solution, Ultrasonic Treated Salmon Sperm DNA (50 μg / ml), 0.1 Washing with 0.2 × SSC (sodium chloride / sodium citrate) at 42 ° C and 50% formamide at 55 ° C, followed by washing with% SDS and 10% dextran sulfate at 42 ° C, followed by 55 ° C. It is a high stringency wash consisting of 0.1 x SSC containing EDTA. Those skilled in the art will recognize methods of adjusting temperature, ionic strength, etc. as needed to adapt to factors such as probe length.

It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, there are many nucleotide sequences encoding the polypeptides described herein. Some of these polynucleotides have minimal homology with the nucleotide sequence of any native gene. Nevertheless, different polynucleotides due to differences in codon usage are specifically contemplated by the present invention. In addition, alleles of genes containing the polynucleotide sequences provided herein are within the scope of the invention. Alleles are endogenous genes that have been altered as a result of one or more mutations such as nucleotide deletions, additions and / or substitutions. The resulting mRNA and protein may, but need not, have altered structure or function. Alleles can be identified using standard techniques (hybridization, amplification and / or comparison of database sequences, etc.).

The polynucleotides of the invention can be obtained using chemical synthesis, recombinant methods, or PCR. Chemical polynucleotide synthesis methods are well known in the art and need not be detailed herein. One of ordinary skill in the art can generate the desired DNA sequence using the sequences provided herein and a commercially available DNA synthesizer.

To prepare a polynucleotide using a recombinant method, a polynucleotide containing the desired sequence can be inserted into the appropriate vector, and instead, as described further herein. The vector can be introduced into a suitable host cell for replication and amplification. The polynucleotide can be inserted into the host cell by any means known in the art. Cells are transformed by introducing foreign polynucleotides by direct uptake, endocytosis, transfection, F-conjugation or electroporation. After introduction, the foreign polynucleotide can be maintained intracellularly as a non-integrating vector (such as a plasmid) or integrated into the host cell genome. The polynucleotide thus amplified can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989, above.

Alternatively, PCR allows replication of DNA sequences. PCR technology is well known in the art and is found in US Pat. Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, and PCR: The Polymerase Chain Reaction, Mullis et al., 1994 (Birkauswer Press, Boston, MA). Are listed.

RNA can be obtained by using the isolated DNA in a suitable vector and inserting it into a suitable host cell. After the cells have been replicated and the DNA transcribed into RNA, RNA can be isolated using methods well known to those of skill in the art, such as Sambrook et al., 1989, described above.

Suitable cloning vectors can be constructed according to standard techniques or selected from a number of cloning vectors available in the art. Although the cloning vector chosen may vary depending on the host cell intended for use, useful cloning vectors are generally capable of self-renewal and may carry a single target for a particular restriction endonuclease. It may carry a gene for a marker that can be used to select clones that are and / or contain the vector. Suitable examples include plasmids and bacterial viruses such as pUC18, pUC19, Bluescript (eg pBS SK +) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and pSA3 and pAT28. Includes shuttle vector. These and many other cloning vectors are available from commercial suppliers such as BioRad, Strategene, and Invitrogen.

The expression vector is generally a replicable polynucleotide construct containing the polynucleotide according to the invention. It is implied that the expression vector must be replicable in the host cell as an episome or as part integrated into chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, adenoviruses, adeno-related viruses, viral vectors including retroviruses, cosmids, and expression vectors disclosed in PCT Publication WO 87/04462 (s). Is included. The components of the vector can include a signal sequence, an origin of replication, one or more marker genes, and one or more of the appropriate transcriptional regulators (promoters, enhancers, terminators, etc.). Expression (ie, translation) usually also requires one or more translational control elements such as a ribosome binding site, a translation initiation site, and a stop codon.

Vectors containing the polynucleotide of interest include electroporation, transfection, microscopic guns, lipofection, and infections with calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances (eg, vector such as vaccinia virus). It can be introduced into the host cell by any of several suitable means, including (if it is an infectious agent). The choice of introducing a vector or polynucleotide often depends on the characteristics of the host cell.

The present invention also provides host cells containing any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used to isolate the gene encoding the antibody, polypeptide or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, NSO, and CHO cells. See also PCT Publication WO 87/04462. Suitable non-mammalian host cells include prokaryotic organisms (such as E. coli or Bacillus subtilis) and yeasts (Saccharomyces ae), fission yeast (S. pombe), or Kay. Lactis (K. lactis, etc.) is included. Preferably, the host cell, if present, is about 5-fold higher, more preferably 10-fold higher, and even more preferably 20-fold higher than the corresponding endogenous antibody or protein of interest in the host cell. Express. Screening of host cells for specific binding to PCSK9 or the PCSK9 domain is accomplished by immunoassay or FACS. Cells that overexpress the antibody or protein of interest can be identified.

C. Composition Compositions used in the methods of the invention include effective amounts of PCSK9 antagonists, polypeptides derived from PCSK9 antagonists, or other PCSK9 antagonists described herein. Examples of such compositions, and methods of formulating them, are also described in the sections already mentioned and below. In one embodiment, the composition further comprises a PCSK9 antagonist. In another embodiment, the composition comprises one or more PCSK9 antagonist antibodies. In other embodiments, the PCSK9 antagonist antibody recognizes human PCSK9. In yet another embodiment, the PCSK9 antagonist antibody has been humanized. In yet another embodiment, the PCSK9 antagonist antibody comprises a constant region that does not initiate an unwanted or unwanted immune response such as antibody-mediated lysis or ADCC. In other embodiments, the PCSK9 antagonist antibody comprises one or more CDRs of the antibody (such as one, two, three, four, five, or in some embodiments all six CDRs). .. In some embodiments, the PCSK9 antagonist antibody is human.

It should be understood that the composition can include multiple PCSK9 antagonistic antibodies (eg, a mixture of PCSK9 antagonistic antibodies that recognize different epitopes of PCSK9). Other exemplary compositions include multiple PCSK9 antagonists that recognize the same epitope (s), or various species of PCSK9 antagonists that bind to different epitopes of PCSK9.

The compositions used in the present invention are pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions (Remington: The Science and Practice of Pharmacy, 20th Edition, 2000, 2000). Lippincott Williams and Wilkins, KE Hoover ed.) Can be further included. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at doses and concentrations and are antioxidants including buffers such as phosphoric acid, citric acid, and other organic acids, ascorbic acid and methionine. Agents, Preservatives (Octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pen Tanol and m-cresol, etc.), low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, or immunoglobulin, hydrophilic polymers such as polyvinylpyrrolidone, glycine, glutamine, asparagine, histidine , Amino acids such as arginine or lysine, monosaccharides including glucose, mannose or dextran, disaccharides and other carbohydrates, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, sodium etc. It may include salt-forming counterions, metal complexes (eg, Zn-protein complexes), and / or nonionic surfactants such as TWEEN , PLURONICS or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

In one embodiment, the antibody has a pH in the range of about 5.0 to about 6.5, an antibody of about 1 mg / ml to about 200 mg / ml, a histidine buffer of about 1 ml to about 100 ml, a histidine buffer of about 0.01 mg / ml. It is administered in the form of a sterile aqueous solution containing ~ about 10 mg / ml of polysorbate 80, about 100 millimorer to about 400 millimorer of trehalose, and about 0.01 millimorer to about 1.0 millimorer of EDTA disodium dihydrate.

