TW201505654A - Methods for reducing remnant cholesterol and other lipoprotein fractions by administering an inhibitor of proprotein convertase subtilisin KEXIN-9 (PCSK9) - Google Patents

Abstract

The present invention provides methods for reducing various lipoprotein fractions in the serum of patients. The methods of the invention include reducing serum remnant cholesterol, and/or the serum concentration of one or more LDL-C subfractions in a patient. The methods of the present invention comprise selecting a patient who exhibits elevated serum lipoproteins, and administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor. In certain embodiments, the PCSK9 inhibitor is an anti-PCSK9 antibody such as the exemplary antibody referred to herein as mAb316P.

Description

Method for reducing residual cholesterol and other lipoprotein fractions by administering a protein converting enzyme subtilisin KEXIN-9 (PCSK9) inhibitor

The present invention relates to the field of treatment of diseases and disorders associated with elevated levels of lipoproteins. More specifically, the invention relates to the administration of PCSK9 inhibitors to reduce the levels of residual cholesterol and other lipoprotein subfractions in the serum of a patient.

Residual cholesterol (also known as residual lipoprotein) is a class of cholesterol that contains only non-HDL and non-LDL. Residual cholesterol is the product of VLDL lipolysis and includes VLDL ₃ and medium density lipoprotein (IDL, a direct precursor that forms LDL). Serum residual cholesterol levels have been identified as predictive risk factors for coronary artery disease. In addition to residual cholesterol, there are several sub-components of low-density lipoprotein cholesterol (LDL-C) that may be associated with cardiovascular health. In particular, LDL-C is composed of LDL particles of various densities and lipid states. Therapeutic reduction of serum residual cholesterol and other lipoprotein sub-component levels can be a means of treating or reducing the risk of cardiovascular disease.

PCSK9 is a proprotein convertase belonging to the proteinase K subfamily of the secreted subtilisin family. The use of PCSK9 inhibitors (anti-PCSK9 antibodies) to reduce serum total cholesterol, LDL cholesterol and serum triglycerides in the United States, 8,062,640, 8,357,371 Both the patent and the US Patent Application Publication No. 2013/0064834 are incorporated herein by reference.

The present invention provides methods for reducing various lipoprotein and lipoprotein components in a patient's serum. The patient may be a patient with hypercholesterolemia or at risk of developing hypercholesterolemia, or a patient with or at risk of cardiovascular disease.

The method of the invention comprises selecting a patient with elevated levels of a particular lipoprotein or lipoprotein component in the serum, and administering to the patient a pharmaceutical composition comprising a proprotein convertase subtilisin 9 (PCSK9) inhibitor. In accordance with certain aspects of the invention, the patient is selected based on elevated residual cholesterol levels in the patient's serum. According to certain other aspects of the invention, according to elevated levels of very low density lipoprotein cholesterol in the serum (eg, elevated serum VLDL-C, VLDL ₁ -C, VLDL ₂ -C, VLDL ₁₊₂ -C) , VLDL ₃ -C, etc.) to select patients. In accordance with further aspects of the invention, the patient is selected based on elevated levels of intermediate density lipoprotein cholesterol (IDL-C) in his serum. According to certain other aspects of the invention, the level of one or more low density lipoprotein cholesterol (LDL-C) sub-fractions in the serum is elevated (eg, elevated serum LDL ₁ -C, LDL ₂ -C) , LDL ₃ -C, LDL ₄ -C, LDL ₃₊₄ -C, etc.) to select patients. In accordance with certain aspects of the invention, elevated Lp(a) levels in serum are used as an additional or replacement criterion to select a patient.

Patients may also be selected based on other risk factors associated with such diseases and disorders as indicated by the patient, where a decrease in lipoprotein levels would be beneficial or helpful in reducing the risk. For example, a patient with hypercholesterolemia (e.g., heFH, non-FH, etc.) can be a suitable candidate for treatment with the methods of the invention.

PCSK9 inhibitors that can be administered in accordance with the methods of the invention include, for example, an anti-PCSK9 antibody or antigen-binding fragment thereof. Specific exemplary anti-PCSK9 antibodies that can be used in the practice of the methods of the invention include any of the antibodies or antigen-binding fragments as described in U.S. Patent No. 8,062,640 and/or disclosed herein.

The PCSK9 inhibitor can be administered to a subject either subcutaneously or intravenously. In addition, the PCSK9 inhibitor can be administered to a patient who is being treated with a statin at the time of a therapeutic intervention.

Other embodiments of the invention will become apparent in the course of the detailed description.

Figure 1 shows the dynamic relationship between mAb316P, free (unbound) PCSK9 and LDL-C levels in a single dose of subcutaneously injected mAb316P 150 mg in healthy subjects who did not receive statin background treatment.

Figure 2 shows the mean concentration of free mAb 316P in HeFH and non-FH patients receiving mAb 316P 50, 100 or 150 mg subcutaneous injection plus atorvastatin, or mAb 316P 150 mg subcutaneous injection plus dietary intervention. The dotted line indicates the quantification limit of free PCSK9 (0.0312 mg/dL).

Figure 3 shows LDL-C from baseline (from HeFH and non-FH combined with n=21, receiving mAb316P 150 mg subcutaneously on days 1, 29 and 43 of study) relative to mean total mAb316P concentration (mg/dL) Average percentage change. Each point represents the sampling time; h represents the hour; d represents the day. As indicated by the arrows, the hysteresis curve shifts with time in a clockwise direction, showing the temporal relationship between mAb 316P concentration and LDL-C change.

Figure 4 shows mean baseline free PCSK9 levels in non-FH patients receiving atorvastatin or only dietary intervention (no statins).

Figure 5 shows LDL-C efficacy curves for patients receiving mAb 316P 150 mg subcutaneously or placebo ± atorvastatin.

Figure 6 shows LDL-C from patients with hypercholesterolemia who received statin therapy at 50 mg, 100 mg, or 150 mg (every 2 weeks), 200 mg, or 300 mg (every 4 weeks) or placebo. The average percentage change from baseline to time. The triangle symbol (Δ) corresponds to the average of the last observed value estimate (LOCF).

Figures 7-9 show VLDL and serum residual lipids before and after treatment with placebo and mAb316P 150 mg (every 2 weeks) in three different clinical studies (A, B, and C, as summarized in Table 1 herein). Protein subcomponent and mean (SD) cholesterol levels in triglycerides. Figure 7 depicts the results of Study A, Figure 8 depicts the results of Study B, and Figure 9 depicts the results of Study C.

Figures 10-12 show that in three different clinical studies (A, B, and C, as summarized in Table 1 herein), in the LDL sub-components before and after treatment with placebo and mAb316P 150 mg (every 2 weeks) The average (SD) cholesterol level. Figure 10 depicts the results of Study A, Figure 11 depicts the results of Study B, and Figure 12 depicts the results of Study C.

Before the present invention is described, it is to be understood that the invention is not limited to the specific methods and experimental conditions described, as these methods and conditions may vary. It is also understood that the terminology used herein is for the purpose of illustration and description, and is not intended to

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains, unless otherwise defined. The term "about" as used herein, when used in reference to a particular value, means that the value can vary within a range of no more than 1% from the listed value. For example, the expression "about 100" as used herein includes 99 and 101 and all values between the two (eg, 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference in their entirety.

Method for reducing residual cholesterol and other lipoprotein components

The present invention provides methods for reducing residual cholesterol and other lipoprotein components in the serum of a patient. The method of the invention comprises selecting to display one or more residual cholesterol (also known as "residual lipoprotein cholesterol", "residual lipoprotein" or RLP-C), very low density lipoprotein cholesterol (VLDL-C), medium density lipid Patients with elevated serum levels of protein cholesterol (IDL-C), triglycerides (TG), and/or Lp(a). In certain embodiments, the method of the invention comprises selecting to display VLDL ₁ , VLDL ₂ , VLDL ₁₊₂ , VLDL ₃ , LDL ₁ , LDL ₂ , LDL ₃ , LDL ₄ , LDL ₃₊₄ and/or Patients with elevated serum levels in other combinations. The method of the invention further comprises administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor.

As used herein, the expressions "residual cholesterol", "residual lipoprotein", "RLP", etc., mean cholesterol content in triglyceride-rich lipoproteins consisting of VLDL and IDL in a fasting state, and in a non-fasting state. These two lipoproteins are supplemented with cholesterol in the granules of the chylomicrons. Residual cholesterol can be calculated as follows: total cholesterol minus HDL-C, minus LDL-C (ie, non-[HDL-C+LDL-C]). Residual cholesterol includes VLDL ₃ and IDL.

Other lipoprotein components include, for example, LDL ₁ -C ("large and suspended" LDL), LDL ₂ -C, LDL ₃ -C, and LDL ₄ -C ("small, dense" LDL). The serum levels of the above lipoproteins can be determined by standard lipoprotein fine separation techniques such as a vertical auto profile (VAP) test.

In the context of the present invention, "increased serum lipoprotein levels" (eg, elevated serum residual cholesterol levels, elevated serum VLDL-C levels, elevated serum VLDL ₁ -C levels, elevated serum VLDL ₂ -C levels) Increased serum VLDL ₁₊₂ -C levels, elevated serum VLDL ₃ -C levels, elevated serum IDL-C levels, elevated serum LDL ₁ -C levels, elevated serum LDL ₂ -C levels, serum LDL ₃ Elevated levels of -C, elevated levels of serum LDL ₄ -C, elevated serum LDL ₃₊₄ -C levels, etc.) mean that the corresponding serum lipoprotein levels are above about 8 mg/dL. In certain embodiments, if the specific lipoprotein level measured in the patient's serum is greater than about 9 mg/dL, 10 mg/dL, 11 mg/dL, 12 mg/dL, 13 mg/dL, 14 mg/dL, 15 mg/dL, 16mg/dL, 17mg/dL, 18mg/dL, 19mg/dL, 20mg/dL, 25mg/dL, 30mg/dL, 35mg/dL, 40mg/dL, 45mg/dL, 50mg/dL, then the patient is considered It is an increase in serum lipoprotein levels. Serum lipoprotein levels can be measured after the patient's meal. In certain embodiments, lipoprotein levels are measured after a period of fasting (eg, after fasting for 6 hours, 8 hours, 10 hours, 12 hours, or longer). Any clinically acceptable diagnostic method can be used to measure the patient's serum lipoprotein in the context of the present invention.

According to the present invention, residual cholesterol or other lipoprotein subfractions in serum (for example, VLDL-C, VLDL ₁ -C, VLDL ₂ -C, VLDL ₁₊₂ -C, VLDL ₃ -C, IDL-C, LDL ₁ a decrease in the levels of -C, LDL ₂ -C, LDL ₃ -C, LDL ₄ -C, LDL ₃₊₄ -C, etc., meaning that the patient is serum lipoprotein/lipoprotein after receiving the pharmaceutical composition of the present invention. The component level drops by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 from the baseline. %, 80%, or more. When referring to a particular lipoprotein or lipoprotein subfraction, the term "baseline level" as used herein means a lipoprotein/lipoprotein measured in the serum of a subject prior to receiving the pharmaceutical composition of the invention. Subcomponent level. A decrease in serum lipoprotein/lipoprotein sub-component levels resulting from administration of a pharmaceutical composition of the invention may be one day after initiation of a treatment regimen comprising the use of one or more doses of a pharmaceutical composition comprising a PCSK9 inhibitor. , 2, 3, 4, 5, 6, 1, 2, 4, 6, 8, 8, 10, 12, 14, 16, 18, 20, 22 The weeks are reached and/or observed, and examples thereof are disclosed elsewhere herein.

For example, the present invention includes methods for reducing serum residual cholesterol levels in a patient by selecting a patient whose serum residual cholesterol level is elevated and administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor such that the patient after receiving the pharmaceutical composition The serum residual cholesterol level drops from baseline by at least about 35% to about 45%.

The present invention also includes methods for reducing serum VLDL-C levels in patients. A patient having an elevated serum VLDL-C level is selected and administered to the patient with a pharmaceutical composition comprising a PCSK9 inhibitor such that the patient's VLDL-C level decreases from baseline by at least about 20% after receiving the pharmaceutical composition. 28%.