The PCSK9 antagonist antibody and its composition can also be used in combination with other agents that play a role in enhancing and / or complementing the efficacy of the agent.

D. kit The present invention also provides a kit for use in this method. The kit of the present invention comprises a PCSK9 antagonist antibody (such as a humanized antibody) or a peptide described herein, and instructions for use according to any of the methods of the invention described herein. Includes one or more containers, including. In general, these instructions include a description of administration of a PCSK9 antagonist antibody, peptide, or aptamer for the therapeutic treatment described above.

In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is human. In other embodiments, the antibody is a monoclonal antibody. Instructions for the use of PCSK9 antagonist antibodies generally include information on the intended treatment dose, dosing schedule, and route of administration. The container can be a unit dose, a bulk package (eg, a multi-dose package) or a subunit dose. The instructions supplied in the kit of the present invention are typically instructions written on a label or package insert (eg, a paper sheet included in the kit), but are machine-readable instructions (eg, magnetic or optical memory). Instructions held on disk) are also acceptable.

The kit of the present invention is in a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, soft packages (eg sealed mylar bags or plastic bags) and the like. Packages for use in combination with specific devices such as inhalers, nasal administration devices (eg, atomizers) or infusion devices such as mini pumps are also contemplated. The kit can have a sterile access port (for example, the container can be an intravenous solution bag or vial with a stopper that can be pierced by a hypopodermic needle). The container can also have a sterile access port (eg, the container can be an intravenous solution bag or vial with a stopper that can be pierced by a hypopodermic needle). At least one active agent in the composition is a PCSK9 antagonist antibody. The container (eg, prefilled syringe or self-injector) may further contain a second pharmaceutically active agent.

The kit may optionally provide additional components such as buffers and information for interpretation. Kits typically include a container and a label or package insert (s) on or associated with the container.

Mutations and modifications To express the PCSK9 antibody of the invention, first V using any of the methods described above._HAnd V_LA DNA fragment encoding the region can be obtained. Various modifications, such as mutations, deletions, and / or additions, can also be introduced into the DNA sequence using standard methods known to those of skill in the art. For example, mutagenesis is a standard method such as PCR-mediated mutagenesis, in which mutated nucleotides are incorporated into PCR primers so that the PCR product contains the desired mutation or site-specific mutagenesis. Can be carried out using.

For example, one possible substitution is to change one or more cysteines in an antibody that may be chemically reactive to another residue, such as, but not limited to, alanine or serine. For example, there may be non-canonical cysteine substitutions. Substitution can be performed in the CDR or framework region of the variable domain or in the constant domain of the antibody. In some embodiments, cysteine is canonical.

Antibodies can also be modified, for example, in the variable domains of heavy and / or light chains, for example to alter the binding properties of the antibody. For example, to increase or decrease the KD of an antibody against PCSK9, k_offMutations in one or more of the CDR regions can be made to increase or decrease the antibody's binding specificity or to alter the binding specificity of the antibody. Techniques for site-specific mutagenesis are well known in the art. See, for example, Sambrook et al. And Ausubel et al., Above.

Modifications or mutations can also be made in the framework region or constant domain to increase the half-life of the PCSK9 antibody. See, for example, PCT Publication WO 00/09560. It also modifies the immunogenicity of the antibody, provides sites for covalent or non-covalent binding with other molecules, or exhibits properties such as complement fixation, FcR binding and antibody-dependent cellular cytotoxicity. Mutations in the framework region or constant domain can also be made to modify. According to the present invention, a single antibody can have mutations in any one or more of the CDRs or framework regions of the variable domain or in the constant domain.

In a process known as "reproductive sequence", V_HAnd V_LSpecific amino acids in the sequence, germline V_HAnd V_LIt can be mutated to match what is naturally found in the sequence. Specifically, V_HAnd V_LThe amino acid sequence of the framework region in the sequence can be mutated to match the germline sequence to reduce the risk of immunogenicity when the antibody is administered. Human V_HAnd V_LGermline DNA sequences of genes are known in the art (eg, "Vbase" Human Germline Sequence Database, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, USDepartment 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 substitution that can be done is to remove potential proteolytic sites in the antibody. Such sites can be in the CDR or framework region of the variable domain or in the constant domain of the antibody. Substitution of cysteine residues and removal of proteolytic sites can reduce the risk of heterogeneity in the antibody product and thus increase its homogeneity. Another type of amino acid substitution eliminates the asparagine-glycine pair that forms a potential deamidation site by altering one or both of the residues. In another example, the heavy chain C-terminal ricin of the PCSK9 antibody of the invention can be cleaved. In various embodiments of the invention, the heavy and light chains of the PCSK9 antibody may optionally contain a signal sequence.

V of the present invention_HAnd V_LAfter the DNA fragments encoding the segments are obtained, these DNA fragments are standard recombinant DNA techniques for converting, for example, variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. Can be further manipulated by. In these operations, V_LOr V_HThe DNA fragment encoding is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operably linked" means that two DNA fragments are linked so that the amino acid sequence encoded by the two DNA fragments is preserved in-frame. Intended to be.

V_HThe isolated DNA encoding the region is V_HThe DNA encoding is operably linked to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3) so that it can be converted to a full long heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (eg Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, USD department of Health and Human). Services, NIH Publication No. 91-3242), DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably IgG1 or IgG2 constant region. The IgG constant region sequence is any of various alleles known to occur between different individuals or allotypes such as Gm (1), Gm (2), Gm (3), and Gm (17). Can be. These allotypes represent naturally occurring amino acid substitutions in the IgG1 constant region. In the Fab fragment heavy chain gene, V_HThe DNA encoding the can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region. The CH1 heavy chain constant region can be derived from any of the heavy chain genes.

V_LThe isolated DNA encoding the region is V_LThe DNA encoding the light chain constant region C_LCan be converted to a full-length light chain gene (and Fab light chain gene) by operably linking with another DNA molecule encoding. The sequences of human light chain constant region genes are known in the art (eg Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242. (See), DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. The kappa constant region can be any of the various alleles known to occur between different individuals, such as Inv (1), Inv (2), and Inv (3). The lambda constant region can be derived from any of the three lambda genes.

To make the scFv gene, V_HAnd V_LThe sequence is expressed as a continuous single chain protein, V_LAnd V_HV so that the regions can be bound by a flexible linker_HAnd V_LA DNA fragment encoding a flexible linker, eg, an amino acid sequence (Gly)₄-Ser)₃Operatively linked with another fragment encoding (eg 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. Single chain antibodies are single Vs._HAnd V_LIf you use only, it is monovalent, and two Vs_HAnd V_LIs divalent when used, and more than two Vs_HAnd V_LCan be multivalued when used. Bispecific or multivalent antibodies that specifically bind to PCSK9 and other molecules can be made.