The invention also includes methods for reducing serum VLDL _1+2- C levels in a patient by administering a patient with elevated serum VLDL _1+2- C levels and administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor, such that the patient After receiving the pharmaceutical composition, the VLDL _1+2- C level decreased from baseline by at least about 20% to about 32%.

The invention also includes methods for reducing serum VLDL ₃ -C levels in a patient by administering a patient having elevated serum VLDL ₃ -C levels and administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor such that the patient is receiving the drug After the composition, the VLDL ₃ -C level drops from baseline by at least about 20% to about 27%.

The invention also includes methods for reducing serum IDL-C levels in a patient by administering a patient having an elevated serum IDL-C level and administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor such that the patient is receiving the pharmaceutical combination After the event, the IDL-C level decreased from baseline by at least about 50% to about 56%.

The invention also includes methods for reducing serum LDL ₁ -C levels in a patient by administering a patient having elevated serum LDL ₁ -C levels and administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor such that the patient is receiving the drug After the composition, the LDL ₁ -C level drops from baseline by at least about 65% to about 78%.

The invention also includes methods for reducing serum LDL ₂ -C levels in a patient by administering a patient having elevated serum LDL ₂ -C levels and administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor such that the patient is receiving the drug After the composition, the LDL ₂ -C level drops from baseline by at least about 75% to about 85%.

The invention also includes methods for reducing serum LDL ₃₊₄ -C levels in a patient by administering a patient having elevated serum LDL ₃₊₄ -C levels and administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor such that the patient After receiving the pharmaceutical composition, the LDL _3+4- C level drops from baseline by at least about 45% to about 70%.

The present invention also encompasses a method of reducing the level of apolipoprotein (apo) CII and/or CIII in a patient's serum by administering a patient with hypercholesterolemia and administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor. According to certain embodiments, the patient has non-familial hypercholesterolemia (non-FH); in certain other embodiments, the patient has a heterozygous congener hypercholesterolemia (he-FH). In accordance with certain embodiments of this aspect of the invention, after administration of the pharmaceutical composition, the patient's apo CII level decreases from baseline by at least about 9% to about 30%, and the patient apo CIII level decreases by at least about 20% to about 25%.

The invention also includes methods for reducing serum Lp(a) levels in a patient by administering a patient having an elevated serum Lp(a) level and administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor such that the patient is receiving the Following the pharmaceutical composition, serum-Lp(a) levels are reduced from baseline by at least about 10% to about 40% (e.g., about 25%, about 30%, or about 35%).

The present invention also encompasses a method of reducing the concentration of lipoprotein particles in a patient by selecting a patient having a high concentration of hypercholesterolemia and/or a patient having a high serum lipoprotein particle concentration and administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor. The method according to this aspect of the invention can be used to reduce low density lipoprotein particles (LDL-P), medium density lipoprotein particles (IDL-P), and very low density lipoprotein particles (VLDL-P), among other uses. Concentrations, including, for example, small VLDL-P (29 to 42 mm in diameter); medium VLDL-P (42 to 60 nm in diameter); large VLDL-P (>60 nM in diameter); small LDL-P (18 to 20.5 in diameter) Nm); large LDL-P (20.5 to 23 nm in diameter); and serum concentration of IDL-P (23 to 29 nm in diameter).

Patient population

The method of the present invention can be used to reduce residual cholesterol and other lipoprotein components in patient serum (eg, VLDL-C, VLDL ₁ -C, VLDL ₂ -C, VLDL ₁₊₂ -C, VLDL ₃ -C, IDL-C, The level of LDL ₁ -C, LDL ₂ -C, LDL ₃ -C, LDL ₄ -C, LDL ₃₊₄ -C, etc.). In some cases, the patient is a substantially healthy patient in addition to showing elevated levels of one or more of the aforementioned serum lipoproteins. For example, a patient may not show any other risk factors for cardiovascular, thrombotic, or other diseases or disorders at the time of treatment. In other cases, however, the patient is selected to be diagnosed as having a disease caused by or associated with elevated levels of residual cholesterol in the serum or elevated levels of other lipoprotein or lipoprotein sub-components or Disordered, or at risk of developing the disease or disorder. For example, at or before the administration of a pharmaceutical composition of the invention, the patient may be diagnosed with a cardiovascular disease or disorder or identified as having a risk of developing such a disease or disorder, such as coronary artery disease, acute myocardial infarction, no Symptoms Carotid atherosclerosis, stroke, and peripheral arterial occlusive disease. In some cases, the cardiovascular disease or disorder is hypercholesterolemia. For example, if a patient is diagnosed with hypercholesterolemia or is identified as having a risk of developing hypercholesterolemia, such as a heterozygous congenital hypercholesterolemia (heFH), homozygous familial hypercholesterolemia (hoFH) , autosomal dominant hypercholesterolemia (ADH, such as ADH associated with one or more functionally acquired mutations in the PCSK9 gene), and hypercholesterolemia different from familial hypercholesterolemia (non-FH), It may be selected to be treated by the method of the invention.

In other cases, the patient may be diagnosed with an arterial disease such as a cerebrovascular occlusive disease, a peripheral vascular disease, a peripheral arterial disease, or the like at or before administration of the pharmaceutical composition of the present invention, or is identified as having these The risk of illness or disorder. In certain embodiments, the patient is selected based on, or has a risk of developing, having or suffering from two or more of the above diseases or disorders.

In other instances, a patient to be treated by the method of the invention is selected according to one or more of the following factors: age (eg, greater than 40, 45, 50, 55, 60, 65, 70, 75 or 80 years old), race, gender (male or female), exercise habits (eg regular exercise, no exercise), other pre-existing conditions (eg type 2 diabetes, high blood pressure, etc.), and current medication status (eg current Taking statins [eg cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin) , lovastatin (pravastatin), p-statin blockers, niacin, etc.). The invention also includes methods for reducing serum residual cholesterol and/or other lipoprotein sub-component levels in patients for treatment of patients who are intolerant, unresponsive or underreactive to conventional statin therapy. Potential patients may be selected/screened (e.g., via a questionnaire, diagnostic assessment, etc.) based on one or more of these factors prior to treatment with the methods of the invention.

PCSK9 inhibitor

The method of the invention comprises administering to a patient a therapeutic composition comprising a PCSK9 inhibitor. The term "PCSK9 inhibitor" as used herein is any agent that binds to or interacts with human PCSK9 in vitro or in vivo and inhibits the normal biological function of PCSK9. Non-limiting examples of classes of PCSK9 inhibitors include small molecule PCSK9 antagonists, peptide-based PCSK9 antagonists (eg, "peptide antibody" molecules), and antigen-binding fragments of antibodies or antibodies that specifically bind to human PCSK9.

The term "human proprotein convertase subtilisin 9", or "human PCSK9" or "hPCSK9" as used herein, refers to a nucleic acid sequence of sequence 754 and an amino acid sequence of sequence 755 or a biologically active fragment thereof. PCSK9.

The term "antibody" as used herein means an immunoglobulin molecule composed of four polypeptide chains, ie, two heavy (H) chains and two light (L) chains, which are linked by a disulfide bond, and a multimer thereof (for example) IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or _VH ) and a heavy chain constant region. The heavy chain constant region comprises three domains, C _H 1, C _H 2 and C _H 3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or V _L) and a light chain constant region. The light chain constant region comprises a domain (C _L 1). And V _L, V _H regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), which is mixed with the more conserved regions referred to as framework regions (FR) of. Each V _H and V _L, three by four FR and CDR, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4. In various embodiments of the invention, the FR of an anti-PCSK9 antibody (or antigen binding portion thereof) may be identical to a human germline sequence, or may be modified naturally or artificially. An amino acid consensus sequence can be defined by side-by-side analysis of two or more CDRs.

The term "antibody" as used herein also includes antigen-binding fragments of intact antibody molecules. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically obtained, synthetic or genetically modified, which specifically binds to an antigen to form a complex. Polypeptide or glycoprotein. The antigen-binding fragment of the antibody can be produced, for example, from intact antibody molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering involving manipulation and expression of DNA encoding the variable and constant regions of the antibody (optional). . Such DNA is known and/or readily available, for example, from commercially available sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated by chemical or molecular biological techniques, such as aligning one or more variable regions and/or constant regions into a suitable configuration, or introducing codons to produce cysteine residues. Base, modify, add or remove amino acids, and the like.

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) a dAb fragment; and (vii) consisting of an amino acid residue that mimics the hypervariable region of the antibody (eg, an isolated complementarity determining region CDR, such as a CDR3 peptide) or an FR3-CDR3-FR4 configuration-binding peptide The smallest unit of recognition. The term "antigen-binding fragment" as used herein also includes other engineered molecules, such as domains. Specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, trimeric antibodies, tetrameric antibodies, minibodies, Nanobodies (eg, monovalent Nanobodies, bivalent Nanobodies, etc.) , Small Module Immunopharmaceutical (SMIP), and shark variable IgNAR domain.

An antigen-binding fragment of an antibody typically comprises at least one variable region. The variable domain can be of any size or of any amino acid composition and typically comprises at least one CDR adjacent to or within one or more of the framework sequences. In conjunction with the V _H region comprising associated antigenic fragment of the V _L region, the relative position of the region and V _L, V _H regions can be any suitable arrangement. For example, the variable region may be dimerized and contain _{V H -V H, V H -V} L or V _L -V _L dimer. Alternatively, antigen-binding fragments of antibodies may contain a single V _H region or V _L region.

In certain embodiments, an antigen-binding fragment of an antibody can contain at least one variable region covalently linked to at least one constant region. In the antigen-binding fragment of an antibody of the present invention may be found in non-limiting exemplary configuration of the variable and constant region _{comprising: (i) V H -C H} 1; (ii) V H -C H 2; ( Iii) V _H -C _H 3; (iv) V _H -C _H 1-C _H 2; (v) V _H -C _H 1-C _H 2-C _H 3; (vi) V _H -C _H 2 -C _H 3; (vii) V _H - C _L ; (viii) V _L - C _H 1; (ix) V _L - C _H 2; (x) V _L - C _H 3; (xi) V _L - _{C H 1-C H 2;} (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V _L -C _L. In any configuration of the variable and constant regions, including any of the exemplary configurations described above, the variable and constant regions may be directly linked to each other or may be joined by a single or partial hinge region or junction region. A hinge region may contain at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids formed between adjacent variable and/or constant regions in a single peptide molecule A flexible or semi-flexible connection. Furthermore, the antigen-binding fragment of the antibody of the present invention may comprise a homodimer or a heterodimer (or other multimer) of any configuration of the variable region and the constant region described above, the variable region and the constant region being non-common valence bond interconnecting and / or with one or more single V _H region or V _L region (e.g., through disulfide) is connected.

Like intact antibody molecules, antigen-binding fragments can be monospecific or multi-specific Heterosexual (eg bispecific). A multispecific antigen-binding fragment of an antibody typically comprises at least two distinct variable regions, each of which is capable of specifically binding to another antigen or a different epitope of the same antigen. For any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, it can be adapted to the use of antigen-binding fragments of the antibodies of the invention using conventional techniques available in the art.

The constant region of an antibody is important for the ability of the antibody to fix complement and to modulate cell-dependent cellular cytotoxicity. Therefore, the isotype of the antibody can be selected depending on whether it is necessary to regulate cytotoxicity by the antibody.

The term "human antibody" as used herein is intended to include antibodies comprising variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may still include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random mutagenesis or site-specific mutagenesis in vitro or by somatic mutation in vivo) Such amino acid residues can be included, for example, in the CDRs, particularly in CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from another mammalian species, such as mice, are grafted onto human framework sequences.

The term "recombinant human antibody" as used herein is intended to include all human antibodies produced, expressed, produced or isolated by recombinant methods, such as antibodies expressed by recombinant expression vectors transfected into a host cell (described further below) An antibody isolated from a recombinant, combined human antibody library (described further below), an antibody isolated from a human immunoglobulin gene transgenic animal (eg, a mouse) (see, eg, Taylor et al. (1992) Nucl .Acids Res. 20:6287-6295), or any other antibody produced, expressed, produced or isolated by methods involving the splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies contain variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies subjected to in vitro mutagenesis (or, when using the sequence of human Ig transgenic animal, subjected to in vivo somatic mutagenesis), so that the region of the V _H and V _L, recombinant antibodies amino acid sequence region of the sequences that become: although they are derived from sequences of human germline V _H and V _L, and the sequence associated with them, but may not naturally exist in the human germline antibody repertoire in vivo.

Human antibodies can exist in two forms that are associated with hinge heterogeneity. In the first form, an immunoglobulin molecule comprises a stable four-stranded construct having a molecular weight of about 150-160 kDa, wherein the two dimers are linked together by a heavy interchain disulfide bond. In the second form, the dimer is not linked by a heavy chain inter-disulfide bond, but forms a molecular weight of approximately 75- consisting of a covalently coupled light and heavy chain (half antibody). 80kDa molecule. Separation of these forms has been extremely difficult, even after affinity purification.

The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with isotypes of the hinge region of the antibody. The single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the frequency of occurrence of the second form (Angal et al. (1993) Molecular Immunology 30: 105) to levels normally observed with human IgGl hinges. The present invention includes antibodies comprising one or more mutations in the hinge region, C _H 2 or C _H 3 region area; the mutation may be desirable, for example, in production to improve the yield of the desired antibody form.

As used herein, "isolated antibody" means an antibody that is recognized, isolated, and/or collected from at least one component of its natural environment. For example, an antibody that is isolated or removed from at least one component of an organism or from a tissue or cell in which the antibody naturally occurs or naturally occurs is an "isolated antibody" consistent with the objectives of the present invention. "Isolation antibodies" also include in situ antibodies in recombinant cells. An isolated antibody is one that undergoes at least one purification or isolation step. According to certain embodiments, the isolated antibody may be substantially free of other cellular material and/or chemicals.

"Specific binding" or like terms means that an antibody or antigen-binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include methods such as equilibrium dialysis, surface plasmonic resonance, and the like. For example, as the context of the present invention to PCSK9 term "specifically binds to" an antibody or portion thereof comprises an antibody binding to PCSK9, and resonance by measuring surface plasmon, which K _D of less than about 1000 nM, less than about 500 nM, Less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM. However, isolated antibodies that specifically bind to human PCSK9 may have cross-reactivity to other antigens, such as PCSK9 molecules derived from other (non-human) species.

An anti-PCSK9 antibody useful in the methods of the invention may comprise one or more amino acid substitutions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibody is derived. , inserts and/or missing. Such mutations can be readily demonstrated by comparing the amino acid sequences disclosed herein to germline sequences obtainable, for example, from public antibody sequence databases. The invention includes methods involving the use of antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein one or more of the one or more amino acid regions within the framework regions and/or CDR regions are mutated to a derivative The corresponding residue of the germline sequence of the antibody, or the corresponding residue of another human germline sequence, or a conservative amino acid substitution that is mutated to the corresponding germline sequence residue (such sequence changes are collectively referred to herein) It is a "species mutation"). Starting from the heavy and light chain variable region sequences disclosed herein, one of ordinary skill in the art to which the present invention pertains readily produces a variety of antibodies and antigen-binding fragments thereof comprising one or more single germline mutations or a combination thereof. In certain embodiments, the V _H and / or all of the framework and / or CDR residues V _L region were back-mutated residues found in the original sequence derived from the germline antibody. In other embodiments, only certain residues are back-mutated to the original germline sequence, eg, a mutant residue found only in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or Mutant residues found only in CDR1, CDR2 or CDR3 are back-mutated to the original germline sequence. In other embodiments, one or more of the framework and/or CDR residues are mutated to the corresponding residues of a different germline sequence (ie, a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, an antibody of the invention may comprise any combination of two or more germline mutations in the framework regions and/or CDR regions, for example, wherein some of the residues are mutated to the corresponding residues of a particular germline sequence, respectively. Other residues other than the original germline sequence remain unchanged or mutated to the corresponding residues of a different germline sequence. Once an antibody and antigen-binding fragment containing one or more germline mutations are obtained, it is easy to test one or more of the desired properties, such as improved binding specificity, increased binding affinity, enhanced or enhanced biological properties of antagonism or resistance. (Conditional), decreased immunogenicity, etc. The use of antibodies and antigen-binding fragments obtained in this general manner is included within the scope of the invention.

The invention also encompasses methods involving the use of an anti-PCSK9 antibody comprising a variant of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein, the amino acid sequence comprising one or more conservative amino acid substitutions. For example, the invention encompasses the use of an anti-PCSK9 antibody comprising an HCVR, LCVR and/or CDR amino acid sequence, the amino acid sequence comprising, for example, 10 relative to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein. One or less, 8 or less, 6 or less, 4 or less, and the like are replaced by a conservative amino acid.

The term "surface plasmon resonance" as used herein, refers to an optical phenomenon that may be utilized based on the optical phenomenon, for example, BIAcore ^TM system (Biacore Life Sciences division of GE Healthcare , Piscataway, NJ) to detect protein concentration in a biological matrix of the sensor Change to analyze real-time interactions.

As used herein, the term "K _D" means that a particular antibody - antigen interaction equilibrium dissociation constant.

The term "epitope" refers to an epitope that interacts with a specific antigen binding site in the variable region of the antibody molecule known as the paratope of the antigen. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions of an antigen and can produce Different biological effects. Epitopes can be either conformational or linear. A conformational epitope is produced by an amino acid juxtaposed by different fragment spaces of a linear polypeptide chain. Linear epitopes are formed by adjacent amino acid residues on the polypeptide chain. In some cases, an epitope may include a group such as a glycosyl group, a phosphonium group, or a sulfonyl group on the antigen.

According to certain embodiments, an anti-PCSK9 antibody for use in the methods of the invention is an antibody having pH dependent binding characteristics. The expression "pH-dependent binding" as used herein means that the antibody or antigen-binding fragment thereof exhibits the characteristics of "the binding strength to PCSK9 at acidic pH is lower than under neutral conditions" (for the present disclosure) For the purpose, the two expressions are used interchangeably). For example, an antibody having a "pH-dependent binding characteristic" includes an antibody having an affinity to PCSK9 under neutral conditions higher than that under acidic pH conditions or an antigen-binding fragment thereof. In certain embodiments, the antibodies of the invention and antigenic fragments thereof bind to PCSK9 at neutral pH conditions at least 3, 5, 10, 15, 20, 25 above the acidic pH. 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 times or more.

In accordance with this aspect of the invention, an anti-PCSK9 antibody having pH-dependent binding characteristics may have one or more amino acid variations relative to the parent anti-PCSK9 antibody. For example, an anti-PCSK9 antibody having a pH-dependent binding profile may comprise one or more histidine substitutions or insertions, for example, in one or more CDRs of a parent anti-PCSK9 antibody. Thus, in accordance with certain embodiments of the invention, methods are provided, comprising administering an anti-PCSK9 antibody comprising a CDR-amino acid sequence (eg, a heavy chain and a light chain CDR), in addition to one of the one or more CDRs of the parent antibody The CDR amino acid sequence is identical to the CDR amino acid sequence of the parent anti-PCSK9 antibody, except that the plurality of amino acids are replaced by a histidine residue. An anti-PCSK9 antibody having pH-dependent binding characteristics may have, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more histidine replacements, which may be present in the parent The single CDR of the antibody, or is dispersed in a plurality (eg, 2, 3, 4, 5 or 6) of the CDRs of the parent anti-PCSK9 antibody. For example, the invention encompasses the use of pH-dependent binding An anti-PCSK9 antibody comprising one or more histidine substitutions in the HCDR1 of the parent anti-PCSK9 antibody, one or more histidine substitutions in the HCDR2, one or more histidine substitutions in the HCDR3, LCDR1 One or more histidine replacements are included, one or more histidine substitutions are included in LCDR2, and/or one or more histidine substitutions are included in LCD3.

As used herein, the expression "acidic pH" means a pH of 6.0 or less (e.g., less than about 6.0, less than about 5.5, less than about 5.0, etc.). The expression "acid pH" includes about 6.0, 5.95, 5.90, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0. , or lower pH. As used herein, the expression "neutral pH" means a pH of from about 7.0 to about 7.4. The expression "neutral pH" includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

Preparation of human antibody

Methods for producing human antibodies in transgenic mice are known in the art. Any such known method can be used in the context of the present invention to produce a human antibody that specifically binds to human PCSK9.

Using a VELOCIMMUNE ^(TM) technology (see, e.g., U.S. Patent No. 6,596,541 to Regeneron Pharmaceuticals, Inc.) or any other known technique for producing monoclonal antibodies, a high affinity for PCSK9 is initially isolated containing a human variable region and a mouse constant region. Chimeric antibody. The VELOCIMMUNE® technique involves the development of a transgenic mouse that contains a number of human heavy and light chain variable regions operably linked to an endogenous mouse constant region locus, allowing the mouse to respond to the antigen. Stimulation produces an antibody comprising a human variable region and a mouse constant region. DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to DNA encoding human heavy and light chain constant regions. The DNA is then expressed in cells that are capable of expressing a fully human antibody.

Usually, challenge the VELOCIMMUNE® mouse with the antigen of interest and The antibody-expressing mouse recovers lymphocytes (such as B cells). The lymphocytes can be fused with a myeloma cell line to prepare an immortal hybridoma cell line, and then the hybridoma cell line is screened to identify a hybridoma cell line capable of producing an antibody specific for the relevant antigen. DNA encoding the heavy and light chain variable regions can be isolated and ligated to the desired constant region of the heavy and light chains. This antibody protein can be produced in a cell such as CHO cells. Alternatively, DNA encoding antigen-specific chimeric antibodies or light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

First, a high affinity chimeric antibody comprising a human variable region and a mouse constant region was isolated. The antibody is identified and selected according to the desired characteristics, including affinity, selectivity, epitope, etc., using standard procedures known to those of ordinary skill in the art to which the invention pertains. The mouse constant region is replaced with a desirable human constant region to produce a fully human antibody of the invention, such as wild-type or modified IgGl or IgG4. The selected constant region can vary depending on the particular use, while high affinity antigen binding properties and target specific features remain in the variable region.

In general, antibodies useful in the methods of the invention have high affinity as described above when assayed in a manner that binds to an antigen immobilized in a solid or liquid phase. The mouse constant region is replaced with a desirable human constant region to produce a fully human antibody of the invention. The selected constant region can vary depending on the particular use, while high affinity antigen binding properties and target specific features remain in the variable region.

Specific examples of antigen-binding fragments of human antibodies or antibodies that specifically bind to PCSK9 useful in the context of the methods of the invention, including any of the three heavy chain CDRs (HCDR1, HCDR2) within the heavy chain variable region (HCVR) An antibody or antigen-binding fragment of HCDR3), and the amino acid sequence of the heavy chain variable region is selected from the group consisting of: 2, 18, 22, 26, 42, 46, 50, 66 , 70, 74, 90, 94, 98, 114, 118, 122, 138, 142, 146, 162, 166, 170, 186, 190, 194, 210, 214, 218, 234, 238, 242, 258, 262, 266, 282, 286, 290, 306, 310, 314, 330, 334, 338, 354, 358, 362, 378, 382, 386, 402, 406, 410, 426, 430, 434, 450, 454, 458, 474, 478, 482, 498, 502, 506, 522, 526, 530, 546, 550, 554, 570, 574, 578, 594, 598, 602, 618, 622, 626, 642, 646, 650, 666, 670, 674, 690, 694, 698, 714, 718, 722, 738 and 742, or a sequence that is substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. The antibody or antigen-binding fragment may comprise three light chain CDRs (LCVR1, LCVR2, LCVR3) within the light chain variable region (LCVR), and the light chain variable region comprises an amino acid sequence selected from the group consisting of , whose serial numbers are: 10, 20, 24, 34, 44, 48, 58, 68, 72, 82, 92, 96, 106, 116, 120, 130, 140, 144, 154, 164, 168, 178, 188, 192, 202, 212, 216, 226, 236, 240, 250, 260, 264, 274, 284, 288, 298, 308, 312, 322, 332, 336, 346, 356, 360, 370, 380, 384, 394, 404, 408, 418, 428, 432, 442, 452, 456, 466, 476, 480, 490, 500, 504, 514, 524, 528, 538, 548, 552, 562, 572, 576, 586, 596, 600, 610, 620, 624, 634, 644, 648, 658, 668, 672, 682, 692, 696, 706, 716, 720, 730, 740 and 744, or a substantially similar thereto having at least A sequence of 90%, at least 95%, at least 98% or at least 99% sequence identity.