In another embodiment, a fusion antibody or immunoadhesin can be made that comprises all or part of the PCSK9 antibody of the invention linked to another polypeptide. In another embodiment, only the variable domain of the PCSK9 antibody is linked to the polypeptide. In another embodiment, the PCSK9 antibody V_HThe V of the PCSK9 antibody, while the domain is linked to the first polypeptide_LDomain is V_HAnd V_LIt is linked to a second polypeptide that associates with the first polypeptide in such a way that the domains can interact with each other to form an antigen binding site. In another preferred embodiment, V_HAnd V_LV so that domains can interact with each other_HDomain is V by linker_LSeparated from the domain. Then V_H-Linker-V_LThe antibody is linked to the polypeptide of interest. Furthermore, it is possible to prepare a fusion antibody in which two (or more) single chain antibodies are linked to each other. This is useful when making divalent or multivalent antibodies on a single polypeptide chain, or when making bispecific antibodies.

In other embodiments, other modified antibodies can be prepared using nucleic acid molecules encoding PCSK9 antibodies. For example, "Kappa Body" (Ill et al., 1997, Protein Eng., 10: 949-57), "Mini Body" (Martin et al., 1994, EMBO J., 13: 5303-9), "Diabody" (Holliger). Et al., 1993, Proc.Natl.Acad.Sci.USA, 90: 6444-6448), or "Janushin" (Traunecker et al., 1991, EMBO J., 10: 3655 ~ 3659 and Traunecker et al., 1992, Int.J. Cancer (Addendum), 7: 51-52) can be prepared using standard molecular biology techniques according to the teachings herein.

Bispecific antibody or antigen binding fragments can be produced by a variety of methods, including fusion of hybridomas or ligation of Fab'fragments. See, for example, Songsivilai and Lachmann, 1990, Clin.Exp.Immunol., 79: 315-321, Kostelny et al., 1992, J.Immunol., 148: 1547-1553. In addition, bispecific antibodies can be formed as "diabodies" or "janusins." In some embodiments, the bispecific antibody binds to two different epitopes of PCSK9. In some embodiments, the modified antibodies described above are prepared using one or more of the variable domains or CDR regions from the human PCSK9 antibodies provided herein.

Preparation of antigen-specific antibody Over 500 polyclonal and monoclonal antibodies produced against recombinant full-length human PCSK9, recombinant full-length mouse PCSK9, and various synthetic peptides, downregulating all LDLR proteins in cultured Huh7 human hepatocytes. Evaluated ability. These antibodies are, among other things, antibodies that are produced and reactive against a set of 12-20 amino acid residue polypeptides that are predicted to cover most of the protein surface based on the structure of PCSK9. There was a pair of. At the highest concentration, the best antibody showed only about 60% blocking activity.

Therefore, an alternative and previously undeveloped approach was used: the production of monoclonal antibodies by immunizing PCSK9 null mice with recombinant full-length PCSK9 protein. As shown in Example 7, this mode of antibody preparation included complete blockade of binding of PCSK9 to LDLR, complete blockade of PCSK9-mediated lowering of LDLR levels in Huh7 cells, and that in mice. An antagonistic antibody was obtained that showed a decrease in LDLc in vivo to levels comparable to those found in PCSK9-/-mice.

A representative antibody (hybridoma) of the present invention was deposited with the American Type Culture Collection (ATCC) on February 28, 2008, and was assigned the accession number shown in Table 3. Hybridomas have deposited antibodies 4A5, 5A10, 6F6 and 7D4. DNA samples of the heavy light chain variable region of antibody L1L3 (antibody designations RN316-PUC19-VH and RN316-PUC19-VL) were deposited with the ATCC on August 25, 2009 and assigned the accession numbers in Table 3.

<tables num="3"><img id="000004" he="26" wi="52" file="JP5750421B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

<p num="0218">(Example 1) Preparation and screening of PCSK9 antagonist antibodies General Procedures for Immunizing Animals to Make Monoclonal Antibodies: Balb / c or 129 / bl6 pcsk9-/-mice were injected 5 times with 100 μg of antigen on a 13-day schedule. PCSK9-/-(ie null or knockout mice) can be obtained from or as described by Rashid et al., 2005, Proc Natl Acad Sci USA, 102: 5374. See also U.S. Pat. No. 7,300,754. For the first four injections, the antigen was prepared by mixing the recombinant protein with an adjuvant. The immunogen was given by injection into the nape, footpad and abdominal cavity approximately every 3 days for 11 days, with the final boost given intravenously without adjuvant. On day 13, the mice were euthanized and their spleen removed. Using standard hybridoma techniques, lymphocytes were immortalized by fusion with an established cell line to generate hybridoma clones and distributed in 96-well plates. Clones were grown and then selected by ELISA screening with immunized antigens as follows.</p><p num="0219"> Elisa screening of antibodies: Supernatant media from growing hybridoma clones were screened separately for their ability to bind recombinant human PCSK9 or recombinant mouse PCSK9. The assay was performed on 96-well plates coated overnight with a 1 μg / ml solution of one of 100 μl of antigen. Excess reagent was washed from the wells during each step using PBS containing 0.05% Tween-20. The plate was then blocked with PBS containing 0.5% BSA. The supernatant was added to the plate and incubated at room temperature for 2 hours. Horseradish peroxidase (HRP) -conjugated goat anti-mouse Fc was added and bound to antigen-bound mouse antibody. Then, tetramethylbenzidine was added as a substrate for HRP to detect the amount of mouse antibody present in the supernatant. The reaction was stopped and the relative amount of antibody was quantified by reading the absorbance at 450 nm. Hybridoma clones that secreted antibodies capable of binding to either mouse or human PCSK9 were selected for further analysis.</p><p num="0220"> PCSK9-mediated LDLR downregulation in Huh7 cells: Hybridoma clones secreting human or mouse PCSK9 binding antibody were expanded and supernatants were collected. Total IgG was purified from approximately 10 ml of supernatant using protein A beads and dialyzed against PBS buffer to reduce the final volume to give a solution with 0.7-1 mg / ml antibody. Purified antibodies were then tested for their ability to inhibit the ability of PCSK9 to mediate LDLR downregulation in Huh7 cells. Huh7 cells were plated and grown to 80% confluent in 96-well plates in RPMI medium containing 10% FBS, 4 mM glutamine, and penicillin and streptavidin. The medium was changed to one containing 10% degreased FBS for 8-16 hours to induce LDLR expression. Cells are then used for 8-16 hours with or without 70-100 μg / ml test antibody in 40 μl / well 293 expression medium supplemented with 6 μg / ml human (preferably) or mouse PCSK9. Incubated. At the end of the incubation, medium containing PCSK9 and antibody was removed and cells were lysed by shaking at 4C for 1 hour with 17 μl lysis buffer. The lysis buffer consisted of 50 mM glycerol phosphate, 10 mM HEPES, pH 7.4, 1% Triton X-100, 20 mM NaCl, and a protease inhibitor cocktail (Roche). Cytolysis was collected and analyzed for LDLR protein levels by staining Western blots after SDS polyacrylamide gel electrophoresis. Hybridoma clones that produce antibodies that can partially or completely rescue LDLR levels were selected for further analysis. "LDLR downregulation assay" means the above assay using Huh7 cells.</p><p num="0221"> Figure 1 illustrates the effect of anti-PCSK9 antagonistic monoclonal antibodies 7D4.4, 4A5.G3, 6F6.G10.3 and 5A10.B8 on the ability of human and mouse PCSK9 to downregulate LDLR in cultured Huh7 cells. A serial dilution of 100 nM mouse or human recombinant PCSK9 and 25-800 nM antibody was used. A) Mouse PCSK9.B) Human PCSK9. The figure is a Western blot showing that antibodies are generally more effective at blocking the function of human PCSk9 than mouse PCSK9. Some antibodies generally have similar affinities for human PCSK9, but differ in their affinities for murine PCSK9.</p><p num="0222">(Example 2) Determining antibody binding affinity The affinity of PCSK9 antibody for PCSK9 was measured using HBS-EP running buffer (Biacore AB, Uppsala, Sweden, now GE Healthcare) on a surface plasmon resonance Biacore 3000 biosensor equipped with a research grade sensor chip. .. Rabbit polyclonal anti-Ms IgG was amine-coupled onto the chip at saturation levels using standard N-hydroxysuccinimide / ethyldimethylaminopropylcarbodiimide (NHS / EDC) chemistry. The buffer was replaced with HBS-EP + 1 mg / mL BSA + 1 mg / mL CM-dextran. Full length PCSK9 IgG was diluted to about 15 μg / mL and supplemented with 5 μL / min for 1 minute to obtain levels of about 500 RU / flow cell, leaving one blank as a reference channel. 3.73 to 302 nM hPCSK9 or 2.54 to 206 nM mPCSK9 were injected at 100 μL / min for 1 minute as a triple series of 5 members. Dissociation was monitored for 5 minutes. After the final injection of each titer, the chips were regenerated with two 30 second pulses with 100 mM phosphate. The buffer cycle provided a blank for double reference of the data, which was then applied comprehensively to the simple binding model using Biaevaluation software v.4.1. Affinity is the kinetic rate constant (K)_D= k_off/ k_on) Deduced from the index. The results of Example 2 are shown in Table 4. These data indicate that the antibody has excellent affinity for murine PCSK9 or human PCSK9 as shown.</p><p num="0223"><tables num="4"><img id="000005" he="84" wi="160" file="JP5750421B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0224">(Example 3) Analysis of the effect of PCSK9 antibody on PCSK9-LDLR interaction PCSK9 has been shown to bind to LDLR with an affinity of 180 nM under neutral pH (Cunningham et al., 2007, Nat Struct Mol Biol, 14 (5): 413-9). Recombinant mouse or human PCSK9 protein was biotin-labeled using the Pierce reagent according to production instructions. 1 μg / ml recombinant LDLR extracellular domain (R &amp; D) in each well with an ELISA plate (Corning Mixisorb) Coated overnight at 4C with Systems) solution, shut off at room temperature for 2 hours with 2% BSA + PBS, then 5 times with wash buffer (1 x PBS + 0.05% Tween-20). Washed. Wells were incubated with 50 μl of the indicated concentration of biotin-labeled PCSK9 protein for 1 hour at room temperature. The binding of LDLR-PCSK9 could be stabilized by adding 50 μl of 4% FDH + 4% sucrose + PBS solution and incubated for 5 minutes. Wells were washed 5 times with wash buffer, incubated with Strepavidin (Invitrogen) conjugated with HRP at a dilution of 1: 2000 for 1 hour at room temperature, and washed 5 times with wash buffer. The TMB substrate was added to the wells and the solution was incubated for 20-30 minutes at room temperature and the reaction was stopped with 1 M phosphate. The signal was read at 450 nm.</p><p num="0225"> Figure 2 shows anti-PCSK9 antagonistic monoclonal antibodies 6F6.G10.3, 7D4. For blocking in vitro binding of recombinant biotin-labeled human PCSK9 and mouse PCSK9 to the immobilized recombinant LDLR extracellular domain. Illustrate the dose response of 4, 4A5.G3, 5A10.B8, negative control antibody 42H7, and PBS. Part A) shows that human PCSK9 binding to the human LDLR extracellular domain, and 7D4, 4A5, 5A10, and 6F6 are effective in blocking the binding, while 42H7 and PBS are not. Part B) shows mouse PCSK9 binding to the human LDLR extracellular domain.