In certain embodiments of the invention, the antibody or antigen-binding fragment thereof comprises 6 CDRs derived from heavy and light chain variable region amino acid sequence pairs (HCVR/LCVR) (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3), the amino acid sequence pair is selected from the group consisting of: 2/10, 18/20, 22/24, 26/34, 42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118/120, 122/130, 138/140, 142/144, 146/ 154, 162/164, 166/168, 170/178, 186/188, 190/192, 194/202, 210/212, 214/216, 218/226, 234/236, 238/240, 242/250, 258/260, 262/264, 266/274, 282/284, 286/ 288, 290/298, 306/308, 310/312, 314/322, 330/332, 334/336, 338/346, 354/356, 358/360, 362/370, 378/380, 382/384, 386/394, 402/404, 406/408, 410/418, 426/428, 430/432, 434/442, 450/452, 454/456, 458/466, 474/476, 478/480, 482/ 490, 498/500, 502/504, 506/514, 522/524, 526/528, 530/538, 546/548, 550/552, 554/562, 570/572, 574/576, 578/586, 594/596, 598/600, 602/610, 618/620, 622/624, 626/634, 642/644, 646/648, 650/658, 666/668, 670/672, 674/682, 690/ 692, 694/696, 698/706, 714/716, 718/720, 722/730, 738/740 and 742/744.

In certain embodiments of the invention, an anti-PCSK9 antibody or antigen-binding fragment thereof useful in the methods of the invention comprises an HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 amino acid sequence comprising a set of sequences selected from the group consisting of The serial numbers are: 76/78/80/84/86/88 (mAb 316P) and 220/222/224/228/230/232 (mAb 300N) (see U.S. Patent Application Publication No. 2010/0166768).

In certain embodiments of the invention, the antibody or antigen-binding fragment thereof comprises a pair of HCVR/LCVR amino acid sequences selected from the group consisting of: 2/10, 18/20, 22 /24, 26/34, 42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118/120 , 122/130, 138/140, 142/144, 146/154, 162/164, 166/168, 170/178, 186/188, 190/192, 194/202, 210/212, 214/216, 218 /226, 234/236, 238/240, 242/250, 258/260, 262/264, 266/274, 282/284, 286/288, 290/298, 306/308, 310/312, 314/322 , 330/332, 334/336, 338/346, 354/356, 358/360, 362/370, 378/380, 382/384, 386/394, 402/404, 406/408, 410/418, 426 /428, 430/432, 434/442, 450/452, 454/456, 458/466, 474/476, 478/480, 482/490, 498/500, 502/504, 506/514, 522/524 , 526/528, 530/538, 546/548, 550/552, 554/562, 570/572, 574/576, 578/586, 594/596, 598/600, 602/610, 618/620, 622/624, 626/634, 642/ 644, 646/648, 650/658, 666/668, 670/672, 674/682, 690/692, 694/696, 698/706, 714/716, 718/720, 722/730, 738/740 and 742/744.

Pharmaceutical composition and method of administration

The invention includes methods for administering a PCSK9 inhibitor to a patient, the PCSK9 inhibitor being included in a pharmaceutical composition. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients, and other agents which impart various desirable properties to the drug, such as transfer, delivery, tolerance, and the like. A number of suitable formulations can be found in the following formulas known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (cationic or anionic) vesicles (such as LIPOFECTIN ^TM), DNA conjugates, anhydrous absorbent pastes, An oil-in-water or water-in-oil emulsion, a latex-like carbon wax (polyethylene glycol of various molecular weights), a semi-solid gel, and a semi-solid mixture containing a carbon wax. See also Powell et al. "Compendium of excipients for parenteral formulations", PDA (1998) J Pharm Sci Technol 52:238-311.

Various drug delivery systems are known for administration of the pharmaceutical compositions of the present invention, such as liposome encapsulation, microparticles, microcapsules, recombinant cells capable of expressing a mutant virus, and endocytosis of a receptor vector (see, for example, Wu et Al., 1987, J. Biol. Chem. 262: 4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral. The composition can be administered by any convenient route, for example, infusion or bolus injection, absorbed through the epithelium or mucosa (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered with other bioactive agents.

The pharmaceutical composition of the present invention can be subcutaneously or statically treated with a standard needle and syringe Pulse administration. Further, for subcutaneous administration, a pen type drug delivery device can be conveniently used for administration of the pharmaceutical composition of the present invention. Such pen type drug delivery devices can be of the reusable or disposable type. Reusable pen-type drug delivery devices typically employ a replaceable cartridge containing a pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been delivered and the cartridge is empty, the empty cartridge can be conveniently disposed of and replaced with a new cartridge containing the pharmaceutical composition. Then, the pen type drug delivery device can be used repeatedly. In a single-use pen type drug delivery device, there is no replaceable cartridge. Rather, the single use pen type drug delivery device has a reservoir that is prefilled with a pharmaceutical composition. Once the pharmaceutical composition in the reservoir is used up, the entire device is disposed of.

Many reusable pen type and automatic injectable drug delivery devices have been used for subcutaneous administration of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPENT ^(TM) (Owen Mumford, Inc., Woodstock, UK), DISETRONIC ^(TM) pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25 ^(TM) pen, HUMALOG ^(TM) pen, HUMALIN 70/30 ^(TM) Pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN ^TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR ^TM (Novo Nordisk, Copenhagen, Denmark), BD ^TM pen (Becton Dickinson, Franklin) ^{Lakes, NJ), OPTIPEN TM,} OPTIPEN PRO TM, OPTIPEN STARLET TM, and ^{OPTICLIK TM (sanofi-aventis, Frankfurt} , Germany), to name just a few. Examples of disposable pen delivery devices for subcutaneous pharmaceutical compositions of the present invention include, but are not limited to SOLOSTAR ^TM pen ^{(sanofi-aventis), FLEXPEN TM} (Novo Nordisk), KWIKPEN TM (Eli Lilly), SURECLICK self ^TM syringe (Amgen, Thousand Oaks, CA) , PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and of HUMIRA pen ^TM (Abbott Labs, Abbott Park IL) , to name a few.

In some cases, the pharmaceutical composition can be administered using a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit.Ref.Biomed.Eng.14:201). In another embodiment, polymeric materials can be employed; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed adjacent to the target of the composition such that only a small portion of the systemic dose is required (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol .2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249: 1527-1533.

Formulations for injection may include intravenous, subcutaneous, intradermal and intramuscular injections, infusion solutions, and the like. These injectable preparations can be prepared by known methods. For example, the above antibody or a salt thereof can be dissolved, suspended or emulsified in a sterile aqueous medium or an oil medium conventionally used for injection to prepare the preparation for injection. As the aqueous medium for injection, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, and the like, and a suitable solubilizing agent such as an alcohol (such as ethanol) or a polyhydric alcohol (such as propylene glycol or polyethylene glycol) may be used in combination. A nonionic surfactant [for example, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and the like. As the oily medium, for example, sesame oil, soybean oil or the like can be used, and a solubilizing agent such as benzyl benzoate or benzyl alcohol can be used in combination. The injection thus prepared is preferably filled into a suitable ampoule.

Advantageously, the above-described oral or parenteral pharmaceutical compositions are prepared in unit dosage form to contain a single dose of the active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injections (in ampoules), suppositories, and the like.

dose

The amount of PCSK9 inhibitor (e.g., anti-PCSK9 antibody) administered to a subject in accordance with the methods of the invention is generally a therapeutically effective amount. The term "therapeutically effective amount" as used herein means a dose of a PCSK9 inhibitor which results in a selected group including residual cholesterol (RLP-C), VLDL-C, VLDL ₁ -C, VLDL ₂ -C, VLDL ₁₊ Detectable decrease in one or more parameters of ₂ -C, VLDL ₃ -C, IDL-C, LDL ₁ -C, LDL ₂ -C, LDL ₃ -C, LDL ₄ -C, LDL ₃₊₄ -C (At least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% from baseline) or above). Alternatively, an animal model can be utilized to determine if a particular amount of a candidate PCSK9 inhibitor is a therapeutically effective amount.

For anti-PCSK9 antibodies, a therapeutically effective amount thereof can be from about 0.05 mg to about 600 mg, such as about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, About 40 mg, about 50 mg, about 60 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg About 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, About 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, or about 600 mg.

The amount of anti-PCSK9 antibody contained in each dose can be expressed as milligrams of antibody per kilogram of patient body weight (i.e., mg/kg). For example, an anti-PCSK9 antibody can be administered to a patient at a dose of from about 0.0001 mg/kg to about 10 mg/kg, depending on the patient's weight.

Combination therapy

According to certain embodiments, the method of the invention may comprise administering to a patient a pharmaceutical composition comprising an anti-PCSK9 antibody, which is receiving a hypercholesterolemia at or just prior to administration of the pharmaceutical composition of the invention. Treatment. For example, patients previously diagnosed with hypercholesterolemia may already be on prescription and in anti-PCSK9 antibodies. A stable treatment of another drug is being received before and/or at the same time as the pharmaceutical composition is administered. The above-mentioned previous or simultaneous treatment may include, for example, (1) an agent which induces depletion of cholesterol-synthesized cells by inhibiting 3-hydroxy-3-methylpentadienyl-coenzyme A (HMG-CoA) reductase, for example, Statins (eg, cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, etc.); (2) inhibition of cholesterol absorption And/or an inhibitor of bile acid reuptake; (3) an agent that promotes lipoprotein catabolism (such as niacin); and/or (4) LXR transcription that plays a role in the clearance of cholesterol such as 22-hydroxycholesterol Activator of the factor. In certain embodiments, the patient is receiving a fixed combination of therapeutic agents, such as a combination of ezetimibe and simvastatin, a statin and a bile resin, prior to or concurrent with administration of the anti-PCSK9 antibody (eg, a combination of enamine, colestipol, and colesevelam; a combination of niacin with a statin such as lovastatin niacin; or with other lipid lowering agents such as omega-3-fatty acid ethyl ester A combination of (eg omacor).

Dosing regimen

In accordance with certain embodiments of the invention, multiple doses of a PCSK9 inhibitor (e.g., a pharmaceutical composition comprising a PCSK9 inhibitor) can be administered to a subject over a period of time. A method according to this aspect of the invention comprises sequentially administering to a subject multiple doses of the same PCSK9 inhibitor. The term "sequential administration" as used herein refers to the administration of a subject per PCSK9 inhibitor at different time points, for example, at predetermined intervals (eg, hours, days, weeks, or months). . The invention includes methods comprising sequentially administering to a subject an initial dose of a PCSK9 inhibitor, and then administering one or more doses of a second dose of a PCSK9 inhibitor, and then optionally administering a dose or Multiple doses of the third dose of PCSK9 inhibitor.

The terms "initial dose", "second dose" and "third dose" refer to the chronological order in which the pharmaceutical compositions containing the PCSK9 inhibitor are administered. Therefore, the "initial dose" refers to the dose administered at the beginning of the treatment regimen (also known as the "baseline dose"); The "dose" refers to the dose administered after the initial dose; the "third dose" refers to the dose administered after the second dose. The initial, second and third doses may all contain the same amount of PCSK9 inhibitor, but the frequency of administration may generally differ from one another. However, in certain embodiments, the amount of PCSK9 inhibitor contained in the initial, second, and/or third doses varies from one another during treatment (e.g., up- or down-regulated, as appropriate). In certain embodiments, two or more doses (eg, 2, 3, 4, or 5 doses) of "loading dose" are administered at the beginning of the treatment regimen, and subsequent doses are administered on a lower frequency basis ( That is, "maintenance dose").