</p><p num="0226"> Interactions can also be evaluated in free solution at neutral pH. Figure 3 shows resistance to blocking in-solution, neutral pH, and in vitro binding between recombinant biotin-labeled human PCSK9 (30 nM) and europium-labeled recombinant LDLR extracellular domain (10 nM). Illustrates the dose response of PCSK9 monoclonal antagonist 6F6.G10.3, 7D4.4, 4A5.G3 and 5A10.B8. This assay measures binding in free solution at neutral pH.</p><p num="0227">(Example 4) Epitope mapping / binding of antibodies using the crystal structure, Biacore, and mutagenesis of the L1L3: PCSK9 complex Crystal structure of the complex of L1L3: PCSK9. Residues are identified by calculating the difference in accessible surface area between the crystal structure of L1L3: PCSK9 and the PCSK9 structure alone. PCSK9 residues, which indicate the surface area buried during complex formation with the L1L3 antibody, are included as part of the epitope. The solvent accessible surface of a protein is defined as the position of the center of a probe sphere (representing a solvent molecule with a radius of 1.4 Å) as it rolls over the van der Waals surface of the protein. The solvent accessible surface area was run by the program AREAIMOL to create surface points on the expanding sphere around each atom (at a distance from the atomic center equal to the sum of the atoms and probe radii) and associated with adjacent atoms. Calculated by excluding those in the equivalent sphere (Briggs, PJ, 2000, CCP4 Newsletter, No. 38, CCLRC, Daresbury).</p><p num="0228"> The result of crystal structure analysis is shown in FIG. FIG. 23A shows the crystal structure of PCSK9 (displaying a light gray surface) bound to the L1L3 antibody (displaying a black image). Epitopes for binding L1L3 to PCSK9 include residues 153-155, 194, 197, 237-239, 367, 369, 374-379 and 381 of the PCSK9 amino acid sequence. For comparison, the epitope of binding of the LDLR EGF domain to PCSK9 contains residues 153 to 155, 194, 238, 367, 369, 372, 374 to 375, and 377 to 381 (Kwon et al., 2008, PNAS, 105). 1820 ~ 1825).</p><p num="0229"> b. A group of antibodies and epitopes based on competition in PCSK9 binding. Full-length IgG was amine-coupled onto a CM5 sensor chip (3 / chip, final about 7000 RU) using standard EDC / NHS mediated amine coupling chemistry. One flow cell was left unmodified to provide a reference channel. Human-PCSK9 (100 nM) was premixed with an array of IgG (final 500 nM) and these complexes were injected onto the chip using a 1 minute injection at 10 μL / min. Antibodies that bind to competing epitopes block the binding of PCSK9 to antibodies immobilized on the chip. Alternatively, first inject human-PCSK9 at 50 nM for 1 minute at 10 μL / min (to anchor it via IgG on the chip) and then bind to an array of IgG (each final 500 nM) for 2 minutes each. By doing so, we used the traditional sandwich technique. The immobilized IgG was regenerated with a weak acid (Pierce mild elution buffer + 1M NaCl). Antibodies to various known epitopes were used as controls for positive sandwich formation in this assay.</p><p num="0230"> c. Structure-guided mutagenesis for mapping antibody-binding epitopes. Based on the crystal structure of PCSK9 in LDLR binding and the potential of D374 (Cunningham et al., 2007, Nat Struct Mol Biol, 14 (5): 413-419), 19 PCSK9 surface residues near or far from the position of D374 Group mutants (F379A, I369A, R194A, D374Y, D238R, T377R, K222A, R199A, F216A, R218A, R237A, D192R, D367R, R165A, R167A, A443T, A53V, I474V, H449A) selected for mutation Then, the antibody-binding epitope was mapped.</p><p num="0231"> d. Mutant and antibody production. Using standard DNA techniques, 19 single point mutants were generated from the previously described wild-type DNA constructs (Cunningham et al., 2007, supra). Mutant proteins were expressed using transient transfection into HEK293T cells and secreted into cell culture medium. Mutant proteins are Ni using a high throughput AKTA Xpress system (GE Healthcare) using conditions similar to those previously described.²⁺And purified by size exclusion chromatography step. Protein concentration was determined using a LabChip instrument (Bio-Rad). Transfection into HEK293F cells was used to express PCSK9-blocking murine antibodies 4A5, 7D4, 5A10 and 6F6 by elution at 0.1 M glycine buffer with a protein G column at pH 2.8. Purified and neutralized with 1.0 M Tris, pH 9.0.</p><p num="0232"> e. Regions of PCSK9 contacted by monoclonal antibodies 5A10 and 7D4 (preparations are described below herein) were determined by protein tomography (Sidec AB, Stockholm, Sweden). The loops at positions 186-200, 371-379, 176-181, 278-283, 449-453, 402-406, and 236-245 at PCSK9 were proximal to the amino acid residues of the antibody. The sequences corresponding to the loops are shown in Table 5, and in a preferred embodiment, the antagonist of the invention binds to one or more of these sequences in PSCK9.</p><p num="0233"><tables num="5"><img id="000006" he="55" wi="139" file="JP5750421B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0234"> f. Biacore binding between mutant and fixed LDLR. Recombinant LDLR extracellular domain protein was immobilized on a Biacore SA chip. Each mutant protein was injected into Biacore-3000M) in pairs at five concentrations from 25 mM to 0.012 mM (from 1 ° C to 50 mM Tris, pH 7.5, 2 mM CaCl).₂, 200 mM NaCl, 0.02% P20 and 1 mg / ml BSA running buffer were used. All results fit well into the 1: 1 coupled kinetic model. As expected, mutations at residues in direct contact with the EGF-A domain (F379A, R194A, I369A, T377R, D238R) significantly weaken LDLR binding (10-100 fold). In addition, the three mutants (R199A, R218A, K222A) that were not in contact with EGF-A showed weaker binding (5-15 fold). This new finding suggests that they are involved in the binding of other domains in the LDLR. Overall, these experiments confirmed the integrity and activity of the mutants for subsequent epitope mapping experiments.</p><p num="0235"> g. Binding of mutants to fixed 4A5, 7D4, 5A10 and 6F6 antibodies. Biotin-labeled anti-PCSK9 antibody was immobilized on an SA chip using standard methods. Mutant binding experiments were performed using Biacore 3000 at 25 ° C. with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.02% P20 running buffer. Mutants were tested in pairs at concentrations of 333 nM or 111 nM, and those that gave weaker binding compared to wild-type were defined as residues involved in mAb binding (described below). Descending binding residues of the mAb mutant effect 4A5 R237, F379, 369, R194, R199 and D238 5A10 R194, R237, I369, D238, R199 6F6 R237, R194, F379, D238, I369, T377, R199 7D4 R237, R194, F379, I369, R199</p><p num="0236">(Example 5) Antibody cloning and sequencing One million hybridoma cells were homogenized using the QIAshredder spin column and total RNA was extracted according to QIAGEN's RNA easy Micro kit. Invitrogen SuperScript III CDNA was synthesized using the RT kit. The variable region from the PCSK9 antibody was cloned using Novagen's mouse IgG-primer set consisting of a mouse IgG heavy chain gene and a degenerate primer for cloning the mouse kappa or lambda light chain. The PCR cycle conditions are as follows: 92C for 1 cycle, 94C for 30 seconds, 44C for 30 seconds and 72C for 2 minutes for 2 cycles, 94C for 30 seconds, 46C for 30 seconds and 72C for 2 minutes. 