In accordance with some exemplary embodiments of the present invention, each dose of the second dose and/or the third dose is between 1 and 26 weeks after the administration of the previous dose (eg, 1, 11⁄2, 2, 21⁄2, 3, 31⁄2) 4, 41⁄2, 5, 51⁄2, 6, 61⁄2, 7, 71⁄2, 8, 81⁄2, 9, 91⁄2, 10, 101⁄2, 11, 11⁄2, 12, 121⁄2, 13, 131⁄2, 14, 141⁄2, 15, 151⁄2, 16, Dosing when 161⁄2, 17, 171⁄2, 18, 181⁄2, 19, 191⁄2, 20, 201⁄2, 21, 211⁄2, 22, 221⁄2, 23, 231⁄2, 24, 241⁄2, 25, 251⁄2, 26, 261⁄2 weeks, or more. As used herein, the term "previous dose" refers to a dose of antigen-binding molecule administered to a patient prior to the next dose of administration during sequential multiple administrations, with no other dose between the two doses.

A method according to this aspect of the invention may comprise administering to the patient any number of second and/or third PCSK9 inhibitor doses. For example, in certain embodiments, only one dose of the second dose is administered to the patient. In other embodiments, the patient is administered a second dose of two or more doses (eg, 2, 3, 4, 5, 6, 7, 8 or more doses). Also, in certain embodiments, only one dose of the third dose is administered to the patient. In other embodiments, the patient is administered two or more doses (eg, 2, 3, 4, 5, 6, 7, 8 or more doses) of the third dose.

In some embodiments involving multiple doses of the second dose, each dose of the second dose can be administered at the same frequency as the other second dose. For example, each dose of the second dose can be administered to the patient at 1 to 2, 4, 6, 8 or more weeks after the previous dose. Again, in some involve more In the third dose of the embodiment, each dose of the third dose can be administered at the same frequency as the other third dose. For example, a third dose per dose can be administered to a patient at 1 to 2, 4, 6, 8 or more weeks after the previous dose. Alternatively, the frequency of administration of the second and/or third dose to the patient can be varied during the course of the treatment regimen. During the course of treatment, the frequency of administration can also be adjusted by the doctor after the clinical examination according to the needs of each patient.

Instance

The following examples are given to provide a complete disclosure and description of how to make use of the methods and compositions of the present invention, and are not intended to limit the scope of the invention. Efforts have been made to ensure the accuracy of the data used (eg, dose, temperature, etc.), but certain experimental errors and deviations should also be considered. Unless otherwise indicated, parts refer to parts by weight, molecular weight refers to average molecular weight, temperature refers to degrees Celsius, and pressure refers to atmospheric pressure or near atmospheric pressure.

Example 1: Production of human antibodies to human PCSK9

Human antibodies against PCSK9 are produced as described in U.S. Patent No. 8,062,640. An exemplary PCSK9 inhibitor used in the following examples is a human anti-PCSK9 antibody (also referred to as "alirocumab" in the scientific literature) called "mAb316P". mAb316P is characterized by the following amino acid sequence: heavy chain variable region (HCVR) containing sequence 90; light chain variable region (LCVR) containing sequence 92; heavy chain complementarity determining region 1 containing sequence 76 ( HCDR1); HCDR2 containing sequence 78; HCDR3 containing sequence 80; light chain complementarity determining region 1 (LCDR1) containing sequence 84; LCDR2 containing sequence 86; and LCDR3 containing sequence No. 88.

Example 2: Dynamic relationship between anti-PCSK9 antibody levels and low-density lipoprotein cholesterol (LDL-C) levels introduction

Pharmacokinetic parameters generated for Phase I and Phase II mAb316P studies Narrative analysis was performed with pharmacodynamic data to characterize the relationship between mAb316P, PCSK9 and LDL-C levels. This analysis included three different clinical trials:

(1) Single-dose study: Phase I, single-dose study in healthy subjects who did not receive statin background treatment, in which subjects received a single dose of subcutaneous (SC) injection of mAb316P 50, 100, 150 And 250mg or placebo.

(2) Multi-dose study: in patients with heterozygous familial hypercholesterolemia (HeFH), non-familial hypercholesterolemia (FH), and non-FH patients receiving only dietary interventions treated with atorvastatin Phase I group study. Patients treated with atorvastatin were randomized to either the mAb316P group (50, 100 or 150 mg) or the placebo group, subcutaneously on days 1, 29 and 43 respectively. Non-FH patients who received only dietary intervention were randomized to either the mAb316P group (150 mg) or the placebo group, subcutaneously on days 1 and 29, respectively.

(3) Phase II study: a 12-week study of hypercholesterolemia patients treated with atorvastatin: patients were randomized to a subcutaneous injection of 50, 100 every 2 weeks (Q2W), or The 150 mg mAb 316P group was injected subcutaneously with a 200 or 300 mg mAb 316P group or a placebo group every 4 weeks (Q4W).

result

A single dose of subcutaneously injected 150 mg of mAb 316P rapidly bound to PCSK9 and rapidly reduced the level of free PCSK9 in the blood circulation; followed by a decrease in LDL-C. It has been noted that free PCSK9 rises back to its original level prior to LDL-C (Figure 1). On day 3 after subcutaneous injection of mAb316P 50, 100 or 150 mg in HeFH or non-FH patients, free PCSK9 levels reached their lowest point (Figure 2). Compared with patients receiving mAb316P plus atorvastatin, patients receiving mAb316P with only dietary intervention showed a slower rise in free PCSK9 to the initial level (Figure 2). The mean decrease in mean LDL-C occurred on day 8 for the 50 mg and 100 mg dose groups and on day 15 for the 150 mg dose group; this delay relative to free PCSK9 concentration may be due to new generation Caused by the time required for LDL-C receptors. A representative hysteresis curve showing the relationship between total mAb 316P and LDL-C after administration at a dose of 150 mg is shown in FIG.

Higher baseline free PCSK9 levels were observed in patients receiving statins compared to patients receiving only dietary intervention (Figure 4). Compared with mAb316P treatment alone with dietary intervention, the target-mediated clearance of mAb316P increased with statin therapy (due to higher free PCSK9 levels, it is likely to reflect an increase in production rate). In a 4-week dosing interval (Day 1 to Day 29), this appears to affect the duration of LDL-C decline compared to LDL-C reduction with only dietary intervention and statin therapy. However, this effect was not observed in a 2-week dosing interval (day 29 to day 43) (Fig. 5).

In hypercholesterolemia (non-FH) patients receiving concurrent statin therapy, mAb316P administration every 2 weeks resulted in a sustained and steady decrease in LDL-C at week 12. LDL-C response showed less variability at week 12 compared to once every 4 weeks for patients who received dosing once every 2 weeks (Figure 6); 150 mg administered every 2 weeks vs. 4 The coefficient of variation for 300 mg administered once a week was 16% and 49%, respectively.

in conclusion

The dynamic relationship between mAb316P, free PCSK9 and LDL-C levels is evident. Treatment with mAb316P resulted in a decrease in free PCSK9 levels within 3 days after administration and a decrease in LDL-C at 8-15 days after administration.

The clearance rate of mAb316P is accelerated due to its binding to free PCSK9 (target-mediated clearance). Lower levels of free PCSK9 result in a longer therapeutic phase due to lower target-mediated clearance rates. Previous studies have shown that administration of statins increases the production of free PCSK9. In the study analyzed in this study, patients receiving mAb316P plus only dietary intervention had lower baseline than those receiving mAb316P plus atorvastatin. Free PCSK9 levels and higher mAb316P plasma concentrations.

In patients receiving concurrent statins, mAb 316P administration every 2 weeks achieved a small change in the magnitude of LDL-C decline at the end of the dosing period compared to the dosing regimen once every 4 weeks. In the regimen of administration once every 4 weeks, the difference in target-mediated clearance between the patient and the patient (among other factors) appears to result in LDL- between the 2nd week and the 4th week after administration. The difference in the duration of the highest level of C resulted in a large difference in the efficacy achieved after 4 weeks of administration. These differences were not observed in the regimen of administration once every 2 weeks.

Example 3A: Anti-PCSK9 antibody reduces cholesterol concentration in serum residual lipoprotein fraction, very low density lipoprotein, triglyceride and lipoprotein (a) [Lp(a)] introduction

Elevated very low density lipoprotein (VLDL) levels form part of the symptoms of atherosclerotic dyslipidemia and tend to cause premature atherosclerosis. Residual cholesterol is a VLDL lipolysis product, including VLDL ₃ and medium density lipoprotein (IDL, a direct precursor that forms LDL).

Lipoprotein (a) [Lp(a)] consists of apolipoprotein (a) [apo(a)] covalently bound to the apo B component of a cholesterol-rich lipoprotein, the size and low density of the lipoprotein Lipoprotein (LDL) particles are approximately the same. The metabolism of Lp(a) is roughly regulated by heredity, and more than 90% of its plasma concentration is affected by the quantitative and qualitative polymorphism of the apo(a) gene ( LPA ). Elevated Lp(a) levels are considered to be an independent risk factor for cardiovascular disease, and as this level rises, the risk will continue to increase. Although elevated Lp(a) promotes atherosclerosis due to cholesterol deposition in the intima, it also promotes thrombus formation due to its high homology to plasminogen, despite its lack of protease activity. In patients undergoing coronary angiography, Lp(a) concentrations above 30 mg/dL have been shown to be associated with increased risk of angiographic stenosis and major coronary events. In addition, Lp(a) levels and genotypes have been shown to be associated with calcification of the aortic valve, suggesting that it may play a role in the etiology.

The purpose of this example was to test the hypothesis that mAb316P reduces serum levels of VLDL and lipoprotein remnants as a fully human-derived monoclonal antibody against the proprotein convertase subtilisin-9 (PCSK9) The ability to lower serum levels of LDL-C and non-HDL-C and demonstrate that mAb316P reduces the activity of Lp(a) levels in patients.

Three multicenters were performed in patients with primary hypercholesterolemia (study A, n=183; study B, n=92) or patients with heterozygous familial hypercholesterolemia (study C, n=77) , double-blind, parallel-group, placebo-controlled clinical trials. The design and administration profiles of these three clinical trials are shown in Table 1.

In Study B, all patients received atorvastatin (ATV) 10 mg prior to randomization, and titration increased to 80 mg ATV at the start of randomized treatment in the placebo and one mAb 316P groups.

Patients receiving background treatment with atorvastatin or statin +/- ezetimibe are given subcutaneously (SC) once every 2 weeks or once every 4 weeks (Q2W, Q4W) mAb316P 50-300 mg, depending on the study. For all three trials, 150 mg of mAb 316P was administered once every 2 weeks (Q2W).

In the post hoc attribution analysis, the lipoprotein was finely separated by the vertical automatic analysis (VAP) method. The VAP method is a simple, straightforward ultracentrifugation test that separates lipoprotein fractions in a vertical rotor according to lipoprotein density, which allows for the high separation of each lipoprotein and each sublipoprotein. The bottom of the tube was pierced and the cholesterol in each layer was measured using a spectrophotometer after adding an enzymatic cholesterol reagent. At week 12 (study A), week 8 (study B), and week 6 (study C), VLDL-study of patients treated with mAb316P 150 mg once every 2 weeks was analyzed using ANCOVA compared to patients receiving placebo. C, VLDL ₁₊₂ -C (a measure of cholesterol in "large and suspended" VLDL particles), triglycerides (TG), VLDL ₃ -C, IDL-C, and total residual lipoprotein cholesterol levels (RLP-C) ; % change in VLDL ₃ -C+IDL-C).

In addition, baseline and treatment Lp(a) levels were assessed in patients who received mAb316P 150 mg (every 2 weeks) or placebo in these three different Phase 2 studies. In all Phase 2 studies, Lp(a) levels were measured in the same laboratory using the same method. Data from baseline and end-of-treatment Lp(a) levels of the modified intent-to-treat population obtained from these three studies were summarized (values in treatment at week 8/12 or values from the last available treatment); Covariate analysis was performed with treatment groups and studies as fixed effects, with baseline Lp(a) as covariates, and percent change from baseline was compared for both mAb316P 150 mg (every 2 weeks) and placebo. The p-values associated with these exploratory analyses are provided for the purposes of the description only and have not been adjusted for multiplicity. The relationship between the percentage change of both Lp(a) and LDL-C from baseline was evaluated using linear regression and the Spearman correlation coefficient was calculated.