2 cycles, 94C for 30 seconds, 48C for 30 seconds and 72C for 2 minutes for 2 cycles, 94C for 30 seconds, 50C for 30 seconds and 72C for 2 minutes for 2 cycles, 94C for 30 seconds, 52C for 30 seconds and Two cycles of 72C for 2 minutes, followed by 35 cycles of 94C for 30 seconds, 54C for 30 seconds and 72C for 45 seconds. The resulting PCR product was cloned and sequenced into a Topo-TA cloning vector from Invitrogen. The cloned antibody sequence was confirmed by N-terminal sequencing of the first 10 amino acids of the original antibody produced from ascites.</p><p num="0237">(Example 6) Preparation of antigen for immunization Cunningham et al., 2007, Nat Struct Mol Biol, 14 (5): 413-9, who produced recombinant human PCSK9 protein as reported. To produce recombinant mouse PCSK9 protein, the mouse PCSK9 cDNA was cloned into the mammalian expression vector PRK5 by adding a 6-His tag to the C-terminus by a method known in the art and transient. Was transfected into HEK293 cells and expressed in HEK293 cells. Recombinant proteins were purified from conditioned medium using a Ni column.</p><p num="0238"> Human and mouse PCSK9 surface peptides were selected based on the PCSK9 protein structure and synthesized by Elim Biopharmaceuticals.</p><p num="0239">(Example 7) Antibodies specific to PCSK9 as PCSK9 antagonists 1. Identification of PCSK9-specific antagonist antibodies Identification of antibodies that block PCSK9 Mouse antibodies against human and / or mouse PCSK9 were obtained by immunizing mice with the human-PCSK9 and mouse-PCSK9 synthetic peptides or recombinant proteins prepared in Example 6 and recombinant the human and / or mouse PCSK9 described in Example 1. It was produced by screening the antibody by an ELISA assay using the protein as an antigen as well as other standard hybridoma procedures. Over 500 positive clones were obtained and grown to confluence in 6-well plates using 10 ml of medium. Collect medium supernatant and mAb Total IgG in conditioned medium was purified using 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 method described in Example 1. Hybridoma clones expressing IgG that showed some degree of blockade were expanded and retested. Sixty promising clones were subcloned, expanded and injected into either Balb / c or nude mice to produce ascites. Antibodies purified from ascites were retested in Huh7 cells for their ability to inhibit LDLR downregulation by human or mouse PCSK9. It has been identified that four hybridoma clones, 4A5, 5A10, 6F6, and 7D4, can completely inhibit the function of human PCSK9 and at least partially inhibit the function of mouse PCSK9. Each IC of these blocking antibodies₅₀A serial dilution of IgG starting at 100 μg / ml and up to 3.125 μg / ml was used in the assay to keep the concentration of human and mouse PCSK9 constant at 6 μg / ml.</p><p num="0240"> b. Effect of PCSK9 antagonists on the binding of PCSK9-LDLR PCSK9 has been shown to co-localize in cellular compartments with LDLR (Lagace et al., 2006, J Clin Inv, 116 (11): 2995-3005. Also, the recombinant PCSK9 protein is LDLR in vitro. It also binds to extracellular domains (Fisher et al., 2007, JBC, 282 (28): 20502-12. Determines the relationship between PCSK9-mediated inhibition of LDLR downregulation and antibody-induced inhibition of PCSK9-LDLR binding. In order to do so, we tested representative examples of PCSK9 antibodies that partially or completely blocked the function of PCSK9 against LDLR and those that did not, and all but one partially antagonistic antibody were the LDLR extracellular domain and PCSK9. Binding to PCSK9 was also partially inhibited. Antagonistic antibodies capable of completely blocking the function of PCSK9, namely 4A5, 5A10, 6F6 and 7D4, also completely inhibited binding of the LDLR extracellular domain to PCSK9 (Table). 5). ICs of these four antibodies₅₀The value correlates with its binding affinity for PCSK9.</p><p num="0241"> c. Determination of blocking antibody epitopes FIG. 4 illustrates the epitope binning of an anti-PCSK9 antibody. Part A) presents epitope information for anti-PCSK9 mAbs determined by binding to synthetic 13-18mer peptides or epitope bindings by Biacore. Part B) shows the ability of the fixed antibodies 6F6, 5A10 and 4A5 to bind to human PCSK9 premixed with mAbs shown on the y-axis by the Biacore assay.</p><p num="0242"> Another monoclonal anti-PCSK9 antibody, called 6G7, binds recombinant mouse PCSK9 but not human PCSK9. See Table 6. 6G7, 4A5, 5A10, 6F6, and 7D4 mutually eliminate binding to each other with mouse PCSK9. Chimeric analysis between mouse and human PCSK9 reveals that the binding of 6G7 to PCSK9 requires a catalytic domain. See Table 6. Therefore, the binding sites of 4A5, 5A10, 6F6, and 7D4 overlap with the catalytic sites and / or the epitopes bound by 6G7.</p><p num="0243"><tables num="6"><img id="000007" he="62" wi="159" file="JP5750421B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0244"> d. Determination of sequence species specificity for anti-PCSK9 antibody To determine the species specificity of the anti-PCSK9 antibody, the antibody was incubated with plasma from various species, the resulting complex was purified and probed with an independent anti-PCSK9 antibody on Western blots. Antibodies 4A5, 5A10, 6F6, and 7D4 recognized human, cynomolgus monkey, mouse, and rat PCSK9. See Figure 5. Antibody 6G7 recognized only murine PCSK9, and unrelated control antibody 42H7 did not recognize any of the PCSK9 tested. Same as above.</p><p num="0245"> e. Sequence of antagonist PCSK9 antibody The amino acid sequences of the variable domains of PCSK9 antibodies 4A5, 5A10, 6F6, and 7D4 were determined using the method described in Example 5. The sequences indicate that the antibodies are related but different from each other. Table 1 shows the amino acid sequences of the variable regions of each antibody. Table 7 shows the light and heavy chain CDR sequences identified by the Kabat and Cotia methods.</p><p num="0246"><tables num="7"><img id="000008" he="204" wi="159" file="JP5750421B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0247"> Anti-PCSK9 IgG 4A5, 5A10 and 6F6 were amine-coupled with the Biacore chip. hPCSK9 (100 nM) was mixed with 500 nM 4A5, 5A10, 6F6 or 7D4 in various ratios and injected at 10 μl / min for 1 minute. The four antibodies mutually blocked each other regardless of the assay orientation tested, suggesting that they all bind to competing epitopes. In contrast, they can form sandwich complexes with other non-blocking antibodies mapped to specific regions using synthetic peptides.