Result/conclusion

In these three studies, a decrease in cholesterol levels in TG, VLDL-C, and residual lipoprotein was observed in comparison with placebo with mAb316P. The results are summarized in Table 2 and Figures 7-9.

In patients with heFH/non-FH, all atherosclerotic lipoproteins (LDL-C, IDL-C, VLDL-C, and RLP subgroups) were compared to background lipid-lowering therapy (statin ± ezetimibe) The fractions were all reduced by mAb316P. Thus, this example shows that in addition to significantly reducing the cholesterol concentration of the residual lipoprotein fraction that can be separated by VAP, including VLDL ₃ -C and IDL-C, mAb 316P also significantly reduced serum TG and VLDL-C.

Data from baseline and treatment Lp(a) were included in 102 of 108 patients who received mAb316P 150 mg (every 2 weeks) and 74 of 77 patients who received placebo in the pooled analysis population. The baseline values are shown in Table 3.

As shown in Table 3, 36 (35%) patients treated with mAb316P and 25 (33%) patients receiving placebo had a baseline Lp(a) > 50 mg/dL, which was considered by the European Society of Arteriosclerosis guidelines. A high-risk segmentation point.

The median Lp(a) absolute decline from the baseline and the percent decrease are summarized in Table 4.

As shown in Table 4, the median decrease in the median Lp(a) from baseline in patients receiving mAb 316P 150 mg (every 2 weeks) was -30.3% ( P < 0.0001) compared to -0.3% of placebo. The absolute decrease in the median Lp(a) from baseline in patients with higher baseline Lp(a) was also significantly higher. In summary, analysis of the summary data from three Phase 2 trials with mAb316P 150 mg (every 2 weeks) showed a significant reduction in Lp(a) relative to placebo, including baseline Lp(a) > 50 mg/dL of patients. In patients with a higher risk of cardiovascular disease due to elevated Lp(a), the percentage decrease in Lp(a) appears to be of similar magnitude, resulting in a larger absolute decrease in Lp(a).

Example 3B: Anti-PCSK9 antibody reduces levels of apolipoprotein CII and CIII in serum introduction

Apolipoproteins (apo) CII and CIII activate and inhibit lipoprotein lipase (LPL), respectively. LPL hydrolyzes the triglyceride moiety of VLDL and its residue. In this example, the ability of the anti-PCSK9 antibody, mAb316P, to reduce VLDL-C and its remnants by affecting serum levels of apolipoproteins CII and CIII was investigated.

In patients with primary non-familial hypercholesterolemia (non-FH) (study A, n=183; study B, n=92) or heterozygous familial hypercholesterolemia (heFH) patients (study C, n Three multicenter, double-blind, parallel-group, placebo-controlled clinical trials were performed in =77). Depending on the study protocol, patients treated with mAb316P 50-150 mg once every 2 weeks (Q2W) or 150-300 mg once every 4 weeks (Q4W) (accepting atorvastatin or statin +/- ezetimibe) . For all three trials, 150 mg of mAb 316P was administered once every 2 weeks (Q2W). (See Example 3A, Table 1).

In the post hoc attribution analysis, apo CII and CIII were determined by immunoassay. At week 12 (Study A), Week 8 (Study B), and Week 6 (Study C), apo CII and apo were analyzed in the placebo group using ANCOVA compared with patients treated with mAb316P 150 mg every 2 weeks. Percent change in CIII from baseline.

result

In all three studies, apo CII was reduced by 21% (p<0.00001), 29.3% in patients treated with mAb316P, compared with placebo (mean at weeks 12, 8, and 6 respectively). p = 0.0067) and 9.4% (p = 0.08) (all are average values). Apo CIII decreased by 24.7% (p<0.00001), 22.9% (p=0.017), and 20.2% (p=0.019), respectively.

in conclusion

Treatment with the anti-PCSK9 antibody mAb316P was associated with a significant decrease in serum apo CII and CIII. In non-FH patients, both apo CII and CIII had a nearly 1:1 decline. In patients with heFH, the decrease in apo CIII was approximately twice as greater than the decrease in apo CII. Apo CII and CIII The decrease may be a manifestation of a decrease in VLDL particle production/secretion, and the VLDL particle is a known carrier of apolipoprotein.

Example 4: Anti-PCSK9 antibody reduces cholesterol concentration in all serum low density lipoprotein fractions introduction

Low density lipoprotein (LDL-C) is composed of LDL particles of various densities and lipid states. Analysis of the effects of lipid-modulating therapy on the composition of LDL particles can help to understand the mode of action of the treatment. The purpose of this example was to test the hypothesis that mAb 316P reduces LDL-C levels by reducing multiple LDL components by acting as a fully human monoclonal antibody against the proprotein convertase subtilisin-9 (PCSK9).

Three multicenters were performed in patients with primary hypercholesterolemia (study A, n=183; study B, n=92) or patients with heterozygous familial hypercholesterolemia (study C, n=77) , double-blind, parallel-group, placebo-controlled clinical trials. Depending on the study protocol, use mAb316P 50-150mg every 2 weeks (Q2W) or 150-300mg (every 4 weeks (Q4W) treatment (receive atorvastatin or statin +/- ezetimibe) Patients. For all three trials, 150 mg of mAb 316P was administered every 2 weeks (Q2W) (see Example 3A, Table 1).

In the post hoc attribution analysis, the lipoprotein was finely separated by the vertical automatic analysis (VAP) method. LDL ₁ -C ("large and suspended" LDL), LDL ₂ -C, LDL ₃ -C, and LDL ₄ -C were evaluated in patients who received mAb316P 150 mg once every 2 weeks, compared with patients receiving placebo. The percentage change of "small and dense" LDL levels from baseline to week 12 (Study A), Week 8 (Study B), and Week 6 (Study C). The LDL ₃ -C and LDL ₄ -C components were separately analyzed and analyzed in a pooled analysis. This is done because LDL ₄ -C is usually present at much lower concentrations than LDL _3-4 -C. There was a much more significant change in the percentage decrease in LDL ₄ -C. The sum of LDL ₃₊₄ -C represents the sum of the two smallest and most dense LDL-C components.

Result/conclusion

In these three studies, patients who received 150 mg of mAb316P every 2 weeks were observed to have a significant decrease in cholesterol levels from all baseline VDL-separated LDL subfractions compared with placebo. The results are summarized in Table 5 and Figures 10-12.

Thus, this example shows that treatment with a monoclonal antibody (mAb316P) targeting PCSK9 significantly reduces the cholesterol concentration in the LDL fraction that can be separated by VAP. The LDL-C decline caused by the previously shown mAb316P was confirmed on all LDL-C subcomponents.

Example 5: Effect of anti-PCSK9 antibody on lipoprotein particle concentration background

The cholesterol levels of low density lipoprotein particles (LDL-P) and high density lipoprotein particles (HDL-P) can vary widely from individual to individual. For example, one person may have a larger cholesterol-rich LDL-P, while another person has a smaller cholesterol-depleted LDL-P. Therefore, the measurement of serum levels of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) is often independent of the number of LDL-P and HDL-P in the blood circulation.

Epidemiological studies have shown that the risk of atherosclerotic cardiovascular disease is associated with both LDL-C and LDL-P. However, in a population of patients whose measurement results are inconsistent (such as diabetes or metabolic syndrome patients), it is shown that this risk is often more correlated with LDL-P (rather than LDL-C). Therefore, many expert groups and consensus statements advocate the addition of LDL-P as a therapeutic target in the management of high-risk patients.

Nuclear magnetic resonance (NMR) technology accurately measures lipoproteins in the blood circulation The number of granules to identify patients who require intensive treatment with elevated levels of LDL-P inconsistency.

The anti-PCSK9 antibody mAb316P has been shown to reduce LDL-C by 72.4% in patients receiving stable doses of atorvastatin during the 12-week period, and the incidence of adverse events was similar between the placebo group and the mAb316P-treated group. mAb316P reduced apolipoprotein B by 56%, which is directly proportional to the change in LDL-C. To date, the effect of mAb316P on the number of lipoprotein particles in hypercholesterolemic patients has not been reported.

This example evaluates the effect of mAb316P on the concentration and size of lipoprotein particles.

method

For LDL-C In a 100 mg/dL patient, a randomized, double-blind, phase II trial was conducted to receive a placebo in addition to daily stable treatment with atorvastatin (10, 20 or 40 mg per day). =31) or mAb316P 150mg subcutaneous injection (SC) every 2 weeks (Q2W) (n=28). Blood lipid and lipoprotein tests were performed using samples taken after fasting for 12 hours overnight. LipoSite-3 algorithm was used at LipoScience (Raleigh, NC) to measure lipoprotein particle data using an NMR spectrometer. Based on the amplitude of the unique lipid methyl NMR signal of the spectrometer, the LDL-P and HDL-P subclasses were quantified and the sum of the diameters of each subclass from its relative mass percentage based on the amplitude of the methyl NMR signal, A weighted average of the LDL and HDL sizes is derived.

The estimated diameter ranges for each subclass are as follows: VLDL-P: small VLDL-P = 29 to 42 nm; medium VLDL-P = 42 to 60 nm; large VLDL-P > 60 nm; LDL-P: small LDL-P = 18 to 20.5 Nm; large LDL-P = 20.5 to 23 nm; medium density lipoprotein particles (IDL-P) = 23 to 29 nm; HDL-P: small HDL-P = 7.3 to 8.2 nm; medium HDL-P = 8.2 to 9.4 nm; And large HDL-P = 9.4 to 14 nm.

Total VLDL-P, LDL-P, and HDL-P are equal to the sum of the particle number concentrations of each of the VLDL, LDL, and HDL subclasses, respectively. The endpoint of the analysis was the percent change in LDL-P, VLDL-P, and HDL-P concentrations from baseline to week 12.

For all statistical tests, any variable of the non-normal distribution is converted, but the untransformed variables are listed in the table in this article. The mean (standard deviation [SD]) is reported for variables with continuous normal distribution, while the median (internal interquartile range [IQR]) is reported for any variable with a non-normal distribution. For category variables, counts and percentages are reported. To determine if the treatment group was significantly different from the placebo group, Fisher's exact test was performed and the associated P value was reported. To determine if each treatment group was significantly different from the placebo group, a t- test was performed and the associated P value was reported. To determine if there was a significant difference in the percentage change between baseline and week 12 in the placebo group compared to the treatment group, covariate analysis was performed using baseline values as covariates.

result

Table 6 shows the concentration of lipoprotein particles before and after subcutaneous injection of mAb 316P 150 mg every 2 weeks compared to placebo.

Compared with the 1% reduction in placebo, mAb316P decreased the mean LDL-P by 63% (P < 0.0001); mAb 316P decreased the median VLDL-P by 36% relative to the 33% increase in placebo ( P < 0.0001). Compared with the 1% increase in placebo, mAb316P increased HDL-P levels by 11% (P < 0.05). All particle subclass changes are similar in direction.

in conclusion

mAb316P significantly reduces LDL-P and other lipoprotein particles in a manner The previously reported effects on LDL-C and apolipoprotein B are similar.

The scope of the invention is not limited by the specific embodiments described herein. It is to be understood that various modifications of the invention will be apparent to those skilled in the <RTIgt; These modifications are also within the scope of the attached patent application.