</p><p num="0248"> 2. Effect of PCSK9-specific antibody as an in vivo PCSK9 antagonist PCSK9 antagonist antibody lowers serum cholesterol in mice To determine if a PCSK9 antagonistic monoclonal antibody can affect cholesterol levels in vivo by inhibiting the function of extracellular PCSK9, the effect of 7D4 on mouse PCSK9 in vitro to mice Tested in serum cholesterol when injected in vitro. Male C57 / bl6 mice aged 6-7 weeks were kept on a 12 hour light / dark cycle and bleeding on day 7 to collect approximately 70 μl of serum. The antagonist PCSK9 antibody 7D4 and control isotypes matching the monoclonal antibody were injected into male 7-week-old C57 / bl6 mice by intraperitoneal injection on days 0, 1, 2, and 3. Mice were sacrificed on day 4 without fasting and serum samples were collected. All frozen serum samples IDEXX for total cholesterol, triglyceride, HDL cholesterol and LDL cholesterol measurements Sent to laboratories. Figure 6 shows that 7D4 lowered serum cholesterol by 48%, while the control antibody had no significant effect. Both the amount and the percentage of reduction are similar to those reported in PCSK9-/-mice (PCSK9 knockout mice), which are full or near complete function of PCSK9 by blocking only extracellular PCSK9. Inhibition can be achieved, suggesting that intracellular PCSK9 plays little or no role in downregulation of LDLR under normal physiological conditions. As expected, liver LDLR levels were induced in animals treated with 7D4 compared to those treated with control antibody (Figure 6).</p><p num="0249"> b. Partially blocking antibodies did not affect blood cholesterol levels FIG. 7 illustrates that the partial antagonist polyclonal anti-PCSK9 mAb CRN6 does not affect cholesterol levels in mice. -Bleeding 8 week old C57 / bl6 mice (n = 10 mice / group) in 2 groups on day 7 and testing for cholesterol levels, 15 mg / kg on days 0, 1, 2 and 3 CRN6 or control antibody / day was administered intravenously, followed by bleeding and tested for cholesterol levels 24 hours after final dose. FIG. 7A shows that the CRN6 antibody partially blocks PCSK9-mediated downregulation of LDLR in Huh7 cells in vitro. FIG. 7B shows that administration of CRN6 antibody does not affect serum cholesterol levels in mice.</p><p num="0250"> c. Sustained effect of antagonistic PCSK9 mAb on serum cholesterol in mice. A time course study was performed to determine the onset and duration of the cholesterol-lowering effect of PCSK9 antagonist antibodies in mice. Forty-eight 6-week-old C57 / bl6 mice were intravenously injected with mAb 7D4 or saline control at 10 mg / kg or 3 ml / kg, respectively. Eight mice from each treatment group were sacrificed on days 1, 2, 4, 7, 14 and 21 after injection. A single injection of 7D4 produced a rapid and sustained lowering effect on serum cholesterol. A 25% reduction in serum cholesterol was seen 24 hours after injection. See Figure 8. A maximum drop in serum cholesterol was observed at 7 days. At 21 days, the decrease in cholesterol was no longer statistically significant. Part B) shows HDL cholesterol. LDL cholesterol levels were very low.</p><p num="0251"> FIG. 9 illustrates that anti-PCSK9 antagonist mAb 7D4 reduces serum total cholesterol, HDL, and LDL in a dose-dependent manner in mice. Bleeding 8-week-old C57 / bl6 mice (n = 8 / group) in 6 groups, testing for basal cholesterol levels on days 7 and intraperitoneal bolus on days 0, 1, 2, and 3. The dose indicated by injection of antibody or saline was administered. Serum samples were collected and tested for cholesterol levels 24 hours after the last dose. FIG. 9A shows total cholesterol levels reduced to less than 60% of controls after administration of 3-30 mg / kg / day. The maximum effect on total cholesterol was seen at 10 mg / kg, and a statistically significant reduction was seen at 1 mg / kg. FIG. 9B shows HDL levels reduced to less than 70% after administration of 3-30 mg / kg / day. FIG. 9C shows LDL levels reduced to near 0 at all tested doses above 0.3 mg / kg / day.</p><p num="0252"> d. Dose response of PCSK9-specific antagonist antibody in mice FIG. 10 illustrates that anti-PCSK9 antagonist antibody 5A10 lowers cholesterol levels in mice in a dose-dependent manner. Figure 10A shows 6 groups of 8-week-old C57 / bl6 mice receiving the indicated doses of antibody or saline by intravenous bolus injection once daily on days 0, 1, 2, and 3. n = 8 animals / group) is shown. Serum samples were collected and tested for cholesterol levels 24 hours after the last dose, showing a gradual decrease with increasing doses of antibody. FIG. 10B shows 5 groups of 8-week-old C57 / bl6 mice (n = 8 animals / group) receiving the indicated doses of antibody or saline by intraperitoneal bolus injection on day 0. Serum samples were collected and tested for cholesterol levels on day 7 and also showed a gradual decrease with increasing doses of antibody.</p><p num="0253"> FIG. 11 illustrates that anti-PCSK9 antagonist antibodies 4A5 and 6F6 lower cholesterol levels in mice in a dose-dependent manner. Eight-week-old C57 / bl6 mice (n = 8 / group) were administered the indicated doses of antibody or saline by intraperitoneal bolus injection on day 0. Serum samples were collected and tested for cholesterol levels on day 7. In Figure 11A, antibody 4A5 showed a gradual decrease in total serum cholesterol with increasing doses of antibody. In FIG. 11B, antibody 6F6 showed a decrease in total serum cholesterol at 10 mg / kg / day.</p><p num="0254"> Anti-PCSK9 antagonist antibodies 4A5, 5A10, 6F6 and 7D4 increased liver LDLR levels in mice, which was found by Western blot analysis. See Figure 12. For 4A5, 5A10 and 6F6, 8-week-old C57 / bl6 mice were given 10 mg / kg antibody or saline by intravenous bolus injection on day 0, and the animals were sacrificed on day 7 and 3 animals. Whole liver lysates of individual animals were analyzed for LDLR and GAPDH protein levels by Western. In 7D4, 8-week-old Bl6 / c57 mice were given 10 mg / kg of antibody by intraperitoneal bolus injection on days 0, 1, 2, and 3, and the animals were sacrificed on day 4 to kill 3 animals. Whole liver lysates of individual animals were analyzed for LDLR and GAPDH protein levels by Western blotting. Mice treated with all antibodies showed higher levels of LDLR compared to PBS control mice.</p><p num="0255"> FIG. 13 illustrates that the anti-PCSK9 antagonist antibody had no effect in LDLR-/-mice. Eight-week-old LDLR-/-mice (LDLR KO mice) were administered 10 mg / kg of 4A5 or saline by intraperitoneal bolus injection on day 0. Serum samples (n = 9-10 mice) were collected and tested for cholesterol levels on day 7. Antibody administration did not change the level of total serum cholesterol, HDL, or LDL sensibly.</p><p num="0256"> FIG. 14 illustrates that multiple treatments of anti-PCSK9 antagonist antibodies in mice can substantially reduce total serum cholesterol. Eight-week-old C57 / bl6 mice received the indicated doses of antibody or PBS by intravenous bolus injection on days 0, 7, 14 and 21. Serum samples (n = 5-11 mice) were collected and tested for cholesterol levels on day 28.</p><p num="0257">(Example 8) PCSK9 antagonist antibody lowers serum LDL in non-human primates To test the in vivo effect of the antibody on PCSK9, antibody 7D4 was tested in cynomolgus monkeys. Four 3-4 year old cynomolgus monkeys were injected with vehicle (PBS + 0.01% Tween 20) on day 0 and 10 mg / kg 7D4 on day 7. Plasma lipid profiles were analyzed on days 0, 2, 7, 9, 11, 14, 21 and 28 after an overnight fast. A single injection of 7D4 at 10 mg / kg resulted in a dramatic reduction in plasma LDL (60%) (Fig. 15A) and LDL particle number (Fig. 15D) in all four animals, while their HDL levels. (Fig. 15B) and the number of HDL particles (Fig. 15E) were minimally affected. Total cholesterol (Fig. 15C) was also reduced after 7D4 treatment, while triglyceride levels (Fig. 15F) were not significantly affected. All 7D4 (G) and all PCSK9 levels (H) were also measured.</p><p num="0258"> FIG. 16 illustrates the dose response of anti-PCSK9 antibody 7D4 to serum cholesterol levels in cynomolgus monkeys. Two male and two female cynomolgus monkeys aged 3-5 years in each group were given the indicated dose of 7D4 on day 7 and the same volume of saline on day 0, intravenous bolus. Given by injection. Plasma samples were collected at the time indicated and plasma LDL levels were measured.</p><p num="0259"> FIG. 17 illustrates a comparison of anti-PCSK9 antibodies 4A5, 5A10, 6F6 and 7D4 to serum cholesterol levels in cynomolgus monkeys. Two male and two female cynomolgus monkeys aged 3 to 6 years in each group were given 1 mg / kg of the indicated antibody by intravenous bolus injection on day 0. Plasma samples were taken at the time indicated, plasma LDL levels were measured and normalized to those on day -2.</p><p num="0260"> FIG. 18 illustrates the effect of anti-PCSK9 antagonist antibody 7D4 on plasma cholesterol levels in cynomolgus monkeys fed a 33.4% kcal fat diet supplemented with 0.1% cholesterol. Six cynomolgus monkeys aged 3 to 5 years were fed a high-fat diet for 16 weeks. On the indicated day, 3 monkeys were treated with 10 mg / kg 7D4 and 3 were treated with saline. LDL levels in individual monkeys were measured and normalized to those on the day of treatment.</p><p num="0261">(Example 9) Humanized anti-PCSK9 antibody The murine monoclonal antibody 5A10 was humanized and affinity maturated to yield the L1L3 antibody. L1L3 has an affinity for murine PCSK9 of 200 pM and an affinity for human PCSK9 of 100 pM as measured by Biacore. L1L3 completely inhibits PCSK9-mediated downregulation of LDLR in cultured Huh7 cells when incubated with 100 nM human or murine PCSK9 antibody. See Figure 19.</p><p num="0262"> FIG. 20 shows L1L3, mouse precursor 5A10, and negative control antibody 42H7 for blocking in vitro binding of recombinant biotin-labeled human PCSK9 and mouse PCSK9 to the immobilized recombinant LDLR extracellular domain. Illustrate the dose response. FIG. 20A shows the binding of human PCSK9 to the human LDLR extracellular domain at pH 7.5. FIG. 20B shows the binding of human PCSK9 to the human LDLR extracellular domain at pH 5.3. FIG. 20C shows the binding of mouse PCSK9 to the human LDLR extracellular domain at pH 7.5. Figure 20D shows the binding of mouse PCSK9 to the human LDLR extracellular domain at pH 5.3.</p><p num="0263"> FIG. 21 shows the effect of treatment with 10 mg / kg L1L3 on serum cholesterol in mice. Two groups (n = 8 / group) of 8-week-old C57 / bl6 mice received 10 mg / kg of L1L3 or the same volume of saline by intraperitoneal injection on day 0. Serum samples were collected and assayed for cholesterol levels on days 2, 4 and 7. L1L3 reduced total serum cholesterol by about 40% on days 2 and 4. In another study, serum cholesterol levels were treated with saline when 10 mg / kg L1L3 was administered as a single intraperitoneal (IP) dose to C57BL / 6 mice (n = 10) fed a normal diet. There was a 47% reduction 4 days after treatment compared to the control. Serum when L1L3 was administered as a single intraperitoneal dose of 0, 0.1, 1, 10 and 80 mg / kg (n = 6 animals / group) in a dose-response experiment in normal-fed male Sprague-Dolly rats. Cholesterol levels decreased in a dose-dependent manner, with a maximum effect of 50% at 10 and 80 mg / kg 48 hours after dosing. The duration of cholesterol suppression was also dose-dependent, ranging from 1 to 21 days.</p><p num="0264"> The amino acid sequence of the fully humanized heavy chain of L1L3 (SEQ ID NO: 15) is shown in Table 8. The variable region array is underlined (SEQ ID NO: 54).</p><p num="0265"><tables num="8"><img id="000009" he="55" wi="158" file="JP5750421B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0266"> The amino acid sequence of the L1L3 fully humanized light chain (SEQ ID NO: 14) is shown in Table 9. The variable region is underlined (SEQ ID NO: 53).</p><p num="0267"><tables num="9"><img id="000010" he="36" wi="159" file="JP5750421B2_D0001.tif" img-format="tif" img-content="drawing" /></tables></p><p num="0268"> FIG. 22 shows the effect of intravenous administration of an effective dose (3 mg / kg) of antibody 5A10 (solid circle) or antibody L1L3 (solid square) to each of the four cynomolgus monkeys on day 0. Changes in serum HDL (Fig. 22A) and serum LDL (Fig. 22B) were measured from -2 days to +28 days. Both antibodies resulted in a reduction in serum LDL levels of more than about 70% by about 7 days, a effect that was substantially sustained for an additional 6 days in animals receiving L1L3. All animals showed normal liver and kidney function as well as near normal hematocrit.</p><p num="0269"> L1L3 reduced LDL-C in a dose-dependent manner, with maximum effect observed in the 10 mg / kg group, with a 70% reduction in LDL-C levels maintained until 21 days post-dose, by 31 days. Completely recovered. In all dose groups, HDL-C levels were unaffected by treatment with L1L3. Animals in the 3 mg / kg dose group (n = 4) were also given two additional intravenous doses of 3 mg / kg L1L3 on days 42 and 56 (2 week intervals). These two additional doses lowered LDL-C again, keeping LDL-C levels below 50% for 4 weeks. LDL-C levels returned to normal after 2 weeks. Serum HDL-C levels remained unchanged throughout the study.</p><p num="0270"> We investigated the efficacy of L1L3 in non-human primates suffering from hypercholesterolemia and the pharmacodynamic interaction between L1L3 and statin-inhibiting HMG-CoA reductase. Normal average LDL-C levels in the cynomolgus monkey cohort (n = 12) by feeding a diet containing 35% fat (weight / weight) and 600 ppm cholesterol for 18 months prior to starting the study. It was increased to an average of 120 mg / dL compared to the level of 50 mg / dL. Surprisingly, after a moderate dose (10 mg / animal) of Crestor® (rosuvastatin calcium) daily for 6 weeks followed by a high dose (20 mg / kg) for 2 weeks daily No effect on serum total cholesterol or LDL-C levels was observed. A 2-week treatment with a single dose of 3 mg / kg L1L3 and Cholesterol® or vehicle effectively reduced serum LDL-C levels to 56% by day 5 after treatment, 2.5-3. Gradually recovered over the course of the week, but HDL-C levels were unaffected. After changing the animals to a once-daily dose of 50 mg / kg Zocor® (simvastatin), their LDL-C levels reached a maximum drop of 43% on day 5 and then stabilized. After administration of 50 mg / kg / day of Zocor® for 3 weeks, these animals were treated with a single dose of 3 mg / kg of L1L3, still given 50 mg / kg / day of Zocor®. .. By day 5, administration of L1L3 reduced LDL-C by an additional 65% in addition to a 43% reduction by Zocor®, returning to pre-dose levels within 2 weeks.</p><p num="0271"> Other CDR amino acid substitutions were performed on 5A10 to achieve specific properties during humanization and affinity maturation. The sequences of the modified CDRs and the PCSK9 binding capacity of the antibodies containing these modified CDRs are shown in FIGS. 24A-G. The number after each sequence in FIGS. 24A to 24 represents the sequence number of that sequence.</p><p num="0272"> The disclosure of all references cited herein is incorporated herein by reference.</p>

ATCC PTA-8986 ATCC PTA-8985 ATCC PTA-8984 ATCC PTA-8983