<110> Gary Swergold William J.Sasiela Robert C.Pordy

<120> Method for reducing residual cholesterol and other lipoprotein components by using pre-protein converting enzyme subtilisin-9 (PCSK9) inhibitor

<130> 7008P5

<140> To be specified

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<400> 60

<210> 61

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 61

<210> 62

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 62

<210> 63

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 63

<210> 64

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 64

<210> 65

<211> 342

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 65

<210> 66

<211> 114

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 66

<210> 67

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 67

<210> 68

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 68

<210> 69

<211> 342

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 69

<210> 70

<211> 114

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 70

<210> 71

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 71

<210> 72

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 72

<210> 73

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 73

<210> 74

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 74

<210> 75

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 75

<210> 76

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 76

<210> 77

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 77

<210> 78

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 78

<210> 79

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 79

<210> 80

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 80

<210> 81

<211> 342

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 81

<210> 82

<211> 114

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 82

<210> 83

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 83

<210> 84

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 84

<210> 85

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 85

<210> 86

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 86

<210> 87

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 87

<210> 88

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 88

<210> 89

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 89

<210> 90

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 90

<210> 91

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 91

<210> 92

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 92

<210> 93

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 93

<210> 94

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 94

<210> 95

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 95

<210> 96

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 96

<210> 97

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 97

<210> 98

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 98

<210> 99

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 99

<210> 100

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 100

<210> 101

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 101

<210> 102

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 102

<210> 103

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 103

<210> 104

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 104

<210> 105

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 105

<210> 106

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 106

<210> 107

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 107

<210> 108

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 108

<210> 109

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 109

<210> 110

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 110

<210> 111

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 111

<210> 112

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 112

<210> 113

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 113

<210> 114

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 114

<210> 115

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 115

<210> 116

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 116

<210> 117

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 117

<210> 118

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 118

<210> 119

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 119

<210> 120

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 120

<210> 121

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 121

<210> 122

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 122

<210> 123

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 123

<210> 124

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 124

<210> 125

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 125

<210> 126

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 126

<210> 127

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 127

<210> 128

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 128

<210> 129

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 129

<210> 130

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 130

<210> 131

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 131

<210> 132

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 132

<210> 133

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 133

<210> 134

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 134

<210> 135

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 135

<210> 136

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 136

<210> 137

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 137

<210> 138

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 138

<210> 139

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 139

<210> 140

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 140

<210> 141

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 141

<210> 142

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 142

<210> 143

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 143

<210> 144

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 144

<210> 145

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 145

<210> 146

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 146

<210> 147

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 147

<210> 148

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 148

<210> 149

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 149

<210> 150

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 150

<210> 151

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 151

<210> 152

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 152

<210> 153

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 153

<210> 154

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 154

<210> 155

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 155

<210> 156

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 156

<210> 157

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 157

<210> 158

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 158

<210> 159

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 159

<210> 160

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 160

<210> 161

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 161

<210> 162

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 162

<210> 163

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 163

<210> 164

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 164

<210> 165

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 165

<210> 166

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 166

<210> 167

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 167

<210> 168

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 168

<210> 169

<211> 375

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 169

<210> 170

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 170

<210> 171

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 171

<210> 172

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 172

<210> 173

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 173

<210> 174

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 174

<210> 175

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 175

<210> 176

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 176

<210> 177

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 177

<210> 178

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 178

<210> 179

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 179

<210> 180

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 180

<210> 181

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 181

<210> 182

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 182

<210> 183

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 183

<210> 184

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 184

<210> 185

<211> 375

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 185

<210> 186

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 186

<210> 187

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 187

<210> 188

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 188

<210> 189

<211> 375

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 189

<210> 190

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 190

<210> 191

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 191

<210> 192

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 192

<210> 193

<211> 375

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 193

<210> 194

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 194

<210> 195

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 195

<210> 196

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 196

<210> 197

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 197

<210> 198

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 198

<210> 199

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 199

<210> 200

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 200

<210> 201

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 201

<210> 202

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 202

<210> 203

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 203

<210> 204

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 204

<210> 205

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 205

<210> 206

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 206

<210> 207

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 207

<210> 208

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 208

<210> 209

<211> 375

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 209

<210> 210

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 210

<210> 211

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 211

<210> 212

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 212

<210> 213

<211> 375

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 213

<210> 214

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 214

<210> 215

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 215

<210> 216

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 216

<210> 217

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 217

<210> 218

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 218

<210> 219

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 219

<210> 220

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 220

<210> 221

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 221

<210> 222

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 222

<210> 223

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 223

<210> 224

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 224

<210> 225

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 225

<210> 226

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 226

<210> 227

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 227

<210> 228

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 228

<210> 229

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 229

<210> 230

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 230

<210> 231

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 231

<210> 232

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 232

<210> 233

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 233

<210> 234

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 234

<210> 235

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 235

<210> 236

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 236

<210> 237

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 237

<210> 238

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 238

<210> 239

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 239

<210> 240

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 240

<210> 241

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 241

<210> 242

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 242

<210> 243

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 243

<210> 244

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 244

<210> 245

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 245

<210> 246

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 246

<210> 247

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 247

<210> 248

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 248

<210> 249

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 249

<210> 250

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 250

<210> 251

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 251

<210> 252

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 252

<210> 253

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 253

<210> 254

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 254

<210> 255

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 255

<210> 256

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 256

<210> 257

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 257

<210> 258

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 258

<210> 259

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 259

<210> 260

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 260

<210> 261

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 261

<210> 262

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 262

<210> 263

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 263

<210> 264

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 264

<210> 265

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 265

<210> 266

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 266

<210> 267

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 267

<210> 268

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 268

<210> 269

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 269

<210> 270

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 270

<210> 271

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 271

<210> 272

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 272

<210> 273

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 273

<210> 274

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 274

<210> 275

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 275

<210> 276

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 276

<210> 277

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 277

<210> 278

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 278

<210> 279

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 279

<210> 280

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 280

<210> 281

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 281

<210> 282

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 282

<210> 283

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 283

<210> 284

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 284

<210> 285

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 285

<210> 286

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 286

<210> 287

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 287

<210> 288

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 288

<210> 289

<211> 372

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 289

<210> 290

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 290

<210> 291

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 291

<210> 292

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 292

<210> 293

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 293

<210> 294

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 294

<210> 295

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 295

<210> 296

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 296

<210> 297

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 297

<210> 298

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 298

<210> 299

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 299

<210> 300

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 300

<210> 301

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 301

<210> 302

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 302

<210> 303

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 303

<210> 304

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 304

<210> 305

<211> 372

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 305

<210> 306

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 306

<210> 307

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 307

<210> 308

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 308

<210> 309

<211> 372

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 309

<210> 310

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 310

<210> 311

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 311

<210> 312

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 312

<210> 313

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 313

<210> 314

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 314

<210> 315

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 315

<210> 316

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 316

<210> 317

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 317

<210> 318

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 318

<210> 319

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 319

<210> 320

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 320

<210> 321

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 321

<210> 322

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 322

<210> 323

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 323

<210> 324

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 324

<210> 325

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 325

<210> 326

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 326

<210> 327

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 327

<210> 328

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 328

<210> 329

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 329

<210> 330

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 330

<210> 331

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 331

<210> 332

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 332

<210> 333

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 333

<210> 334

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 334

<210> 335

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 335

<210> 336

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 336

<210> 337

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 337

<210> 338

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 338

<210> 339

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 339

<210> 340

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 340

<210> 341

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 341

<210> 342

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 342

<210> 343

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 343

<210> 344

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 344

<210> 345

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 345

<210> 346

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 346

<210> 347

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 347

<210> 348

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 348

<210> 349

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 349

<210> 350

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 350

<210> 351

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 351

<210> 352

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 352

<210> 353

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 353

<210> 354

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 354

<210> 355

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 355

<210> 356

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 356

<210> 357

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 357

<210> 358

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 358

<210> 359

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 359

<210> 360

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 360

<210> 361

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 361

<210> 362

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 362

<210> 363

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 363

<210> 364

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 364

<210> 365

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 365

<210> 366

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 366

<210> 367

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 367

<210> 368

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 368

<210> 369

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 369

<210> 370

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 370

<210> 371

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 371

<210> 372

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 372

<210> 373

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 373

<210> 374

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 374

<210> 375

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 375

<210> 376

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 376

<210> 377

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 377

<210> 378

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 378

<210> 379

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 379

<210> 380

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 380

<210> 381

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 381

<210> 382

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 382

<210> 383

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 383

<210> 384

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 384

<210> 385

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 385

<210> 386

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 386

<210> 387

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 387

<210> 388

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 388

<210> 389

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 389

<210> 390

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 390

<210> 391

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 391

<210> 392

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 392

<210> 393

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 393

<210> 394

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 394

<210> 395

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 395

<210> 396

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 396

<210> 397

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 397

<210> 398

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 398

<210> 399

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 399

<210> 400

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 400

<210> 401

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 401

<210> 402

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 402

<210> 403

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 403

<210> 404

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 404

<210> 405

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 405

<210> 406

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 406

<210> 407

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 407

<210> 408

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 408

<210> 409

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 409

<210> 410

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 410

<210> 411

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 411

<210> 412

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 412

<210> 413

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 413

<210> 414

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 414

<210> 415

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 415

<210> 416

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 416

<210> 417

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 417

<210> 418

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 418

<210> 419

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 419

<210> 420

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 420

<210> 421

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 421

<210> 422

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 422

<210> 423

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 423

<210> 424

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 424

<210> 425

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 425

<210> 426

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 426

<210> 427

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 427

<210> 428

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 428

<210> 429

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 429

<210> 430

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 430

<210> 431

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 431

<210> 432

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 432

<210> 433

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 433

<210> 434

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 434

<210> 435

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 435

<210> 436

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 436

<210> 437

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 437

<210> 438

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 438

<210> 439

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 439

<210> 440

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 440

<210> 441

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 441

<210> 442

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 442

<210> 443

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 443

<210> 444

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 444

<210> 445

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 445

<210> 446

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 446

<210> 447

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 447

<210> 448

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 448

<210> 449

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 449

<210> 450

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 450

<210> 451

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 451

<210> 452

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 452

<210> 453

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 453

<210> 454

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 454

<210> 455

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 455

<210> 456

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 456

<210> 457

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 457

<210> 458

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 458

<210> 459

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 459

<210> 460

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 460

<210> 461

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 461

<210> 462

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 462

<210> 463

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 463

<210> 464

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 464

<210> 465

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 465

<210> 466

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 466

<210> 467

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 467

<210> 468

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 468

<210> 469

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 469

<210> 470

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 470

<210> 471

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 471

<210> 472

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 472

<210> 473

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 473

<210> 474

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 474

<210> 475

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 475

<210> 476

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 476

<210> 477

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 477

<210> 478

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 478

<210> 479

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 479

<210> 480

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 480

<210> 481

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 481

<210> 482

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 482

<210> 483

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 483

<210> 484

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 484

<210> 485

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 485

<210> 486

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 486

<210> 487

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 487

<210> 488

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 488

<210> 489

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 489

<210> 490

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 490

<210> 491

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 491

<210> 492

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 492

<210> 493

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 493

<210> 494

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 494

<210> 495

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 495

<210> 496

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 496

<210> 497

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 497

<210> 498

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 498

<210> 499

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 499

<210> 500

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 500

<210> 501

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 501

<210> 502

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 502

<210> 503

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 503

<210> 504

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 504

<210> 505

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 505

<210> 506

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 506

<210> 507

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 507

<210> 508

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 508

<210> 509

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 509

<210> 510

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 510

<210> 511

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 511

<210> 512

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 512

<210> 513

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 513

<210> 514

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 514

<210> 515

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 515

<210> 516

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 516

<210> 517

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 517

<210> 518

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 518

<210> 519

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 519

<210> 520

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 520

<210> 521

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 521

<210> 522

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 522

<210> 523

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 523

<210> 524

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 524

<210> 525

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 525

<210> 526

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 526

<210> 527

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 527

<210> 528

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 528

<210> 529

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 529

<210> 530

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 530

<210> 531

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 531

<210> 532

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 532

<210> 533

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 533

<210> 534

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 534

<210> 535

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 535

<210> 536

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 536

<210> 537

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 537

<210> 538

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 538

<210> 539

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 539

<210> 540

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 540

<210> 541

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 541

<210> 542

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 542

<210> 543

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 543

<210> 544

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 544

<210> 545

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 545

<210> 546

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 546

<210> 547

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 547

<210> 548

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 548

<210> 549

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 549

<210> 550

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 550

<210> 551

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 551

<210> 552

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 552

<210> 553

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 553

<210> 554

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 554

<210> 555

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 555

<210> 556

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 556

<210> 557

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 557

<210> 558

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 558

<210> 559

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 559

<210> 560

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 560

<210> 561

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 561

<210> 562

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 562

<210> 563

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 563

<210> 564

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 564

<210> 565

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 565

<210> 566

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 566

<210> 567

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 567

<210> 568

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 568

<210> 569

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 569

<210> 570

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 570

<210> 571

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 571

<210> 572

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 572

<210> 573

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 573

<210> 574

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 574

<210> 575

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 575

<210> 576

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 576

<210> 577

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 577

<210> 578

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 578

<210> 579

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 579

<210> 580

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 580

<210> 581

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 581

<210> 582

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 582

<210> 583

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 583

<210> 584

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 584

<210> 585

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 585

<210> 586

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 586

<210> 587

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 587

<210> 588

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 588

<210> 589

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 589

<210> 590

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 590

<210> 591

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 591

<210> 592

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 592

<210> 593

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 593

<210> 594

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 594

<210> 595

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 595

<210> 596

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 596

<210> 597

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 597

<210> 598

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 598

<210> 599

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 599

<210> 600

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 600

<210> 601

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 601

<210> 602

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 602

<210> 603

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 603

<210> 604

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 604

<210> 605

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 605

<210> 606

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 606

<210> 607

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 607

<210> 608

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 608

<210> 609

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 609

<210> 610

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 610

<210> 611

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 611

<210> 612

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 612

<210> 613

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 613

<210> 614

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 614

<210> 615

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 615

<210> 616

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 616

<210> 617

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 617

<210> 618

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 618

<210> 619

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 619

<210> 620

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 620

<210> 621

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 621

<210> 622

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 622

<210> 623

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 623

<210> 624

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 624

<210> 625

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 625

<210> 626

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 626

<210> 627

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 627

<210> 628

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 628

<210> 629

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 629

<210> 630

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 630

<210> 631

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 631

<210> 632

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 632

<210> 633

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 633

<210> 634

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 634

<210> 635

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 635

<210> 636

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 636

<210> 637

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 637

<210> 638

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 638

<210> 639

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 639

<210> 640

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 640

<210> 641

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 641

<210> 642

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 642

<210> 643

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 643

<210> 644

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 644

<210> 645

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 645

<210> 646

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 646

<210> 647

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 647

<210> 648

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 648

<210> 649

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 649

<210> 650

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 650

<210> 651

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 651

<210> 652

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 652

<210> 653

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 653

<210> 654

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 654

<210> 655

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 655

<210> 656

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 656

<210> 657

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 657

<210> 658

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 658

<210> 659

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 659

<210> 660

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 660

<210> 661

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 661

<210> 662

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 662

<210> 663

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 663

<210> 664

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 664

<210> 665

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 665

<210> 666

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 666

<210> 667

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 667

<210> 668

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 668

<210> 669

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 669

<210> 670

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 670

<210> 671

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 671

<210> 672

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 672

<210> 673

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 673

<210> 674

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 674

<210> 675

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 675

<210> 676

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 676

<210> 677

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 677

<210> 678

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 678

<210> 679

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 679

<210> 680

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 680

<210> 681

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 681

<210> 682

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 682

<210> 683

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 683

<210> 684

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 684

<210> 685

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 685

<210> 686

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 686

<210> 687

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 687

<210> 688

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 688

<210> 689

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 689

<210> 690

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 690

<210> 691

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 691

<210> 692

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 692

<210> 693

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 693

<210> 694

<211> 127

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 694

<210> 695

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 695

<210> 696

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 696

<210> 697

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 697

<210> 698

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 698

<210> 699

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 699

<210> 700

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 700

<210> 701

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 701

<210> 702

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 702

<210> 703

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 703

<210> 704

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 704

<210> 705

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 705

<210> 706

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 706

<210> 707

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 707

<210> 708

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 708

<210> 709

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 709

<210> 710

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 710

<210> 711

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 711

<210> 712

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 712

<210> 713

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 713

<210> 714

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 714

<210> 715

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 715

<210> 716

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 716

<210> 717

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 717

<210> 718

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 718

<210> 719

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 719

<210> 720

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 720

<210> 721

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 721

<210> 722

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 722

<210> 723

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 723

<210> 724

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 724

<210> 725

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 725

<210> 726

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 726

<210> 727

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 727

<210> 728

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 728

<210> 729

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 729

<210> 730

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 730

<210> 731

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 731

<210> 732

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 732

<210> 733

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 733

<210> 734

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 734

<210> 735

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 735

<210> 736

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 736

<210> 737

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 737

<210> 738

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 738

<210> 739

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 739

<210> 740

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 740

<210> 741

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 741

<210> 742

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 742

<210> 743

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 743

<210> 744

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 744

<210> 745

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221> variant

<222> (1)...(8)

<223> Xaa = any amino acid

<400> 745

<210> 746

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221> variant

<222> (1)...(8)

<223> Xaa-any amino acid

<400> 746

<210> 747

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221> variant

<222> (1)...(20)

<223> Xaa = any amino acid

<400> 747

<210> 748

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221> variant

<222> (1)...(12)

<223> Xaa = any amino acid

<400> 748

<210> 749

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221> variant

<222> (1)...(3)

<223> Xaa = any amino acid

<400> 749

<210> 750

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221> variant

<222> (1)...(9)

<223> Xaa = any amino acid

<400> 750

<210> 751

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 751

<210> 752

<211> 327

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 752

<210> 753

<211> 327

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 753

<210> 754

<211> 2076

<212> DNA

<213> Homo sapiens

<400> 754

<210> 755

<211> 692

<212> PRT

<213> Homo sapiens

<400> 755

<210> 756

<211> 692

<212> PRT

<213> Macaque

<400> 756

<210> 757

<211> 694

<212> PRT

<213> Mus musculus

<400> 757

<210> 758

<211> 653

<212> PRT

<213> Homo sapiens

<400> 758

<210> 759

<211> 753

<212> PRT

<213> Homo sapiens

<400> 759

<210> 760

<211> 785

<212> PRT

<213> Homo sapiens

<400> 760

<210> 761

<211> 692

<212> PRT

<213> Crab-eating macaque

<400> 761

<210> 762

<211> 698

<212> PRT

<213> Golden Hamster

<400> 762

<210> 763

<211> 691

<212> PRT

<213> Brown Rat

<400> 763

Claims (39)

A use of a pharmaceutical composition comprising a PCSK9 inhibitor for the manufacture of a medicament for reducing residual cholesterol in a patient's serum. The use of claim 1, wherein after administration of the pharmaceutical product, residual cholesterol in the serum of the patient decreases from at least about 35% to about 45% from baseline. Use of a pharmaceutical composition comprising a PCSK9 inhibitor for the manufacture of a medicament for reducing VLDL in a patient's serum. The use of claim 3, wherein the VLDL in the serum of the patient drops from the baseline by at least about 20% to about 28% after administration of the pharmaceutical product. Use of a pharmaceutical composition comprising a PCSK9 inhibitor for the manufacture of a medicament for lowering VLDL _1+2- C in a patient's serum. The use of claim 5, wherein the VLDL _1+2- C in the serum of the patient drops from the baseline by at least about 20% to about 32% after administration of the pharmaceutical product. Use of pharmaceutical composition comprising the PCSK9 inhibitors, for the manufacture of reducing serum VLDL ₃ -C pharmaceuticals. The use of claim 7 wherein the VLDL _3- C in the serum of the patient drops from the baseline by at least about 20% to about 27% after administration of the medicament. Use of a pharmaceutical composition comprising a PCSK9 inhibitor for the manufacture of a medicament for lowering IDL-C in a patient's serum. The use of claim 9, wherein the IDL-C level in the patient's serum is decreased from the baseline by at least about 50% to about 56% after administration of the pharmaceutical product. Use of a pharmaceutical composition comprising a PCSK9 inhibitor for the manufacture of a medicament for lowering the serum concentration of at least one low density lipoprotein cholesterol (LDL-C) subcomponent of a patient. For example, the application of the scope of claim 11 wherein the LDL-C subcomponent is LDL ₁ -C. The use of claim 12, wherein the LDL ₁ -C in the serum of the patient drops from the baseline by at least about 65% to about 78% after administration of the pharmaceutical product. The use of the scope of claim 11 wherein the LDL-C subcomponent is LDL ₂ -C. The use of claim 14 wherein the LDL ₂ -C in the serum of the patient drops from the baseline by at least about 75% to about 85% after administration of the medicament. For example, the application of the scope of claim 11 wherein the LDL-C subcomponent is LDL ₃ -C. The use of the scope of claim 11 wherein the LDL-C subcomponent is LDL ₄ -C. For example, the application of the scope of claim 11 wherein the LDL-C subcomponent is LDL _3+4- C. The use of claim 18, wherein the LDL _3+4- C in the serum of the patient drops from the baseline by at least about 45% to about 70% after administration of the pharmaceutical product. The use of any one of claims 1 to 19, wherein the PCSK9 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to PCSK9. The use of claim 20, wherein the pharmaceutical composition comprises from 20 mg to 200 mg of the PCSK9 inhibitor. The use of claim 21, wherein the pharmaceutical composition comprises from 50 mg to 150 mg of the PCSK9 inhibitor. The use of claim 22, wherein the pharmaceutical composition comprises 50 mg of a PCSK9 inhibitor. The use of claim 22, wherein the pharmaceutical composition comprises 75 mg of a PCSK9 inhibitor. The use of claim 22, wherein the pharmaceutical composition comprises 100 mg of a PCSK9 inhibitor. The use of claim 22, wherein the pharmaceutical composition comprises 150 mg of a PCSK9 inhibitor. The use of any one of clauses 20 to 26, wherein the antibody or antigen-binding fragment thereof comprises a weight selected from the group consisting of HCVR/LCVR amino acid sequence pairs of SEQ ID NOs: 90/92 and 218/226 Strand and light chain CDR sequences. The use of claim 27, wherein the antibody or antigen-binding fragment thereof comprises heavy and light chain CDR amino acid sequences of SEQ ID NO: 220, 222, 224, 228, 230 and 232. The use of claim 28, wherein the antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 218, and an LCVR comprising the amino acid sequence of SEQ ID NO: 226. The use of claim 27, wherein the antibody or antigen-binding fragment thereof comprises heavy and light chain CDR amino acid sequences of SEQ ID NO: 76, 78, 80, 84, 86 and 88. The use of claim 30, wherein the antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 90, and an LCVR comprising the amino acid sequence of SEQ ID NO: 92. The use of the scope of claim 20, wherein the antibody or antigen-binding fragment thereof and an antibody comprising a heavy chain and a light chain CDR amino acid sequence bind to the same epitope on PCSK9, and the amino acid sequence has the sequence number of 220, 222, 224, 228, 230, and 232; or 76, 78, 80, 84, 86, and 88. The use of claim 20, wherein the antibody or antigen-binding fragment thereof, when bound to PCSK9, competes with an antibody comprising a heavy chain and a light chain CDR amino acid sequence, the amino acid sequence having the sequence number of 220 , 222, 224, 228, 230, and 232; or 76, 78, 80, 84, 86, and 88. The use of claim 20, wherein the antibody or antigen-binding fragment thereof exhibits a pH-dependent characteristic when bound to PCSK9. The use of the invention in the scope of claim 34, wherein the antibody or antigen-binding fragment thereof is at a neutral pH The binding affinity to PCSK9 is higher than that under acidic pH conditions. The use of any of claims 1, 3, 5, 7, 9 and 11 wherein the patient is receiving treatment with a therapeutic statin at or just prior to administration of the medicament. For use in claim 36, wherein the therapeutic statin treatment comprises a statin selected from the group consisting of cerivastatin, atorvastatin, simvastatin ( Simvastatin), pitavastatin, rosuvastatin, fluvastatin, lovastatin, and pravastatin. For example, the application of the scope of claim 36, wherein the statin is atorvastatin. The use of any of claims 1, 3, 5, 7, 9 and 11 wherein the patient is not receiving therapeutic statin therapy at the time of administration of the medicament.